Toxicology in Vitro xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier P. Bellwon a,⇑,1, M. Culot b,c,1, A. Wilmes d,1, T. Schmidt a,1, M.G. Zurich e,1, L. Schultz f,1, O. Schmal a, A. Gramowski-Voss f, D.G. Weiss f, P. Jennings d, A. Bal-Price g, E. Testai h, W. Dekant a a
Department of Toxicology, University of Wuerzburg, Wuerzburg, Germany Univ Lille Nord de France, F59000 Lille, France UArtois, BBB Laboratory, EA 2465, F62300 Lens, France d Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck 6020, Austria e Department of Physiology, University of Lausanne, Lausanne, Switzerland f Department of Animal Physiology, Institute of Biological Sciences, University of Rostock, Rostock, Germany g ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra, VA, Italy h Mechanism of Toxicity Unit, Environment and Primary Prevention Department, Istituto Superiore di Sanità, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 14 July 2014 Accepted 5 January 2015 Available online xxxx Keywords: Cyclosporine A uptake Neurotoxicity Brain cell cultures BBB model Passive diffusion Cyclophilin B
a b s t r a c t There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood–brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The time and cost of compounds coming to market are increasing world-wide and this has been attributed to the drug failures late in discovery either to lack of efficacy (56%) or safety issues (28%) (Arrowsmith and Miller, 2013; DiMasi et al., 2010). There Abbreviations: CsA, Cyclosporine A; Cyp-B, cyclophilin B; BBB, blood–brain barrier; LY, lucifer yellow; TC10, 10% toxic concentration; AB, apical-to-basolateral; BA, basolateral-to-apical; PAMPA, parallel artificial membrane assay; PK, pharmacokinetics; CNS, central nervous system; EC, endothelial cells. ⇑ Corresponding author at: Department of Toxicology, University Wuerzburg, Versbacher Str. 9, 97078 Wuerzburg, Germany. Tel.: +49 931 31 86367. E-mail addresses: [email protected], patricia.bellwon@yahoo. com (P. Bellwon). 1 Equal contributing authors.
is now a consensus that improvement of preclinical phase testing would drastically alleviate this burden. Neurotoxicity has been identified as a frequent adverse drug effect contributing to the termination of up 22% of drug candidates (Watkins, 2011). The evaluation of potential neurotoxicity is recommended to be performed in rodents during the preclinical safety assessment of drugs and chemicals (Bolon et al., 2010; OECD Test Guideline No. 424), however in vivo data can be affected by variables such as neuronal, hormonal and immunological stimuli (Harry and TiffanyCastiglioni, 2005). To date, in vitro approaches using neuronal and glial cells are applied to investigate the mechanisms of neurotoxicity, but such systems may also be considered as alternative methods for risk assessment to meet the needs for predictive, time-saving and cost-reducing methods (Bal-Price et al., 2010). Within the
http://dx.doi.org/10.1016/j.tiv.2015.01.003 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
2
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
‘‘ACuteTox’’ Project it was shown that brain cell cultures, e.g. primary cultures of cortical neurons and primary aggregating rat brain cells, may serve as prediction models for acute neurotoxic effects (Bal-Price et al., 2008; Forsby et al., 2009; Suñol et al., 2008). Since pharmaceutical compounds are usually given in repeated exposures, often life-long, the prediction models for toxic effects after repeated exposures are required. To address this issue,the 7th Framework Program ‘‘Predict-IV’’ aimed to improve the predictability of drug-induced toxicity in the early stage of drug development. Pharmacodynamics in combination with pharmacokinetics (PK) was characterized in well-established in vitro models representing three target organs, namely liver, kidney and central nervous system (CNS). As PK evaluation is necessary to describe the systemic exposure and its relationship to observed (adverse) effects, PK data enhance the understanding of mechanisms of action and/or toxicity induced by xenobiotics and moreover help to assess the risk and safety in humans (ICH S3A Guideline; OECD Test Guideline No. 417). The cyclic peptide cyclosporine A (CsA), which is a widely used immunosuppressive compound, was chosen as a cross-organ test compound in the ‘‘Predict-IV’’ project as it has been shown to induce hepatotoxicity (Atkinson et al., 1983; Klintmalm et al., 1981), nephrotoxicity (Bennett and Pulliam, 1983; Klintmalm et al., 1985; Palestine et al., 1984) and also neurotoxicity (Calne et al., 1979; Gijtenbeek et al., 1999). As the therapeutic index of CsA is narrow, therapy requires drug monitoring to avoid toxicity. Nevertheless, neurotoxicity was also observed in patients with CsA blood levels within the therapeutic range (Serkova et al., 2004). Various symptoms of CsA-induced neurotoxicity have been reported: tremor, headache, seizures, visual hallucinations and delusions (Gijtenbeek et al., 1999). In general, neurotoxicity is reversible after termination of CsA administration (Gijtenbeek et al., 1999). Whilst a major focus in the literature has been to the mechanisms of the neurotoxicity of CsA, there has been less focus on the capacity of CsA to cross the blood–brain barrier (BBB). Indeed the weight of evidence is that CsA poorly crosses the BBB. CsA determination in brain tissue of humans (Atkinson et al., 1982; Lensmeyer et al., 1991), rats (Lemaire et al., 1988; Niederberger et al., 1983; Wagner et al., 1987) and mice (Boland et al., 1984) revealed very low levels of the drug, indicating that only minor CsA amounts are able to cross the BBB. The aim of this paper was to investigate whether CNS exposures to the model compound CsA can be predicted using an integrated in vitro testing strategy. In order to achieve this, kinetics and dynamics were investigated in two rodent brain cell cultures: (i) primary neuronal culture of mouse cortex (2D model) and (ii) primary aggregating rat brain cells (3D model). In order to obtain CsA kinetic profiles after single and repeated exposure to a low (nontoxic) and high (10% toxic) CsA concentration, CsA levels were determined in treatment solutions, cell lysates, cell culture media and plastic adsorption samples. To further determine whether CsA is taken up by the cell systems, evaluation of a CsA-specific pharmacodynamic endpoint (cyclophilin B [Cyp-B] depletion by western blot analysis) was included (Fearon et al., 2011; Price et al., 1994; Wilmes et al., 2013). In order to investigate the ability of CsA to cross the BBB, CsA permeability across an established BBB model based on co-culture of bovine brain capillary endothelial cells (EC) and rat astrocytes was studied (Fabulas-da Costa et al., 2013). In addition, in order to get insight into the cellular uptake mechanism transporter experiments were performed using a parallel artificial membrane assay (PAMPA) and Caco-2 cells. PAMPA determines a possible uptake by passive diffusion (Kansy et al., 1998). While the predicted permeability can be over- or underestimated, PAMPA in combination with cell systems that possess transporter activities is recommended (Di and Kerns, 2003). Thus
Caco-2 cells were used as a standard reference cell line for active cellular extrusion (Augustijns et al., 1993; Gan et al., 1996). 2. Materials and methods 2.1. Materials (specified by partner) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dodecane, dimethylsulfoxid (DMSO) and lucifer yellow (LY) were obtained from Sigma Aldrich (Taufkirchen, Germany). CsA was obtained from Calbiochem (Darmstadt, Germany). The internal standard deuterium-labeled CsA (CsA-d12) was purchased from Analytical Services International Ltd (London, United Kingdom). For the mobile phase preparation ammonium acetate, formic acid, methanol (HPLC grade) and water (HPLC grade) were purchased from Carl Roth (Karlsruhe, Germany). For Caco-2 cell culture DMEM (Cat. No. P04-03500) was purchased from PAN Biotech (Aidenbach, Germany) and all other cell culture reagents were purchased from PAA (Coelbe, Germany). 2.2. Isolation and cell culture conditions 2.2.1. Primary neuronal mouse cells (2D model) Primary frontal cortex cultures were derived from dissociated embryonic frontal cortex tissue from NMRI mice (Charles River, Sulzfeld, Germany) on day 16. The culture was processed according to Ransom et al. (1977). The method was modified by the usage of DNase I (8000 units/mL, Roche, Mannheim, Germany) and papain (10 U/mL, Roche, Mannheim, Germany) for tissue dissociation (Huettner and Baughman, 1986) and further mechanical cell dissociation. Cells were suspended in primary neurobasal medium (PNB, Lonza, Verviers, Belgium) at a density of 0.5 106 cells/mL, seeded into 6 well plates and incubated under defined conditions (37 °C, 5% CO2; Binder, Tuttlingen Germany). The cell culture networks formed from a mixture of different types of neurons and glial cells can be cultured for several months due to their stability by co-cultivation (Gramowski et al., 2006; Weiss, 2011). Glial cells were allowed to proliferate for 3–5 days after seeding until reaching approximately 80% confluency. Growth of glial cells was stopped by 5-fluoro-20 -deoxyuridine (25 lM) treatment. Serum free cell culture medium was used and refreshed three times a week. The repeated exposure experiments started 4 weeks after seeding. 2.2.2. Primary aggregating rat brain cell cultures (3D model) The aggregating rat brain cultures were prepared from 16-day old Sprague–Dawley rat embryos (Janvier, St Berthevin, France) as described by Honegger et al. (2011). Briefly, the brains of the embryos were dissected and dissociated into a single cell suspension by sequential passage through nylon sieves of 200 lm and 115 lm pores (Sefar, Heiden, Switzerland). The cells were washed by centrifugation, resuspended in cold serum-free culture medium (modified Dulbecco’s Modified Eagle Medium (DMEM)), and incubated in modified Erlenmeyer culture flasks. The spontaneously generated three-dimensional cultures were maintained as suspension culture under constant gyratory agitation (80 rpm) at 37 °C, in an atmosphere of 10% CO2 and 90% humidified air. Culture media were replenished with fresh medium every third day until day 14 by exchanging 5 mL (of a total of 8 mL per flask). After 14 days in vitro (DIV), the culture media were replenished every second day. The repeated exposure experiments started at DIV 18. 2.2.3. In vitro model of the BBB Cells were isolated and cultured as described previously (Fabulas-da Costa et al., 2013). Briefly, ECs isolated from capillary fragments were co-cultured with primary mixed glial cells from
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
newborn Sprague Dawley rats. The glial cells were isolated according to the method of Booher and Sensenbrenner (1972) and cultured for 3 weeks, plated on the bottom of cell culture clusters containing six wells each. The EC were seeded onto cell culture inserts, which were placed in the wells containing glial cells. The medium used for the co-culture was Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS), 10% horse serum, 2 mM glutamine, 50 mg/L gentamicin and 1 lg/L basic fibroblast growth factor (Sigma). The medium was changed every second day. Under these conditions, ECs formed a confluent monolayer after 7 days. Treatments with CsA were initiated 5 days after confluence. 2.2.4. Human intestinal cell line (Caco-2) Cells were cultured in DMEM supplemented with 2 mM L-glutamine, 1 nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 lg/mL streptomycin and 10% FCS. Cell culture medium was refreshed twice a week and Caco-2 cells were passaged 1:5 by with trypsin after reaching 70–85% confluence. 2.3. Kinetics and dynamics – experimental design and sampling The 2D model was exposed to a 10% toxic concentration (TC10, 2 lM) as determined by the extracellular LDH activity measured routinely with every medium change during the time of repeated exposure and 1/20th of the TC10 (0.1 lM). The 1000-fold concentrated stock solutions were prepared in DMSO. On the first day of treatment, 3 lL of the concentrated stock solution were pipetted directly into the cell medium of each well of the replicate culture. The culture medium was replenished (1.5 mL out of a total of 3 mL) every second day and the stock solutions were pipetted into the fresh medium prior to the replenishment and then given to the cell cultures. The 3D model was exposed to the 10% toxic concentration (TC10, 1 lM) as determined by the intracellular LDH activity measured at the end of the repeated exposure time and 1/5th of the TC10 (0.2 lM). The 2000-fold concentrated stock solutions were prepared in DMSO. On the first day of treatment, 4 lL of the respective 2000-fold concentrated stocks were pipetted directly into the cell culture medium of each replicate culture. Every second day after medium replenishment (5 mL out of a total of 8 mL) cultures were allowed to re-equilibrate the pH of their medium during 2 h. Then, 2.5 lL of the 2000-fold stock solutions were pipetted directly into the cell culture medium of each replicate culture. Cells were treated every second day with a low and high CsA concentration for one and 13 (3D model) or 14 days (2D model). Samples were generated on the first and the last treatment day at five time points (30 min, 1 h, 3 h, 6 h and 24 h). In addition, CsA concentration was determined in the treatment solution. Four biological replicates were performed for the 2D model and three biological replicates (2 replicate cultures each) for the 3D model. To determine the CsA kinetic profile, samples were generated from the cell culture media and cell lysates. After removal of the cell culture media, cells were washed twice with PBS. Cells from the 2D model were scraped in 0.25 mL methanol, transferred to a glass vial and rinsed with 0.1 mL methanol. For the 3D model, 1 mL methanol was added to the cells. After sonication, lysates were transferred to LoBind tubes. In order to determine the CsA amount adsorbed to plastic plates used for the 2D model, wells were washed twice with PBS and 1.5 mL methanol was added and incubated under gentle shaking at room temperature for two hours. No plastic adsorption samples were generated for the 3D model as cells were cultured in glass flasks. All samples were stored at 80 °C until analysis (see Section 2.5.3).
3
2.4. CsA permeability across a BBB in vitro model 2.4.1. Experimental design After 12 days of co-culture with glial cells, CsA (1 lM in 0.25% DMSO) was applied to the apical compartment containing ECs. As controls, ECs were treated only from the apical side with 0.25% of DMSO without drugs. The basolateral compartments containing glial cells were filled with fresh medium. Treatments and media were renewed every 2 days for 14 days. The permeability of CsA and of the non-permeable fluorescent marker LY, used as a marker of the integrity of the EC monolayer, was assessed in ECs on day 0, 7 and 14. Briefly, ECs monolayers were transferred to six-well plates containing 2.5 mL Ringer– HEPES solution (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2 (6 H2O), 6 mM NaHCO3,5 mM HEPES, 2.8 mM glucose, pH 7.4) per well. 1.5 mL Ringer-HEPES solution containing 10 lM CsA and 20 lM LY was then added to the apical compartment. Incubations were performed at 37 °C. After 1 h, aliquots were taken from both compartments and the concentrations of CsA and LY were measured by LC/MS or with a fluorescence spectrophotometer (Synergy H1, Biotek, Winooski, USA). For each experiment three inserts with cells and three without cells were assayed to enable the calculation of the endothelial permeability coefficient (Pe). Pe was calculated (in cm/min). In this calculation, both filter without cells permeability (PSf = insert filter + collagen coating) and filter plus cell permeability (PSt = filter + collagen + EC) were taken into account, according to the formula:
1 1 1 ¼ PSe PSt PSf PSe Pe ¼ A
ð6Þ ð7Þ
where A is the surface area of the filter (i.e. 4.7 cm2). When BBB integrity was evaluated over time, cell culture inserts were filled with fresh medium at the end of the 1 h transport experiment and treatment solutions were renewed. As previously established (Fabulas-da Costa et al., 2013) a compound was considered to have generated a toxic effect (i.e. impaired BBB function) when the Pe (LY) value of treated cells was increased 2-fold compared to control cells (treated with carrier solvent in the absence of drug) at the same time points. 2.4.2. LC–MS/MS analysis CsA concentrations in the luminal and abluminal compartments were determined by LC–MS/MS. Analyses were performed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, West Palm Beach, FL, USA) equipped with a TurboIonspray with an Ion Max source and coupled to Thermo Scientific Accela HPLC autosampler and pump (Thermo Scientific, West Palm Beach, FL, USA). A fusion column (80 Å, 150 4.6 mm, 4 lm – Phenomenex Synergi Fusion) was used for chromatographic separation at 50 °C. The separation was isocratic using a mixture of acetonitrile/water/formic acid (90/9/1, v/v/v) as mobile phase. An injection volume of 10 lL was used with a flow rate of 0.4 mL/min. CsA was detected in positive ion mode setting the spray voltage and capillary temperature were set at 4.0 kV and 300 °C, respectively. The pressures for the nitrogen sheath gas, auxiliary gas and sweep gas were maintained at 30, 10 and 5 units. Data acquisition was performed in selected reaction monitoring (SRM) mode detecting two transitions (m/z 1225.1 ? 1203.15; m/z 1225.1 ? 1185.1) with collision energy of 30 eV and S-lense RF amplitude voltage set to 90 V. Argon was used as collision gas at 0.2 Pa. Instrument control and data processing were carried out by means of Xcalibur Software 2.0.7 SP1 (Thermo Electron, San José, USA).
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
4
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
F(cum) is the cumulative fraction transported (pmol/cm3) and was determined as:
2.5. Transport experiments Transport experiments on Caco-2 cells and a parallel artificial membrane were performed in triplicates and for each experiment corresponding controls were generated by using cell culture medium containing 0.1% DMSO. 2.5.1. Caco-2 Cells were seeded at a density of 6 105 cells/mL cell culture medium on PET filter inserts (Thinserts, 0.4 lm pore size, translucent; Greiner Bio-One) in a 12-well plate and blank cell culture medium was added to the basolateral side. After 2 weeks of cell culture FCS was reduced from 10% to 5% and after 3 weeks the transport experiment was performed at 0% FCS. CsA was dissolved in DMSO (1 mM and 10 mM) and further diluted with cell culture medium to final concentrations of 1 lM and 10 lM with 0.1% DMSO content in all treatment solutions. The transport of CsA was tested in both directions apical-tobasolateral (AB) and basolateral-to-apical (BA). Therefore, after removing the cell culture medium, pre-warmed CsA solutions were either added to the apical (0.5 mL) or to the basolateral side (1.5 mL) – called donor well - while the corresponding opposite sides were filled with pre-warmed cell culture medium containing 0.1% DMSO – called acceptor well. An aliquot of the treatment solutions was taken for determination of the actual CsA amount given to the cells. After 30 and 60 min of incubation, aliquots were taken from the acceptor well (apical: 0.25 mL; basolateral: 0.75 mL) and replaced by fresh cell culture medium containing 0.1% DMSO. The incubation was terminated after 3 h and cell culture medium from both donor and acceptor wells were collected separately into glass vials. Prior to cell lysis the paracellular integrity of the cell monolayer was tested by usage of the integrity marker lucifer yellow (LY) as described earlier by Broeders et al. (2012). Therefore, cells were washed with PBS and 0.5 mL LY dissolved in cell culture medium (0.2 mM) was added to each well from the apical side and blank medium from the basolateral side. After 1 hour incubation aliquots (0.1 mL) were taken from the basolateral compartments, transferred into a 96-well plate and fluorescence was measured at 485 nm (excitation) and 535 nm (emission) using the Mithras LB 940 Multimode Mikroplate Reader (Berthold Technologies, Bad Wildbad, Germany). Cell culture medium was set as the blank reference and 0.2 mM LY solution in cell culture medium as 100%. Cell lysate and plastic adsorption samples were generated in order to determine the CsA mass balance and further, to investigate whether potential CsA loss occurs due to biotransformation or adsorption to the plastic of the well walls. Therefore, after removal of the cell culture medium, cells were washed with PBS and 0.1 mL trypsin was added to each well for cell harvesting. Samples were transferred to glass vials, wells were washed with 0.4 mL methanol and added to the same vials. Afterwards, 0.5 mL and 1.5 mL methanol were added to the apical and basolateral side, respectively. Plates were covered with parafilm and stored at room temperature under gentle shaking. After 1 h samples from one well (apical and basolateral) were pooled in order to determine the CsA amount adsorbed to the plastic of the well walls. All samples were stored at 20 °C until LC–MS/MS quantification (see Section 2.5.3). Papp was calculated according to Broeders et al. (2012):
Papp ¼
DF ðcumÞ A1 V D Dt
ð1Þ
where DF(cum)/Dt is the slope of the graphs representing the cumulative CsA fraction transported over time, A (cm2) is the filter insert area (1.131 cm2 for 12-well inserts), VD is the CsA solution volume (cm3) filled into the donor well (apical: 0.5 cm3; basolateral: 1.5 cm3).
F ðcumÞ ¼
X bC AðTPxÞ f C AðTPx1Þ c V A CD V D
ð2Þ
CD and CA are the measured CsA concentrations (pmol/cm3) at each time point (TPx) in the donor and acceptor well, respectively; VA is the volume (cm3) of cell culture medium in the acceptor well and f is the replacement factor and was calculated as:
f ¼1
VS VA
ð3Þ
where VS is the volume of the sample. 2.5.2. Parallel artificial membrane permeability assay (PAMPA) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was dissolved in dodecan with a final concentration of 1 mg/mL. After adding 0.05 mL of the 0.1% DOPC solution to the PET filter inserts, plates were shaken for 5 min at room temperature. Passive diffusion was tested from the apical to basolateral side. Therefore, CsA treatment solutions were added to the apical compartment and blank cell culture medium to the basolateral compartment. After 30 min incubation aliquots (0.75 mL) were taken from the acceptor well and replaced by fresh cell culture medium. The incubation was terminated after 3 h and samples were taken from the donor and acceptor well separately and stored at 20 °C until analysis (see Section 2.5.3). Inserts were washed with PBS. The integrity of the artificial membrane was tested by LY after 3 h and plastic adsorption samples were taken as described above. In order to investigate the effect of the PET filter insert on CsA diffusion, the same experiment was performed without DOPC layer. As the experiments were not performed under sink conditions and also the effect of mass loss has to be considered, Papp was calculated with the modified ‘‘two-way flux’’ equation according to Liu et al. (2003):
Papp ¼ 2:303
V AV D V A þ V D C AðtÞ log10 1 ðV A þ V D Þ A t VD S C Dð0Þ ð4Þ
where S (fraction of sample remaining in the wells) was determined:
S¼
V A C AðtÞ C DðtÞ þ V D C Dð0Þ C Dð0Þ
ð5Þ
2.5.3. CsA quantification by LC–MS/MS The LC–MS/MS method is described by Bellwon et al. (2014) (submitted in this issue). Per analysis batch three independent calibration curves were prepared with 11 standard concentrations in a range from 10 nM to 2500 nM. Samples and calibration standards were evaporated at room temperature using the Heraeus Instruments vacuum centrifuge (Osterode, Germany) for CsA determination in cell culture media. The dry samples were re-dissolved in 50 lL of the corresponding cell culture medium prior to analysis. Samples were prepared in duplicates and each replicate was measured twice. The method was linear in a range from 10 nM to 2500 nM with R2 values ranging from 0.98 to 0.99. The limits of quantification (LOQ) and detection (LOD) were determined according DIN 32645 (Table 1). 2.6. Cylophilin B (Cyp-B) detection by western blot analysis Cyp-B secretion was determined by measurements of the Cyp-B content in the cell lysates and cell culture medium of the 3D model
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx Table 1 Determined limits of detection (LOD), limits of quantification (LOQ) and accuracies of analytical quality controls for CsA dissolved in the specific cell culture media and methanol. Matrix
LOD (nM)
LOQ (nM)
Accuracy of quality controls (%)
Cell culture medium 2D Cell culture medium 3D Cell culture medium Caco-2 Methanol (cell lysates, plastic adsorption)
0.64 ± 0.07 0.75 ± 0.03 2.71 ± 0.41 0.80 ± 0.02
2.23 ± 0.22 2.59 ± 0.08 9.98 ± 1.49 2.73 ± 0.06
102 ± 3 109 ± 14 103 ± 11 100 ± 10
by western blotting as described in the Bellwon et al. (2014) (submitted in this issue) with the following modifications. Cell culture medium samples were concentrated by evaporating an aliquot (150 lL) using a Savant™ SC110 SpeedVac concentrator (ThermoScientific) and reconstitution in 10 lL 1 LDS sample buffer (Life technologies) supplemented with 50 mM DTT. 2.7. Statistical analyses Statistical analyses for kinetic experiments were performed by 2 way ANOVA followed by Turkey’s multiple comparisons test using Graph Pad Prism 6. For kinetic experiments on the 2D model 4 biological replicates were conducted (n = 4) and for the 3D model 3 biological replicates (2 replicate cultures each) (n = 3). For transporter experiments three biological replicates (n = 3) were performed and statistical significance was tested by an unpaired, two-tailed Student’s t-test. 3. Results 3.1. Kinetics and dynamics In order to describe the kinetic profile of CsA, concentrations were measured in the treatment solutions, cell culture media, cell lysates and plastic adsorption samples of the 2D and 3D models. In addition, aliquots of the same samples from the 3D model were used to analyze the intra- and extracellular Cyp-B content. 3.1.1. 2D model Although the measured CsA concentrations in the stock solutions were comparable to the nominal concentrations, strong aberrations were observed for the treatment solutions. The CsA amounts in the treatment solutions were approximately 18% (low concentration) and 46% (high concentration) of the nominal concentration. For both low and high concentration treatment CsA concentrations reached the concentration-dependent steady state level in cell culture media (low concentration: 0.03 ± 0.01 lM; high concentration: 0.85 ± 0.21 lM) and in cell lysates (low concentration: 0.03 ± 0.01 nmole/well; high concentration: 0.14 ± 0.03 nmole/ well) within 30 min on day 0 (Fig. 1). For low concentration samples the total CsA recovery was 187 ± 53% of the administered CsA amount after 30 min on day 0. This can be attributed to the determined aberrations in the treatment solution. However, no statistical significant changes were observed neither within 24 h (p-value = 0.999, 2 way ANOVA with Turkey’s multiple comparisons test) nor 14 days (p-value = 0.996, 2 way ANOVA with Turkey’s multiple comparisons test). For high concentration treatment total CsA recovery was 103 ± 22% of the administered CsA amount and no statistical significant change was determined within 24 h (p-value = 0.259, 2 way ANOVA with Turkey’s multiple comparisons test). Although total CsA recovery was significantly
5
lower after repeated exposure (91 ± 15%, p-value = 0.041, 2 way ANOVA with Turkey’s multiple comparisons test) this seems not to be of biological relevance. Overall, neither CsA bioaccumulation nor biotransformation was observed. The recovered CsA amount adsorbed to plastic was 6.5 ± 1.2% of the nominal low concentration after 3 h and did not change within 13 days. Similar observations were made for the high concentration treatment with the recovered CsA amount being 3.4 ± 0.8% of the nominal concentration after 30 min and no further changes within the following 13 days of treatment. 3.1.2. 3D model The determined CsA concentrations in the treatment solutions did not show aberrations from the nominal concentrations. CsA concentrations in the cell culture medium decreased within 30 minutes, but remained stable for the following 23.5 h (low concentration: 0.09 ± 0.02 lM; high concentration: 0.61 ± 0.13 lM). CsA steady state levels in cell lysates were reached within 30 minutes after low (0.42 ± 0.08 nmole/flask) and high concentration treatment (1.37 ± 0.30 nmole/flask) on day 0 and remained stable for the following 13 treatment days (Fig. 2). The total CsA recovery was 94 ± 16% and 70 ± 21% of the administered low and high CsA amount, respectively, on day 0 after 30 min. No statistical significant changes were determined neither within 24 h nor 13 day of treatment (p-values > 0.05, 2 way ANOVA with Turkey’s multiple comparisons test). This resulted in missing bioaccumulation and biotransformation. Additionally, Cyp-B contents were measured in the cell culture medium and cell lysates for each sampling time point (Fig. 3). After low concentration treatment no remarkable Cyp-B decrease was detected in the cell lysates within 24 h exposure, although a detectable amount of Cyp-B was secreted to the cell culture medium. After 12 days of low concentration treatment Cyp-B was decreased in the cell lysates, but not detectable in the cell culture medium samples anymore. For high concentration treatment the Cyp-B amount decreased significantly in the cell lysate samples within 24 h and repeated treatment revealed complete depletion between 1 and 12 days. 3.2. CsA permeability across a BBB in vitro model As previously reported (Fabulas-da Costa et al., 2013), repeated exposure to 1 lM CsA did not have any effect on BBB integrity. The Pe (LY) values for cells treated with carriers (DMSO) in the absence of drugs remained low and were similar to those recorded with untreated cells (Table 2). The permeability coefficients of ECs for CsA Pe (CsA) were low despite its high lipophilicity and no changes in the distribution of CsA across the BBB were observed following one or two weeks of repeated treatment with 1 lM CsA. Furthermore, Pe values were in the same magnitude as the integrity threshold for LY. 3.3. Transport experiments 3.3.1. Results of the LY integrity test The determined LY amount in the acceptor wells was less than 0.5% of the added LY amount for untreated and treated Caco-2 cells after three hours of incubation. Therefore, exposure to 1 lM and 10 lM CsA did not influence the barrier integrity. For PAMPA and blank wells the detected LY amount was 1–3% and 3–11%, respectively, indicating that the small amount of recovered LY for Caco-2 cells was not due to impermeable filter inserts. 3.3.2. Results in Caco-2 cells Fig. 4 illustrates the determined CsA mass balances obtained from transport experiments using Caco-2 cells. CsA was
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
6
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
(A) day 0
(B) day 13
*
Fig. 1. Intracellular and extracellular CsA concentrations over time in the primary mouse 2D brain cell culture model. Primary frontal cortex cultures were derived from dissociated embryonic frontal cortex tissue from NMRI mice as described in the methods section. CsA concentrations were determined by LC–MS/MS in treatment solutions, cell culture media and cell lysates after low (0.1 lM) and high (2 lM) concentration treatment for one and 14 days. Samples were generated after 0 min (treatment concentration), 30 min, 1 h, 3 h, 6 h and 24 h on day 0 (A) and day 13 (B). Mean (± SD) of 4 biological replicates is shown. Statistical significance was tested by 2 way ANOVA followed by Turkey’s multiple comparisons test and asterisk (⁄) indicates p-values
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier P. Bellwon a,⇑,1, M. Culot b,c,1, A. Wilmes d,1, T. Schmidt a,1, M.G. Zurich e,1, L. Schultz f,1, O. Schmal a, A. Gramowski-Voss f, D.G. Weiss f, P. Jennings d, A. Bal-Price g, E. Testai h, W. Dekant a a
Department of Toxicology, University of Wuerzburg, Wuerzburg, Germany Univ Lille Nord de France, F59000 Lille, France UArtois, BBB Laboratory, EA 2465, F62300 Lens, France d Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck 6020, Austria e Department of Physiology, University of Lausanne, Lausanne, Switzerland f Department of Animal Physiology, Institute of Biological Sciences, University of Rostock, Rostock, Germany g ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra, VA, Italy h Mechanism of Toxicity Unit, Environment and Primary Prevention Department, Istituto Superiore di Sanità, Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 14 July 2014 Accepted 5 January 2015 Available online xxxx Keywords: Cyclosporine A uptake Neurotoxicity Brain cell cultures BBB model Passive diffusion Cyclophilin B
a b s t r a c t There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood–brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The time and cost of compounds coming to market are increasing world-wide and this has been attributed to the drug failures late in discovery either to lack of efficacy (56%) or safety issues (28%) (Arrowsmith and Miller, 2013; DiMasi et al., 2010). There Abbreviations: CsA, Cyclosporine A; Cyp-B, cyclophilin B; BBB, blood–brain barrier; LY, lucifer yellow; TC10, 10% toxic concentration; AB, apical-to-basolateral; BA, basolateral-to-apical; PAMPA, parallel artificial membrane assay; PK, pharmacokinetics; CNS, central nervous system; EC, endothelial cells. ⇑ Corresponding author at: Department of Toxicology, University Wuerzburg, Versbacher Str. 9, 97078 Wuerzburg, Germany. Tel.: +49 931 31 86367. E-mail addresses: [email protected], patricia.bellwon@yahoo. com (P. Bellwon). 1 Equal contributing authors.
is now a consensus that improvement of preclinical phase testing would drastically alleviate this burden. Neurotoxicity has been identified as a frequent adverse drug effect contributing to the termination of up 22% of drug candidates (Watkins, 2011). The evaluation of potential neurotoxicity is recommended to be performed in rodents during the preclinical safety assessment of drugs and chemicals (Bolon et al., 2010; OECD Test Guideline No. 424), however in vivo data can be affected by variables such as neuronal, hormonal and immunological stimuli (Harry and TiffanyCastiglioni, 2005). To date, in vitro approaches using neuronal and glial cells are applied to investigate the mechanisms of neurotoxicity, but such systems may also be considered as alternative methods for risk assessment to meet the needs for predictive, time-saving and cost-reducing methods (Bal-Price et al., 2010). Within the
http://dx.doi.org/10.1016/j.tiv.2015.01.003 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
2
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
‘‘ACuteTox’’ Project it was shown that brain cell cultures, e.g. primary cultures of cortical neurons and primary aggregating rat brain cells, may serve as prediction models for acute neurotoxic effects (Bal-Price et al., 2008; Forsby et al., 2009; Suñol et al., 2008). Since pharmaceutical compounds are usually given in repeated exposures, often life-long, the prediction models for toxic effects after repeated exposures are required. To address this issue,the 7th Framework Program ‘‘Predict-IV’’ aimed to improve the predictability of drug-induced toxicity in the early stage of drug development. Pharmacodynamics in combination with pharmacokinetics (PK) was characterized in well-established in vitro models representing three target organs, namely liver, kidney and central nervous system (CNS). As PK evaluation is necessary to describe the systemic exposure and its relationship to observed (adverse) effects, PK data enhance the understanding of mechanisms of action and/or toxicity induced by xenobiotics and moreover help to assess the risk and safety in humans (ICH S3A Guideline; OECD Test Guideline No. 417). The cyclic peptide cyclosporine A (CsA), which is a widely used immunosuppressive compound, was chosen as a cross-organ test compound in the ‘‘Predict-IV’’ project as it has been shown to induce hepatotoxicity (Atkinson et al., 1983; Klintmalm et al., 1981), nephrotoxicity (Bennett and Pulliam, 1983; Klintmalm et al., 1985; Palestine et al., 1984) and also neurotoxicity (Calne et al., 1979; Gijtenbeek et al., 1999). As the therapeutic index of CsA is narrow, therapy requires drug monitoring to avoid toxicity. Nevertheless, neurotoxicity was also observed in patients with CsA blood levels within the therapeutic range (Serkova et al., 2004). Various symptoms of CsA-induced neurotoxicity have been reported: tremor, headache, seizures, visual hallucinations and delusions (Gijtenbeek et al., 1999). In general, neurotoxicity is reversible after termination of CsA administration (Gijtenbeek et al., 1999). Whilst a major focus in the literature has been to the mechanisms of the neurotoxicity of CsA, there has been less focus on the capacity of CsA to cross the blood–brain barrier (BBB). Indeed the weight of evidence is that CsA poorly crosses the BBB. CsA determination in brain tissue of humans (Atkinson et al., 1982; Lensmeyer et al., 1991), rats (Lemaire et al., 1988; Niederberger et al., 1983; Wagner et al., 1987) and mice (Boland et al., 1984) revealed very low levels of the drug, indicating that only minor CsA amounts are able to cross the BBB. The aim of this paper was to investigate whether CNS exposures to the model compound CsA can be predicted using an integrated in vitro testing strategy. In order to achieve this, kinetics and dynamics were investigated in two rodent brain cell cultures: (i) primary neuronal culture of mouse cortex (2D model) and (ii) primary aggregating rat brain cells (3D model). In order to obtain CsA kinetic profiles after single and repeated exposure to a low (nontoxic) and high (10% toxic) CsA concentration, CsA levels were determined in treatment solutions, cell lysates, cell culture media and plastic adsorption samples. To further determine whether CsA is taken up by the cell systems, evaluation of a CsA-specific pharmacodynamic endpoint (cyclophilin B [Cyp-B] depletion by western blot analysis) was included (Fearon et al., 2011; Price et al., 1994; Wilmes et al., 2013). In order to investigate the ability of CsA to cross the BBB, CsA permeability across an established BBB model based on co-culture of bovine brain capillary endothelial cells (EC) and rat astrocytes was studied (Fabulas-da Costa et al., 2013). In addition, in order to get insight into the cellular uptake mechanism transporter experiments were performed using a parallel artificial membrane assay (PAMPA) and Caco-2 cells. PAMPA determines a possible uptake by passive diffusion (Kansy et al., 1998). While the predicted permeability can be over- or underestimated, PAMPA in combination with cell systems that possess transporter activities is recommended (Di and Kerns, 2003). Thus
Caco-2 cells were used as a standard reference cell line for active cellular extrusion (Augustijns et al., 1993; Gan et al., 1996). 2. Materials and methods 2.1. Materials (specified by partner) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dodecane, dimethylsulfoxid (DMSO) and lucifer yellow (LY) were obtained from Sigma Aldrich (Taufkirchen, Germany). CsA was obtained from Calbiochem (Darmstadt, Germany). The internal standard deuterium-labeled CsA (CsA-d12) was purchased from Analytical Services International Ltd (London, United Kingdom). For the mobile phase preparation ammonium acetate, formic acid, methanol (HPLC grade) and water (HPLC grade) were purchased from Carl Roth (Karlsruhe, Germany). For Caco-2 cell culture DMEM (Cat. No. P04-03500) was purchased from PAN Biotech (Aidenbach, Germany) and all other cell culture reagents were purchased from PAA (Coelbe, Germany). 2.2. Isolation and cell culture conditions 2.2.1. Primary neuronal mouse cells (2D model) Primary frontal cortex cultures were derived from dissociated embryonic frontal cortex tissue from NMRI mice (Charles River, Sulzfeld, Germany) on day 16. The culture was processed according to Ransom et al. (1977). The method was modified by the usage of DNase I (8000 units/mL, Roche, Mannheim, Germany) and papain (10 U/mL, Roche, Mannheim, Germany) for tissue dissociation (Huettner and Baughman, 1986) and further mechanical cell dissociation. Cells were suspended in primary neurobasal medium (PNB, Lonza, Verviers, Belgium) at a density of 0.5 106 cells/mL, seeded into 6 well plates and incubated under defined conditions (37 °C, 5% CO2; Binder, Tuttlingen Germany). The cell culture networks formed from a mixture of different types of neurons and glial cells can be cultured for several months due to their stability by co-cultivation (Gramowski et al., 2006; Weiss, 2011). Glial cells were allowed to proliferate for 3–5 days after seeding until reaching approximately 80% confluency. Growth of glial cells was stopped by 5-fluoro-20 -deoxyuridine (25 lM) treatment. Serum free cell culture medium was used and refreshed three times a week. The repeated exposure experiments started 4 weeks after seeding. 2.2.2. Primary aggregating rat brain cell cultures (3D model) The aggregating rat brain cultures were prepared from 16-day old Sprague–Dawley rat embryos (Janvier, St Berthevin, France) as described by Honegger et al. (2011). Briefly, the brains of the embryos were dissected and dissociated into a single cell suspension by sequential passage through nylon sieves of 200 lm and 115 lm pores (Sefar, Heiden, Switzerland). The cells were washed by centrifugation, resuspended in cold serum-free culture medium (modified Dulbecco’s Modified Eagle Medium (DMEM)), and incubated in modified Erlenmeyer culture flasks. The spontaneously generated three-dimensional cultures were maintained as suspension culture under constant gyratory agitation (80 rpm) at 37 °C, in an atmosphere of 10% CO2 and 90% humidified air. Culture media were replenished with fresh medium every third day until day 14 by exchanging 5 mL (of a total of 8 mL per flask). After 14 days in vitro (DIV), the culture media were replenished every second day. The repeated exposure experiments started at DIV 18. 2.2.3. In vitro model of the BBB Cells were isolated and cultured as described previously (Fabulas-da Costa et al., 2013). Briefly, ECs isolated from capillary fragments were co-cultured with primary mixed glial cells from
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
newborn Sprague Dawley rats. The glial cells were isolated according to the method of Booher and Sensenbrenner (1972) and cultured for 3 weeks, plated on the bottom of cell culture clusters containing six wells each. The EC were seeded onto cell culture inserts, which were placed in the wells containing glial cells. The medium used for the co-culture was Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS), 10% horse serum, 2 mM glutamine, 50 mg/L gentamicin and 1 lg/L basic fibroblast growth factor (Sigma). The medium was changed every second day. Under these conditions, ECs formed a confluent monolayer after 7 days. Treatments with CsA were initiated 5 days after confluence. 2.2.4. Human intestinal cell line (Caco-2) Cells were cultured in DMEM supplemented with 2 mM L-glutamine, 1 nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 lg/mL streptomycin and 10% FCS. Cell culture medium was refreshed twice a week and Caco-2 cells were passaged 1:5 by with trypsin after reaching 70–85% confluence. 2.3. Kinetics and dynamics – experimental design and sampling The 2D model was exposed to a 10% toxic concentration (TC10, 2 lM) as determined by the extracellular LDH activity measured routinely with every medium change during the time of repeated exposure and 1/20th of the TC10 (0.1 lM). The 1000-fold concentrated stock solutions were prepared in DMSO. On the first day of treatment, 3 lL of the concentrated stock solution were pipetted directly into the cell medium of each well of the replicate culture. The culture medium was replenished (1.5 mL out of a total of 3 mL) every second day and the stock solutions were pipetted into the fresh medium prior to the replenishment and then given to the cell cultures. The 3D model was exposed to the 10% toxic concentration (TC10, 1 lM) as determined by the intracellular LDH activity measured at the end of the repeated exposure time and 1/5th of the TC10 (0.2 lM). The 2000-fold concentrated stock solutions were prepared in DMSO. On the first day of treatment, 4 lL of the respective 2000-fold concentrated stocks were pipetted directly into the cell culture medium of each replicate culture. Every second day after medium replenishment (5 mL out of a total of 8 mL) cultures were allowed to re-equilibrate the pH of their medium during 2 h. Then, 2.5 lL of the 2000-fold stock solutions were pipetted directly into the cell culture medium of each replicate culture. Cells were treated every second day with a low and high CsA concentration for one and 13 (3D model) or 14 days (2D model). Samples were generated on the first and the last treatment day at five time points (30 min, 1 h, 3 h, 6 h and 24 h). In addition, CsA concentration was determined in the treatment solution. Four biological replicates were performed for the 2D model and three biological replicates (2 replicate cultures each) for the 3D model. To determine the CsA kinetic profile, samples were generated from the cell culture media and cell lysates. After removal of the cell culture media, cells were washed twice with PBS. Cells from the 2D model were scraped in 0.25 mL methanol, transferred to a glass vial and rinsed with 0.1 mL methanol. For the 3D model, 1 mL methanol was added to the cells. After sonication, lysates were transferred to LoBind tubes. In order to determine the CsA amount adsorbed to plastic plates used for the 2D model, wells were washed twice with PBS and 1.5 mL methanol was added and incubated under gentle shaking at room temperature for two hours. No plastic adsorption samples were generated for the 3D model as cells were cultured in glass flasks. All samples were stored at 80 °C until analysis (see Section 2.5.3).
3
2.4. CsA permeability across a BBB in vitro model 2.4.1. Experimental design After 12 days of co-culture with glial cells, CsA (1 lM in 0.25% DMSO) was applied to the apical compartment containing ECs. As controls, ECs were treated only from the apical side with 0.25% of DMSO without drugs. The basolateral compartments containing glial cells were filled with fresh medium. Treatments and media were renewed every 2 days for 14 days. The permeability of CsA and of the non-permeable fluorescent marker LY, used as a marker of the integrity of the EC monolayer, was assessed in ECs on day 0, 7 and 14. Briefly, ECs monolayers were transferred to six-well plates containing 2.5 mL Ringer– HEPES solution (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2 (6 H2O), 6 mM NaHCO3,5 mM HEPES, 2.8 mM glucose, pH 7.4) per well. 1.5 mL Ringer-HEPES solution containing 10 lM CsA and 20 lM LY was then added to the apical compartment. Incubations were performed at 37 °C. After 1 h, aliquots were taken from both compartments and the concentrations of CsA and LY were measured by LC/MS or with a fluorescence spectrophotometer (Synergy H1, Biotek, Winooski, USA). For each experiment three inserts with cells and three without cells were assayed to enable the calculation of the endothelial permeability coefficient (Pe). Pe was calculated (in cm/min). In this calculation, both filter without cells permeability (PSf = insert filter + collagen coating) and filter plus cell permeability (PSt = filter + collagen + EC) were taken into account, according to the formula:
1 1 1 ¼ PSe PSt PSf PSe Pe ¼ A
ð6Þ ð7Þ
where A is the surface area of the filter (i.e. 4.7 cm2). When BBB integrity was evaluated over time, cell culture inserts were filled with fresh medium at the end of the 1 h transport experiment and treatment solutions were renewed. As previously established (Fabulas-da Costa et al., 2013) a compound was considered to have generated a toxic effect (i.e. impaired BBB function) when the Pe (LY) value of treated cells was increased 2-fold compared to control cells (treated with carrier solvent in the absence of drug) at the same time points. 2.4.2. LC–MS/MS analysis CsA concentrations in the luminal and abluminal compartments were determined by LC–MS/MS. Analyses were performed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, West Palm Beach, FL, USA) equipped with a TurboIonspray with an Ion Max source and coupled to Thermo Scientific Accela HPLC autosampler and pump (Thermo Scientific, West Palm Beach, FL, USA). A fusion column (80 Å, 150 4.6 mm, 4 lm – Phenomenex Synergi Fusion) was used for chromatographic separation at 50 °C. The separation was isocratic using a mixture of acetonitrile/water/formic acid (90/9/1, v/v/v) as mobile phase. An injection volume of 10 lL was used with a flow rate of 0.4 mL/min. CsA was detected in positive ion mode setting the spray voltage and capillary temperature were set at 4.0 kV and 300 °C, respectively. The pressures for the nitrogen sheath gas, auxiliary gas and sweep gas were maintained at 30, 10 and 5 units. Data acquisition was performed in selected reaction monitoring (SRM) mode detecting two transitions (m/z 1225.1 ? 1203.15; m/z 1225.1 ? 1185.1) with collision energy of 30 eV and S-lense RF amplitude voltage set to 90 V. Argon was used as collision gas at 0.2 Pa. Instrument control and data processing were carried out by means of Xcalibur Software 2.0.7 SP1 (Thermo Electron, San José, USA).
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
4
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
F(cum) is the cumulative fraction transported (pmol/cm3) and was determined as:
2.5. Transport experiments Transport experiments on Caco-2 cells and a parallel artificial membrane were performed in triplicates and for each experiment corresponding controls were generated by using cell culture medium containing 0.1% DMSO. 2.5.1. Caco-2 Cells were seeded at a density of 6 105 cells/mL cell culture medium on PET filter inserts (Thinserts, 0.4 lm pore size, translucent; Greiner Bio-One) in a 12-well plate and blank cell culture medium was added to the basolateral side. After 2 weeks of cell culture FCS was reduced from 10% to 5% and after 3 weeks the transport experiment was performed at 0% FCS. CsA was dissolved in DMSO (1 mM and 10 mM) and further diluted with cell culture medium to final concentrations of 1 lM and 10 lM with 0.1% DMSO content in all treatment solutions. The transport of CsA was tested in both directions apical-tobasolateral (AB) and basolateral-to-apical (BA). Therefore, after removing the cell culture medium, pre-warmed CsA solutions were either added to the apical (0.5 mL) or to the basolateral side (1.5 mL) – called donor well - while the corresponding opposite sides were filled with pre-warmed cell culture medium containing 0.1% DMSO – called acceptor well. An aliquot of the treatment solutions was taken for determination of the actual CsA amount given to the cells. After 30 and 60 min of incubation, aliquots were taken from the acceptor well (apical: 0.25 mL; basolateral: 0.75 mL) and replaced by fresh cell culture medium containing 0.1% DMSO. The incubation was terminated after 3 h and cell culture medium from both donor and acceptor wells were collected separately into glass vials. Prior to cell lysis the paracellular integrity of the cell monolayer was tested by usage of the integrity marker lucifer yellow (LY) as described earlier by Broeders et al. (2012). Therefore, cells were washed with PBS and 0.5 mL LY dissolved in cell culture medium (0.2 mM) was added to each well from the apical side and blank medium from the basolateral side. After 1 hour incubation aliquots (0.1 mL) were taken from the basolateral compartments, transferred into a 96-well plate and fluorescence was measured at 485 nm (excitation) and 535 nm (emission) using the Mithras LB 940 Multimode Mikroplate Reader (Berthold Technologies, Bad Wildbad, Germany). Cell culture medium was set as the blank reference and 0.2 mM LY solution in cell culture medium as 100%. Cell lysate and plastic adsorption samples were generated in order to determine the CsA mass balance and further, to investigate whether potential CsA loss occurs due to biotransformation or adsorption to the plastic of the well walls. Therefore, after removal of the cell culture medium, cells were washed with PBS and 0.1 mL trypsin was added to each well for cell harvesting. Samples were transferred to glass vials, wells were washed with 0.4 mL methanol and added to the same vials. Afterwards, 0.5 mL and 1.5 mL methanol were added to the apical and basolateral side, respectively. Plates were covered with parafilm and stored at room temperature under gentle shaking. After 1 h samples from one well (apical and basolateral) were pooled in order to determine the CsA amount adsorbed to the plastic of the well walls. All samples were stored at 20 °C until LC–MS/MS quantification (see Section 2.5.3). Papp was calculated according to Broeders et al. (2012):
Papp ¼
DF ðcumÞ A1 V D Dt
ð1Þ
where DF(cum)/Dt is the slope of the graphs representing the cumulative CsA fraction transported over time, A (cm2) is the filter insert area (1.131 cm2 for 12-well inserts), VD is the CsA solution volume (cm3) filled into the donor well (apical: 0.5 cm3; basolateral: 1.5 cm3).
F ðcumÞ ¼
X bC AðTPxÞ f C AðTPx1Þ c V A CD V D
ð2Þ
CD and CA are the measured CsA concentrations (pmol/cm3) at each time point (TPx) in the donor and acceptor well, respectively; VA is the volume (cm3) of cell culture medium in the acceptor well and f is the replacement factor and was calculated as:
f ¼1
VS VA
ð3Þ
where VS is the volume of the sample. 2.5.2. Parallel artificial membrane permeability assay (PAMPA) 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was dissolved in dodecan with a final concentration of 1 mg/mL. After adding 0.05 mL of the 0.1% DOPC solution to the PET filter inserts, plates were shaken for 5 min at room temperature. Passive diffusion was tested from the apical to basolateral side. Therefore, CsA treatment solutions were added to the apical compartment and blank cell culture medium to the basolateral compartment. After 30 min incubation aliquots (0.75 mL) were taken from the acceptor well and replaced by fresh cell culture medium. The incubation was terminated after 3 h and samples were taken from the donor and acceptor well separately and stored at 20 °C until analysis (see Section 2.5.3). Inserts were washed with PBS. The integrity of the artificial membrane was tested by LY after 3 h and plastic adsorption samples were taken as described above. In order to investigate the effect of the PET filter insert on CsA diffusion, the same experiment was performed without DOPC layer. As the experiments were not performed under sink conditions and also the effect of mass loss has to be considered, Papp was calculated with the modified ‘‘two-way flux’’ equation according to Liu et al. (2003):
Papp ¼ 2:303
V AV D V A þ V D C AðtÞ log10 1 ðV A þ V D Þ A t VD S C Dð0Þ ð4Þ
where S (fraction of sample remaining in the wells) was determined:
S¼
V A C AðtÞ C DðtÞ þ V D C Dð0Þ C Dð0Þ
ð5Þ
2.5.3. CsA quantification by LC–MS/MS The LC–MS/MS method is described by Bellwon et al. (2014) (submitted in this issue). Per analysis batch three independent calibration curves were prepared with 11 standard concentrations in a range from 10 nM to 2500 nM. Samples and calibration standards were evaporated at room temperature using the Heraeus Instruments vacuum centrifuge (Osterode, Germany) for CsA determination in cell culture media. The dry samples were re-dissolved in 50 lL of the corresponding cell culture medium prior to analysis. Samples were prepared in duplicates and each replicate was measured twice. The method was linear in a range from 10 nM to 2500 nM with R2 values ranging from 0.98 to 0.99. The limits of quantification (LOQ) and detection (LOD) were determined according DIN 32645 (Table 1). 2.6. Cylophilin B (Cyp-B) detection by western blot analysis Cyp-B secretion was determined by measurements of the Cyp-B content in the cell lysates and cell culture medium of the 3D model
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx Table 1 Determined limits of detection (LOD), limits of quantification (LOQ) and accuracies of analytical quality controls for CsA dissolved in the specific cell culture media and methanol. Matrix
LOD (nM)
LOQ (nM)
Accuracy of quality controls (%)
Cell culture medium 2D Cell culture medium 3D Cell culture medium Caco-2 Methanol (cell lysates, plastic adsorption)
0.64 ± 0.07 0.75 ± 0.03 2.71 ± 0.41 0.80 ± 0.02
2.23 ± 0.22 2.59 ± 0.08 9.98 ± 1.49 2.73 ± 0.06
102 ± 3 109 ± 14 103 ± 11 100 ± 10
by western blotting as described in the Bellwon et al. (2014) (submitted in this issue) with the following modifications. Cell culture medium samples were concentrated by evaporating an aliquot (150 lL) using a Savant™ SC110 SpeedVac concentrator (ThermoScientific) and reconstitution in 10 lL 1 LDS sample buffer (Life technologies) supplemented with 50 mM DTT. 2.7. Statistical analyses Statistical analyses for kinetic experiments were performed by 2 way ANOVA followed by Turkey’s multiple comparisons test using Graph Pad Prism 6. For kinetic experiments on the 2D model 4 biological replicates were conducted (n = 4) and for the 3D model 3 biological replicates (2 replicate cultures each) (n = 3). For transporter experiments three biological replicates (n = 3) were performed and statistical significance was tested by an unpaired, two-tailed Student’s t-test. 3. Results 3.1. Kinetics and dynamics In order to describe the kinetic profile of CsA, concentrations were measured in the treatment solutions, cell culture media, cell lysates and plastic adsorption samples of the 2D and 3D models. In addition, aliquots of the same samples from the 3D model were used to analyze the intra- and extracellular Cyp-B content. 3.1.1. 2D model Although the measured CsA concentrations in the stock solutions were comparable to the nominal concentrations, strong aberrations were observed for the treatment solutions. The CsA amounts in the treatment solutions were approximately 18% (low concentration) and 46% (high concentration) of the nominal concentration. For both low and high concentration treatment CsA concentrations reached the concentration-dependent steady state level in cell culture media (low concentration: 0.03 ± 0.01 lM; high concentration: 0.85 ± 0.21 lM) and in cell lysates (low concentration: 0.03 ± 0.01 nmole/well; high concentration: 0.14 ± 0.03 nmole/ well) within 30 min on day 0 (Fig. 1). For low concentration samples the total CsA recovery was 187 ± 53% of the administered CsA amount after 30 min on day 0. This can be attributed to the determined aberrations in the treatment solution. However, no statistical significant changes were observed neither within 24 h (p-value = 0.999, 2 way ANOVA with Turkey’s multiple comparisons test) nor 14 days (p-value = 0.996, 2 way ANOVA with Turkey’s multiple comparisons test). For high concentration treatment total CsA recovery was 103 ± 22% of the administered CsA amount and no statistical significant change was determined within 24 h (p-value = 0.259, 2 way ANOVA with Turkey’s multiple comparisons test). Although total CsA recovery was significantly
5
lower after repeated exposure (91 ± 15%, p-value = 0.041, 2 way ANOVA with Turkey’s multiple comparisons test) this seems not to be of biological relevance. Overall, neither CsA bioaccumulation nor biotransformation was observed. The recovered CsA amount adsorbed to plastic was 6.5 ± 1.2% of the nominal low concentration after 3 h and did not change within 13 days. Similar observations were made for the high concentration treatment with the recovered CsA amount being 3.4 ± 0.8% of the nominal concentration after 30 min and no further changes within the following 13 days of treatment. 3.1.2. 3D model The determined CsA concentrations in the treatment solutions did not show aberrations from the nominal concentrations. CsA concentrations in the cell culture medium decreased within 30 minutes, but remained stable for the following 23.5 h (low concentration: 0.09 ± 0.02 lM; high concentration: 0.61 ± 0.13 lM). CsA steady state levels in cell lysates were reached within 30 minutes after low (0.42 ± 0.08 nmole/flask) and high concentration treatment (1.37 ± 0.30 nmole/flask) on day 0 and remained stable for the following 13 treatment days (Fig. 2). The total CsA recovery was 94 ± 16% and 70 ± 21% of the administered low and high CsA amount, respectively, on day 0 after 30 min. No statistical significant changes were determined neither within 24 h nor 13 day of treatment (p-values > 0.05, 2 way ANOVA with Turkey’s multiple comparisons test). This resulted in missing bioaccumulation and biotransformation. Additionally, Cyp-B contents were measured in the cell culture medium and cell lysates for each sampling time point (Fig. 3). After low concentration treatment no remarkable Cyp-B decrease was detected in the cell lysates within 24 h exposure, although a detectable amount of Cyp-B was secreted to the cell culture medium. After 12 days of low concentration treatment Cyp-B was decreased in the cell lysates, but not detectable in the cell culture medium samples anymore. For high concentration treatment the Cyp-B amount decreased significantly in the cell lysate samples within 24 h and repeated treatment revealed complete depletion between 1 and 12 days. 3.2. CsA permeability across a BBB in vitro model As previously reported (Fabulas-da Costa et al., 2013), repeated exposure to 1 lM CsA did not have any effect on BBB integrity. The Pe (LY) values for cells treated with carriers (DMSO) in the absence of drugs remained low and were similar to those recorded with untreated cells (Table 2). The permeability coefficients of ECs for CsA Pe (CsA) were low despite its high lipophilicity and no changes in the distribution of CsA across the BBB were observed following one or two weeks of repeated treatment with 1 lM CsA. Furthermore, Pe values were in the same magnitude as the integrity threshold for LY. 3.3. Transport experiments 3.3.1. Results of the LY integrity test The determined LY amount in the acceptor wells was less than 0.5% of the added LY amount for untreated and treated Caco-2 cells after three hours of incubation. Therefore, exposure to 1 lM and 10 lM CsA did not influence the barrier integrity. For PAMPA and blank wells the detected LY amount was 1–3% and 3–11%, respectively, indicating that the small amount of recovered LY for Caco-2 cells was not due to impermeable filter inserts. 3.3.2. Results in Caco-2 cells Fig. 4 illustrates the determined CsA mass balances obtained from transport experiments using Caco-2 cells. CsA was
Please cite this article in press as: Bellwon, P., et al. Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood–brain barrier. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.01.003
6
P. Bellwon et al. / Toxicology in Vitro xxx (2015) xxx–xxx
(A) day 0
(B) day 13
*
Fig. 1. Intracellular and extracellular CsA concentrations over time in the primary mouse 2D brain cell culture model. Primary frontal cortex cultures were derived from dissociated embryonic frontal cortex tissue from NMRI mice as described in the methods section. CsA concentrations were determined by LC–MS/MS in treatment solutions, cell culture media and cell lysates after low (0.1 lM) and high (2 lM) concentration treatment for one and 14 days. Samples were generated after 0 min (treatment concentration), 30 min, 1 h, 3 h, 6 h and 24 h on day 0 (A) and day 13 (B). Mean (± SD) of 4 biological replicates is shown. Statistical significance was tested by 2 way ANOVA followed by Turkey’s multiple comparisons test and asterisk (⁄) indicates p-values
