Nine- to 11-week-old male Sprague-Dawley rats were purchased from Charles River Laboratories (Wilmington, MA) and housed in microisolator cages with rodent chow and autoclaved water ad libitum. All experiments were in accordance with National Institutes of Health guidelines and approved by the University of California, Davis, Institutional Animal Care and Use Committee. For i.v. injection SKA-31 was dissolved at 10 mg/ml in a mixture of 10% Cremophor®EL and 90% saline and injected at 10 mg/kg. For i.p. application SKA-31 was dissolved at 10 mg/ml in Miglyol 812 neutral oil (caprylic/capric triglyceride; Tradename Neebee M5®, Spectrum Chemicals, Gardena, CA). Following tail vein injection of the aqueous solution or i.p. administration of the oily solution approximately 200 μl of blood were collected from the tail into EDTA blood sample collection tubes at various time points. For very early time points (3 min, 5 min and 10 min) following i.v. administration blood samples were obtained by cardiac puncture under deep isoflurane anesthesia. Plasma was separated by centrifugation and stored at −80°C pending analysis. After determining that SKA-31 plasma concentrations peaked 2 h after i.p. application (10 mg/kg), we took blood samples under deep isoflurane anesthesia by cardiac puncture from a group of 3 rats before sacrificing the animals to remove brain, heart, liver, spleen and fat. Tissue samples were homogenized in 1 ml of H2O with a Brinkman Kinematica PT 1600E homogenizer and the protein precipitated with 1 ml of acetonitrile. The samples were then centrifuged at 3000 rpm and supernatants concentrated to 1 ml. Plasma and homogenized tissue samples were purified using C18 solid phase extraction (SPE) cartridges. Elution fractions corresponding to SKA-31 were evaporated to dryness under nitrogen and dissolved in acetonitrile.
LC/MS analysis was performed with a Hewlett-Packard 1100 series HPLC stack equipped with a Merck KGaA RT 250–4 LiChrosorb RP-18 column interfaced to a Finnigan LCQ Classic MS. The mobile phase consisted of acetonitrile/water with 0.2% formic acid. The flow rate was 0.5 ml min−1 and the gradient was ramped from 80/20 for 5 minutes to 70/30 over 15 min. With the column temperature maintained at 30°C, SKA-31 eluted at 5.7 min and was detected with a variable wavelength detector (VWD) set to 254 nm and the MS in series. Using electrospray ionization MS/MS (capillary temp. of 350°C, capillary voltage of 26 V, tube lens offset of 20V, positive ion mode) SKA-31 was detected at a mass of 201.35 (MW plus H+). SKA-31 concentrations were calculated with a five-point calibration curve from 500 nM to 8 μM. Concentrations above 1 μM were determined by their UV absorption using a second calibration curve from 1 μM to 250 μM. Riluzole (retention time 13.5 min; mass 235.35 [MW plus H+]) was used as an internal standard.
The percentage of plasma protein binding for SKA-31 was determined by ultrafiltration. Rat plasma was spiked with 10 μM SKA-31 in 1% DMSO and the sample loaded onto a Microcon YM-100 Centrifugal Filter (Millipore Corporation, Bedford, MA) and centrifuged at 14000 rpm for 15 min at RT. The centrifugate (= free SKA-31) was directly analyzed for SKA-31 by HPLC-MS. The retentate was collected by inverting the filter into an Eppendorf tube and spinning at 14000 rpm for 15 min. The retentate then underwent sample preparation as per the above-described procedure for determining total SKA-31 concentration in plasma. The plasma protein binding of SKA-31 was found to be 39 ± 0.8 % (n = 3). The unbound (= free) fraction was 61 ± 1.7%.
LC/MS analysis was performed with a Hewlett-Packard 1100 series HPLC stack equipped with a Merck KGaA RT 250–4 LiChrosorb RP-18 column interfaced to a Finnigan LCQ Classic MS. The mobile phase consisted of acetonitrile/water with 0.2% formic acid. The flow rate was 0.5 ml min−1 and the gradient was ramped from 80/20 for 5 minutes to 70/30 over 15 min. With the column temperature maintained at 30°C, SKA-31 eluted at 5.7 min and was detected with a variable wavelength detector (VWD) set to 254 nm and the MS in series. Using electrospray ionization MS/MS (capillary temp. of 350°C, capillary voltage of 26 V, tube lens offset of 20V, positive ion mode) SKA-31 was detected at a mass of 201.35 (MW plus H+). SKA-31 concentrations were calculated with a five-point calibration curve from 500 nM to 8 μM. Concentrations above 1 μM were determined by their UV absorption using a second calibration curve from 1 μM to 250 μM. Riluzole (retention time 13.5 min; mass 235.35 [MW plus H+]) was used as an internal standard.
The percentage of plasma protein binding for SKA-31 was determined by ultrafiltration. Rat plasma was spiked with 10 μM SKA-31 in 1% DMSO and the sample loaded onto a Microcon YM-100 Centrifugal Filter (Millipore Corporation, Bedford, MA) and centrifuged at 14000 rpm for 15 min at RT. The centrifugate (= free SKA-31) was directly analyzed for SKA-31 by HPLC-MS. The retentate was collected by inverting the filter into an Eppendorf tube and spinning at 14000 rpm for 15 min. The retentate then underwent sample preparation as per the above-described procedure for determining total SKA-31 concentration in plasma. The plasma protein binding of SKA-31 was found to be 39 ± 0.8 % (n = 3). The unbound (= free) fraction was 61 ± 1.7%.
