Document a4vZRgzyVmLq2j7NkE6b3ZwDy

-2- Absorption of FC-143-14 C In Rats After a Single Or&) 0060 December 28* 1979 WC#\ JAN I Pi1'980 Conducted at: During: Conducted by; Report by: Reviewed by: Riker Laboratorl*s,Inc. Subsidiary of 3M St. Paul, Minnesota 55101 October to November, 1979 S. J. Gibson and J. D. Johnson 6@VRC) ems D nson, "S Date @s:SrSeion@iroDir BIaeoical Pharmacologist *Wert E. Ober, Ph.u. Manager, Drug Metabolism Surm-a i fter a single oral dose of FC-143-14C (mean dose, 11.0 awkg) In solution to groups of three mie rats, at )east 93% of the total carbon-14 Is absorbed at 24 hours. The half-life for *Ilminatlon of total carbon-14 from plasma Is 4.8 days. introduction + c 7 F 15to2-um 4 FC-143 Denotes Position of Carbon-14 FC-143 Is the ammonium salt of a perfluorinated 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 absorption, elimination from plasma, and excretion date were noc ssa,ry ttC form a basis for efficient design of these experiments. This ;C- 43-1 oral dozing experiment (FC-Experimnt 5) which was designed to provide total carbon-14 absorption and plasma elimination data was paired with an FC-143-14C iv dosing experiment (FC-Experiment6) which was designed to provide data on the route and extent of total carbon-14 excretion. Methods 14 Radiolabeled FC-143- C The carbon-14 label Is at the carbony) carbon (see above structure). The specificactivity of this lot of FC-143-lht(Riker Isotope Inventory Number 459) Is 0.5071 UCI/mg. The FC-143-1 C was found to be suitable for metabolism studies;details-of the specific activity determination, chemical characterization, and redlochomical purity determination was reported separately.(4) AnitiIbsa Male Charles Rivera CD rats, ton weeks old, were conditioned to Individual metal metabolism cages for 24 hours prior to dosing. The body weights rang---d from 304 to 398 9, mean 362 9. The rats war* allowed free access to Purin;k Ground Chow and water before and Immediately after dosing. Dosing Each non-fasted rat was weighed Immediately before being given a sinqle oral dose of FC-143-19C. The dose was 2.0 mi of a 0.9% NaC) solution containirici 4.1412 mg FC-143-14C/2.0 mi. The average dose was 11.4 mg/kg. The dose was administeredwith a 2.0 cc glass syringe (TrylonOL)fitted with a stainless 14C steel intubationtube. The dosing solution was prepared by weighing FC-143into a 100 mi Class A volumetric flask, adjusting to volume with 0.9% NaC). 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 biologicalmaterial. The homogenates Charles River breeding Laboratories, Wilmington. Mass. Purina Lab Chow, Ralston Purine Company, St. Louis, Missouri. Aloe Medical, Division of Brunswick. St. Louis, Missouri. were weighed into combustion cones2- In triplicate on a top-loading balance by taring the cone and adding 1.0 9 of the homogenate. Care was taken to mix the homogenate between sampling&. 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 and direct counting. Plasma samples (including the 24 and 48 hour post dose groups) re counted directly by transferring 1.0 ml of plasma and 15 mi of Aquasof 4r to a scintillationvial and counting In a liquid scintillation spectrometer. tkmwgenate, plesm, urine and red blood -eelI samples from the 24 and 48 hour post dose group war* combusted with a Packard Model 306 Oxidizer. Combustion recovery of carbon-14 from biological sevies was determined by combusting ank fecal homogenates and blank plasm spiked with dilutions of FC-143-4k,C dosing solution at the beginning, middle. and end of the analysis of the experimental s&Wio set (see Appendix 2). All radiamtric measurements weredoneusinga PackardModel3385Tri-Carb Liquid Scintillation Spect. ter. For p)asm sawles 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 sampl*s. counting efficiency for each sample was determined by use of the AES (Automatic External Standardization) ratio method. To calibrate the external standard, Internal standard was added to selected s&Mles 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 1700Computer System In the 3h Central Research Date Processing Laboratory. Data reduction to dpa was accomplished with the Biological Automatic External Standardization computer program. Sa!Ele Collection Groups of three rats were sacrificed by exsanguination at 1, 2, 6. )2. 24. 48, 96, and 144 hours post dose. Rats were anesthetized with diethyl ether and blood was drawn from the descending sorts of each rat and Immediately transferredto a heparinizedtube. Plasma was prepared promptlyby centrifugation.In additionto plasmaand red bloodcells, totalurine, total faces, spleen,digestivetract plus contents (esophagus,stomach. small intestine,large Intestine,and colon),and remainderof 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 resultsof the urine, feces,digestivetractplus contents,and carcass analybesare shown in Table 1. At 24 hours post dose, the digestivetract 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 digestivetract plus contents is % 7% of the dose. Packard InstrumentCompany, Inc., 2200 Warrenville Rd., Downers Grove. lllinc New England Nuclear, Boston, Mass. Some of this 7% likely represents systemically absorbed carbon-14 present eithe@rin 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 consit ent with the 24 hour post do** data. Thus, at least 93% of the FC-143-14C dose was absorbed from solution after &"Inlstratton 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 homogenstes were necessary and hanagoneity of sample aliquots from carcass homo"nat*s was difficult to assure. The quantity of total carbon-14 excreted via urine was on the average 9.2% of the dose at 24 hours and 14.8of the dose at 43 hour This Is a much higher rot* of excretion than that oosorved for FC-95-Itiat 24 hours post dose (1.6%) (1) or for FC-607-14Cat 24 hours (0.02;) (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 dos* In the whole organ was % 0.1%. This Is much lower than_t 6% of the dose In sploon observed at 124 days post [v dose for FC-307li@c.(2) It Is similar to the A4'0.2% of the ose found In spleen at 24 and 48 hours post oral dosing with FC-95-ItC. The concentrations of total carbon-14 In red blood cells and plasm@ were conpared (Table 2). The mean ratios of rod blood call to plasm& concen- tration at 24 and 48 hours are 0.25 and 0.22. respectively. Thus, at 24 and 48 hours after a single oral dos* of FC-143-'qCt,hereIs 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-143-14C are shown in Table 3. The log of mean concentration versus time Is plotted in Figure ). The least squares line through the Individual points from 24 to 144 hours for these date fits the equation: Cp - 64.64e-0-006005t; cp T@e is plasma concentration.The half-life of elimination from plasma is 115 hours(4.8 days). half-life of elimination of total carbon-14 after oral dosing with FC-95- 'C Is 7.5days (1). Thus, ellmi?ttlon from plasma of total carbon-14aftera singleor&) doseof FC-143- C Is slowbut mofl rapid than the eliminationof total carbon-14after oral dosingwith FC-95- C. References (1) Johnson JO: Absorptionof FC-95-14C In Rats After a Single Oral Dose (Report),October 26, 1979. (2) Johnson JD: Extent and Route of Excretionand Tissue Distributionof Total Carbon-)4 In Rats After a Single IV Dose of FC-807-14C (Report), September 29, 1979. (3) Johnson JD: Absorptionof FC-807-14C in Rats After a Single Oral Dose (Report),July 10, 1979. (4) Behr FE, Johnson JO: Synthesisand Characterizationof FC-143-14C (Report),December 28, 1979. -7- List of Tables and Figures Table 1: Percent Recover f Total Carbon-14 After a Single Oral Dose of FC-143-y4*C to Rats (Mean Dose, 11.4 mg/kg) at 24 amd 48 Hours Post Dose. MI 525&k. p. 27. Table 2: Comparison of Total Carbon-14 Content of Red ood Calls to Plasma After a Single Ora) Dose of FC-111134_C to Rats (Mean Dose, 11.0 mg/kg) at 24 and 48 Hours Post Dose. UB 52584, p. 25. Table 3: Concentration of Carbon-14 In Rat Plasm After a Single Oral Dose of FC-143-14C (Mean Do**. 11.4 mg/kg) at 1, 2, 6, 12, 24, 48. 96, and 144 Hours Post Dose. NO 52584, P. 30. Figure 1: Mean Concentration of Carbon-14 In Rat Plasm After a Single Oral Dose of FC-143-14C (Mean Dose, 11.4 mg/kg). Appendix Table 1: Determination of Carbon-14 Content of Dosing Solution. US 51807, P. 32. Appendix Table 2: Recovery of Total Carbon-14 from Blank Biological Samples Spiked with FC-143-14C. NO 52584, P. 28. Table I Percent Recovery! of Total Carbon-14 After a 14 Single Oral Dos* of FC-143- C to Rats (OWen Dos*, 11.4 mg/kg) at 24 and 46 Hours Post Do$* 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 51.60 5S.04 53-67 Percent of Do" Digustivo Tract plus Contents Foces 2.56 2.62 2.53 2.58 1.68 ).49 i.so 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 17.20 11.69 14.81 lotii recovi.-i-yis 75.9 atid 7.r,.8foo- tl)t,2)o ii)(Ilig ii(iterciris4iii-.,#-t!sl)i-ciivi-ly; tlii!>it,likuly iircl*lec;tiuoifi Llicililiul'uisiiilai;c;ui-oilj'LL;lyiC text). dalil(tCL: Table 2 Comparison of Total Carbon-14 Content! of Red Blood Calls and Pies" After a Single Oral Dose of FC-143-14 C to Rats (Mean Dos*, 11.0 mg/kg) at 24 and 48 Hours Post Dose Rat Number 1 2 3 Mean 4 5 6 Mean Time Post Dos* (Hours) 24 24 24 48 48 48 Red blood Cells g.16 14.60 14-45 10.20 12.69 7.50 Plazas 46-34 60.68 48.54 50.66 42-76 43-72 Ratio Red Blood Colls/Plasoo 0.20 0.24 0.30 0.25 0.20 0.30 0.17 0.22 -@ Data are expressed as pq equivalents of FC-143-14c/g. Table 3 Concentration of Carbon-14! In Rat Plasma After a Single Oral Dose of FC-143- 14C (Haan Dose, 11.4 Wkg) Hours Post Dose 2 6 12 24 48 96 144 A b 27-75=68.J#l 64-67 63-96 49.89 54-63 34-91 29.87 30-04 48.40 58.29 59-39 64-53 45-74 42.42 23-79 c 24.12 41.58 64.62 77-10 53.66 c 29-73 29-51 Average t so 27-30 -t 2.96 52.80 * 13-94 62-53 1 3.67 66-78 1 9.21 56-03 t 7.61 50-19 t 6.29 35-69 1 27-72 1 6.38 3.41 Data were obtained by_direct counting; data are expressed as )19 equivalents of FC-143 14C/mi 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. 8 7 6 5 4 3 E 2 LL 0 vi Figure Mean Concentration of Carbon-14 In Rat plasma After a Single Oral Dose of FC-143-14 C (Mean Dose, 11.4 mg/kg) Cp-64.64*-0-006005t ----(Least Squares Line) th 115 hours (4-8days) cm 0 0 #u E 0 24 72 96 120 Hours Post Dose 144 Append Ix _I Determination of Carbon-14 Content of Dosing Solution 14 Just prior to dosing of rats. the FC;lt3- C dosing solution was sampled with calibrated-micropipetto s-id-irectly Into counting vials; six 10 ul and six 50 Ul allquots war* pipettod. &w of of water b and 15 mi of Aquasol* were added:--,corrections were mde for background and counting efflcl*ncy orW. using the specific activity of FC-143-14C already detorminedr-, the UCI/2.0 ol was calculated. The data are shown In Appendix Table 1. The dose administavd each rat was 2.0 ol of so:utign containing 2.100 mCi of FC-143-1 C which Is 4.1412 mg of FC-143-1*C. This Is a mean dose of 11.4 og/kg for the whole group of rats and 11.0 mg/kg for the 24 and 48 hour groups of rats. L/I Micropipettor, Lab Industries, berkeley, California. b Aqtiasol*was found to be a suitable solvent-scintillent for FC-1,.5-314C during specific activity determination (to be reported separately). Specific activity - 0.5071 PCI/mg. (Specific activity determination to be reported separately.) -13- Appendix Table I Carbon-14 Content of Dosing Solution 10 mi Aliquot PCI/ Imi 1.0393 1.0449 1.0598 ).0427 1.0709 1.0473 - Overall average - 2.100 uCi/2.0 mi dose 2.100 uci/OZ 4.1412 mg FC-)43)I-C#/2.0 mi 0.507-1tiCi 50 pi Aliquot P-cI/ imi 1.0804 1.0365 1.0329 1.'0655 1.0447 1.0348 Appendix 2 Recovery of Total Carbon-14 From Blank Biological Samples Spiked with FC-143-11#C 14 Good r overy of total carbon-14 from combustod FC-607- C and FC-95-ItC has been reported (2, 3). In the** labeled molecules, the carbon-14 label Is a to the sulfur &too and Is fluorinated. For FC-143-14Cth,e carbonyl carbon Is labeled, and the carbon-14 atom should be readily released by con6ustion. Thus. in view of the good refovery of total carbon-14 observed for FC-607-14C and FC-95-14C, It was anticipated that good recovery of total carbon-14 from combusted FC-143-14C would be realized. For the set of Z;71*s four replicates of 10 pi, 50 ul. and 100 ul of diluted FC-1 - 4C dosing solution were aliquoted with calibrated micropipettors directly Into scintillation vials. At the some tine using the same solution and pipettes. either three or four replicates Of 10 IJ), 50 Pland 100 01 were aliquoted directly Into combustion cones which already contained blank biological mt4wial (fecal homogenat*s or plasna). 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-143-14C had been addld directly. One each of the 10 pi. 50 ul, and 100 IA FC-143-'qC 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 man recovery is 97.3%. Thus, egipbustion Is a suitable means of preparing sawpies containing FC-143-'qC for counting In a liquid scintillation counter. Appendix Table 2 Recovery of Total Carbon-14 From Focal Havogonate 14 of Plosma Samples Spiked with FC-143- c io uil 1244@- 1159 1)27 1172 1176 49 10 JAI 1149 1195 1197 1164 1-17'6 24 Direct Addition 50 ti1 6171 6088 5982 6155 wg-g ;t86 Spiked and Oxidized @o pi 5969 5964 5572 5734 W9 193 100 111 12284 12233 12132 12158 12202 t 70 100 jAl 11689 ll$l6 11798 11793 11774 58 1176 x 100 100% 1176 5809 x 100 - 95.3 "609-9 Overall Average a 97.3 11774 x 100 96.5 12201 14 Amount of FC-143- C spiking solution added. Data are expressed as dpm. DISTRIBUTICO LZBT m. T. Case S. F. ChAng G. J. conard H. E. PrOiSr L. 1. Harrison T. L. Kerley J. D. L&Urte L. J. PAgill R. A. Nelson R. z. ober J. A. Pendergrass R. A. Prokop A. L. Rosenthal P. A. ubal P. Venkateswarl'U DMG Central File li@achCommunications W. P. 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