Document ymaGRoDpZ7jk5Da6Lnz7kQokE

2. Absorption of PC-95-.14C in Rats After a Single Oral Dose October 26, 1979 Conducted at: During: Conducted by: Report by: Reviewed by: Riker Laboratories, Subsidiary of 3H St. Paul, Minnesota Inc. 55101 June to July,.1979 S. J. Gibson and J. D. Johnson S:aimeossrDD.l A@h@s@6n-, ms Date Senior %#6hemical Pharmacologist Rbbert E. Ober, Ph.D. Manager, Drug Metabolism Date 3. Summarv After a single oral dose of FC-95-14C (mean dose, 4.2 @m@g) in solution to groups of three male rats, at least 95% of the total carbon-14 is systemically absorbed at 24 hours. The half-life for eli-ination of total carbon-14 from plasma is 7.5 days. Introduction 4. c7Flg C-SO 3 K+ p FC-95 *Denotes Position of Carbon-14 FC-95 is the potassium salt of a Perfluorinated sulfonic acid. A series of experiments has been planned to investigate possible means of increasing the rate of elimination of FC-95 from the body. FC-95 absorption, elimination from plasma, and excretion data were neceflary to form a basis for efficient design of these experiments. This PC-95- C oral dosing experiment (FC-Experiment 4) which was designed to provide totall@@bon-14 absorption and plasma elimination data was paired with an FC-95- C iv dosing experiment (FC-Experiment 3) which was designed to provide data on the route and extent of total carbon-14 excretion. In addition, these FC-95 absorption, plasma elimination, and excretion data will be used to interpret and guide other experiments on the Possible biotransfo-mtion of FC-807 to FC-95. Methods Radiolabeled FC-95- 14 c The carbon-14 label is at the position a to the sulfur a M. (See above structure) . The specific activity of this lot of PC-95-12 C (Riker Isotope Inventory Number 442) is 0.459 UCi/mg. The FC-95-14C was found to be suitable for metabolism studies' ; details of the specific activity determination, chemical characterization, and radiochemical purity determination will be reported separately. Animals Male Charles River'! CD rats, eight weeks old, were conditioned to individual metal metabolism cages for 24 hours prior to dosing. The body weights ranged from 243 to 315g; mean 285g. The rats were allowed free access to Purinab-Ground Chow and water before and after dosing. Dosinct Each non-fasted rat was weiahed immediately before beina given a single oral dose of FC-95-14C. The dose was 2.0 ml of a 0.9% NaCl solution containing 1.21 mg FC-95-14C/2.0 ml. The average dose was 4.2 mg/kg. The dose was delivered with a 2.0 cc glass syrinqe (TrylonSc-)fitted with a stainless steel intubation tube. The dosing solution was prepared by adding '1,200 mg of FC-95-14C to 0.9% NaCl, shaking for one half hour at moderate speed in a mechanical shaker, and centrifuging. The supernatant was removed and used for dosing solution. The carbon-14 content of the dosing solution was determined by direct counting (see Appendix 1). --------------------------- @lCharles River Breeding Laboratories, WilnLington, Mass. b Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. Aloe Medical, Division of Brunswick, St. Louis 3, Missouri. Sample Analysis for Carbon-14 Homoqenizinq of feces and tissue was done in Warina blenders bv addina nine varts of water (w/w) to one Dart of biolocical material. The homocenates wexe weiahed into combustion conesg in trildlicate on a too-loadina balance bv tarina the cone and addina 1-Oa of the homoaenate. Care was taken to mix the homocenate between sa-lincs. Urine. red blood cells. and PlasTnA samnles (from the 24 and 4'Bhour Dost dose aroulas) were also measured into combustion cones bv weicht. Mie concentration of carbon-14 in clasma for the 3 rats in the 24 and 48 hour crouns was determined bv both combustion and direct countina. Plasma samoles (includina the 24 and 48 hour nost dose arouns) were counted directly by transferring 1.0 ml of plasma and 15 ml of Aquasolft to a scintillation vial and counting in a liquid scintillation spectrometer. Homogenate, plasma, urine and red blood cell samples from the 24 and 48 hour post dose group were combusted with a Packard Model 306 Oxidizer. Combustion recovery of carbon-14 from biological samples was determined by combusting blank fecal homogenates and blank red blood cells spiked with dilutions of*rc-95-14C dosing solution at the beginning, middle, and end of the analysis of the experimental sample set (see Appendix 2). All radiometric measurements were done using a Packard Model 3385 Tri-Carb Liquid Scintillation Spectrometer. For plasma samples counted directly, the efficiency for each sample was determined by adding internal standard to each sample and recounting, correcting for background from appropriate blanks, and comparing the results to a sealed standard of known activity. After correcting for background with appropriate blanks and for efficiency, the carbon-14 content of each sample was calculated. For oxidized samples, counting efficiency for each sample was determined by use of the ABS (Automatic External Standardization) ratio method. To calibrate the external standard, internal standard was added to selected samples from the group of samples (three with low AES ratios and three with high ratios) and these samples were recounted along with a sealed standard. Data were collected on punch tape and processed by the CDC 1700 Computer System in the 3M Central Research Data Processing Laboratory. Data reduction to dpm was accomplished with the Biological Automatic External Standardization computer program. Sample Collection Groups of three rats were sacrificed by exsanguination at 1, 2, 6, 12, 24, 48, 96,and 144 hours post dose. Rats were anesthetized with diethyl ether and blood was drawn from the descending aorta of each rat and i-diately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. In addition to plasma and red blood cells, total urine, total feces, spleen, digestive tract plue contents (esophagus, stomach, small intestine, large intestine, and colon) and remainder of carcass were saved from each of the three rats in the 24 'and 48 hours post dose groups for carbon-14 analysis. ------------------------- a Packard Instrument Company, Inc., 2200 Warrenville Rd., Downers Grove, Illinois. b New England Nuclear, Boston, Mass. 6. Results and Discussion The results of the urine, feces, digestive tract plus contents, and ca cass analyses are shown in Table 1. The digestive tract and contents contained on the average, 3.45% of the dose. The mean fecal excretion is 1.55%.of the dose at 24 hours and 3.24% at 48 hours. At 24 hours, the mean sum of total carbon-14 in feces and digestive tract plus contents is 5% of the dose. Some of this 5% likely represents Systemically absorbed carbon-14 present either in the digestive tract tissues or in the digestive tract contents as a result of excretion. The data from the 48 hour post dose group of rats are consistent with the 24 hour post dose data. Thus, at least'95% of the FC-95-14C dose was absorbed from solution after administration to non-fasted rats. The major portion of the radioactivity recovered was found in the carcass. The carcass data are not as reliable as the other tissue data since large volume homogenates were necessary and homogeneity of sample aliquots was difficult to assure. There is some excretion'of total carbon-14 in urine (1-2%/day); this is in contrast to the much sl wer rate of urinary excretion of total carbon-14 after an iv dose of FC-807-i4C (0.0671,/day)(1). The spleens from the 24 hour and 48 hour post dose rats were analyzed for total carbon-@14 content, and the percent of the dose in the whole organ was P- 0.2%. This is much lower than the 6% of the dose in spleen observed at 124 days post iv dose for FC-807-14C (1). The concentrations of total carbon-14 in red blood cells and pla- were compared (Table 2). The mean ratio of red blood cell to plasma concentration at 24 and 48 hours is 0.25 and 0.39, respectively. Thus, at 24 and 48 hours after a single oral dose of pC-95-14C, there is no selective retention of carbon-14 in red blood cells. The results from analysis of plasma samples from groups of three rats at 1, 2, 6, 12, 24, 48, 96, and 144 hours after a single oral dose of FC-95-14C are shown in Table 3. The log of mean concentration versus time for these data is plotted in Figure 1. The least squares line through the individual points from 24 to 144 hours for these data fits the equation: C = 15.65e-0.00387t ;C is plasma concentration. The half-life of elimination p p from plasma is 179 hoars (7.5 days). Thus, elimination from plasma of total carbon-14 after a si.ngleoral dose of pC-95-14C is slow. References 1. Johnson, JD: Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Rats after a Single IV Dose of FC-807-14C (Report), September 29, 1979. 2. Johnson, JD: Absorption of FC-807-14C in Rats after a Single Oral Dose (Report), July 10, 1979. 7. List of Tables and Figures Table 1: Percent Recovery of Total Carbon-14 after a pC-95-14C to Rats (Mean Dose, 4.2 mg/.kg) at Dose. NB 52584 p. 7. Single 24 and Oral Dose of 48 Hours Post Table 2: Comparison of Total Carbon-14 Content of Red Plasma after a Single Oral Dose of M-95-14C 4.2 mg/kg) at 24 and 48 Hours Post Dose. NB 52584 p. 7. Blood Cells to to Rats (Mean Dose, Table 3: Concentration of Carbon-14 in Rat Plasma after a Single Oral Dose of FC-95-14C (Mean Dose, 4.2 pg/kg) at 1, 2, 6', 12, 24, 48, 96,tand 144 Hours Post Dose. NB 52584 p. 10. Figure 1: Mean Concentration of Carbon-14 in Rat Plasma after Oral Dose of FC-95-14C (Mean Dose, 4.2 mg/kg). NB 52584 p. 10. a Singla Appendix, Determination of Carbon-14 Content of Dosing Solution. Table 1: NB 51312 p. 35. Appendix, Recovery of Total Carbon-14 Table 2A: Spiked with FC-95-14C. NB 52584 p. 12. from Blank Fecal Homogenate Samples Appendix, Recovery of Total Carbon-14 from Blank Fecal Homogenate and Table 2B: Red Blood Cell Samples Spiked with FC-95-14C. NB 52584 p. 8. Appendix, Table 3: Comparison of Oxidation with from Rats at 24 and 48 Hours FC-95-14C. NB 52584 p. 11. Direct Counting of Plasma Samples after a Single Oral Dose of Table 1 Percent Recovery of Total Carbon-14 after a Single Oral Dose of FC-95-14C to Rats (mean' Dose, 4.2 ;mg/kg) at 24 and 48 Hours Post Dose Rat Number 1 2 3 Mean 4 5 6 Mean Time Post Dose (Hours) 24 24 24 48 48 48 Carcass 77.34L' 77.06 82.62 79.01 103.61 86.21 92.63 94.15 Percent of DigeatiVe Tract plus Contents Dose Feces 3.27 3.45 4.02 3.58 3.55 3.17 3.24 3.32 1.68 1.72 1.24 1.55 3.25 3.10 3.37 3.24 Data were adjusted for combustion recovery (see Appendix 2). Urine 1.67 1.94 1.09 1.57 1.91 2.78 2.88 2.52 9. Table 2 Comparison of Total Carbon-14 Content of Red Blood Calls and Plasma after a S@ncile Oral Dose of pC-95-14C to Rats (Mean Dose, 4.2 @ncj^g) at 24 and 48 Hours Post Dose Rat Number 1 2 3 Mean 4 5 6 Mean Time Post Dose (Hours) 24 24 24 48 48 48 Red Blood calls, 3.63!Jb4.15 3.18 2.80 7.85 4.94 Plasma 16.3413 14.62 13.40 14.27 14.25 11.76 Ratio Red Blood Calls/Plasma 0.22 0.28 0.24 0.25 0.20 0.55 0.42 0.39 a Data are expressed as ug equivalents of PC-95-14C/g. b The data were obtained by combustion and were adjusted for recovery (see Appendix 2). 10. Table 3 Concentration of Carbon-14 in Rat Pla after a Single Oral-Dose of-.FC-95-14C (Mean Dose, 4.2.mg@kg) at 1,' 2, 6, 12, 24, 48, 96,and 144 Hours Post Dose Hours Post Dose' 1 2 6 12 24 48 96 144 ... A b 11. 35@- 9.84 11.74 13.20 16.35 13.64 11.05 8.71 Rat! 7.47 12.88 11.59 14.14 14.43 13.98 9.67 9.53 ..... c 7.62 6.12 12.60 12.61 13.02 11.06 11.00 9.09 Average + SD 8.81 + 2.20 9.61 + 3.39 11.98 + 0.55 13.32 + 0.77 14.60 + 1.60 12.89 + 1.60 10.57 + 0.78 9.11 @t 0.41 2-Three rats were dosed and sacrificed for each time interval. b Data were obtained by direct counting; data are expressed as pg equivalents of FC-95-14C/ml of plasma. Figure mean Concentration of Carbon-14 in Rat Plasma After a Single Oral Dose of FC-95-14C (mean Dose, 4.2 mg/kg) 3 - - - - (Least Squares Line) 24 t _1/2 179 hours (7.5 days) ic, C 15.65e -0.00387t 7. p 65. 4. 3. 2 0 ;4. 4'8 7@ 96 120 14"4 Hours Post Dose 12. Appendix 1 Determination of Carbon-14 Content of Dosing Solution Just prior to dosing of rats, the PC-95- 14 C dosing solution was sampled with calibrated micropipettors! directly into counting vials; six 10 ul and six So Ul aliquots were pipetted. One ml of water and 15 ml of AquasolOO were added b, corrections were made for background and counting efficiency and, using the specific activity of PC-95- 14 C 'already determinede, the uCi/2.0 ml was calculated. The data are shown in Appendix Table 1. The dose administered each rat was 2.0 ml of solution containing 0.553 lici of FC-9S- 14 C which is 1.21 mg of FC-95- 14 C. This is a mean dose of 4.2 mg/kg. a -;;-L/MIicropipettor, Lab Industries, Berkeley, California. b Aquasol(D was found to be a suitable solvent-scintillent for PC-95- 14 c during specific activity determination (to be reported separately). ESpecific activity - 0.459 uCi/mg. be reported separately). (Specific activity determination to 13. ........... 'Appendix 'Table 1 Carbon-14 Content of Dosi.ng Solution 10 Ul Aliquot uCi/2.,O 'mi 0.5602 0.5554 0.5614 0.5628 0.5612 0.5554 50 ul Aliquot 0.5624 0.5532 0.5290 0.5390 0.5382 0.5534 Overall average 0.5526 0.553 0.4586 uci/mg 14 1.21 uCi P!C-95- C/2.0 ml 14. APPendix 2 Recovery of Total Caxbon-14 from Blank Biological Samples Spiked with pc-gs-14C The good recovery of total carbon-14 from combusted FC-807- 14 C in biological samples has been reported (2). Since the'carbon label is a to the sulfur 14 atom in both PC-95- C and the components of FC-807- 14 C, it is reasonable to 14 expect that good recovery of total carbon-14 from FC-9S- C and/or its metabolites in biological samples can be attained. For each set of samples combusted, four replicates of 10 Ul, SO ul, and 100 Ul of diluted PC-9S- 14 C dosing solution were aliquoted with calibrated micropipettors directly into scintillation vials. At the same +-i--using the same solution and pipettes, either three or four replicates of 10 91, 50 ul or 100 ul were aliquoted directly into combustion cones which already contained blank biological material (fecal homogenates, pla-, or red blood cells). The combustion cones were dried and then pelletized with 5 cm ashless filter paper. Blank filter paper pellets were combusted and the solvents collected in the vials to which the FC:-95-14 c had been added directly. One each of the 10 pl, 50 ul, and 100 Ul PC-95- 14 c spiked pellets were routinely combusted at the beginning, middle, and end of each set of biological samples. After correction for background and counting efficiency, percent recovery was calculated by comparing mean results from direct addition and combustion. The mean recovery data for three sets of 14 fecal samples that were analyzed on different days for total carbon-14 (FC-95- C) for FC-Experiments 3(to be reported later) are shown in Appendix Table 2A. The mean recovery is 98.0 + 1.5%. Since each entry in the table is the mean of four determinations, this mean recovery is based on 36 determinations. Thus, good recovery of total carbon-14 from FC-95- 14 C spiked biological material is shown. The mean recovery for the set of samples in the report (FC-Experiment 4) which is based on triplicate 10 pl,50 pl, and 100 pl FC-95-14 C spiked blank fecal homoqenates and red blood cells was 93.6%. (See Appendix Table 2B) . This sliqhtly lower recovery was due to a minor malfunction of the Packard Oxidizer and was fairly uniform throuqhout the set of samples. Thus, the total carbon-14 Appendix 2 (Cont'd) data obtained from combustion in this report were adjusted for recovery. There was no apparent differences in recovery between spiked blank red blood cells and spiked fecal homogenates. 16. Appengiix' 'Table '2A Recovery of Total Carbon-14 Fecal Homogenate Samples Spiked from Blank_ with FC-95 14 c ........................ 10 U12@ 50 ul ......... ..100.ul b,c 97.4 99.5 97.7 100.6 97.0 98.1 97.9 98.6 95.4 97.3 98.1 98.0 Overall average-- 98.0 14 Amount of FC-95- C reference solution added. Data are expressed as t recovery . dpm dpm from from combustion x 100) direct addit@ion Each entry is the mean of 4 combustion recovery determinations. 17. Appendix 'Table,2B Recovery of Total Carbon-14 from Fecal Homogenate and Red Blood Cell Samples Spiked with.PC-95--@4c ............................................. 'DIRECT*ADDITION 10 756b756 727 723 741 + 18 so,ul 3868 3688 3719 3671 3737 + 90 '100'ul 7404 ___c '73947400 + 6 10 ul Fecal Homogenates 619 613 677 636 Red Blood Cells 671 d 711 691 SPIKED'AND'OXIDIZED 50 ul Fecal Homogenates 3581 3775 3484 3613 Red Blood Cells 3583 3583 3347 3504 100 Ul Fecal Homogenates 7408 7104 7105 7106 Red Blood Cells 7093 7098 7081 7091 636 i4-l x loo = 85'8% 691 741 x 100 = 93.3% 3613 3737 x 100 = 96.7% 3504 x 100 = 93.8% 3737 Overall average - 93.6% L106 7400 x 100 = 96.0% 7091 7400 x 100 = 95.8% 2@Amount of FC-95 _14 C spiking solution added. Data are expressed as dpm. Sample was not used; cracked vial. Spiking error; sample was not used. Appendix 3 Comparison of Oxidation with Direct Counting of Pla-a Samples from Rats at 24 and 48 Hours after a Single Oral Dose of PC-95- 14 c concentration of Carbon-14 in pl-- for the three rats in both the 24 and 48 hour post dose groulswas determined by both oxidation and direct counting. Samples were counted directly by transferring 1.0 ml of plasma and ISrml of AquasolgD to scintillation vials and counting in the liquid scintillation counter. Corrections for efficiency and background were made for each sample. For determination by oxidation, plasma samples were measured by weight into combustion cones and carbon-14 was determined by combusting and counting. The data from the pla-- oxidation - direct counting comparison are shown in Appendix Table 3. The mean ratio of oxidized to direct counting is 0.97 0.02. Thus, the plasma level data obtained via either of these two methods will be essentially the same. The direct counting method, being much more convenient, was chosen to analyze the pla--.mm,and the data quoted for the plasma concentration versus time (Figure 1) is from direct counting. Though a direct measurement of absolute recovery from plasma was not done, these results indicate that 14 recovery of total carbon-14 from plasma spiked with FC-95- C would.be similar to that found for recovery from fecal homogenates (98%, Appendix 2). 19. ix Table 3 c arison of Oxidation with'Direct Counting of Pla- Sanples from Rats at 24 and 48 Hours after a Single Oral Dose of FC-95-14C Rat 1. (24 hours) 2 (24 hours) 3- (24 hours) 1 (48 hours) 2 (48 hours) 3 (48 hours) Direct Counting oxidation ... .....(ug/g)l 'Ratio Oxidation/ 'Direct Counting 16.35 14.43 13.02 13.64 13.98 11.06 15.30 13.99 12.55 13.36 13.33 11.01 0.94 0.97 0.96 0.98 0.95 1.00 X + SD 0.97 + 0.02 Data are expressed as ug equivalents of PC-95- 14 C.