Document QXKpJw96VN7kaqOMV4YME98D6

A im & -0 0 0 7 2 14 Absorption of FC-95- C xn Rats After a Single Oral Dose October 26, 1979 Conducted at: During: Conducted by: Report by: Reviewed by: Riker Laboratories, Inc. Subsidiary of 3M St. Paul, Minnesota 55101 June to July,.1979 S. J. Gibson and J. D. Johnson Tames D 'Senior "AM nson, MS Date chemical Pharmacologist 000056 Summary After a single oral dose of FC-95-14C (mean dose, 4.2 mg/kg) 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 elimination of total carbon-14 from plasma is 7.5 days. 3 000057 Introduction 4 .* - ,,4C7F1 5 F- S03 ** 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 necessary to form a basis for efficient design of these experiments. This FC-95- c oral dosing experiment (FC-Experiment 4) which was designed to provide total carbon-14 absorption and plasma elimination data was paired with an FC-95- 4C 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 biotransformation of FC-807 to FC-95. Methods Radiolabeled FC-95-^C The carbon-14 label is at the position a to the sulfur atom. (See above structure). The specific activity of this lot of FC-95- 4C (Riker Isotope Inventory Number 442) is 0.459 yCi/mg. The FC-95-^C was found to be suitable for metabolism studies; details of the specific activity deter mination, chemical characterization, and radiochemical purity determina tion 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 Purinak. Ground Chow and water before and after dosing. Dosing Each non-fasted rat was weighed immediately before being 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.o T^e average dose was 4.2 mg/kg. The dose was delivered with a 2.0 cc glass syrinqe (TrvlortDSL) fitted with a stainless steel intubation tube. The dosing solution was prepared by adding ^ 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). - Charles River Breeding Laboratories, Wilmington, Mass. jj - Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. Q - Aloe Medical, Division of Brunswick, St. Louis 3, Missouri. 000058 5 Sample Analysis for Carbon-14 Homogenizing of feces and tissue was done in Waring blenders bv adding nine carts of water (w/w) to one oart of biological material. The homogenates were weighed into combustion cones in triplicate on a too-loading balance bv taring the cone and adding 1.Og of the homogenate. Care was taken to mix the homogenate between samplings. Urine, red blood cells, and plasma samples (from the 24 and 48 hour post dose groups) were also measured into combustion cones bv weight. The concentration of carbon-14 in plasma for the 3 rats in the 24 and 48 hour groups was determined bv both combustion and direct counting. Plasma samples (including the 24 and 48 hour post dose groups) were counted directly by transferring 1.0 ml of plasma and 15 ml of Aquasol^ to a scintilla tion vial and counting in a liquid scintillation spectrometer. Homogenate, plasma, urine and red blood cell saxqples from the 24 and 48 hour post dose group were combusted with a Packard Model 306 Oxidizer. Combustion recovery of carbon-14 from biological sanqples was determined by combusting blank fecal homogenates and blank red blood cells spiked with dilutions of FC-95-^C dosing solution at the beginning, middle, and end of the analysis of the exper imental 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 AES (Automatic External Standardi zation) 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, 96j and 144 hours post dose. Rats were anesthetized with diethyl ether and blood was drawn from the descending aorta of each rat and immediately trans ferred 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 plus contents (esophagus, stomach, small intestine, large intestine, and colony 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. 000059 Results and Discussion 6. The results of the urine, feces, digestive tract plus contents, and carcass 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 toted. 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 h om og en ate s 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 slower rate of urinary excretion of total carbon-14 after an iv dose of FC-807- 4C (0.067%/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 * 0.2%. This is much lower than the 6% of the dose in spleen observed at 124 days post iv dose for FC-807-^-4C (1) . The concentrations of total carbon-14 in red blood cells and plasma were com pared (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 FC-95-^4C, 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-^4C 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: "0 00387t Cp = 15.65e ' ';CP_ is plasma concentration. The half-life of elimination from plasma is 179 hours (7.5 days). Thus, elimination from plasma of total carbon-14 after a single oral dose of FC-95-l4C 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-l4C in Rats after a Single Oral Dose (Report), July 10, 1979. 000060 List of Tables and Figures 7 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. NB 52584 p. 7. Table 2: Comparison of Total Carbon-14 Content of Red Blood Cells to Plasma after a Single Oral Dose of FC-95-14C to Rats (Mean Dose, 4.2 mg/kg) at 24 and 48 Hours Post Dose. NB 52584 p. 7. Table 3: Concentration of Carbon-14 in Rat Plasma 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. NB 52584 p. 10. Figure 1: Mean Concentration of Carbon-14 in Rat Plasma after a Single Oral Dose of FC-95-14C (Mean Dose, 4.2 mg/kg). NB 52584 p. 10. Appendix, Determination of Carbon-14 Content of Dosing Solution. Table 1: NB 51312 p. 35. Appendix, Recovery of Total Carbon-14 from Blank Fecal Homogenate Samples Table 2A: Spiked with FC-95-14C. NB 52584 p. 12. Appendix, Recovery of Total Carbon-14 from Blank Fecal Homogenate and Table 2B: Red Blood Cell Samples Spiked with FC-95-^4C. NB 52584 p. 8. Appendix, Comparison of Oxidation with Direct Counting of Plasma Samples Table 3: from Rats at 24 and 48 Hours after a Single Oral Dose of FC-95-14C. NB 52584 p. 11. 000061 Table 1 Percent Recovery of Total Carbon-14 after a Single Oral Dose of FC-95--*-4C 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 77.34^ 77.06 82.62 79.01 103.61 86.21 92.63 94.15 Percent of Dose Digestive Tract plus Feces Contents 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 Urine 1.67 1.94 1.09 1.57 1.91 2.78 2.88 2.52 -- Data were adjusted for combustion recovery (see Appendix 2). 000062 9. Table 2 Comparison of Total Carbon-14 Content of Red Blood Cells and Plasma 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 Red Blood Cells 3.63-- 3-- 4.15 3.18 2.80 7.85 4.94 Plasma Ratio Red Blood Cells/Plasma 16.3414.62 13.40 14.27 14.25 11.76 0.22 0.28 0.24 0.25 0.20 0.55 0.42 0.39 -- Data are expressed as jjg equivalents of FC-95-^C/g. b The data were obtained by combustion and were adjusted for recovery (see Appendix 2). 000063 10 Table 3 Concentration of Carbon-14 in Rat Plasma 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 11.359.84 11.74 13.20 16.35 13.64 11.05 8.71 Rat-- B 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 0.41 -- Three rats were dosed and sacrificed for each time interval. -- Data were obtained by direct counting; data are expressed as pg equivalents of FC-95-14c/ml of plasma. 000064 I Figure 1 Mean Concentration of Carbon-14 in Rat Plasma After a Single Oral Dose of FC-95-14C (Mean Dose, 4.2 mg/kg) 30. 11 (Least squares Line) 2C C/liUJ i. lujiua ^ u t i - 'i - i i_ u u L e iiL my e t J U J - V a i e l l l t i oL 6 5. 4. 3 2 000065 1 0 24 48 72 96 120 144 Hours Post Dose Appendix 1 Determination of Carbon-14 Content of Dosing Solution Just prior to dosing of rats, the FC-95-14C dosing solution was sampled g with calibrated micropipettors- directly into counting vials; six 10 yl and six 50 yl aliquots were pipetted. One ml of water and 15 ml of Aquasol were added-, corrections were made for background and counting efficiency and, using the specific activity of FC-95-14C already Q determined-, the yCi/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 yCi of FC-gS-^C which is 1.21 mg of FC-gs-^C. This is a mean dose of 4.2 mg/kg. --L/I Micropipettor, lab Industries, Berkeley, California. b 14 --Aquasol was found to be a suxtable solvent-scintillent for FC-95- C during specific activity determination (to be reported separately). ^Specific activity = 0.459 yCi/mg. (Specific activity determination to be reported separately). 000066 13 Appendix Table 1 Carbon-14 Content of Dosing Solution 10 pi Aliquot pCi/2.0 ml 0.5602 0.5554 0.5614 0.5628 0.5612 0.5554 50 yl Aliquot .............. yCi/2.0 ml 0.5624 0.5532 0.5290 0.5390 0.5382 0.5534 Overall average - 0.5526 0.553 0.4586 yCi/mg - 1.21 yCi FC-95-14C/2.0 ml 000067 Appendix 2 14 Recovery of Total Carbon-14 from Blank Biological Samples Spiked with FC-95-14c 14 The good recovery of total carbon-14 from combusted FC-807- C in biological samples has been reported (2). Since the carbon label is a to the sulfur 14 14 atom in both FC-95- C and the components of FC-807- C, it is reasonable to 14 / expect that good recovery of total carbon-14 from FC-95- C and/or its metabolites in biological sasples can be attained. For each set of samples combusted, four replicates of 10 yl, 50 yl, and 100 yl of diluted FC-95-14C dosing solution were aliquoted with calibrated micropipettors directly into scintillation vials. At the same time using the same solution and pipettes, either three or four replicates of 10 yl, 50 yl or 100 yl were aliquoted directly into combustion cones which already contained blank biological material (fecal homogenates, plasma, 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-95had been added directly. One each of the 10 yl, 50 yl, and 100 yl FC-95-14C 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 14 recovery of total carbon-14 from FC-95- 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 yl, 50 yl, and 100 yl FC-95-l4c spiked blank fecal homogenates and red blood cells was 93.6%. (See Appendix Table 2B). This slightly lower recovery was due to a minor malfunction of the Packard Oxidizer and was fairly uniform throughout the set of samples. Thus, the total carbon-14 000068 Appendix 2 (Cont'd) \ ! 15. 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. 000069 I 16. Appendix Table 2A Recovery of Total Carbon-14 from Blank . Fecal Homogenate Samples Spiked with FC-95- 4C 10 yl-- ............5Q u l ......... ......100 Ml 97.4-- '-- 99.5 100.6 97.0 97.9 98.6 95.4 97.3 Overall average = 98.0 1 . 5 % . 97.7 98.1 98.1 98.0 a 14 -- Amount of FC-95- C reference solution added. -- Data are expressed as % recovery dpro from combustion x 100) dpm from direct addition -- Each entry is the mean of 4 combustion recovery determinations. 000070 17 Appendix Table 2B Recovery of Total Carbon-14 from Fecal Homogenate and Red Blood Cell Samples Spiked with FC-9 DIRECT ADDITION 10 yl- 756^756 727 723 741 + 18 50 yl 3868 3688 3719 3671 3737 + 90 SPIKED AND OXIDIZED 10 yl Fecal Homogenates Red Blood Cells 619 671d 613 677 711 X 636 691 50 pi Fecal Homogenates Red Blood Cells 3581 3775 3484 3583 3583 3347 3613 3504 100 til 7403 7404 __ c_ 7394 7400 + 6 100 ul Fecal Homogenates Red Blood Cells 7408 7104 7105 7093 7098 7081 7106 7091 636 74l X 100 = 85.8% 691 741 X 100 = 93.3% 3613 3737 X 100 = 96.7% 3504 X 100 = 93.8% 3737 Overall average = 93.6% 7106 7400 X 100 = 96.0% 7091 100 = 95.8% 7400 X -- Amount of FC-95-^C spiking solution added. -- Data are expressed as dpm. c -- Sample was not used; cracked vial, -- Spiking error; sample was not used. 000071 18. Appendix 3 Comparison of Oxidation with Direct Counting of Plasma Samples from Bats at 24 and 48 Hours after a Single Oral Dose of FC-95-14C Concentration of Carbon-14 in plasma for the three rats in both the 24 and 48 hour post dose group3was determined by both oxidation and direct counting. Samples were counted directly by transferring 1.0 ml of plasma and 15- ml of Aquasol 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 plasma 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 plasma, 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 recovery of total carbon-14 from plasma spiked with FC-95-14C would be similar to that found for recovery from fecal homogenates (98%, Appendix 2). 000072 i I 19 Appendix Table 3 Comparison of Oxidation with Direct Counting of Plasma Samples from Rats at 24 and 48 Hours after a Single Oral Dose of FC-^95-^C Rat 1 (24 hours) 2 (24 hours) 3. (24 hours) 1 (48 hours) 2 (48 hours) 3 (48 hours) Direct Counting (yq/ml) 16.35 14.43 13.02 13.64 13.98 11.06 Oxidation (ng/g>- Ratio Oxidation/ Direct Counting 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 --ci Data are expressed as yg equivalents of FC-95-24C. 000073