Document 31pZanDV235KOo0r6VX1owpO

-2- Absorption of FC-l43-*^C In Rets After e Single Orel Dose December 28, J979 WC/*\ JAN 17 1980 Conducted et: During: Conducted by: Report by: Reviewed by: Rlker Leboretories, Inc. Subsidiery of 3M St. Paul, Minnesota 55101 October to November, 1979 S. J. Gibson end J. 0. Johnson l/l/S s - )ernes D. Johnson, NS ' Date 'Senior Biooiemicel Phermecologist (1?. <?. ^ ilnleo robert i. ObirTVh.O. '--- T t Manager, Drug Metabolism 002855 r.. BEST COPY AVAtl ABLE Summary 1 )i| A f t e r a s i n g l e oral dose of FC-lk3" C (mean dose, 11.0 mg/kg) In s o l u t i o n to groups of three male rats, at least 93* of the total c a r b o n - Ik Is absorbed at 2k hours. The half-11 fa for elimination o f total carbon-lk from plasma Is k .8 days. 002856 Int.rod uct ion -H - BEST COPY AVA ABLE CFI502 'H< F C - 143 * Denotes Position of Carbon-1A FC-143 is the ammonium salt of a perftuorInatad carboxylic acid. A series of experiments has been planned to Investigate possible means of increasing the rate of elimination of FC-143 from the body. FC-143 ab sorption, elimination from plasma, and excretion data Mere necessary to form a basis for efficient design of these experiments. This FC-I43-**C oral dosing experiment (FC-Experlment 5) which was designed to provide total carbon-14 absorption and plasma elimination data was paired with an FC-l43-14c iv dosing experiment (FC-Experlment 6) which was designed to provide data on the route and extent of total carbon-IA excretion. hethods IA Radiolabeled FC-143- C The carbon-14 label is at the carbonyl carbon (see above structure). The specific activity of this lot of FC-I43-^C (Rlker Isotope Inventory Number 459) is 0.5071 pCi/mg. The FC-)43~^C was found to be suitable for metabolism studies; details of the specific activity determination, chemical characterization, and radiochemical purity determination was reported separately. (4) An i mo1s Hale Charles Rlver^- CO rats, ten weeks old, were conditioned to individual metal metabolism cages for 24 hours prior to dosing. The body weights ranged from 304 to 398 g, mean 362 g. The rats were allowed free access to Purina^Ground Chow and water before and immediately after dosing. Posing Each non-fasted rat was weighed immediately before being given a single oral dose of FC-143-^C. The dose was 2.0 ml of a 0.91 NaCI solution containing 4.1412 mg FC-143*^0/2.0 ml. The average dose was 11.4 mg/kg. The dose was administered with a 2.0 cc glass syringe (Trylon^t) fitted with a stainless . steel intubation tube. The dosing solution was prepared by weighing FC-l43-'*C into a 100 ml Class A volumetric flask, adjusting to volume with 0.91 NaCI, and mixing by inverting by hand. The carbon-14 content of the dosing solution was determined by direct counting (see Appendix 1). Sample Analysis for Carbon-14 Homogenizing of feces and tissues was done in Waring blenders by adding nine parts of water (w/w) to one part of biological material. The homogenates -- Charles River Breeding Laboratories, Wilmington, Hass. -- Purina Lab Chow, Ralston Purina Company, St. Louis, Hissouri. -- Aloe Hedical, Division of Brunswick, St. Louis, Hissouri. C02SSV were weighed into combustion cones-- in triplicate on a top-loading balance by taring the cone and adding 1.0 g 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 by weight. The concentration of carbon-14 in plasma for the 3 rats in the 24 and 48 hour group was determined by both combustion end direct counting. Plasma samples (including the 24 and 48 hour post dose groups) were counted directly by transferring 1.0 ml of plasma and IS ml of A q u s s o l * 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 plasma spiked with dilutions of F C - 143- t dosing solution at the beginning, middle, and end of the analysis of the exper(mental sample set (see Appendix 2). All radiometric measurements were done using a Packard Model 3385 Tri-Carb Liquid Scintillation Spectrometer. For plasme 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 (Auto matic 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 Croups of three rats were sacrificed by exsanguination at 1, 2, 6 , 12, 24, 48, 36, and 144 hours post dose. Rats were anesthetized with diethyl ether and blood was drawn from the descending aorta of each rat and immediately 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 plus contents (esophagus, stomach, small intestine, large intestine, and coion), 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. Results and Discussion The results of the urine, feces, digestive tract plus contents, and carcass analyses are shown in Table 1. At 24 hours post dose, the digestive tract and contents contained 2.6$ of the dose. The mean fecal excretion is 4.3$ of the dose, at 24 hours and 5.0$ at 48 hours. At 24 hours, the mean sum of total carbon-14 in feces and digestive tract plus contents is % 7$ of the dose. -- Packard Instrument Company, inc., 2200 Warrenville Rd., Downers Grove, lllint -- New England Nuclear, Boston, Mass. 002858 -0- BEST COPY A VM f llll Some^of thi, 72 likely represents systemcelly absorbed carbon-14 present either in the digestive tract tissues or in the digestive tract or feces as a result of elimination. The data from the 48 hour post dose group of rats are consistent with the 24 hour post dose data. Thus, at least 931 of the FC-I43-'*C dose was absorbed from solution after administration to non-fasted rats. The major portion of the radioactivity recorded was found In the carcass. The carcass data are not as accurate as the other tissue data since large volume homogenates were necessary and homogeneity of sample aliquots from carcass homogenates was difficult to assure. The quantity of total carbon-14 excreted via urine was on the average 9.28 of the dose at 24 hours and 14.8 of the dose at 48 hours. This is a much higher rate of excretion than that observed for F C - 9 5 - a t 24 hours post dose (1.68) (l) or for FC-807-**C at 24 hours (0.02.8) (3). The spleens from the 24 and 48 hour post dose group of rats were analyzed for total carbon-14 content, and the percent of the dose In the whole organ was \ 0.12. This is much lower than the 62 of the dose In spleen observed at 124 days post Iv dose for FC-807-**C. (2 ) It is similar to the % 0.22 of the dose found In spleen at 24 and 48 hours post oral dosing with Fc-95-lic. The concentrations of total carbon-14 In red blood cells and plasma were compared (Table 2). The mean ratios of red blood cell to plasma concen tration at 24 and 48 hours are 0.25 and 0.22, respectively. Thus, at 24 and 48 hours after a single oral dose of FC-l43-^C, 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-143-**C are shown in Table 3> The log of mean concentration versus time is plotted in Figure I. The least squares line through the individual points from 24 to 144 hours for these data fits the equation: Cp 6^.640* '" ^ . Cp is plasma concentrt ion. The half-life of elimination from plasma is 115 hours(4.8 days). The half-life of elimination of total carbon-14 after oral dosing with FC-95- C is 7.5 days (1). Thus, elimination from plasma of total carbon-14 after a single oral dose of FC-143- C Is slow but rapid than the elimination of total carbon-14 after oral dosing with FC-95- C. References (1) Johnson JD: Absorption of FC- 9 5 " ^ C in Rats After a Single Oral Oose (Report), October 26, 1979. (2) Johnson JD: Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 In Rats After a Single IV Dose of FC-807-^C (Report), September 29, 1979. (3) Johnson JD: Absorption of FC-807-^C in Rats After a Single Oral Dose (Report), July 10, 1979- (4) Behr FE, Johnson JD: Synthesis and Characterization of FC-143-^C (Report), December 28, 1979- 002859 -7List of Tables and Figures Table 1: Percent Recovery of Total Carbon-14 After a Single Oral Dose of FC-143-'*C to Rats (Mean Dose, 11.4 mg/kg) at 24 amd 48 Hours Post Dose. NB $2584, p. 27. Table 2: Comparison of Total Carbon-14 Content of Red Blood Cells to Plasma After a Single Oral Dose of FC-143~**C to Rats (Mean Dose, 11.0 mg/kg) at 24 and 48 Hours Post Dose. NB 52584, p. 25. Table 3: Concentration of Carbon-14 In Rat Plasma After a Single Oral Dose of FC-143-^C (Mean Dose, 11.4 mg/kg) at 1, 2, 6, 12, 24, 48, 96, and 144 Hours Post Dose. NB 52584, p. 30. Figure 1: Mean Concentration of Carbon-14 In Rat Plasma After a Single Oral Dose of FC-I43" **C (Mean Dose, 11.4 mg/kg). Appendix Table 1: Determination of Carbon-14 Content of Dosing Solution. NB 51807. p. 32. Appendix Table 2: Recovery of Total Carbon-14 from Blank Biological Samples Spiked with FC-143-^C. NB 52584, p. 28. C028S0 BPy$BESTtt- f Jfs 2* Table I Percent Recovery-- of Total Carbon-lb After a 14 Single Ora) Dose of FC-143- C to Rat (Mean Dote, II.4 mg/kg) at 24 and 46 Hours Post Dose Rat Number 1 2 3 Mean 4 5 6 Mean Time Post Dose (Hours) 24 24 24 48 48 48 Carcass 56.04 60.90 62.58 59.84 54.38 5 1 .60 55.04 53-67 Percent of Dose Digestive Tract plus Feces Contents 2.58 2.62 2.53 2.58 1.68 1.49 1.80 1.66 4.49 3.43 4.92 4.28 5.32 4.37 5.32 5.00 Urine 13.98 7.36 6.29 9.21 15.54 1720 11.69 14.81 0 Moan coin I re c o v e ry i s 75.9 and 75.8 fo r tho 24 and 48 Itour ijrouio., r e s p e c t i v e l y ; t i n s it. l i k e l y a r e f l e c t i o n o f Lite inlkcreitl in a c c u ra c y o f the c a r c a s s d a t a ( i c c text). 002861 -9- Teble 2 Comparison of Total Carbon"!*! Content-- of Red flood Cells and Plasma After a Single Oral Dose of F C - U j ' ^ C to Rats (Mean Dose, 11.0 mg/kg) at 2k and k8 Hours Post Dose Rat Number 1 2 3 Mean k 5 6 Mean Time Post Dose (Hours) 2k 2k 2k k8 k8 k8 Red Blood Cells 9.16 lk. 80 Ik.k5 10.20 12.69 7.50 PlasaM k6 .3k 60.68 k8.5k 50.66 k2.76 k3.72 Ratio Red Blood Cells/Plasma 0.20 0 .2k 0.30 05 0.20 0.30 0.17 02 -- Data are expressed as pg equivalents of FC-lk3-^C/g. 002862 Table 3 Concentration of Carbon-14-- In Rat Plasma After a Single Oral Dose of FC-143-**C (Mean Dose, 11.4 ng/kg) Hours Post Dose 1 2 6 12 24 48 96 144 A ----R-a-t--s----- C 27.75^68.41 64.67 63.86 49.89 54.63 34.91 29.87 30.04 48.40 58.29 59.39 64.53 45.74 42.42 23-79 24.12 41.58 64.62 77.10 53.66 c 29.73 29.51 Average 1 SO 27.30 2.98 52.80 t 13-94 62.53 t 3.67 66.78 t 9.21 56.03 t 7.61 50.19 6.29 35.69 t 6.38 27.72 3.41 ** Data were obtained by direct counting; data are expressed as pg equivalents of FC-l43" ^C/ml of plasma. -- Three rats were dosed and sacrificed for each time interval. -- Sample was very hemolized; data for this sample (obtained by direct counting) was not used. Plasma Carbon-Ut Content (yg equivalents of FC -1^*3- C/ml) -12- Apptndl I Determ ination o f Carbon-14 Content of Dosing Solution I4 Just prior to dosing of rats, the FC-143- C dosing solution was sampled with calibrated mlcroplpettors^- directly Into counting vials; six 10 pi and six 50 pi aliquots were pipetted. One ml of water and 15 ml of Aquasol* were added^-, corrections were made for back ground and counting efficiency and, using the specific activity of FC-143-^C already determined^, the pCt/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 2.100 pCi of FC-I43-1*C which is 4.1412 mg of FC-I4J-1*C. This is a mean dose of 11.4 mg/kg for the whole group of rats and 1 1 .0 mg/kg for the 24 and 48 hour groups of rats. -- L/l Micropipettor, Lab Industries, Berkeley, California. -- Aquasol* was found to be a suitable solvent-scintl)lent for FC-95- ^ C during specific activity determination (to be reported separately). -- Specific activity 0.5071 pCi/mg. (Specific activity determination to be reported separately.) C028S5 - 13- Append Ix TbU 1 Carbon-)* Content of Dosing Solution 10 h I Aliquot $0 Aliquot _______________________________________ H i 1/-!*!____ 1.0393 1 .OkkS I.0598 1 .080*i 1.0385 I .0328 1.0427 1 .0709 1.0*i73 I 6 5 5 1.0**7 I .0368 Overall average - 2.100 uCi/2.0 ml dose ld 0 2 _ y C I . *. ,*12 mg FC-1*3*^C/2.0 ml 0 .5 0 7 I|iC i/mg ^ C02866 -Ik - Append ix 2 Recovery of Total Carbon-11 Fro Blank Biological Samples Spiked with F C - 143-^C Good recovery of total carbon-14 from combusted FC-807- 1 C and FC-95-'*C has been reported (2, 3). In these labeled molecules, the carbon-14 label is a to the sulfur atom and Is fluorlnated. For FC-l43-^C, the carbonyl carbon is labeled, and the carbon-14 atom should be readily released by combustion. Thus, in view of the good recovery of total carbon-14 observed for FC-807-t^C and FC-95-^C, it was anticipated that good recovery of total carbon-14 from combusted FC-143-^C would be realized. For the set of samples four replicates of 10 pi, SO pi, and 100 pi of diluted FC-US-'^C dosing solution were al(quoted with calibrated micropipettors directly Into scintillation vials. At the same time using the same solution and pipettes, either three or four replicates of 10 pi, 50 pi and 100 pi were al(quoted directly Into combustion cones which already contained blank biological material (fecal homogenates or plasma). 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-143-1^C had been added directly. One each of the 10 pi, 50 pi, and 100 ii FC-l43~'<iC spiked pellets were 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 are shown in Appendix Table 2. The mean recovery is 97-38. Thus, combustion is a suitable means of preparing samples containing FC-lkJ-'^C for counting in a liquid scintillation counter. 002867 -15Appendix Tabic 2 BEST COPY AVAIlABLE Recovery of Total Carbon-1% From Fecal Homogenate 1fc of Plasma Samples Spiked with FC-H3" C 10 pl 121 1159 1127 1172 T75 - 1*9 Direct Addition 50jlL 6171 6088 5982 6155 5099 86 100 ul 1Z28A 12233 12132 12158 12202 * 70 10 pi 1 li*9 1195 1197 1161 TT75 21 Spiked and Oxidized 50jii 5969 5961 5572 5731 5509 193 100 pi 11689 11816 11798 11793 T T 7 7 T * 58 1176 TITS' X 100 loot 5809 x 100 95.3 5099 Overall Average 9 7 - 3 177M 12201 96.5 a i I^ -- Amount of FC-Jh 3" C spiking solution added. -- Data are expressed as dpm. C02868 -16- DISTRIBUTION LIST M. T. Case S. F. Chang G. J. Conard H. E. Fraiar L. I. Harriaon T. L. Kerlay J. D. LaZarta L. J. Magill R. A. Nelson R. E. Ober J. A. Pandargraaa R. A. Prokop A. L. Roaanthal P. A. Ubal P. 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