SUPPLEMENTARY INFORMATION
1.0 Plasmodium Falciparum SYBR Green Assay:
The Malaria SYBR Green I - Based Fluorescence (MSF) Assay is a microtiter plate drug sensitivity assay that uses the presence of malarial DNA as a measure of parasitic proliferation in the presence of antimalarial drugs or experimental compounds. As the intercalation of SYBR Green I dye and its resulting fluorescence is relative to parasite growth, a test compound that inhibits the growth of the parasite will result in a lower fluorescence. D6 (CDC/Sierra Leone), TM91C235 (WRAIR, Thailand), and W2 (CDC/Indochina III) laboratory strains of P. falciparum were used for each drug sensitivity assessment. The parasite strains were maintained continuously in long-term cultures as previously described in Johnson 2007. Pre-dosed microtiter drug plates for use in the MSF assay were produced using sterile 384-well black optical bottom tissue culture plates containing quadruplicate 12 two-fold serial dilutions of each test compound or mefloquine hydrochloride (Sigma-Aldrich Co., Catalog #M2319) suspended in dimethyl sulfoxide. The final concentration range tested was 0.5 – 1000 ng/ml for all assays. Predosed plates were stored at 4°C until used, not to exceed five days. No difference was seen in drug sensitivity determinations between stored or fresh drug assay plates (data not shown). A batch control plate using Chloroquine (Sigma-Aldrich Co., Catalog #C6628) at a final concentration of 2000 ng/ml was used to validate each assay run. The Tecan Freedom Evo liquid handling system (Tecan US, Inc., Durham, NC) was used to produce all drug assay plates. Based on modications of previously described methods by Plouffe 2008 and Johnson 2007, P. falciparum strains in late-ring or early-trophozoite stages were cultured in the predosed 384-well microtiter drug assay plates in 38 µl culture volume per well at a starting parasitemia of 0.3% and a hematocrit of 2%. The cultures were then incubated at 37°C within a humidified atmosphere of 5% CO2, 5% O2 and 90% N2, for 72 hours. Lysis buffer (38 µl per well), consisting of 20mM Tris HCl, 5mM EDTA, 1.6% Triton X, 0.016% saponin, and SYBR green I dye at a 20x concentration (Invitrogen, Catalog #S-7567) was then added to the assay plates for a final SYBR Green concentration of 10x. The Tecan Freedom Evo liquid handling system was used to dispense malaria cell culture and lysis buffer. The plates were then incubated in the dark at room temperature for 24 hours and examined for the relative fluorescence units (RFU) per well using the Tecan Genios Plus (Tecan US, Inc., Durham, NC). Each drug concentration was transformed into Log[X] and plotted against the RFU values. The 50% and 90% inhibitory concentrations (IC50s and IC90s, respectively) were then generated with GraphPad Prism (GraphPad Software Inc., SanDiego, CA) using the nonlinear regression (sigmoidal dose-response/variable slope) equation.
References:
- Johnson, J.D., et. al. (2007) Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening. Antimicrob Agents Chemother. 51:1927.
- Plouffe, D., et. al. (2008) In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen. PNAS. 105: 9059.
2.0 Permeability Across MDCK-MDR1 Cell Monolayers:
Permeability was determined under contract by Absorption Systems. MDR1-MDCK monolayers were grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well Costar Transwell plates. Data are considered valid for a specific assay plate if TEER values are < 1400 Ω.cm2, the Papp of propanolol is between 10-30 x 10-6 cm/s and the Papp of atenolol is < 0.5 x 10-6 cm/s. The permeability assay buffer was Hanks Balanced Salt Solution containing 10 mM HEPES and 15 mM glucose at a pH of 7.4. A known p-glycoprotein inhibitor cyclosporin A (CSA) was also added to the assay buffer at 10 microM. The dosing solution concentrations of the test compounds were 5.0 micro M in the assay buffer. All cell monolayers were first pre-incubated for 30 minutes with assay buffer to saturate any P-glycoprotein sites with test compound. After 30 minutes, the buffer was removed, replaced with fresh buffer, and time was recorded as 0. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37°C with 5% CO2 in a humidified incubator. After 2 hours, aliquots were taken from the receiver chambers. Samples were taken from the donor chamber at 0 and 2 hours. Each determination was performed in duplicate. The lucifer yellow flux was also measured for each monolayer to ensure no damage was inflicted to the cell monolayers during the flux period. All samples were assayed by LC/MS/MS using electrospray ionization.
The apparent permeability, Papp, and percent recovery were calculated as follows:
Papp = (dCr /dt) x Vr/(A x C0) (1)
Percent Recovery = 100 x ((Vr x Crfinal) + (Vd x Cdfinal))/(Vd x CN) (2)
where,
dCr /dt is the slope of the cumulative concentration in the receiver compartment versus time in microM s-1.
Vr is the volume of the receiver compartment in cm3.
Vd is the volume of the donor compartment in cm3.
A is the area of the cell monolayer (1.13 cm2 for 12-well Transwell).
C0 is the measured concentration of the donor chamber at time 0 in microM.
CN is the nominal concentration of the dosing solution in microM.
Crfinal is the cumulative receiver concentration in microM at the end of the incubation period.
Cdfinal is the concentration of the donor in microM at the end of the incubation period.
3.0 In Vivo P. Berghei Efficacy Methods:
Compounds were evaluated for their potential activity in vivo against P. berghei in mice as previously described(Dow 2006). Mefloquine was formulated compounds was formulated in 0.5% hydroxyethylcellulose and 0.1% Tween 80 (HECT). WR621308 was formulated in 5% (v/v) ethanol, 5% (v/v) chremophor EL in HECT. The compounds were administered as a single dose PO to groups of n=5 mice on Day 3 after a blood stage inoculation. For mefloquine cumulative data from all available experiments is presented. Efficacy is expressed in terms of the mean time in days to human euthanasia, the % of mice surviving through Day 31 and the % of mice surviving through Day 31 that were parasite free with no evidence of typical malaria legions (cures).
References:
Dow GS, Heady TN, Bhattacharjee AK, Caridha D, Gerena L, Gettayacamin M, Lanteri CA, Obaldia N 3rd, Roncal N, Shearer T, Smith PL, Tungtaeng A, Wolf L, Cabezas M, Yourick D, Smith KS.(2006)Utility of alkylaminoquinolinyl methanols as new antimalarial drugs.Antimicrob Agents Chemother. 50:4132-43
4.0. Synthetic Methods
Materials:
All anhydrous reactions were performed using oven-dried glassware, which was then cooled under vacuum and purged with nitrogen gas. Tetrahydrofuran (THF), dichloromethane (CH2Cl2), toluene, and diethyl ether (Et2O) were filtered through activated alumina under nitrogen. Absolute ethanol (anhydrous; with a sure-seal) was purchased from Sigma Aldrich and used without further purification. Unless otherwise noted, all materials were used as received from a commercial supplier without further purification. 4-(oxiran-2-yl)-2,8-bis(trifluoromethyl)quinoline (S-5) was constructed in-house (Figure 1) and is commercially available from Bioblocks (San Diego, California). The amines were purchased from Maybridge and Sigma Aldrich.
The microwave-assisted amino alcohol synthesis was performed in a CEM Discover LabMate microwave fitted with a 24-place autosampler and with the IntelliVent™ Pressure Control System and CEM's SynergyT software.Reactions were magnetically stirred and monitored by thin layer chromatography using Analtech HL and HLF glass-backed 250um uniplates and analyzed with 254 nm UV light and ceric ammonium molybdate stain (CAM; Hanessian’s stain), vanillin, p-anisaldehyde, phosphomolybdic acid stain (PMA), dinitrophenylhydrazine (DNP), or potassium permanganate treatment. Flash chromatography was performed with 60 angstrom (200-425 mesh, surface area 470-530 m2/g, pH = 6.5-7.0) silica gel supplied by Sigma Aldrich. Preparative thin layer chromatography was performed utilizing Analtech silica gel GF 2000 um 20x20cm glass-backed plates with calcium sulfate binder-soft layer. Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise noted.
Proton nuclear magnetic resonance (NMR) spectra were recorded using an internal deuterium lock at ambient temperature on Bruker 300 MHz and 600 MHz spectrometers. Internal references of δH7.26 and δH4.78 were respectively used for CDCl3and MeOD-d4. Data are presented as follows: chemical shift (in ppm on the δ scale relative to δTMS= 0), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintuplet, m = multiplet, br. = broad, app. = apparent), coupling constant (J/Hz) and integration. Resonances that are either partially or fully obscured are denoted obscured (obs.). Carbon-13 NMR spectra were recorded at 75 MHz. Internal references of δC77.16 and δC39.52 was respectively used for CDCl3and methanol-d4.
An LC-UV/Vis/Ion Trap MS system was used to confirm mass and ascertain purity of the compounds. The system consisted of an Agilent (Foster City, CA) 1100 Series LC-UV/VIS system online with a ThermoFinnigan (now ThermoFisher; Walthan, MA) LCQ MS equipped with an electrospray ionization source. Chromatographic separation was achieved using a shallow acetonitrile: 0.1% (v:v) formic acid gradient through an XTerra MS C18, 3.5 µM, 2.1x50 mm column (Waters Milford, MA) at 0.3 ml/min. Mass analysis following positive ionization was done in full scan (100-650 amu) and ms2/ms3 of the most abundant ions using the data dependent scan mode and a collision energy of 35 volts. UV/VIS data was collected through contact closure using a photo-diode array (190-800 nm) detector. All data was processed by the Xcalibur Quan Browser software.
Construction of the 4-position library epoxide scaffold (3):
Scheme 1. Synthesis of quinoline methanol 4-position diamine librarya
aReagents and conditions: (a) POBr3, 75C – 150C, 2h, 91%; (b) (i) Pd2(dba)3, P(t-Bu)3, toluene, tributylvinylstannane, 4h, 62%; (ii) m-CPBA, K2CO3, CH2Cl2, reflux overnight, 74%. (c) HNR2, 200 proof EtOH, microwave for 5-30 min hold time (150 watts, 275 PSI, 130 oC, stirring, no-cool), R = H, alkyl amines, pyrrolidines, piperidines, and bicyclic diamines.
4-bromo-2,8-bis(trifluoromethyl)quinoline (2): Phosphorous oxybromide (4eq.; 42mmol; 12g) was added to an oven-dried round-bottomed flask fitted with a reflux condenser and nitrogen gas inlet. The flask was heated to 75 oC until the solid melted. 2,8-bis(trifluoromethyl)quinolin-4-ol (S-1) (1.0eq; 10.5mmol; 3g) was added to the molten reagent while stirring. The flask was heated to 150 oC for 2h. The reaction mixture was cooled to room temperature followed by the addition of ice-water (100 mL), which initiated the formation of a white precipitate. The precipitate was filtered through a fritted-funnel while washing with water. The white solid was dried under vacuum was isolated in 91% yield (9.24mmol; 3.18g) and used without further purification. 1H NMR (300 MHz, CDCl3): δ 8.51 (d, J = 8.5Hz, 1H), 8.26 (d, J = 7.2 Hz, 1H), 8.15 (s, 1H), 7.82 (t, J = 7.6 Hz, 1H). 13C NMR (300 MHz, CDCl3): δ 148.5, 144.4, 136.3, 131.4, 130.3, 129.2, 128.8, 125.2. LRMS (ESI): calcd for C11H4BrF6N m/z 342.94, found 342.91.
Blumbergs, P.; Ao, M. S.; LaMontagne, M. P.; Markovac, A. J. Med. Chem. 1975, 18, 1122.
2,8-bis(trifluoromethyl)-4-vinylquinoline: 4-bromo-2,8-bis(trifluoromethyl) quinoline (2) (0.344g, 1.0 eq., 1.0 mmol) was dissolved in toluene under nitrogen. Pd2(dba)3 (4.6mg, 0.005eq., 0.005 mmol), P(t-Bu)3 (0.06 mL of 0.2M solution in 1mL toluene, 0.012 eq., 0.012 mmol), tributylvinylstannane (0.31mL, 1.05 eq., 1.05 mmol) were added and stirred at room temperature for 4h. Potassium fluoride (1mL, 1M in H2O) and 1 mL EtOAC were added and stirred for 30 minutes. The reaction matrix was passed through a plug of silica (eluted with ethyl acetate), concentrated in vacuo, and purified via preparative thin layer chromatography with 3:1 hexanes:ethyl acetate and the desired product was isolated in 62% yield (0.62 mmol; 0.18g). 1H NMR (300 MHz, CDCl3): δ 8.31 (d, J = 8.7 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 7.83 (s, 1H), 7.69 (t, J = 8.1 Hz, 1H), 7.45-7.35 (dd, J = 11.1 Hz, J = 6.0 Hz, 1H), 6.08 (d, J = 16.8 Hz, 1H), 5.81 (d, J = 10.5 Hz, 1H). NMR (300 MHz, CDCl3): δ 148.5, 146.5, 144.3, 129.3, 129.2, 129.1, 127.3, 125.6, 123.3, 122.0, 119.7, 114.4. LRMS (ESI): calcd for C12H5F6NO m/z 291.05, found 291.11.
A) Littke, A.; Schwarz, L.; Fu, G. J. Am. Chem. Soc.2002, 124, 6343. B) Duncton, M.; Pattenden, G. J. Chem. Soc., Perkin Trans.1 1999, 1235.
4-(oxiran-2-yl)-2,8-bis(trifluoromethyl)quinoline (3): 2,8-bis(trifluoromethyl)-4-vinylquinoline (4) (0.18g, 1.0eq., 0.62 mmol) was dissolved in dichlormethane. Meta-chloroperbenzoic acid (77% m-CPBA, 3.5g, 2.5 eq., 1.6 mmol), and K2CO3 (9mg, 1.2 eq., 0.74mmol) were added. The reaction mixture was heated to reflux overnight. The reaction matrix was cooled to room temperature, passed through a plug of silica (eluted with 4:1 dichlomethane:methanol), concentrated in vacuo, and purified via preparative thin layer chromatography with 3:1 hexanes:ethyl acetate and the desired product was isolated in 74% yield (0.46 mmol; 0.141g). 1H NMR (300 MHz, CDCl3): δ 8.39 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.80 (s, 1H), 4.55 (s, 1H ), 3.44 (d, J = 2.4 Hz, 1H), 2.83 (d, J = 2.4 Hz, 1H). 13C NMR (300 MHz, CDCl3): δ 148.5, 146.3, 142.9, 130.0, 128.8, 127.2, 126.6, 124.3, 121.5, 119.3, 112.9, 50.4, 48.8. LRMS (ESI): calcd for C13H7F6NO m/z 307.19, found 307.21.
Ruano, J. L. G.; Fajardo, C.; Fraile, A.; Martin, M. R. Journal of Organic Chemistry,2005, 70, 4300.
General procedure for constructing 4-position amino alcohols (4):
Figure 2: Microwave-assisted synthesis of 4-position amino alcohol empirical library
In a microwave vial, 4-(oxiran-2-yl)-2,8-bis(trifluoromethyl)quinoline (4) (1.0eq; 0.065mmol; 20mg) was dissolved in absolute EtOH. The requisite amine (5-10eq) was added and the reaction was placed in the microwave for 5-30 min hold time (150 watts, 275 PSI, 130 oC, stirring, no-cool). Once judged complete via TLC (3:1 EtOAc:Hexanes to view epoxide disappearance and 95:5 CH2Cl2:MeOH to view amino alcohol formation) the solution was concentrated in vacuo. Smaller scale reactions (< 20mg) were purified via preparatory thin layer chromatography and eluted in 95:5 CH2Cl2:MeOH. The desired UV-active compound(s) were removed from the plate, transferred to a fine-fritted funnel, and rinsed with 4:1 CH2Cl2:MeOH. Larger scale reactions (> 20mg) were purified via flash column chromatography. The column was eluted with 95:5 CH2Cl2:MeOH. The compounds were isolated in > 70% yield. The library of compounds was characterized via LCMS and/or 1H NMR prior to in vitro assays. For the compounds tested in vivo, compounds were scaled-up and 13C NMR spectra were also obtained and are provided below.
1-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-(2-((2-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-hydroxyethyl)(methyl)amino)ethylamino)ethanol
WR319691 (67%) 1HNMR (600MHz, CDCl3) δ 8.27 (d, J=8.4 Hz, 1H), 8.18 (d, 7.2 Hz, 2H), 8.13 (d, J=6.0 Hz, 2H), 7.98 (d, J=8.4 Hz, 1H), 7.67 (t, J=7.8 Hz, 2H), 5.80 (d, J=9.6 Hz, 1H), 5.47 (d, J=9.6 Hz, 1H), 3.20 (d, J=10.8 Hz, 1H), 2.96 (m, 1H), 2.88 (dd, J=4.2, 13.2 Hz, 2H), 2.80 (m, 2H), 2.75 (dd, J=1.8, 21.0 Hz, 1H), 2.68 (m ,1H), 2.61 (dd, 3.6, 10.2 Hz, 2H), 2.46 (s, 3H)
MS, calc’d for C29H24F12N4O2 = 688.17. Obsv’d m/z: 689.14 (M+1), 394.26, 351.02, 307.99, 266.07
2-((2-aminoethyl)(methyl)amino)-1-(2,8-bis(trifluoromethyl)quinolin-4-yl)ethanol
WR319670 (43%)1HNMR (300MHz, CDCl3) δ 2.43 (s, 3H), δ 2.55-2.63 (m, 2H), δ 2.74-2.86 (m, 2H), δ 2.99-3.05 (m, 2H), 5.58 (d, J= 8.4Hz, 1H), δ 7.66 (t, J= 8.0 Hz, 1H), δ 8.08-8.15 (m, 3H)
13CNMR (75MHz, MeOD) δ 154.9, 149.4, 149.2, 144.7, 130.2, 129.3, 129.0, 128.7, 128.5, 126.0, 124.2, 122.4, 121.9, 116.2, 68.6, 65.8, 58.9, 42.9, 39.2
MS, calc’d for C16H17F6N3O = 381.13. Obsv’d m/z: 382.0 (M+1), 330.65
1-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-(2-(methylamino)ethylamino)ethanol
WR318973 (61%)1HNMR (300MHz, CDCl3) δ 2.37 (s, 3H), δ 2.73-2.92 (m, 5H), δ 3.09 (dd, J= 2.4, 12.6 Hz, 1H), δ5.63 (d, J= 7.2 Hz, 1H), δ 7.28 (t, J= 7.8 Hz), δ 8.12-8.18 (m, 2H), δ 8.29 (d, J=8.7 Hz, 1H)
13CNMR (75MHz, MeOD) δ 151.8, 130.7, 129.5, 129.2, 127.6, 116.0, 66.5, 57.4, 54.2, 45.7, 44.6, 33.9
MS, calc’d C16H17F6N3O = 381.13. Obsv’d m/z: 382.12 (M+1), 365.22, 308.24, 288.05
1-(2,8-bis(trifluoromethyl)quinolin-4-yl)-2-(methylamino)ethanol
(WR308245): 93% yield. 1H NMR (300 MHz, MeOD) δ 8.56 (d, J = 8.7 Hz, 1H), 8.29 (d, J = 7.2 Hz), 8.17 (s, 1H), 8.90 (t, J = 8.1 Hz, 1H), 5.71 (dd, J = 3 Hz, 8.7 Hz, 1H), 2.97 (dd, J = 3 Hz, 12.3 Hz, 1H), 2.84 (dd, J = 8.7 Hz, 12.6 Hz, 1H), 2.5 (s, 3H). 13C NMR (300 MHz, MeOD) δ 148.4, 144.5, 142.4, 129.4, 128.8, 128.5, 126.7, 126.1, 122.0, 120.1, 113.3, 70.7, 60.3, 37.8. LRMS (ESI): calcd for C14H12F6N2O [M + H]+ 338.28, found 339.23.
2-(2-aminoethylamino)-1-(2,8-bis(trifluoromethyl)quinolin-4-yl)ethanol
WR319707(51%) 1HNMR (600MHz, CDCl3) δ 2.07, (s, 4H), 2.78 (d, J=10.8 Hz, 2H), 2.87 (d, J=10.8 Hz, 1H), 2.90 (d, J=5.4 Hz, 2H), 3.20 (d, J=5.4 Hz, 1H), 5.55 (d, J=6 Hz, 1H), 7.73 (t, J=7.8 Hz, 1H), 8.15 (s, 1H), 8.17 (d, J=7.2 Hz, 1H), 8.25(d, J=8.4 Hz, 1H)
13CNMR (75MHz, CDCl3) δ 151.87, 148.75, 143.86, 129.43, 128.79, 127.22, 126.79, 125.53, 123.32 , 121.90, 119.67, 118.28, 116.02, 114.83, 68.35, 56.12, 51.24, 41.56
MS, calc’d for C15H15F6N3O = 367.11. Obsv’d m/z: 367.84 (M+1)
2-amino-1-(2,8-bis(trifluoromethyl)quinolin-5-yl)ethanol
(WR308314) 94% yield. 1H NMR (300 MHz, MeOD) δ 8.52 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 7.2 Hz, 1H), 8.15 (s, 1H), 7.85 (t, J = 7.8 Hz, 1H), 5.68 (dd, J = 1.8Hz, 8.7 Hz, 1H), 3.17 (dd, J = 3.9 Hz, 12.6 Hz, 1H), 13C NMR (300 MHz, MeOD) δ 156.8, 151.4, 146.5, 134.3, 129.9, 128.4, 126.6, 124.2, 122.1, 119.9, 116.2, 70.7, 45.1. LRMS (ESI): calcd for C13H10F6N2O [M + H]+ 325.10, found 325.12.
5.0PK Parameter Determination
Drug concentrations were generated for test drug. A measured plasma drug concentration vs. time curve were produced, in graphic and tabular form, for each subject on both linear/linear and log/linear scales, for the parent compound. Mean plasma drug concentration vs. time curves were also presented separately. Summary statistics (i.e. mean, standard deviation, minimum, maximum, n and coefficient of variation) were calculated for plasma concentrations for each time point and each dose level. Pharmacokinetic parameter values were estimated using WinNonlin pharmacokinetic software (version 5.2). Unless otherwise agreed with the sponsor, a non-compartmental model and/or compartment model were used to generate parameter estimates.
Quantified plasma test compound concentrations were utilized for the calculation of the following pharmacokinetic parameters for each subject administered active treatment:
AUCinf.: Area under the plasma (AUC) concentration-time curve extrapolated to infinite time was calculated by extrapolation of the elimination slope from t to infinity, thus: AUC(inf) = AUC(0-last) + (Ct/Kel). Where Ct = the plasma drug concentration at time t, and Kel = the elimination rate constant for the drug, determined from the terminal elimination slope.
Cmax: The mean predicted test drug concentration at the peak of the administration was determined by calculation from the log-linear regression of WinNonlin program.
t½ beta: Terminal half-life of test drug was determined by regression analysis of the terminal elimination slope.
Cl:Plasma apparent clearance of test compound, calculated as Dose/AUCinfwhere Dose is the dose of test compound. This parameter is used for i.v. dosing, and is adjusted based on the fraction absorbed for PO dosing as below.
Cl/FApparent clearance (Cl) adjusted with oral bioavailability (F). This is for oral dosing where the fraction absorbed is known. Where it is not known bioavailability is assumed to be 100%. Since bioavailability after oral dosing is rarely 100%, the derived value reflects the maximum possible clearance.
Vss: Volume of distribution of test drug at steady state = volume of the central compartment (Vc) + volume of the tissue compartment (Vt), calculated as CL x t½/Ln(2). This can only be determined after i.v. dosing so is not relevant for PO dosing.
Vz/FApparent volume of distribution based on the terminal phase adjusted with oral bioavailability (F). This is for oral dosing where the fraction absorbed is known. Where the oral bioavailability is not known it is assumed to be 100%. Since bioavailability after oral dosing is rarely 100$, the derived value reflects the maximum possible volume of distribution.
MRT: Mean residence time of test agent is the average time the drug stays in the body, which was determined by the ratio of AUME/AUC following administration.
Note: Pharmacokinetic analysis of test agent data was only conducted using a non-compartmental analysis (NCA) by using WinNonlin program for plasma, WinNonlin Version 5.2 (Pharsight Corporation). The mean modeling curve of test agent concentration-time is shown in Table 1 below. All times used in the pharmacokinetic analysis were actual times, with the exception of pre-dosing samples which were designated a nominal time of 0 hr.
~ S-1 ~