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Home , McN5652, Reuptake

Fluorescence-based Transporter Uptake Assay in Primary Neuronal Cultures. Parsa Safa, MDS Analytical Technologies, Sunnyvale, CA 94089 & Lesley Radov, AstraZeneca Pharmaceuticals, CNSP Discovery, Wilmington, DE 19803

Abstract Results

The use of a fluorescence-based assay kit for the measurement of neurotransmitter Fig. 1. Dye loading occurs in a time and Fig. 4. Cultures from several but not all brain regions show significant Table 2. IC50 values are reproducible in primary transporter (NT) uptake activity in cell lines stably expressing the human DAT, NET and SERT gene has been previously reported. We describe here the application of this temperature dependent manner in primary dye uptake. neuronal cultures of the same age. fluorescence-based method to measure NT uptake activity in primary E-18 rat neuronal neuronal cultures and is sensitive to external Nucleus accumbens 7000 Striatum cultures. Real-time changes in NET, DAT & SERT transport activity were monitored in 7000 2 sodium and chloride concentrations. 6000 Compound Confidence R Days in these . Since these 3 biogenic amine transporters show overlapping localization and 6000 37°C 5000 IC50 (nM) Interval (nM) Culture expression in the brain, NT uptake in sister cultures derived from the hippocampus, cortex, a) Time and Temperature 5000 midbrain, striatum and nucleus accumbens were compared.. Hippocampal cultures were 4000 4000 Week 1 Day 1 10.5 5.8 - 58.9 0.9863 Day 7 used to assess NET uptake activity; nucleus accumbens cultures were used to assess DAT 7000 3000 RFU uptake activity; and midbrain cultures were used to assess SERT uptake activity. Reference RFU 3000 Day 2 8.6 6.1 - 12.1 0.9272 Day 8 37°C 2000 NET, DAT & SERT inhibitors from various pharmacological classes were evaluated for their 6000 2000 RT 37°C ability to inhibit transport in these neuronal cultures and the IC50 values generated 1000 1000 compared to literature values. 5000 0 0 500 1000 1500 2000 2500 3000 3500 4000 Week 2 Day 1 11.3 4.8 - 26.7 0.9130 Day 7 Time (seconds) RT

RFU -1000 4000 RT -1000 Day 2 6.8 2.4 - 19.0 0.9914 1000 2000 3000 4000 5000 6000 7000 8000 Day 8 Time (seconds) 3000 Week 3 Day 1 15.7 11.1 - 22.3 0.9852 Day 7 2000 Hippocampus Midbrain 7000 Day 2 6.8 3.0 - 15.0 0.9113 Day 8

7000 6000 1000 37°C 0 500 1000 1500 2000 2500 3000 3500 4000 6000 This table illustrates that the results are reproducible from week to week when Time (seconds) 5000 5000 cultures of the same age are used. The confidence limits overlap between all 6 4000 4000 studies, showing that there is no significant difference among the 6 the IC50 RT RFU 3000 values.

Introduction RFU 3000 37°C 2000 b) Replacing Sodium or Chloride in the Buffer 2000 1000 1000 RT 6500 Depression is a common metal disorder. It is estimated that the lifetime prevalence of major Normal Table 3. Comparison of IC results from primary 6000 0 0 50 1 500 1000 1500 2000 2500 3000 3500 4000 depressive disorder (MDD) is ~7%. Approximately two dozen marketed are 5500 Time (seconds) -1000 -1000 500 1000 1500 2000 2500 3000 3500 4000 neuronal cultures with reported K values. currently available worldwide. Many of these increase the concentration of the 3 5000 i Time (seconds) biogenic amine most closely linked to depression (, , 4500 4000 The dye uptake signal/noise window in primary cultures of hippocampus (NET), midbrain (SERT), and norephinephrine/noradrenaline) in the synaptic cleft. This is accomplished by inhibiting their 3500 reuptake back into the presynaptic . Drugs targeting a single neurotransmitter 3000 nucleus accumbens (DAT) is sufficient and reliable enough for these tissues to be used for transporter Syn 1° Cell Syn 1° Cell Syn 1° Cell RFU 2500 studies. The dye uptake signal/noise window in primary cultures of striatal cultures is NOT large enough NET NET SERT SERT DAT DAT transporter, such as the selective serotonin reuptake inhibitors (SSRIs) or selective 2000 noradrenalin reuptake inhibitors (SRNIs), were developed to limit the untoward side effects of 1500 Chloride Replacement to allow these cultures to be used on a routine basis – especially with the variability inherent in the system. nM nM nM nM nM nM the tricyclic antidepressants while mimicking their efficacy. The next step forward in 1000 500

depression treatment was the development of dual uptake inhibitors (SSRI/SSNI). These 0 Sodium Replacement agents have gained acceptance in the clinic, but are not totally satisfactory due to delayed onset, -500 15.6 22.8 4.6 4.3 369.2 496 -1000 low response rates and their side effect profile. The latest advance in depression therapy has 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 Fig. 5. The of a triple uptake inhibitor: McN5652 281 306 12 11.6 1600 2961 Time (seconds)

been the development of the triple uptake inhibitors (SSRI/SSNI/selective dopamine uptake IC50 Curve for McN5652 in the NT Dye Uptake IC50 Curve for McN5652 in the NT Dye Uptake Inhibition 4000 6147 1.3 2.8 28000 33970 inhibitor). Inhibition Assay in Prim ary Cortical Cultures Assay in Primary Hippocampal (NET) Cultures Top: uptake proceeds faster at 37°C than at room temperature. This 620 709 7 3.3 5000 5293 is consistent with the ‘rule of thumb’ that rate of uptake 9000 81 121 0.29 0.47 5100 5800 The most common technique used to estimate biogenic amine transport has been monitoring 8500 14000 approximately doubles for every increase of 10°C. 160 197 0.19 0.11 48 66.1 the accumulation of radiolabeled substrate or inhibitor. One major drawback to the use of 8000 Bottom: sodium and chloride are essential for transport into 12500 these radioactive methods is the difficulty in monitoring real-time changes. A second 7500 GBR12909 440 242 170 301 1 1.6 primary neurons confirming that transport is via Na+ /Cl- coupled consideration is the increased pressure to minimize the use of radiolabeled materials due to 7000 11000 1.3 1.7 310 351 510 1000 transporters 6500

public safety concerns and increasing disposal costs. An alternative, non-radioactive approach RFU RFU 9500 5 15.5 1280 1660 51 65.1 for determining biogenic amine transport is available. The use of a fluorescence-based assay 6000 5500 0.9 0.24 340 632 5200 6700 kit for the measurement of neurotransmitter transporter uptake activity in cell lines stably 8000 expressing the human DAT, NET and SERT gene has been previously reported. A question 5000 2300 2200 15600 11540 630 826 6500 that constantly arises is the relevance of the use of cell lines over-expressing the recombinant 4500 8.2 15.8 1070 1636 >100000 4000 10 -1 0 10 -9 10 -8 10 -7 10 -6 5000 SERT, DAT or NET vs. the use of native tissue protein to determine uptake. We describe here Fig. 2. The Neurotransmitter Transporter Uptake 10 -12 .5 10 -12.0 10 -11.5 10-1 1.0 10 -1 0.5 10 -10 .0 10 -9.5 10 -9.0 10-8.5 10-8 .0 10-7 .5 10-7 .0 10 -6 .5 Concentration [M] the application of this fluorescence-based method to measure NT uptake activity in a native Concentration [M] Assay is rapid and robust. McN5652: IC = 27.8nM; CI: 16.6 - 46.8nM; R 2 = 0.9607 2 13 26 42 33.8 5110 8238 tissue system. The native tissue system we have chosen to employ is the primary E-18 rat 50 McN5652: IC 50 = 2.8nM; CI: 1.5 - 5.4nM; R = 0.9429 Control Control McN5652 2.9 2.8 0.68 0.68 36.8 44.9 neuronal culture model. Data in Excel Spreadsheet Plate 1° neuronal cells 5-9 [Conc] 60' Pre-dye Difference [Conc] 60' Pre-dye Difference [Conc] 60' Pre-dye Difference 1.67E-07 9493.9 9167.7 326.1 6.17E-09 10476.0 8490.4 1985.7 2.28624E-10 12115.6 9452.4 2663.2 IC50 Curve for McN5652 in the NT Dye Uptake Inhibition IC50 Curve for McN5652 in the NT Dye Uptake Inhibition days before assay 10755.3 10215.3 540.0 10849.3 9008.8 1840.5 11312.5 8588.7 2723.7 There was excellent agreement between published literature Ki values for 11342.8 10998.0 344.9 10035.7 7958.9 2076.8 10938.9 8298.8 2640.1 Assay in Primary Midbrain (SERT) Cultures Assayin Primary Nucleus Accumbens (DAT) Cultures 10667.7 10152.4 515.3 10804.5 8723.9 2080.6 10671.4 8021.8 2649.6 10945.8 10755.7 190.1 10770.8 9085.6 1685.1 10964.4 8356.3 2608.1 the 14 drugs evaluated and the IC50 values generated in the primary 10314.7 9896.7 418.0 11108.3 9048.4 2059.8 10069.3 7442.2 2627.1 1200 12000 1100 neuronal culture system. The Spearman r for each transporter is as 5.56E-08 10346.6 9103.2 1243.4 11361.2 9269.3 2091.8 7.62079E-11 10350.1 7553.9 2796.1 Materials & Methods 12881.1 12305.8 575.3 2.06E-09 10479.5 8338.9 2140.6 11179.8 8435.0 2744.8 1000 Take initial reading 10263.6 9138.8 1124.8 10136.4 8037.3 2099.0 10712.6 7801.7 2910.9 11000 follows: NET = 0.9801; SERT = 0.9956; & DAT = 0.9835. 10437.0 9837.9 599.1 10886.0 8513.6 2372.4 11836.3 8945.2 2891.2 900 10814.8 9887.3 927.5 10810.8 8665.8 2145.1 11144.1 8363.3 2780.8 10588.1 9863.7 724.4 10928.9 8609.4 2319.6 10746.2 7885.6 2860.7 800 10000 1.85E-08 11452.9 9776.5 1676.4 6.86E-10 12015.1 9453.6 2561.6 2.54026E-11 12066.163 8710.2 3355.9 700 Materials: The reference agents Paroxetine, GBR12909, McN5652, Citalopram, 10389.7 8983.9 1405.8 11501.2 9009.7 2491.5 11265.109 7749.0 3516.1 +/- Compounds 15’ @ 37°C 10928.9 9572.9 1355.9 10794.3 8272.1 2522.2 11373.526 7732.0 3641.5 600 RFU Fluvoxamine, Fluoxetine, Nomifensine, Sertraline, Reboxetine, Nisoxetine and Bupropion 12406.4 11059.6 1346.8 11127.0 8683.6 2443.4 11204.594 8268.7 2935.9 RFU 9000 10775.9 9329.0 1446.9 10281.4 7896.5 2384.9 10821.675 7823.2 2998.5 500 11393.3 9797.8 1595.5 10919.9 8401.6 2518.3 10963.865 7214.7 3749.2 were were obtained from Tocris. Duloxetine, Desipramine, and Imipramine were obtained 4000 400 300 8000 from Sigma. All the tissue culture media and supplements were purchased from Invitrogen. Add fluorescent dye – 3500 The cultureware was purchased from Corning. Other biochemicals and buffers were 200 incubate @ 37°C; 30-60’ 3000 100 7000 Conclusion purchased from Mediatech/Thermo Fisher. 0 2500 10 -14 10 -13 10-12 10 -11 10 -10 10-9 10-8 10-7 10-6 10-5 10-10 10 -9 10 -8 10-7 10 -6 Concentration [M] Concentration [M]

2000 2 RFU McN5652: IC = 0.68nM; CI:0.06 - 7.1nM ; R = 0.8728 2 Cell Culture – Primary Neurons: Brains from 18-day-old rat (Holtzman) fetuses (E-18) 50 McN5652: IC50 = 44.9nM; CI: 19.9 - 101nM; R = 0.9779 Take final reading Control Control were aseptically removed from pregnant females and placed into a petri dish containing 1500 •Dye uptake in primary neuronal cultures is time, chilled Hank’s Buffered Saline solution (HBSS). The tissue was digested in a solution of 1000 Neuronal cultures were pretreated with various concentrations of the triple uptake inhibitor McN5652. The IC50 temperature, sodium and chloride dependent. 0.25% tissue culture grade trypsin with DNAse (1 ug/ml) for ~ 10’ at 37°C. Following the 500 Analyze/Graph Data value and 95% confidence intervals for the various tissues were as follows: 1) Cortical IC50 = 27.8nM; the incubation, the tissue was then gently agitated and the material centrifuged at 4°C at 200g 2 0 confidence interval was 16.6 to 46.8nM; and the R was 0.9607. This is a combined NET/SERT/DAT value since 10 -11 10 -1 0 10 -9 10 -8 10 -7 for 10 minutes. The supernatant containing cellular debris was aspirated and the cells were Concentration [M] the tissue contained DAT, NET & SERT. To determine individual DAT, SERT & NET numbers, specific tissues •Assay is reproducible if culture age is kept constant. washed a second time.. At the end of the second centrifugation, the cells were resuspended in and/or procedures must be used to ensure that only a single NT transporter is measured. 2) Hippocampal (NET) neurobasal media containing B-27 and 0.5mM glutamine, and passed through a sterile cell 2 IC50 = 2.8nM; the confidence interval was 1.5 to 2.4nM; and the R was 0.9429; 3) Midbrain (SERT) IC50 = strainer to remove large cellular clumps. The cell suspension was then resuspended with a 0.68nM; the confidence interval was 0.06 to 7.1nM; and the R2 was 0.8728; 4) N. accumbens (DAT) IC = 50 •Tissue source is critical to determine valid NET vs DAT vs large bore 5 ml pipette, followed by a small bore transfer pipette. The cells were counted 44.9nM; the confidence interval was 19.9 to 101nM; and the R2 was 0.9779. with a hemocytometer and plated at a concentration of 125,000 cells/well in poly-L-lysine Fig. 3. The Effect of a 15 Minute Pretreatment with Compound X SERT inhibitor selectivity & potency. coated 96 well-plate. The plates were maintained in a humidified 37°C CO2 incubator and were used after 5-9 days in culture. on Uptake in Day 9 Neuronal Cultures Table 1. The effect of culture age on compound potency. •Excellent correlation exists between the published Ki values Measurement of Neurotransmitter Uptake Inhibition: The assay was conducted Compound Confidence R2 Days in using the MDS Analytical Technologies Neurotransmitter Transporter Uptake Assay Kit®. 13250 for the 14 compounds evaluated and the IC50 values IC50 (nM) Interval (nM) Culture The media was removed from the neuronal cultures by gently blotting the plate onto 35.1 15.4 - 79.9 0.9537 Day 4 generated with the MDS Analytical Technologies absorbent towels. Subsequently, the cultures were washed twice with at least 200µl/well of 25.6 17.2 - 38.1 0.9363 Day 4 assay buffer (1X HBSS/20mM HEPES, pH 7.4) followed by the addition of either 100µl of 10750 Neurotransmitter Transporter Uptake Assay Kit®. 15.9 5.7 - 44.2 0.9696 Day 5 assay buffer alone or assay buffer containing the inhibitor at the desired concentration. The cultures were incubated at 37°C for 15 minutes in the dark and an initial “pre-dye” 15.7 1.8 - 13.5 0.9398 Day 5 8250 Compound X 14.4 8.9 - 23.1 0.9731 Day 6 fluorescence measurement was read using the MDS Analytical Technologies Flexstation II. No Drug

RFU 13.6 1.5 - 126 0.9539 Day 6 The cultures were then loaded with 100µl of the neurotransmitter uptake dye (NT) and the No Dye plates incubated for an additional 30 minutes at 37°C in the dark. After the final 30-minute 12.9 3.3 - 50.2 0.9577 Day 6 5750 Toxic Effect incubation period, the plates were read again in the Flexstation II instrument. For both 11.4 3.6 - 36.6 0.9691 Day 7 readings, the output was mean relative fluorescence units (RFU). 11.3 4.8 - 26.7 0.9130 Day 7 3250 10.6 5.1 - 21.7 0.8489 Day 7 Data Analysis: Data was collected as mean RFU using the SOFTmax Pro software. In all 8.9 5.2 - 15.4 0.9743 Day 8 experiments, the appropriate average buffer control was included. Outliers were detected 8.6 6.1 - 12.1 0.9272 Day 8 750 References using the Grubb’s Test and removed from the calculations if appropriate. IC50 concentration 7.1 3.0 - 17.2 0.9298 Day 8 curves for the transporters were determined for the neurotransmitters. Individual IC 50 10 -8 10-7 10-6 10 -5 10 -4 10-3 5.7 4.4 - 7.3 0.9605 Day 9 values, Hill slopes, 95% confidence intervals, and goodness of fit numbers were computed 1. Waraich, P et al; Can. J. Psych. 49:124 (2004) Concentration [M] 4.3 3.2 - 57.0 0.9615 Day 9 using the Graphpad Prism software nonlinear regression analysis program. For these 2. Jorgensen S. et al; J. Neurosci Methods 169:168 (2008) Mean±SD analyses, constrained values were used when the fits did not converge using unconstrained Compound is really not effective: the reduction of fluorescence is actually 3. Wong, DT; Exp. Opin. Invest. Drugs 7:1691 (1998) parameters. The correlation between the IC value generated in the primary neuronal the result of toxic compound concentrations, causing cell disintegration and 13.4±7.9 50 4. Richelson, E & Pfenning M; Eur. J. Pharm. 104:277 (1984) cultures and the Ki values from the literature was done using the Spearman method. thus the appearance of uptake inhibition. The experimental data shows that as the culture mature over 5. Hytell, J; Int. Clin. Psychopharm. 9 Suppl.1,: 19 (1994) time, the test compound appears more potent, although the 6. Shank et al.; J. Pharmacol. Exp. Ther. 247:1032 (1988) differences (IC values) are not statistically significant since 50 7. Anderson, P; Eur. J. Pharm. 166;493 (1989) the confidence intervals are overlapping. The most consistent results are seen when using cultures between days 6-8 of after 8. Hajós, M et al; CNS Drug Reviews 10:23 (2004) plating.