http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.925919
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RESEARCH ARTICLE
Caco-2 cells permeability evaluation of nifuroxazide derivatives with potential activity against methicillin-resistant Staphylococcus aureus (MRSA) Mariane B. Fernandes1, Jose´ E. Gonc¸alves1, Leoberto C. Tavares2, and Sı´lvia Storpirtis1 1
Pharmacy Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Sa˜o Paulo – SP, Brazil and 2Chemical and Pharmaceutical Technology Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Sa˜o Paulo – SP, Brazil Abstract
Keywords
Throughout the period of evaluation and selection in drug development, the assessment of the permeability potential of a compound to achieve an efficient refinement of the molecular structure has been widely appraised by the transport of substances across cell monolayers. This study aims to develop in vitro assays through Caco-2 cells in order to analyze the permeability of 5-nitro-heterocyclic compounds analogues to nifuroxazide with antimicrobial activity, especially showing promising activity against multidrug-resistant Staphylococcus aureus (MRSA). Caco-2 cell monolayers cultivated for 21 days in Transwell plates were used for the in vitro permeability assays. The quantification of the nifuroxazide derivatives in the basolateral chambers was performed by a validated high performance liquid chromatography with UV (HPLC-UV) method. Apparent permeability values (Papp) show that these compounds can be considered as new drug candidates with the potential to present high absorption in vivo, according to the classifications of Yee and Biganzoli. The thiophenic derivatives showed permeability values higher than the furanic ones, being AminoTIO the compound with the greatest potential for the development of a new drug against MRSA, since it showed the best cytotoxicity, permeability and solubility ratio among all the derivatives.
5-Nitro-heterocyclic, anti-infectives, biopharmacy, drug development, intestinal absorption
Introduction The infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem due to its ease of transmission and wide spread both in hospital and community settings1. The high resistance level that strains of Staphylococcus aureus have shown to the currently available antimicrobial drugs, often as a result of their indiscriminate and improper use, results in the need of search and identification of more effective compounds for the treatment of infections caused by resistant bacteria2. The development of drugs already available in the therapeutics is an affordable alternative able to contribute to promising antimicrobial substances, since the development of drugs with totally new structure is costly and involves years of research3. Nifuroxazide, a synthetic 5-nitrofuran used as second or third choice in the treatment of enteric infection, has excellent features such as a broad spectrum of activity, inability to significantly promote bacterial resistance and chemical structure conducive to change. Thus, this drug is envisioned as a molecule capable of
Address for correspondence: Mariane Ballerini Fernandes, Pharmacy Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 580, bl. 13, Cidade Universita´ria, 05508-900, Sa˜o Paulo – SP, Brazil. Tel: +55 11 3091 36 23. E-mail: [email protected]
History Received 28 March 2014 Revised 11 May 2014 Accepted 12 May 2014 Published online 11 June 2014
leading to the development of new analogs, and possibly lead compounds. 5-Nitro-heterocyclic derivatives of nifuroxazide have shown satisfactory in vitro activity against MRSA strains4,5. The next step in the development of a drug candidate would be the analysis of the processes involved in its pharmacokinetic profile and toxicity, or ADMET (absorption, distribution, metabolism, excretion and toxicity). Once the oral route is the preferred route for the administration of drugs due to its convenience, low cost and high adherence when compared with other routes of administration, adequate screening systems must be implemented in the early stages of drug discovery to evaluate and predict the ability of a substance to be absorbed, avoiding waste of time and resources with compounds with insufficient intestinal absorption6. Several models based on cell cultures for the determination of the in vitro intestinal permeability have been developed and currently acquired great popularity. Among them, the Caco-2 cells, which are cells derived from human colorectal carcinoma, have been extensively employed due to their interesting characteristics, such as their ability to differentiate spontaneously in vitro, expressing many morphological and biochemical features present in the human small intestine. When maintained under appropriate conditions of cultivation, Caco-2 cells grow to form a polarized monolayer of columnar cells with microvilli at the apical side, presenting also the tight junctions between adjacent cells and expressing transport proteins, efflux protein conjugation and phase II enzymes7–11.
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The predictive capability and utility of Caco-2 cells with respect to the analysis of the potential for absorption of substances has been repeatedly demonstrated by various studies8,12–16. Therefore, a compound’s permeability through these cells, called as apparent permeability (Papp), is a valuable index used to estimate the uptake of orally administered drugs6,17. Data obtained by Yee18 using 36 compounds with different structures led the researcher to classify the Papp values according to the absorbed fraction, and as a result a drug can be considered as poorly absorbed (0–20%) when the value of Papp is less than 1 106 cm/s; moderately absorbed (20–70%) when the Papp value is between 1 and 10 106 cm/s; well absorbed (70–100%) when Papp is greater than 10 106 cm/s. Two years later, Biganzoli and colleagues19 obtained Papp data of a number of antimicrobial agents with natural and synthetic origin and compared these values with their oral bioavailability in humans. According to the results, it was suggested a specific classification for antimicrobials, being the Papp values less than 0.2 106 cm/s related to compounds with bioavailability of less than 1%; Papp values between 0.2 and 2 106 cm/s refer to drugs with bioavailability between 1 and 90%; and Papp values higher than 2 106 cm/s correspond to the substances with bioavailability above 90%. Information on the oral absorption of nifuroxazide as well as its intestinal permeability is scarce. However, it is consensus that this drug has low absorption20–22, which explains its therapeutical use as antimicrobial for the treatment of infections in the gastrointestinal tract22. Some researchers, after analyzing human plasma concentrations of the unchanged drug after a single or multi-dose administration, suggest that after its oral administration, nifuroxazide might be extensively metabolized, retained in the tissues or it might not be significantly absorbed. These researchers have also carried out studies on the distribution and elimination of the drug in mice by administrating 10 and 100 mg/kg of nifuroxazide by gastric intubation. Data indicate that approximately 50% of the dose was absorbed and that the nifuroxazide is, in fact, extensively metabolized rather than having low absorption23,24. The use of substances with well established pharmacokinetic characteristics and similar structure to the new drug candidates is of great value in permeability screening assays with Caco-2 cells, increasing the reliability of the results. Nitrofurantoin, a drug with structure similar to nifuroxazide, which is used in the treatment of urinary infections, has been extensively studied with respect to its pharmacokinetics, mainly due to its excretion in human milk. Although it has limited solubility in water, it readily dissolves in alkaline solutions and urine. Buzard and colleagues25 found that this drug is rapidly absorbed by the small intestine, showing increased plasma levels in few minutes. The presence of food in the gastrointestinal tract or the delay in the gastric emptying increase nitrofurantoin absorption by increasing its dissolution rate. The bioavailability is 87% when administered in the absence of food and increases to 94% in its presence. Peak plasma concentrations after a single oral dose of 100 mg is generally achieved within 1–2 h after ingestion and reach 1 mg/mL26. Lin et al.27 evaluated the permeability of some drugs across the Caco-2 cells using inhibitors of efflux transporters for P-gp and BCRP. For nitrofurantoin, the Papp results of the apical to basolateral and basolateral to apical portions were 0.8 and Figure 1. (A) Molecular structure of nitrofurantoin. (B) Molecular structure of nifuroxazide.
Drug Dev Ind Pharm, Early Online: 1–7
18.8 106 cm/s, respectively, in absence of a BCRP inhibitor. In the presence of the inhibitor, the values were 4.0 and 7.2 106 cm/s, respectively, showing the influence of the carrier on the absorption of nitrofurantoin. To date, there is no literature data on nifuroxazide permeability across monolayers of Caco-2 as well as on the series of its analogs present in this study, being necessary their evaluation as next stage in the development of a new antimicrobial drug against MRSA. It is important and necessary to evaluate their bioavailability aiming at an efficient selection and further improvement of the selected molecular structures.
Methods Chemicals Dulbecco’s modified Eagle medium (DMEM) with and without phenol, penicillin, streptomycin, Trypsin–EDTA (0.2%) and fetal calf serum (FCS) and Hank’s balanced salt solution (HBSS) with 200 mM of HEPES salt, with and without phenol and pH adjusted to 7.4 were obtained from Cultilab (Campinas, SP, Brazil). Glutamine, D-glucose, 3-(4,5 dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), nifuroxazide (Figure 1B) and nitrofurantoin (Figure 1A) were purchased from Sigma-Aldrich (St. Louis, MO). The 5-nitro-heterocyclic compounds used in this study were designed and synthesized by the Laboratory of Drug Design and Development of the Chemical and Pharmaceutical Technology Department at the Pharmaceutical Sciences Faculty/University of Sa˜o Paulo. Eight compounds belonging to an analogous series with similar structure to nifuroxazide were selected, as referred in Figure 2. Cell line and culture conditions Human colon adenocarcinoma cell line Caco-2 (ATCC #HTB-37) was purchased from American Type Culture Collection (ATCC, Rockville, MD). Caco-2 cells were cultured in DMEM supplemented with 4.5 g/L D-glucose (Sigma-Aldrich Corp., St. Louis, MO), NaHCO3 (2.2 g/L) (Sigma-Aldrich Corp., St. Louis, MO), 10% FCS (Cultilab, Campinas, SP), 1% non-essential amino acids (Sigma-Aldrich Corp., St. Louis, MO), 100 UI/mL penicillin and 100 mg/mL streptomycin (Cultilab, Campinas, SP) in an atmosphere of 5% CO2 and 90% relative humidity at 37 C. All cells used in this study were between passages 40 and 50. In vitro permeability tests Caco-2 cells were cultured in 12-well or 6-well Transwell (Corning Incorporated, New York, NY) insert filters for 21 days to reach confluence and differentiation at a seeding density of 5 104 cells/cm2. 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 running standard assays using amoxicillin and metoprolol as paracellular and transcellular flux markers, respectively. Only cell monolayers with a TEER above 300 cm2 were used for the transport assays. 5-Nitro-heterocyclic compounds were dissolved in dimethylsulfoxide (DMSO), a solvent frequently used when poorly soluble
DOI: 10.3109/03639045.2014.925919
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Figure 2. Chemical structure of the 5-nitro-heterocyclic derivatives used in this study and their purities.
compounds are evaluated in permeability assays, and subsequently diluted in HBSS buffer at pH 7.4 to prepare the permeability samples in concentrations previously determined by cytotoxic tests28. The compounds EtoTIO and BrFUR had lower concentrations due to their solubility restrictions. The initial concentrations and collection times for each compound studied are described in Table 1. Experiments were initiated with the addition of the 5-nitroheterocyclic solutions in the apical compartment of the Transwell plates, and the basolateral compartment was maintained at pH 7.4. Six replicates for all nitro compounds were assessed. Plates were kept under agitation in an orbital shaker at 37 C (25 rpm). Samples volumes of 200 mL were collected from the basolateral compartment at predetermined times for each compound (Table 1). After each sampling, 200 mL of HBSS buffer was replaced in order to maintain the volume of solution in the compartment. Samples were quantified by high performance liquid chromatography with UV (HPLC-UV) detection on a Shimadzu LC-10A chromatographer (Kyoto, Japan). The methods were developed and validated according to Resolution RE 899 de 29 de maio de 2003 – Guia para validac¸a˜o de me´todos bioanalı´ticos – ANVISA29. Chromatographic separations were obtained using a phenyl-2 HYPERSIL column (150 mm 4.6 mm, 5 mm, Thermo Scientific, Waltham, MA) at 30 C. The mobile phase (1 mL/min) was composed of phosphate buffer 5 mM (pH 3.1) and acetonitrile. The analytical wavelength was set at 370 nm and samples of 50 mL were injected into the HPLC system. The apparent permeability coefficients (Papp, cm/s) were calculated according to the following equation: Papp ¼ ðVR DQ=DtÞ=ðA C0 Þ where VR is the volume of the receiver compartment (basolateral or apical), DQ/Dt is the linear appearance rate of the compound on the receiver chamber (in ng/s), A is the membrane surface area (cm2) and C0 is the initial concentration in the donor compartment (ng/cm3). This calculation requires that the sink conditions are fulfilled; therefore, only receiver concentrations below 10% of the donor concentration were employed in the calculations. Statistics The results are expressed as mean ± standard deviation (SD) of six replicates.
Results Transepithelial electrical resistance results obtained at 10, 15 and 21 days of cell cultivation on the insert filters in DMEM at pH 7.4
Table 1. Initial concentrations and sampling times for each 5-nitro-heterocyclic compound in the permeability experiment through Caco-2 cells monolayers.
Compound Nifuroxazide Nitrofurantoin HFUR MeFUR EtoFUR AminoFUR BrFUR HTIO EtoTIO AminoTIO
Initial concentration (mg/mL)
Sampling times (min)
4.0 4.0 4.0 4.0 4.0 4.0 1.0 4.0 0.5 4.0
15, 30, 45, 60, 90 and 120 15, 30, 45, 60, 90 and 120 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 60, 90, 120 and 180 15, 30, 45 and 60
and TEER results at the beginning and end of the permeability experiment of the derivative EtoTIO in HBSS at pH 7.4 are shown in Figures 3 and 4, respectively. TEER evaluation during cultivation of the membranes showed that the values increase with time until they reach a stable average value of 580–650 V cm2 between 15 and 21 days of the start of the cultivation. TEER values obtained ( cm2) during the permeability experiment for the compound EtioTIO ranged from 652 to 468. Despite the decreased levels of TEER, these did not reach values below 200 cm2. Papp values of the 5-nitro-heterocyclic, shown in Table 2, were determined from the amount permeated through the Caco-2 cells membranes at the apical direction-basolateral. All the nifuroxazide derivatives Papp values calculated in this study were greater than 15 106 cm/s.
Discussion Over the past 20 years there has been growth in the use of Caco-2 cells as a rapid screening tool for in vitro intestinal permeability assessment, supporting the development of drugs and directing the changes of physicochemical characteristics responsible for absorption7,11,12,16,30. During the Caco-2 cells membrane formation, it is necessary to assess its integrity which, in this experiment, was first evaluated through the TEER values. The use of this parameter as an indicator of the membrane integrity formed by Caco-2 cells has been widely used in this technique, and significant correlations between the TEER values and permeability of compounds absorbed by the paracellular route have been obtained31. TEER values obtained in this study demonstrate that the membranes formed had adequate integrity,
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Figure 3. TEER values obtained with the Caco-2 cells membranes at 10, 15 and 21 days of culture in DMEM medium with pH 7.4. Average of six determinations.
Figure 4. TEER values obtained with the membranes of Caco-2 cells under the permeability test conditions of the derived EtoTIO at the beginning (T0) and at the end (180 min) of the experiment. Average of six determinations.
Table 2. Papp values of the 5-nitro-heterocyclic compounds determined from the amount permeated through the Caco-2 cells monolayers. Compound Nifuroxazide Nitrofurantoin HFUR MeFUR EtoFUR AminoFUR BrFUR HTIO AminoTIO
Papp (106 cm/s) 7.86 ± 0.94 2.08 ± 0.20 53.89 ± 0.60 39.66 ± 0.67 31.77 ± 0.42 15.55 ± 2.40 26.03 ± 3.03 61.58 ± 5.10 42.95 ± 3.44
with the formation of junctional intercellular complex. Similar values to those obtained are reported in the literature32–34. TEER assessment was also performed prior to the beginning and at the end of the experiments and only the membranes with TEER values above 200 V cm2 were selected for conducting the tests. It was observed an average reduction of 60 V cm2 in each hour of the experiment; however, the values were never below 200 V cm2, keeping an average of 470 V cm2 at the end of three hours of experiment for all compounds.
Another way of assessing membrane integrity and viability during the permeability experiments is by determining the permeability of compounds with well known Papp data. Many drugs are used for this purpose, especially mannitol, digoxin, cimetidine and fluorescein. Amoxicillin and metoprolol were used in this study as low and high permeability standards, respectively, to assess the adequacy and integrity of the culture conditions of the membranes of Caco-2 cells. Although both present high in vivo absorption, amoxicillin shows low Caco-2 cells permeability due to the low expression of PEPT1, which is the main transporter responsible for its in vivo uptake. Metoprolol, on the other hand, has the passive transcellular via as main absorption mechanism. Both are part of the recommendation list of the guide issued by the Food and Drug Administration (FDA) regulatory agency, which provides information for biowaivers35. The mean of the Papp values obtained for amoxicillin was 0.69 106 cm/s, and for metoprolol, 27.27 106 cm/s. These values are in agreement with those obtained in the literature, indicating adequacy of cultivation conditions of Caco-2 cells and reliability to the results obtained for the 5-nitro-heterocyclic compounds36–38. Given the current knowledge about the intestinal absorption of the drug nitrofurantoin, as well as the structural similarity between this and nifuroxazide and its derivatives, the permeability evaluation of the former was used as a parameter for the other 5-nitro-heterocyclic compounds. Additionally, the existence of Papp values for nitrofurantoin in the literature calculated by assays employing Caco-2 cells enabled comparison of the results obtained in this study. The Papp value calculated in this study (2.08 106 cm/s) is approximately 2–3 times greater than the values reported by Lin and coworkers27 (0.6, 0.8 and 1.1 106 cm/s), being this difference probably attributed to the way how Papp was calculated in both studies. In the former, a minimum of three samples was obtained for all compounds and were used for the construction of the concentration curve relative to the time of the permeation test. In the latter, a single sampling at the end of the experiment was made, being the first point in the curve a zero concentration for time zero (T0). If the Papp value for the present data was calculated using the latter approach, it would result in a Papp value of 1.4 106 cm/s, which is a closer and comparable value to those obtained by Lin and coworkers27. Thus, in addition to the standardization of the culture and experimental conditions in order to get comparable Papp values among different laboratories, the establishment of a protocol for the calculation of this parameter is also a pre-requisite for the robustness of the in vitro permeability tests through Caco-2 cells. Placing the results obtained by both laboratories according to Yee classification, the average Papp values calculated by Lin et al.27 would indicate that nitrofurantoin is a drug with low absorption (between 0 and 20% of absorption), while the Papp values obtained in this study would indicate it as a drug with moderate absorption (between 20 and 70% of absorption) (Figure 5). By performing the same analysis according to the classification of Biganzoli and coworkers19 different results are observed (Figure 5). The former would indicate nitrofurantoin is a drug with moderate absorption (between 1 and 90% of bioavailability), while results from this study would classify nitrofurantoin as a drug with high absorption (bioavailability greater than 90%). Since nitrofurantoin absorption rates found in literature are 87–94%26, it is likely that the Biganzoli and coworkers19 classification of the permeability values through Caco-2 is more suitable to what occurs in vivo with this drug and, hence, it could be suitable to analyze the permeability potential of the other 5-nitro-heterocyclic compounds present in this study. Since there is no evidence to date in the literature of Papp values for the drug nifuroxazide calculated in permeability tests
DOI: 10.3109/03639045.2014.925919
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Figure 5. Representation of Yee’s and Biganzoli’s classification for the Papp values of D (triangle) nitrofurantoin (Lin et al.)27, O (circle) nitrofurantoin, œ (square) nifuroxazide and * (star) AminoFur.
through Caco-2, this study is the first to report this parameter to the drug. Placing the Papp value calculated for nifuroxazide (7.86 106 cm/s) according to the Yee classification, nifuroxazide would be classified as a drug with moderate absorption (between 20 and 70% absorption). According to the Biganzoli classification, however, the drug would be placed in the drugs with high absorption band (absorption 490%) (Figure 5). As previously highlighted, the latter classification would be more suitable to indicate what happens in vivo with 5-nitro-heterocyclic compounds, that is, nifuroxazide could be highly absorbed in vivo. Although little information about the pharmacokinetics of nifuroxazide is available in the literature, it is believed that it has low absorption20–22. However, there is evidence that this drug presents considerable absorption and is extensively metabolized in the liver23, which would explain the low levels of unchanged nifuroxazide found in studies with human plasma after oral administration of the drug20,21. The high Papp value found for nifuroxazide in this study, taking into account that there is no hepatic metabolism in the Caco-2 membranes technique corroborates, therefore, with the hypothesis that this drug has significant absorption and extensive metabolism in vivo. Additionally, according to the Biopharmaceutics Drug Disposition Classification System (BDDCS), a classification developed to predict the in vivo disposition of drugs from measurements of solubility and metabolism (clearance route), nifuroxazide belongs to class II. This means that this drug is classified as a drug with low solubility and high permeability, and is extensively metabolized39; therefore, corroborating with the hypothesis that the drug is considerably absorbed in vivo. All Papp values calculated in this study for the nifuroxazide derivatives are greater than 15 106 cm/s. Therefore, employing either the Yee or the Biganzoli classification, these compounds would have considerable potential for presenting high permeability in vivo. Also, Paap values are closer to metoprolol Papp (27.27 106 cm/s) than to amoxicillin (0.69 106 cm/s), what corroborates the assumption of high permeability in vivo, implying that the mechanism of permeability of these derivatives would be mainly through transcellular transport rather than via PEPT1 or another low expressed transporter in Caco-2 cells. The thiophenic derivatives showed permeability values higher than the furanic ones, with minimum Papp values of 42.95 106 cm/s (AminoTIO) and 15.55 106 cm/s (AminoFUR), respectively. This fact could be explained by the higher lipophilicity of the former in relation to the latter, which would facilitate their passage through the membranes of Caco-2 cells. Additionally, due to an increased permeability of the
thiophenic derivatives by consequence of their lipophilicity, these may have wider distribution in the body, which would make them more suitable than the furanic derivatives in the treatment of the main infections caused by MRSA, which affect skin and soft tissue. In addition to the increased permeability presented by the thiophenic derivatives, these were less toxic in the cytotoxicity assays previously discussed28, noting that the toxicity of the nifuroxazide derivatives may be influenced by both the reactivity of the atom in the ring linked to the nitro group as well as the substituent on the benzene ring. Compounds with more hydrophobic substituents, for imparting greater toxicity to the molecule, should be avoided within this series of compounds. However, it is noteworthy that the highest lipophilicity of the thiophenic derivatives entails lower solubility in aqueous media, as noted in the stage of the methods development for quantifying this study. EtoTIO was the lowest soluble compound, hence a lower concentration was used (0.5 mg/mL) in the permeability tests. However, its Papp could not be calculated since the permeated concentrations were below the quantitation limit of the method developed by HPLC. Despite the problems related to solubility, the thiophenic derivatives presented the best results in terms of cytotoxicity28 and permeability, and have a greater potential to be more promising than the furanic derivatives. Among these derivatives, AminoTIO was the compound identified as the one with the greatest potential for the development of a new drug, that is, the one which has the best relationship between cytotoxicity (410 mg/ mL; nifuroxazide: 56 mg/mL)28, permeability (42.95 106 cm/s; nifuroxazide: 7.86 106 cm/s) and solubility (410 mg/mL; nifuroxazide: 410 mg/mL). In the development of a product containing this compound, the solubility-related problems could be minimized by the use of pharmacotechnical knowledge to improve the solubility, such as micronization, the use of solid dispersions40, the formation of soluble complexes like cyclodextrins, or dendrimers41, as well as use of nanostructured systems42, such as nanoparticles and nanocapsules43. As the next step in the development of a new drug with activity against multidrugresistant strains of S. aureus, tests must be conducted with the derivative AminoTIO aiming at deepening the knowledge acquired regarding its permeability. It is necessary to carry out mechanistic studies related to the absorption of this compound, as the elucidation of the prevalence of active or passive transport and the influence of efflux systems, such as the one which occurs with nitrofurantoin, which is a substrate of BCRP. Additionally, in vitro studies must be conducted aiming at the evaluation of its metabolites, employing, for example, models that use liver cells.
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Conclusions
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The results obtained in this study indicate that the Papp value calculated for nitrofurantoin is in accordance with what occurs in vivo with the drug. The Papp value calculated for nifuroxazide corroborates with the hypothesis that the drug presents a considerable absorption and is extensively metabolized in the liver. Papp values of the nifuroxazide derivatives show that these can be considered as new drug candidates with the potential to present high absorption in vivo. The thiophenic derivatives showed permeability values higher than the furanic derivatives, being the AminoTIO the compound with greater potential for the development of a new drug against multidrug-resistant strains of S. aureus, as it showed the best relationship between cytotoxicity, permeability and solubility.
Acknowledgements We would like to thank Alex Alfredo de Oliveira, Fanny Palace Berl, Joa˜o Sussumu Murayama, Leandro Bortolozzo, Rodrigo Rocha, Saloma˜o Do´ria Jorge from the Laboratory of Drug Design and Development of the Chemical and Pharmaceutical Technology Department at the Pharmaceutical Sciences Faculty/University of Sa˜o Paulo for the synthesis of the nifuroxazide derivatives.
14.
15. 16. 17.
18. 19. 20.
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Declaration of interest The authors report no declarations of interest.
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rat ileum model of the intestinal mucosa. Pharm Res 1993;10: 1710–14. Conradi RA, Wilkinson KF, Rush BD, et al. In vitro/in vivo models for peptide oral absorption: comparison of Caco-2 cell permeability with rat intestinal absorption of rennin inhibitory peptides. Pharm Res 1993;10:1790–2. Rubas W, Villagran J, Cromwell M, et al. Correlation of solute flux across Caco-2 monolayers and colonic tissue in vitro. S.T.p. Pharma Sci 1995;5:93–7. Ginski JM, Polli JE. Prediction of dissolution/absorption relationships from a dissolution/Caco-2 system. Int J Pharm 1999;177: 117–25. Gonc¸alves JE, Souza J, Storpirtis S. Avaliac¸a˜o da permeabilidade de fa´rmacos empregando culturas celulares. In: Storpirtis S, Gonc¸alves JE, Chiann C, Gai MN, eds. Biofarmacote´cnica – se´rie cieˆncias farmaceˆuticas. Rio de Janeiro: Guanabara Koogan; 2009:204–11. Yee S. In vitro permeability across Caco-2 cells (Colonic) can predict in vivo (small intestine) absorption in man: fact or myth. Pharm Res 1997;14:763–6. Biganzoli E, Cavenaghi LA, Rossi R, et al. Use of a Caco-2 cell culture model for the characterization of intestinal absorption of antibiotics. Il Farmaco 1999;54:594–9. Caekenberghe DLV, Breyssens J. In vitro synergistic activity between bismuth subcitrate and various antimicrobial agents against Campylobacter pyloridis (C. pylori). Antimicrob Agents Chemother 1987;31:1429–30. Cedillo-Rivera R, Munoz O. In vitro susceptibility of Giardia lamblia to albendazole, mebendazole and other chemotherapeutic agents. J Med Microbiol 1992;37:221–4. Ercefuryl : nifuroxazide. France: Sanofi-Aventis; 2007. Medicine information Available from: http://urunler.sanofi.com.tr/ Ercefuryl_kapsul_RR.pdf [last accessed 01 Jun 2014]. Guinebault PR, Broquaire M, Thebault J, et al. Determination of nifuroxazide and furazolidone in biological fluids. Application to the comparative study of their intestinal absorption. In: Aiache JM, Hirtz J, eds. First European Congress of Biopharmaceutics and Pharmacokinetics, Paris. Eur J Drug Metabol Pharmacok 1981;8:151–8. Labaune JP, Moreau JP, Byrne R. Comparative physiological disposition of two nitrofuran anti-microbial agents. Biopharmaceut Drug Dispos 1986;7:431–41. Buzard JA, Conklin JD, O’Keefe E, et al. Studies on the absorption, distribution and elimination of nitrofurantoin in the rat. J Pharmacol Exp Ther 1961;131:38–43. Shah RR, Wade G. Reappraisal of the risk/benefit of nitrofurantoin: review of toxicity and efficacy. Adverse Drug React Acute Poisoning Rev 1989;8:183–201. Lin X, Skolnik S, Chen X, et al. Attenuation of intestinal absorption by major efflux transporters: quantitative tools and strategies using a Caco-2 model. Drug Metab Dispos 2011;39:265–74. Fernandes MB, Gonc¸alves JE, Scotti MT, et al. Caco-2 cells cytotoxicity of nifuroxazide derivatives with potential activity against Methicillin-resistant Staphylococcus aureus (MRSA). Toxicol In Vitro 2012;26:535–40. Brasil. Resoluc¸a˜o RDC n.899, de 29 de maio de 2003: Guia para validac¸a˜o de me´todos analı´ticos e bioanalı´ticos. Artursson P, Magnusson C. Epithelial transport of drug in cell culture. II: effect of extracellular calcium concentration on paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J Pharm Sci 1990;79: 595–600. Hidalgo IH. Cultured intestinal epithelial cell models. In: Borchard T, Smith PL, Wilson G, eds. Models of assessing drug absorption and metabolism. New York: Plenum Press; 1996:35–50. Behrens I, Kissel T. Do cell culture conditions influence the carriermediated transport of peptides in Caco-2 cell monolayers? Eur J Pharmaceut Sci 2003;19:433–42. Schmidt E, Kelly SM, Van der Walle CF. Tight junction modulation and biochemical characterization of the zonula occludens toxin C-and N-Termini. FEBS Lett 2007;58:2974–80. Rutishauser BR, Braun A, Gu¨nthert M, et al. Formation of multilayers in the Caco-2 cell culture model: a confocal laser scanning microscopy study. Pharmaceut Res 2000;17:460–5. Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System. U.S.
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Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); 2000. Sun D, Lennerna¨s H, Welage LS, et al. Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequence tags and correlation with permeability of 26 drugs. Pharm Res 2002;19:1400–16. Teksin ZS, Seo PR, Polli JE. Comparison of drug permeabilities and BCS classification: three lipid-component PAMPA system method versus Caco-2 monolayers. AAPS J 2010;12:2507–21. Masungi C, Borremans C, Willems B, et al. Usefulness of a novel Caco-2 cell perfusion system. I. In vitro prediction of the absorption potential of passively diffused compounds. J Pharm Sci 2004;93: 238–41. Wu C-Y, Benet LZ. Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of
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a Biopharmaceutics Drug Disposition Classification System. Pharm Res 2005;22:11–23. Jang D-J, Sim T, Oh E. Formulation and optimization of spray-dried amlodipine solid dispersion for enhanced oral absorption. Drug Dev Ind Pharm 2013;39:1133–41. Jain S, Kesharwani P, Tekade RK, Jain NK. One platform comparison of solubilization potential of dendrimer with some solubilizing agents. Drug Dev Ind Pharm. [Epub ahead of print]. doi:10.3109/03639045.2014.900077. Patel GV, Patel VB, Pathak A, Rajput SJ. Nanosuspension of efavirenz for improved oral bioavailability: formulation optimization, in vitro, in situ and in vivo evaluation. Drug Dev Ind Pharm 2014;40:80–91. Ferraz HG. Novas ferramentas farmacote´cnicas para modular a biodisponibilidade de medicamentos. In: Storpirtis S, Gonc¸alves S, Chiann C, Gai MN, eds. Biofarmacote´cnica – se´rie cieˆncias farmaceˆuticas. Rio de Janeiro: Guanabara Koogan; 2009:66–71.
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RESEARCH ARTICLE
Caco-2 cells permeability evaluation of nifuroxazide derivatives with potential activity against methicillin-resistant Staphylococcus aureus (MRSA) Mariane B. Fernandes1, Jose´ E. Gonc¸alves1, Leoberto C. Tavares2, and Sı´lvia Storpirtis1 1
Pharmacy Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Sa˜o Paulo – SP, Brazil and 2Chemical and Pharmaceutical Technology Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Sa˜o Paulo – SP, Brazil Abstract
Keywords
Throughout the period of evaluation and selection in drug development, the assessment of the permeability potential of a compound to achieve an efficient refinement of the molecular structure has been widely appraised by the transport of substances across cell monolayers. This study aims to develop in vitro assays through Caco-2 cells in order to analyze the permeability of 5-nitro-heterocyclic compounds analogues to nifuroxazide with antimicrobial activity, especially showing promising activity against multidrug-resistant Staphylococcus aureus (MRSA). Caco-2 cell monolayers cultivated for 21 days in Transwell plates were used for the in vitro permeability assays. The quantification of the nifuroxazide derivatives in the basolateral chambers was performed by a validated high performance liquid chromatography with UV (HPLC-UV) method. Apparent permeability values (Papp) show that these compounds can be considered as new drug candidates with the potential to present high absorption in vivo, according to the classifications of Yee and Biganzoli. The thiophenic derivatives showed permeability values higher than the furanic ones, being AminoTIO the compound with the greatest potential for the development of a new drug against MRSA, since it showed the best cytotoxicity, permeability and solubility ratio among all the derivatives.
5-Nitro-heterocyclic, anti-infectives, biopharmacy, drug development, intestinal absorption
Introduction The infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) is a serious problem due to its ease of transmission and wide spread both in hospital and community settings1. The high resistance level that strains of Staphylococcus aureus have shown to the currently available antimicrobial drugs, often as a result of their indiscriminate and improper use, results in the need of search and identification of more effective compounds for the treatment of infections caused by resistant bacteria2. The development of drugs already available in the therapeutics is an affordable alternative able to contribute to promising antimicrobial substances, since the development of drugs with totally new structure is costly and involves years of research3. Nifuroxazide, a synthetic 5-nitrofuran used as second or third choice in the treatment of enteric infection, has excellent features such as a broad spectrum of activity, inability to significantly promote bacterial resistance and chemical structure conducive to change. Thus, this drug is envisioned as a molecule capable of
Address for correspondence: Mariane Ballerini Fernandes, Pharmacy Department, Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Av. Prof. Lineu Prestes, 580, bl. 13, Cidade Universita´ria, 05508-900, Sa˜o Paulo – SP, Brazil. Tel: +55 11 3091 36 23. E-mail: [email protected]
History Received 28 March 2014 Revised 11 May 2014 Accepted 12 May 2014 Published online 11 June 2014
leading to the development of new analogs, and possibly lead compounds. 5-Nitro-heterocyclic derivatives of nifuroxazide have shown satisfactory in vitro activity against MRSA strains4,5. The next step in the development of a drug candidate would be the analysis of the processes involved in its pharmacokinetic profile and toxicity, or ADMET (absorption, distribution, metabolism, excretion and toxicity). Once the oral route is the preferred route for the administration of drugs due to its convenience, low cost and high adherence when compared with other routes of administration, adequate screening systems must be implemented in the early stages of drug discovery to evaluate and predict the ability of a substance to be absorbed, avoiding waste of time and resources with compounds with insufficient intestinal absorption6. Several models based on cell cultures for the determination of the in vitro intestinal permeability have been developed and currently acquired great popularity. Among them, the Caco-2 cells, which are cells derived from human colorectal carcinoma, have been extensively employed due to their interesting characteristics, such as their ability to differentiate spontaneously in vitro, expressing many morphological and biochemical features present in the human small intestine. When maintained under appropriate conditions of cultivation, Caco-2 cells grow to form a polarized monolayer of columnar cells with microvilli at the apical side, presenting also the tight junctions between adjacent cells and expressing transport proteins, efflux protein conjugation and phase II enzymes7–11.
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The predictive capability and utility of Caco-2 cells with respect to the analysis of the potential for absorption of substances has been repeatedly demonstrated by various studies8,12–16. Therefore, a compound’s permeability through these cells, called as apparent permeability (Papp), is a valuable index used to estimate the uptake of orally administered drugs6,17. Data obtained by Yee18 using 36 compounds with different structures led the researcher to classify the Papp values according to the absorbed fraction, and as a result a drug can be considered as poorly absorbed (0–20%) when the value of Papp is less than 1 106 cm/s; moderately absorbed (20–70%) when the Papp value is between 1 and 10 106 cm/s; well absorbed (70–100%) when Papp is greater than 10 106 cm/s. Two years later, Biganzoli and colleagues19 obtained Papp data of a number of antimicrobial agents with natural and synthetic origin and compared these values with their oral bioavailability in humans. According to the results, it was suggested a specific classification for antimicrobials, being the Papp values less than 0.2 106 cm/s related to compounds with bioavailability of less than 1%; Papp values between 0.2 and 2 106 cm/s refer to drugs with bioavailability between 1 and 90%; and Papp values higher than 2 106 cm/s correspond to the substances with bioavailability above 90%. Information on the oral absorption of nifuroxazide as well as its intestinal permeability is scarce. However, it is consensus that this drug has low absorption20–22, which explains its therapeutical use as antimicrobial for the treatment of infections in the gastrointestinal tract22. Some researchers, after analyzing human plasma concentrations of the unchanged drug after a single or multi-dose administration, suggest that after its oral administration, nifuroxazide might be extensively metabolized, retained in the tissues or it might not be significantly absorbed. These researchers have also carried out studies on the distribution and elimination of the drug in mice by administrating 10 and 100 mg/kg of nifuroxazide by gastric intubation. Data indicate that approximately 50% of the dose was absorbed and that the nifuroxazide is, in fact, extensively metabolized rather than having low absorption23,24. The use of substances with well established pharmacokinetic characteristics and similar structure to the new drug candidates is of great value in permeability screening assays with Caco-2 cells, increasing the reliability of the results. Nitrofurantoin, a drug with structure similar to nifuroxazide, which is used in the treatment of urinary infections, has been extensively studied with respect to its pharmacokinetics, mainly due to its excretion in human milk. Although it has limited solubility in water, it readily dissolves in alkaline solutions and urine. Buzard and colleagues25 found that this drug is rapidly absorbed by the small intestine, showing increased plasma levels in few minutes. The presence of food in the gastrointestinal tract or the delay in the gastric emptying increase nitrofurantoin absorption by increasing its dissolution rate. The bioavailability is 87% when administered in the absence of food and increases to 94% in its presence. Peak plasma concentrations after a single oral dose of 100 mg is generally achieved within 1–2 h after ingestion and reach 1 mg/mL26. Lin et al.27 evaluated the permeability of some drugs across the Caco-2 cells using inhibitors of efflux transporters for P-gp and BCRP. For nitrofurantoin, the Papp results of the apical to basolateral and basolateral to apical portions were 0.8 and Figure 1. (A) Molecular structure of nitrofurantoin. (B) Molecular structure of nifuroxazide.
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18.8 106 cm/s, respectively, in absence of a BCRP inhibitor. In the presence of the inhibitor, the values were 4.0 and 7.2 106 cm/s, respectively, showing the influence of the carrier on the absorption of nitrofurantoin. To date, there is no literature data on nifuroxazide permeability across monolayers of Caco-2 as well as on the series of its analogs present in this study, being necessary their evaluation as next stage in the development of a new antimicrobial drug against MRSA. It is important and necessary to evaluate their bioavailability aiming at an efficient selection and further improvement of the selected molecular structures.
Methods Chemicals Dulbecco’s modified Eagle medium (DMEM) with and without phenol, penicillin, streptomycin, Trypsin–EDTA (0.2%) and fetal calf serum (FCS) and Hank’s balanced salt solution (HBSS) with 200 mM of HEPES salt, with and without phenol and pH adjusted to 7.4 were obtained from Cultilab (Campinas, SP, Brazil). Glutamine, D-glucose, 3-(4,5 dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), nifuroxazide (Figure 1B) and nitrofurantoin (Figure 1A) were purchased from Sigma-Aldrich (St. Louis, MO). The 5-nitro-heterocyclic compounds used in this study were designed and synthesized by the Laboratory of Drug Design and Development of the Chemical and Pharmaceutical Technology Department at the Pharmaceutical Sciences Faculty/University of Sa˜o Paulo. Eight compounds belonging to an analogous series with similar structure to nifuroxazide were selected, as referred in Figure 2. Cell line and culture conditions Human colon adenocarcinoma cell line Caco-2 (ATCC #HTB-37) was purchased from American Type Culture Collection (ATCC, Rockville, MD). Caco-2 cells were cultured in DMEM supplemented with 4.5 g/L D-glucose (Sigma-Aldrich Corp., St. Louis, MO), NaHCO3 (2.2 g/L) (Sigma-Aldrich Corp., St. Louis, MO), 10% FCS (Cultilab, Campinas, SP), 1% non-essential amino acids (Sigma-Aldrich Corp., St. Louis, MO), 100 UI/mL penicillin and 100 mg/mL streptomycin (Cultilab, Campinas, SP) in an atmosphere of 5% CO2 and 90% relative humidity at 37 C. All cells used in this study were between passages 40 and 50. In vitro permeability tests Caco-2 cells were cultured in 12-well or 6-well Transwell (Corning Incorporated, New York, NY) insert filters for 21 days to reach confluence and differentiation at a seeding density of 5 104 cells/cm2. 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 running standard assays using amoxicillin and metoprolol as paracellular and transcellular flux markers, respectively. Only cell monolayers with a TEER above 300 cm2 were used for the transport assays. 5-Nitro-heterocyclic compounds were dissolved in dimethylsulfoxide (DMSO), a solvent frequently used when poorly soluble
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Figure 2. Chemical structure of the 5-nitro-heterocyclic derivatives used in this study and their purities.
compounds are evaluated in permeability assays, and subsequently diluted in HBSS buffer at pH 7.4 to prepare the permeability samples in concentrations previously determined by cytotoxic tests28. The compounds EtoTIO and BrFUR had lower concentrations due to their solubility restrictions. The initial concentrations and collection times for each compound studied are described in Table 1. Experiments were initiated with the addition of the 5-nitroheterocyclic solutions in the apical compartment of the Transwell plates, and the basolateral compartment was maintained at pH 7.4. Six replicates for all nitro compounds were assessed. Plates were kept under agitation in an orbital shaker at 37 C (25 rpm). Samples volumes of 200 mL were collected from the basolateral compartment at predetermined times for each compound (Table 1). After each sampling, 200 mL of HBSS buffer was replaced in order to maintain the volume of solution in the compartment. Samples were quantified by high performance liquid chromatography with UV (HPLC-UV) detection on a Shimadzu LC-10A chromatographer (Kyoto, Japan). The methods were developed and validated according to Resolution RE 899 de 29 de maio de 2003 – Guia para validac¸a˜o de me´todos bioanalı´ticos – ANVISA29. Chromatographic separations were obtained using a phenyl-2 HYPERSIL column (150 mm 4.6 mm, 5 mm, Thermo Scientific, Waltham, MA) at 30 C. The mobile phase (1 mL/min) was composed of phosphate buffer 5 mM (pH 3.1) and acetonitrile. The analytical wavelength was set at 370 nm and samples of 50 mL were injected into the HPLC system. The apparent permeability coefficients (Papp, cm/s) were calculated according to the following equation: Papp ¼ ðVR DQ=DtÞ=ðA C0 Þ where VR is the volume of the receiver compartment (basolateral or apical), DQ/Dt is the linear appearance rate of the compound on the receiver chamber (in ng/s), A is the membrane surface area (cm2) and C0 is the initial concentration in the donor compartment (ng/cm3). This calculation requires that the sink conditions are fulfilled; therefore, only receiver concentrations below 10% of the donor concentration were employed in the calculations. Statistics The results are expressed as mean ± standard deviation (SD) of six replicates.
Results Transepithelial electrical resistance results obtained at 10, 15 and 21 days of cell cultivation on the insert filters in DMEM at pH 7.4
Table 1. Initial concentrations and sampling times for each 5-nitro-heterocyclic compound in the permeability experiment through Caco-2 cells monolayers.
Compound Nifuroxazide Nitrofurantoin HFUR MeFUR EtoFUR AminoFUR BrFUR HTIO EtoTIO AminoTIO
Initial concentration (mg/mL)
Sampling times (min)
4.0 4.0 4.0 4.0 4.0 4.0 1.0 4.0 0.5 4.0
15, 30, 45, 60, 90 and 120 15, 30, 45, 60, 90 and 120 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 15, 30, 45 and 60 60, 90, 120 and 180 15, 30, 45 and 60
and TEER results at the beginning and end of the permeability experiment of the derivative EtoTIO in HBSS at pH 7.4 are shown in Figures 3 and 4, respectively. TEER evaluation during cultivation of the membranes showed that the values increase with time until they reach a stable average value of 580–650 V cm2 between 15 and 21 days of the start of the cultivation. TEER values obtained ( cm2) during the permeability experiment for the compound EtioTIO ranged from 652 to 468. Despite the decreased levels of TEER, these did not reach values below 200 cm2. Papp values of the 5-nitro-heterocyclic, shown in Table 2, were determined from the amount permeated through the Caco-2 cells membranes at the apical direction-basolateral. All the nifuroxazide derivatives Papp values calculated in this study were greater than 15 106 cm/s.
Discussion Over the past 20 years there has been growth in the use of Caco-2 cells as a rapid screening tool for in vitro intestinal permeability assessment, supporting the development of drugs and directing the changes of physicochemical characteristics responsible for absorption7,11,12,16,30. During the Caco-2 cells membrane formation, it is necessary to assess its integrity which, in this experiment, was first evaluated through the TEER values. The use of this parameter as an indicator of the membrane integrity formed by Caco-2 cells has been widely used in this technique, and significant correlations between the TEER values and permeability of compounds absorbed by the paracellular route have been obtained31. TEER values obtained in this study demonstrate that the membranes formed had adequate integrity,
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Figure 3. TEER values obtained with the Caco-2 cells membranes at 10, 15 and 21 days of culture in DMEM medium with pH 7.4. Average of six determinations.
Figure 4. TEER values obtained with the membranes of Caco-2 cells under the permeability test conditions of the derived EtoTIO at the beginning (T0) and at the end (180 min) of the experiment. Average of six determinations.
Table 2. Papp values of the 5-nitro-heterocyclic compounds determined from the amount permeated through the Caco-2 cells monolayers. Compound Nifuroxazide Nitrofurantoin HFUR MeFUR EtoFUR AminoFUR BrFUR HTIO AminoTIO
Papp (106 cm/s) 7.86 ± 0.94 2.08 ± 0.20 53.89 ± 0.60 39.66 ± 0.67 31.77 ± 0.42 15.55 ± 2.40 26.03 ± 3.03 61.58 ± 5.10 42.95 ± 3.44
with the formation of junctional intercellular complex. Similar values to those obtained are reported in the literature32–34. TEER assessment was also performed prior to the beginning and at the end of the experiments and only the membranes with TEER values above 200 V cm2 were selected for conducting the tests. It was observed an average reduction of 60 V cm2 in each hour of the experiment; however, the values were never below 200 V cm2, keeping an average of 470 V cm2 at the end of three hours of experiment for all compounds.
Another way of assessing membrane integrity and viability during the permeability experiments is by determining the permeability of compounds with well known Papp data. Many drugs are used for this purpose, especially mannitol, digoxin, cimetidine and fluorescein. Amoxicillin and metoprolol were used in this study as low and high permeability standards, respectively, to assess the adequacy and integrity of the culture conditions of the membranes of Caco-2 cells. Although both present high in vivo absorption, amoxicillin shows low Caco-2 cells permeability due to the low expression of PEPT1, which is the main transporter responsible for its in vivo uptake. Metoprolol, on the other hand, has the passive transcellular via as main absorption mechanism. Both are part of the recommendation list of the guide issued by the Food and Drug Administration (FDA) regulatory agency, which provides information for biowaivers35. The mean of the Papp values obtained for amoxicillin was 0.69 106 cm/s, and for metoprolol, 27.27 106 cm/s. These values are in agreement with those obtained in the literature, indicating adequacy of cultivation conditions of Caco-2 cells and reliability to the results obtained for the 5-nitro-heterocyclic compounds36–38. Given the current knowledge about the intestinal absorption of the drug nitrofurantoin, as well as the structural similarity between this and nifuroxazide and its derivatives, the permeability evaluation of the former was used as a parameter for the other 5-nitro-heterocyclic compounds. Additionally, the existence of Papp values for nitrofurantoin in the literature calculated by assays employing Caco-2 cells enabled comparison of the results obtained in this study. The Papp value calculated in this study (2.08 106 cm/s) is approximately 2–3 times greater than the values reported by Lin and coworkers27 (0.6, 0.8 and 1.1 106 cm/s), being this difference probably attributed to the way how Papp was calculated in both studies. In the former, a minimum of three samples was obtained for all compounds and were used for the construction of the concentration curve relative to the time of the permeation test. In the latter, a single sampling at the end of the experiment was made, being the first point in the curve a zero concentration for time zero (T0). If the Papp value for the present data was calculated using the latter approach, it would result in a Papp value of 1.4 106 cm/s, which is a closer and comparable value to those obtained by Lin and coworkers27. Thus, in addition to the standardization of the culture and experimental conditions in order to get comparable Papp values among different laboratories, the establishment of a protocol for the calculation of this parameter is also a pre-requisite for the robustness of the in vitro permeability tests through Caco-2 cells. Placing the results obtained by both laboratories according to Yee classification, the average Papp values calculated by Lin et al.27 would indicate that nitrofurantoin is a drug with low absorption (between 0 and 20% of absorption), while the Papp values obtained in this study would indicate it as a drug with moderate absorption (between 20 and 70% of absorption) (Figure 5). By performing the same analysis according to the classification of Biganzoli and coworkers19 different results are observed (Figure 5). The former would indicate nitrofurantoin is a drug with moderate absorption (between 1 and 90% of bioavailability), while results from this study would classify nitrofurantoin as a drug with high absorption (bioavailability greater than 90%). Since nitrofurantoin absorption rates found in literature are 87–94%26, it is likely that the Biganzoli and coworkers19 classification of the permeability values through Caco-2 is more suitable to what occurs in vivo with this drug and, hence, it could be suitable to analyze the permeability potential of the other 5-nitro-heterocyclic compounds present in this study. Since there is no evidence to date in the literature of Papp values for the drug nifuroxazide calculated in permeability tests
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Figure 5. Representation of Yee’s and Biganzoli’s classification for the Papp values of D (triangle) nitrofurantoin (Lin et al.)27, O (circle) nitrofurantoin, œ (square) nifuroxazide and * (star) AminoFur.
through Caco-2, this study is the first to report this parameter to the drug. Placing the Papp value calculated for nifuroxazide (7.86 106 cm/s) according to the Yee classification, nifuroxazide would be classified as a drug with moderate absorption (between 20 and 70% absorption). According to the Biganzoli classification, however, the drug would be placed in the drugs with high absorption band (absorption 490%) (Figure 5). As previously highlighted, the latter classification would be more suitable to indicate what happens in vivo with 5-nitro-heterocyclic compounds, that is, nifuroxazide could be highly absorbed in vivo. Although little information about the pharmacokinetics of nifuroxazide is available in the literature, it is believed that it has low absorption20–22. However, there is evidence that this drug presents considerable absorption and is extensively metabolized in the liver23, which would explain the low levels of unchanged nifuroxazide found in studies with human plasma after oral administration of the drug20,21. The high Papp value found for nifuroxazide in this study, taking into account that there is no hepatic metabolism in the Caco-2 membranes technique corroborates, therefore, with the hypothesis that this drug has significant absorption and extensive metabolism in vivo. Additionally, according to the Biopharmaceutics Drug Disposition Classification System (BDDCS), a classification developed to predict the in vivo disposition of drugs from measurements of solubility and metabolism (clearance route), nifuroxazide belongs to class II. This means that this drug is classified as a drug with low solubility and high permeability, and is extensively metabolized39; therefore, corroborating with the hypothesis that the drug is considerably absorbed in vivo. All Papp values calculated in this study for the nifuroxazide derivatives are greater than 15 106 cm/s. Therefore, employing either the Yee or the Biganzoli classification, these compounds would have considerable potential for presenting high permeability in vivo. Also, Paap values are closer to metoprolol Papp (27.27 106 cm/s) than to amoxicillin (0.69 106 cm/s), what corroborates the assumption of high permeability in vivo, implying that the mechanism of permeability of these derivatives would be mainly through transcellular transport rather than via PEPT1 or another low expressed transporter in Caco-2 cells. The thiophenic derivatives showed permeability values higher than the furanic ones, with minimum Papp values of 42.95 106 cm/s (AminoTIO) and 15.55 106 cm/s (AminoFUR), respectively. This fact could be explained by the higher lipophilicity of the former in relation to the latter, which would facilitate their passage through the membranes of Caco-2 cells. Additionally, due to an increased permeability of the
thiophenic derivatives by consequence of their lipophilicity, these may have wider distribution in the body, which would make them more suitable than the furanic derivatives in the treatment of the main infections caused by MRSA, which affect skin and soft tissue. In addition to the increased permeability presented by the thiophenic derivatives, these were less toxic in the cytotoxicity assays previously discussed28, noting that the toxicity of the nifuroxazide derivatives may be influenced by both the reactivity of the atom in the ring linked to the nitro group as well as the substituent on the benzene ring. Compounds with more hydrophobic substituents, for imparting greater toxicity to the molecule, should be avoided within this series of compounds. However, it is noteworthy that the highest lipophilicity of the thiophenic derivatives entails lower solubility in aqueous media, as noted in the stage of the methods development for quantifying this study. EtoTIO was the lowest soluble compound, hence a lower concentration was used (0.5 mg/mL) in the permeability tests. However, its Papp could not be calculated since the permeated concentrations were below the quantitation limit of the method developed by HPLC. Despite the problems related to solubility, the thiophenic derivatives presented the best results in terms of cytotoxicity28 and permeability, and have a greater potential to be more promising than the furanic derivatives. Among these derivatives, AminoTIO was the compound identified as the one with the greatest potential for the development of a new drug, that is, the one which has the best relationship between cytotoxicity (410 mg/ mL; nifuroxazide: 56 mg/mL)28, permeability (42.95 106 cm/s; nifuroxazide: 7.86 106 cm/s) and solubility (410 mg/mL; nifuroxazide: 410 mg/mL). In the development of a product containing this compound, the solubility-related problems could be minimized by the use of pharmacotechnical knowledge to improve the solubility, such as micronization, the use of solid dispersions40, the formation of soluble complexes like cyclodextrins, or dendrimers41, as well as use of nanostructured systems42, such as nanoparticles and nanocapsules43. As the next step in the development of a new drug with activity against multidrugresistant strains of S. aureus, tests must be conducted with the derivative AminoTIO aiming at deepening the knowledge acquired regarding its permeability. It is necessary to carry out mechanistic studies related to the absorption of this compound, as the elucidation of the prevalence of active or passive transport and the influence of efflux systems, such as the one which occurs with nitrofurantoin, which is a substrate of BCRP. Additionally, in vitro studies must be conducted aiming at the evaluation of its metabolites, employing, for example, models that use liver cells.
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Drug Dev Ind Pharm, Early Online: 1–7
Conclusions
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The results obtained in this study indicate that the Papp value calculated for nitrofurantoin is in accordance with what occurs in vivo with the drug. The Papp value calculated for nifuroxazide corroborates with the hypothesis that the drug presents a considerable absorption and is extensively metabolized in the liver. Papp values of the nifuroxazide derivatives show that these can be considered as new drug candidates with the potential to present high absorption in vivo. The thiophenic derivatives showed permeability values higher than the furanic derivatives, being the AminoTIO the compound with greater potential for the development of a new drug against multidrug-resistant strains of S. aureus, as it showed the best relationship between cytotoxicity, permeability and solubility.
Acknowledgements We would like to thank Alex Alfredo de Oliveira, Fanny Palace Berl, Joa˜o Sussumu Murayama, Leandro Bortolozzo, Rodrigo Rocha, Saloma˜o Do´ria Jorge from the Laboratory of Drug Design and Development of the Chemical and Pharmaceutical Technology Department at the Pharmaceutical Sciences Faculty/University of Sa˜o Paulo for the synthesis of the nifuroxazide derivatives.
14.
15. 16. 17.
18. 19. 20.
21.
Declaration of interest The authors report no declarations of interest.
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