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7ri@@ RETRCOEECXEEI3lCtvVrIr-F--yDD@ Enhanced Elimination of 14 FC-143- C in Rats with Cholestyramine OLOG 3m TOXICOLOGVY-- and Treatment March 5, 1980 Conducted at: D-iring: Conducted by: Report by: Reviewed by: Riker Laboratories, Subsidiary of 3M St. Paul, Minnesota Inc. 55101 November,. 1979 to January, 1980 S. J. Gibson and J. D. Johnson James D. nson, ?S: njor JSeni:r BBii@;Vhemical -3127ZEO MS t@a--t-e Pharmacologist @pE-C-q,Q,.- Robert E. Ober, PhD Date Manager, Drug Metabolism -2- Summary In male rats cholestyramine administered (4% w/w) in feed decreased the retention of carbon-14 in liver, plasma, and red blood cells and increase the eliminivon of carbon-14 via feces after iv dosing with el@her FC-95- @4 c or FC-143- C. Groups of five ra were dosed iv with FC-95@l (mean dose, 3.4 mg/kg) or FC-143- c (mean dose, 13.3 mg/kg). C Groups of five control rats were-dosyl similarly but were not treated with cholestyiamine. The FC 95- C rats were sacrificed at 21 days and the FC-143- C rats at 14 days post dose. The mean liver, plasma, and red blood cell concentration as well as fecal and urinary excretion of carbon-14 for choles ixramine-treated rats were compared to mean control rat values. For FC-95- C, mean cholestyramine-treated rat carbon-14 concentrations in liver (9.4 ug/g) , plasma (0.9 ug/ml), and red blood cells (0-3 ug/g) represent a decrease from mean control rat concentrations of 3.8, 7.7, and 6.0 fold, respectively; fecal elimination (75.9 th cholestyramine 14w' treatment) was increased 9.5 fold. For FC-143- C, mean cholestyramine- treated rat carbon-14 concentrations in liver (12.1 ulg/g), plasma (5.1 ug/ml), and red blood cells (1-8 ug/g) represent a decrease from mean control concentrations of 2, 3, and 2 fold, respectively; fecal elimination (43.2%lrth cholestyrnine treatment) was increased 9.8 fold. For both FC-95- C and FC-143- C, the extent of urinary cabon-14 elimination, as a result of the relatively high rate of fecal elimination of carbon-14, was lower in cholestyramine-treated rats. The extent of total elimination of carbon-14 (urine plus feces) was higher in the cholestyrainine-treated rats. Since cholestyramine is approved for use in humans as a cholesterol lowering agent, these results in rats support the concept of testing cholestyramine in humans to promote excretion of FC-95 and FC-143. Introduction Cholestyrarnine, an agent approved for use in humans to lower cholesterol blood levels, is an anion exchange resin which has been administered orally to promote fecal excretion of the pesticide chlordecone (Kepone in rats (1) and man (2). Since both FC-95 and FC-143 are perfluorinated salts and exist as anions at physiologic pH, they would be expected to complex with cholestyramine. Non-ionic binding could also be involved. These two experiments in rats were designed to assess the effect of cholestyramine treatment onliarbon-14 elimination and tissue retei@2on after iv dosing with FC-95- C (FC-Experiment 8) and with FC-143- C (FC-Experiment 9). -3Methods Radiolabeled FC-143- 14 c * c 7F is COO FC-143 + NH 4 Denotes postion of carbon-14 The carbon-14 label is at the carbonyl carbo atom (see above structure). 14 The specific activity of this lot of FC-143- C (Riker Isotope Inventory Number 459) is 0.51 uCi/mg. Details of specific activity, chemical charac- terization, 1 2nd radiochemical purity have been reported separately (3); the FC-143- C was found suitable for metabolism studies. Radiolabeled FC-95- 14 c F *1 - + c F c so x 7 15 3- I F Denotes postion of carbon-14 The carbon-14 label is at the carbon atom oC to the sulfur atom see above structure). The specific activity of this lot ot FC-95- C (Riker Isotope Inventory Number 442) is 0.46 uCi/mg. Details of specific activity, chemical characterization 14 and radiochemical purity have been reported separately (4); the FC-95- C was found suitable for metabolism studies. Animals FC-95- 14 C (FC-Experiment 8) Ten male Charles River a CD rats, twelve weeks old, were randomly divided into a control group of five (weiaht 304 to 331 g, mean 320 g) and a group of five to be treated with cholestyramine (weiaht 322 to 342 g, mean 330 g). For 24 hours prior to dosing, the rats were conditioned to individual metal metabolism cages and fatted with free access to water. The rats were allowed free access to Purina- Ground Chow (with or without added cholestyramine) and water immediately after dosing. a b Charles River Breeding Laboratories, Wilmincrton, Massachusetts. Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. -4- 14 FC-143- C (FC-Experiment 9) a T,en male Charles River CD rats# twelve weeks old, were randomly divided into a control group of five (weight 300 to 341 g, mean 313 g) and a group of five to be treated with cholestyramine (weight 300 to 344 g, mean 317 g). For 24 hours prior to dosing, the rats were conditioned to individual metal metabolism cages and fasted gith free access to water. 7he rats were allowed free access to Purina- Ground Chow (with or without added cholestyramine) and water immediately after dosing. Dosin 14 FC-95- C (FC-Experiment 8) Each rat was weighed, anesthetized licrhtly with disthyl ether, and then given a single iv dose (via tail vein) of FC-95- C The dose was 2.0 ml of a 0.9% NaCl solution containing 1.13 mg FC-95- 14 C/2.0 ml. The average dose administered the cholestyramine-treated rats was 3.4 mg/kg and the average dose for control rats was 3.5 mg/kg e dose was delivered with a 3.0 cc disposable plastic syringe (Monoje*ct i!C) fitted with a 26 gauge 1/42" needle. The dosing solution..was prepared by adding - 150 mg of FC-95- C to 0.9% NaCl, shaking for one hour at moderate speed 721n 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). 14 FC-143- C (FC-Experiment 9) Each rat was weighed, anesthetized lightly with ethyl ether, and then given a single iv dose (via tail vein) of FC-143 @i C. P2e dose was 2.0 ml of a 0.9% NaCl solution containing 4.21 mg of FC-143- C/2.0 ml. The average dose administered the cholestyramine-treated rats was 13.3 mg/kg and the average dose for with a 3.0 cc disposable a 26 gauge 1/1"4 needle. mg of FC-143- C in 0.9% control rats was 13.5 mg/kg. The dose was delivered plastic syringe (Monoject m@-d ) fitted with The dosing solution was prepared by dissolving 225 NaCl. The carbon-14 content of the dosing solution was determined by direct counting (see Appendix 2). a Charles River Breeding Laboratories, Wilmingtonf Massachusetts. -@Purina Lab Chow, Ralston PLIrina Companyf St. Louis, Missouri. 2Sherwood Medical Industries, Inc., Deland,, Florida 32720. Cholestyramine Treatment a Cholestyramine (dried and ground resin Z-620) was mixed 4% by weight with Purina Ground Chow. A weighed portion of the cholestyramine feed was prepared for each rat so that consumption could be individually monitored. Each of the cholestyramine-treated rats in both FC-Experiments 8 and 9 received only treated feed during the cou e of the studies. 7he actual fl mean dose of cholestyramine for the FC-95 C treated rats was 2.7+0.2 14 (SD) g/kg/day. The actual mean dose of cholestyramine for the FC-143- C treated rats was 3.0+0.2 (SD) g/kg/day. Sample Collection 14 FC-95- C (FC-Experiment 8) Urine and feces were collected at intervals (see Tables 3 and 5) for each of the ten rats for 21 days. At 21 days post dose, the rats were anesthetized with diethyl ether; blood was drawn from the descending aorta and immediately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. The rats were sacrificed by exsanguination, and liver was collected as the whole organ. 14 FC 43- C (FC-Ex2eriment 9) Urine and feces were collected at intervals (see Tables 4 and 6) for each of the ten rats for 14 days. At 14 days post dose, the rats were anesthetized with diethyl ether; blood was drawn from the descending aorta and immediately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. The rats were sacrificed by exsanguination, and liver was collected as the whole organ. Sample Analysis for Carbon-14 Feces and whole liver were prepared for carbon-14 analysis by hvnogenizing and aliquoting a sample of the homogenate into combustion cones-. Homogenizing was done in Waring blenders by adding nine parts of water (wlw) to one part of biological material. The homogenates were weighed Into combustion cones in duplicate on a top-loading balance by taring the cone and adding 1.0 g of the homogenate. Red blood cells were measured into combustion cones by weight. -amead Johnson Research Center, -@Packard Instrument Co., Inc., Downers Grove, Illinois. Evansville, Indiana 47721. 220 Warrenville Road, -6- Homogenateas and red blood cells were combusted with a Packard model 306 Cx I i zer . Recovery of carbon-14 from biological samples was determined bv combusting suitable blank homogenates (feces and liver) spiked with dilutions of the appropriate dosing solution at the beginning, middle, and end of the experimental for recovery recovery from sample sets. All mean rec veries were > thus, no T4 ?1%; was made for either Fc-95- C or FC-143- C samples. cywbustion analysis has previously been reported for correction Good FC-95- 14 C (5) and FC-143- C (6). Urine collections were sampled before freezing and were counted directly; duplicate 1.0 ml aliquots of each sample were pipetted directly into scintillation vials and 15 ml Aquasol 0 @As added. It has previously been 1 ieported that direct counting inltquasol is appropriate for FC-95- C urine samples (5) and FC-143- C urine samples (6). All samples were cooled to refrigerator temperature in the dark before counting. All radiometric analyses were done using a Packard Model 3385 Tri-Carb Liquid Scintillation Spectrometer 2. Counting efficiency for each sample was determined by use of the AES (Automatic External Standardization) ratio method. 7b 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. Results and Discussion The results of the liver, plasma, and red bly2d cell analyses for carbon-14 content results after intravenous after intravenous dosing with FC-95-_ are shown in Table 1; the dosing with FC-143 15 C are shown in Table 2. Cholestyramine treatment caused a decrease i mean liver, plasma, and red Y4 blood cell carbon-14 content for both FC-95- C and FC-143- C dosed rats. For Fc-9b- C, mean cholestyramine-treated rat carbon-14 concen- trations in liver (9.4 ug/g), plasma (0.9 ug/ml), and red blood cells (0.3 ug/g) represent a decrease from mean control rat lioncentrations of 3.8, 7.7, and 6.0 fold, respectively. For FC-143- C, mean cholestyramine-treated rat carbon-14 concentrations in liver (12.1 ug/g), plasma (5.1 ug/ml), and red blood cells (1-8 ug/g) represent a decrease from mean control concentrations of 2, 3, and 2 fold, respectively. Thus, oral cholestyramine treatment effectively reduced the liver, plasma, and red blood cell concentrations of these two fluorocarbons and/or their metabolites. The larger decrease in plasma concentration as compared to decrease in liver concentration for both compounds may indicate, however, that some of the liver fluorocarbon is not affected to the same extent as plasma fluorocarbon by cholestyramine treatment. The results of carbon-14 fecal analyses for FC-95- 14 C dosed rats are shown in Table 3 and in Figure 1. The mean cumulative percent of dose eliminated via feces by cholestyramine-treated rats (75.9%) is 9 5_foldlihe mean percent of dose eliminated via feces by control rats. For ;c 143- C APackard Instrument Co., Inc., 220 Warrenville Dc)wners Grove, Illinois. Road, -7- rats, the carbon-14 fecal analyses results are shown in Table 4 and in Figure 2. The mean cumulative percent of dose eliminated via feces by cholestyramine-treated rats (43.2%) is 9.8 fold the mean percent of dose eliminated via feces by control rats. Thus, the decrease of liver, plasma, and red blood cell iirbon-14 concei@rations after oral cholestyramine treatment of FC-95- C and FC-143- C dosed rats is accompanied by a large increase in the extent of fecal elimination of carbon-14. The results of the urine analyses for FC-95-14 C dosed rats are shown in Table 5 and in Figure 3. The cumulative percent of dose eliminated in urine by the cholestyramine-treated rats (5.4%) is about one third of the percent of dose elimi@ ated by the control rats. The results of the urine analyses for FC-143- C dosed rats are shown in Table 6 and Figure 4. The cumulative percent of dose eliminated in urine by cholestyramine-treated rats (41.1%) is about three fifths yi the percent yi dose eliminated by the control rats. Thus for both FC-95- C and FC-143- C dosed rats, the cholestyramine-treated rats eliminate less carbon-14 in urine than do the control rats. The lower urinary excretion by cholestyramine-treated rats is a result of the nearly 10 fold increase in fecal elimination of carbon-14, especially during the first few days post dosing; since in cholestyramine- treated rats much of the carbon-14 is eliminated via feces, it is not available for urinary eliminatiy2@ Total elimination of carbon-14 (urine plus fey2s) is shown for FC-95- dosed rats- Figure 5 and for FC-143- C dosed rats in Figure 6. For FC-95 l@ C dosed rats, after 21 days of cholestyramine treatment, 81.2% of the dose has been eliminated versus only 25.6% of the dose for control rats. The mean percentaoes of dose remaining in the body, 19% for cholestyramine-treated rats and 74% for control rats, are in about the same proportion (1:4) as the differences in liver carbon-14 content (cholestyramine-treated, 9.4 ug/g; control,_21-6 ug/g). The total carbon-14 eliminated (urine plus feces) by FC-14 c dosed rats is 84% of the dose for cholestyramine-treated rats and 72% of the dose for control rats; the percentages of dose rt@aining in the body are 16% and 28%, respectively. Again as with FC-95- C, the mean percent- ages of dose remaining in the body are reflected in the relative mean liver concentrations (cholestyramine-treated, 12.1 ug/g; control, 22.3 ug/g). The individual body weights at time of dosing and the individual body weights and liver weights at time of sacrifice are y@own, along with corresponding contrgl groups, in Table 7 for FC-95- C dosed rats and in Table 8 for FC-143-'qC dosed rats. Cholestyramine does not appear to have caused a change in weight gain or liver weight in either exderiment. Cholestyramine is approved for human use as a cholesterol lowering agent and has been used in humans to promote the excretion of xenobiotics (2). These results In rats with FC-95 and FC-143 support the concept of testing cholestyramine in humans to promote excretion of fluorocarbons. References (1) Boylan JJ, Egle JL, Guzelian PS: Cholestyramine: Use as a New Therapeutic Approach for Chlordecone (Kepone ) Poisoning. Science 199: 893-895, 1978. (2) Cohn WJ, Treatment New Engl Boylan JJ, Blanke RV, Fariss MW, Howell 40 of Chlordecone (Kepone ) Toxicity with J Med 298: 243-278, 1978. JR, Guzelian PS: Cholestyramine. (3) Behr FE, Johnson JD: Synthesis and Characterization (Report) December 28, 1979. of FC-143- 14 c (4) Johnson JD, Behr FE: Synthesis and Characterization (Report) November 2, 1979. of FC-95- 14 c (5) Johnson JD: EKtent and 7btal Carbon-14 in Rats December 28, 1979. Route after of Excretion and a Single IV Dose Tissue D2f@ribution of of FC-95- C (Report) (6) Johnson JD: Mctent and Route of Excretion and Tissue Distribution of 7btal Ctibon-14 in Male and Female Rats after a Single IV Dose of FC-143- C (Report) January 30, 1979. Acknowledgement The preparation of the 4% w/w cholestyramine In Purina Ground Chow by L. J. Sibinski of Pathology-Toxicology Is gratefully aknowledged. Idst of Tables and Figures Table 1: IAver, Plasma, and Red Blood Cell Concentration_of_iarbon-14 in Rats at 21 Days after a Single IV Dose of FC 95 c (Mean Dose of FC-95-"4C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 25-27. Table 2: TAver, Plasma, and Red Blood Cell Concentration_of Clibon-14 in Rats at 14 Days af r a Single IV Dose of FC 143- C @2 (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kcr; Control, 13.5 mg/kg) NB 53102 P 38,39. Table 3: Excretion of Total Carbon-li in Feces in Rats afte a Single 14 Intravenous Dose of FC-95- C (mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 20,21. Table 4: Excretion of Total Carbon-1 n Feces in Rats after Single f4i t4 Intravenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine-7'reated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 30,32. Table 5: Excretion of Total Carbon-11 in Urine in Rats afte a Single 14 Intravenous Dose of FC-95- C (Mean Dose of FC-95- C: Cholestyramine Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 23,24. Table 6: Excretion of Total Carbon-1 in Urine in Rats after Single f4 f4 Intravenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 35,36. Table 7: Body Weights at Dosing and Body and ldver Weights_f@ Sacrifice for Rats after Single Intravenous Dose of FC-95 C (Mean ii Dose of FC-95- c: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 26 Table 8: Body Weights at Dosing and Body and Uver Weights a Sacrifice 14 for Rats after- ingle Intravenous Dose of FC-143- C (Mean 14S rbse of FC-143 C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 38. Figure 1: Cumulative Excretion of Total CarNn-14 in Feces after a Single,intravenous Dose of PC-95- C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mq/kg; Control, 3.5 mg/kg) mean of Five Rats NB 53102 P 28. T@ist of Tables and Figures (continued) Figure 2: Cumulative Excretion of Total Cart)?@-14 in Feces after a Single 12travenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kq; Control, 13.5 mg/kg) Mean of Five Rats NB 53102 P 40. Figure 3: Cumulative Excretion of Total CarNn-14 in Urine after a Single intravenous Dose of FC-95- C (Mean Dose of FC-95-1 C: Cholestyramine-Treated, 3.4 mg/kq; Control, 3.5 mg/kg) Mean of Five Rats NB 53102 P 28. Figure 4: Cumulative Excretion of Total Carb22-14 in Urine after a Single_fRtravenous Dose of FC-143- C (Mean Dose of FC-143 C: Cholestyramine-Treated, 13.3 mg/kg, Control, 13.5 mg/kg) Mean of Five Rats NB 53102 P 40. Figure 5: Cumulative Excretion (Urine + Feces) of !I@@al Carbon-14 after a S12gle Intravenous Dose of FC-95- C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Mean of Five Rats NB 53102 P 28. Figure 6: Cumulative Excretion (Urine + Feces) of Toial Carbon-14 after a Sii@le Intravenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) Mean of Five Rats NB 53102 P 40. Appendix 1: Determination of Carbon-14 Content of FC-95- 14 C Dc)sing Solution NB 51807 P 41,44. Appendix Table 1 Carbon-14 Content of FC-95- 14 C DDsing Solution NB 51807 P 44. Appendix 2: 14 Determination of Carbon-14 Content of FC-143- C Dc)sing Solution NB 51807 P 41-45. Appendix Table 2 14 Carbon-14 Content of FC-143- C Dosing Solution NB 51807 P 45. -12- Tabl e 1 Liver, Plasma, and Red Blood Cell Concentratloriar-of,5arbon-14 in Rats at 21 Days aner a Single IV Dose of FC-95- C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg Ldver Plasma Red Blood Cells Cholestziamine-Treated Rat Number 1 2 3 4 5 9.8 11.5 9.7 7.1 8.8 0.9 1.1 0.8 0.7 0.9 0.2 0.3 0.3 0.2 0.3 Mean + SD 9.4 + 1.6 0.9 + 0.1 0.3 + 0.1 Liver Plasma Red Blood Cells 30.8 7.0 2.1 Control Rat Number 2 3 4 5 33.2 40.6 30.7 42.5 7.4 6.2 6.4 7.5 2.2 1.6 1.7 1.3 Mean + SD 35.6 + 5.6 6.9 + 0.6 l.e + 0.4 !Tbtal carbon-14 concentration for liver anl4red blood cells is expressed as ug equivalents of FC-95- C/g; plqgma carbon-14 concentration is expressed as ug FC-95- @C equivalents/ml. -13- Table 2 Liver, Plasma, and Red Blood Cell Concentration a of @2rbon-14 in Rats at 21 Days af@2 r a Single IV Dose of FC-143- C (Mean DDse of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg raver Plasma Red Blood Cells 1 14.6 7.1 2.7 Cholestyramine-Treated Rat Number 2 3 4 5 9.4 13.5 11.9 10.9 3.0 6.1 5.5 3.9 1.0 2.4 2.0 1.2 Mean + SD 12.1 + 2.1 5.1 + 1.7 1.8 + 0.7 Liver Plasma Red Blood Cells 1 25.4 19.9 6.7 2 31.4 23.9 7.0 Control Rat Number 3 4 5 21.0 17.5 16.3 10.1 8.8 10.7 2.3 2.6 2.4 Mean + SD 22.3 + 6.2 14.7 + 6.8 4.2 + 2.4 !7btal carbon-14 concentration for liver aii red blood cells is expressed as ug equivalents of FC-143- C/g; pl ma ii carbon-14 concentration is expressed as ug FC-143- C equivalents/ml. -14- Table 3 Excretion of Total Ci@bon-14 In FecesA In Rats after a Single Intravenous Dose (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/k Collection Period (Days) 0-0.5 0.5-1 1-2 2-3 3-4 4-5 5-6 6-7 7-9 9-11 11-15 15-18 18-21 Total 1 0.08 Cholestvramine-Treated Rat Number 2 3 4 5 2.90 1.97 2.07 b O.OL@@ 1.49 7.94 11.30 7.58 3.97 6.75 14.27 15.63 17.59 16.47 15.34 11-03 12.68 12.03 14.19 11.85 7.24 8.42 9.96 8.71 8.54 5.26 5.67 3.81 5.77 4.42 6.02 4.61 8.20 5.12 4.54 2.69 3.28 3.67 5.13 5.07 4.48 4.17 4.80 5.30 3.65 3.38 3.37 3.96 3.90 4.36 4.57 4.08 4.76 5.27 2.48 0.48 2.50 0.5s 2.40 0.64 2.63 0.77 2.54 0.98 69.89 71.06 7R.13 80.45 79.7P Mean+SD 1.40+1.30 6.46+3.80 14.14+4.31 13.05+1.72 9.24+1.75 6.84+1.41 4.64+0.58 3.86+0.98 4.76+0.45 3.65+0.28 4.61+0.45 2.51+0.08 0.69+0.19 75.86+5.01 1 0.35 Control Rat Number -2 3 4 0.60 0.40 0. 0.64 0.59 0.57 0.41 0.46 0.86 1.11 0.55 0.72 0. 0.53 0. 0.50 0. 0.48 0.4 0.40 0.35 0.39 0.4 0.35 0.37 0.43 0.5 0.32 0.40 0.31 0.3 0.58 0.56 1.01 0.69 0.64 1.25 0.69 0.7 0.69 0.0 0.95 1.1 0.64 0.48 0.54 0.58 0.65 0.9 0.64 0.7 -@.qO @.40 319 @.6 a Data are expressed as percent of dose excreted during collection period. -@No feces excreted. -15Table 4 Excretion of Total Ca i@On-14 in Feces-@!In Rats after a Single Intravenous Dose (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mq/kg; Control, 13.5 m Collection Period (DAYS) 0-0.5 0.5-1 1-2 2-3 3-4 4-5 5-6 6-7 7-9 9-11 11-14 7btal Cholestyramine-Treated Rat Number 1 2 3 4 5 0.04 0.13 0.01 3.15 1.78 5.06 5.50 6.85 4.96 4.54 3.72 7.21 7.21 4.45 2.79 1.73 7.20 7.74 5.91 5.14 5.6R 8.11 7.52 5.15 3.57 4.63 7.91 5.50 3.84 3.22 3.58 2.54 2.34 2.04 4.09 3.26 3.35 2.56 2.41 1.98 4.87 2.73 5.04 3.74 2.91 3.66 4.01 1.83 1.94 3.87 3.61 2.61 2.82 2.21 2.04 45.61 36.39 47.60 48.26 38.33 Mean+SD 1.02+1.40 4.16+1.54 7.17+1.01 6.96+0.88 4.86+0.78 3.85+0.97 3.15+0.76 2.48+0.52 3.86+1.07 2.84+0.89 2.88+0.92 43.23+5.50 1 0. 0.44 0.32 0.34 0.2S 0.31 0.21 0.16 0.33 0.41 0.27 Control Rat Number 2 3 4 0.09 0.21 0.3 0.54 0.44 0.3 0.51 0.69 0.7 0.54 0.52 0.4 0.33 0.51 0.7 0.33 0.42 0.2 0.54 0.65 0.2 0.25 0.22 0.2 0.97 0.69 0.4 0.48 0.54 0.3 0.59 0.61 0.2 3.07 5.17 5.50 4.3 AData are expressed as percent of dose excreted durinq collection period. .tNo feces excreted. -16- Table 5 EKcretion of Total Ci@bon-14 In Urine-@lIn Rats after a Single Intravenous Dose (Mean DDse of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg Collection Period (Days) 0-0.5 0.5-1 1-2 2-3 3-4 4-5 5-6 6-7 7-9 9-11 11-15 15-18 16-21 Total Cholestyramine-Treated Rat Number 1 2 3 4 5 1.58 1.53 1.75 1.91 1.53 0.49 0.48 0.50 0.30 0.26 0.69 0.61 0.58 0.83 0.61 0.67 0.61 0.52 0.50 0.62 0.59 0.33 0.39 0.48 0.42 0.26 0.20 0.22 0.23 0.32 0.23 0.16 0.19 0.21 0.25 0.22 0.13 0.11 0.15 0.16 0.25 0.19 0.18 0.21 0.24 0.20 0.17 0.18 0.21 0.19 0.28 0.23 0.25 0.26 0.35 0.22 0.17 0.23 0.22 0.22 0.15 0.11 0.14 0.14 0.12 5.83 4.92 5.24 5.65 5.29 Mean+SD 1.66+0.17 0.41+0.12 0.66+0.10 0.58+0.07 0.44+0.10 0.25+0.05 0.21+0.03 0.15+0.04 0.21+0.03 0.19+0.02 0.27+0.05 0.21+0.02 0.13+0.02 5.37+0.36 1 1.98 Control Rat Number 2 3 4 1.61 1.83 1.0 1.06 1.00 0.90 0.8 1.14 1.30 0.94 1.o 1.45 1.31 1.11 1.1 1.13 0.94 0.96 0.76 1.55 1.17 0.87 0.92 0.68 1.62 0.97 1.0 0.88 0.8 0.75 0.9 0.71 0.7 1.26 1.4 1.51 1.41 1.30 0.9 2.62 1.79 1.72 2.51 1.76 1.59 2.18 2.2 1.63 1.( 1.50 1.3 18.51 17.75 15.96 15.3 AD&ta are expressed as percent of dose excreted during collection period. -17Table 6 Excretion of Total Ca n-14 In Urine2 in Rats after a Single Intravenous Dose i@O (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 m Collection Period (Days) 0-0.5 0.5-1 1-2 2-3 3-4 4-5 5-6 6-7 7-9 9-11 11-14 Total Cholest yramine-Treated Rat- Number 1 2 3 4 5 4.72 8.60 5.21 8.79 12.17 2.46 5.11 2.23 2.97 5.00 4.15 9.05 4.09 5.00 8.09 3.50 8.68 3.95 4.73 5.75 2.87 5.74 4.19 3.18 4.18 2.27 3.34 2.65 2.57 2.90 2.44 2.99 2.67 1.88 2.36 1.87 2.15 2.11 1.41 1.66 3.55 3.22 3.48 2.21 3.23 2.38 2.19 2.05 1.54 1.98 2.69 1.98 1.85 1.48 1.78 32.90 53.05 34.48 35.76 49.10 Mean+SD 7.90+3.04 3.55+1.40 6.08+2.33 5.32+2.06 4.03+1.12 2.75+0.40 2.47+0.41 1.84+0.31 3.14+0.54 2.03+0.31 1.96+0.45 41.07+9.31 1 3.85 3.48 6.07 6.35 6.10 5.22 4.83 4.34 7.21 6.09 6.95 Control Rat Number 2 3 4 5.06 4.11 15. 4.15 7.88 5. 7.21 5.54 8.72 ll.( 6.68 9.( 4.76 6.67 7.1 4.20 6.32 4.8 3.69 5.08 4.4 3.40 4.14 3. 6.37 6.37 6.2 5.55 5.62 5.2 6.98 5.57 4.4 60.49 56.91 67.16 77.6 a Data are expressed as percent of dose excreted during collection period. Table 7 Body Weights a @4 Dosing and Body and Lly2r Weights at Sacrifice for Rats after a lbse of FC-95- C (Mean rbse of FC-95- C: Cholestyramine-Treated, 3.4 mg/kql cc Cholestvramine-Treated RaZ Number@ 1 2 3 4 5 Body Weight at Dosing (g) 324 329 322 342 331 Body Weight at Sacrifice (g) 400 422 403 410 402 Percent Change In Body Weight 23.5 28.3 25.2 19.9 21.5 Liver Weight (at Sacrifice) (g) 14.0 15.0 13.9 13.1 11.8 Percent Liver Weight of Body Weight (at Sacrifice) 3.5 3.6 3.4 3.2 2.9 Mean+SD 330+8 407+9 23.7+3.3 13.6+1.2 3.3+0.3 Control Rat Number 1 2 3 316 304 331 321 393 409 394 440 24.4 34.5 19.0 37.1 14.6 13.3 11.7 14.9 3.7 3.3 3.0 3.4 -19- Table 8 Body Weights at,ioslng and Body and fAvef4welghts at Sacrifice for Rats after a DDse of FC-143- C (Mean Dose of FC-143- C: Cholestyrami ne -Treated, 13.3 mg/k Cholestyramine-Treated Rat Number 1 2 3 4 5 Body Weight at Dosing (g) 344 330 302 307 300 Body weight at Sacrifice (g) 414 382 354 347 389 Percent Change In Body Weight 20.4 15.8 17.2 13.0 29.7 laver Weight (at Sacrifice) (9) 17.0 14.1 11.2 11.8 14.9 Percent laver Weight of Body Welqht (at Sacrifice) 4.1 3.7 3.2 3.4 3.8 Mean+SD 317+19 377+27 19.2+6.4 13.8+2.4 3.6+0.4 Control Rat Number 1 2 3 4 341 312 301 300 384 356 383 358 12.6 14.1 27.2 19. 15.5 14.7 15.0 10. 4.0 4.1 3.9 3. 75- u w 44 a to 8 4450" 0 25. Figure I Cumulative Excretion of Totil Carbon-14 in Feces af@er a Single Intravenous Dose of FC-95- C (Mean Dose of FC-95- C: Cholestyramin Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Mean of 5 Rats Cholestyrami 0 5 ;o 1'5 26 Figure 2 Cumulative Excretion of Total Carbon-1 14'n Feces after a Singit Intravenous Dose of FC-143- C (Mean Dose of PC-143- C: Cholestyramine Treated, 13.3 mg/kg: Control, 13.5 mg/kg) Mean of 5 Rats 100 75 u U) 0 so 0 41 25 43.2t5.5 Cholestyramine Treated Rats 0 1 2 34 5 10 Days Post Dose 4.41.0 Control Rats 15 18- 15. 10. 0 41 1 u 104 5. a, r4 0 0 Figure 3 Cumulative Excretion of Total Carbon 44 in Urine After a-Sinyie Intravenous Dose of FC-95- 1 C (Mean Dose of FC 95- C: Cholestyramine Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Mean of 5 Rats Chhoo@lle@ 5 10 15 20 Days Post Dose 100, Figure 4 cumulative Excretion of Total Carbon-lf in Urine after 4 a Single Intravenous Dose of FC-143- C (Mean Dose of FC-143: Cholestyramine Treated, 13.3 mg/kg; Control, 13.5 mg/kg) Mean of 5 Rats '75 - U) a 4, 50 0 u .> u 25 67.48.9 ,@@c,ontrol Rats 41.19.3 Cholestyramine-Treatecl Rats 0 01 2 3 4 5 10 15 Days Post Dose 75. 41 $4 0 x 44500 41 V. 04 4J 0 25. Figure 5 Cumulative Excretion (Urine + Fecesl4of Total Carbon-14 aft a Single Intravenous Dose of FC-95- C (Mean Dose of FC-95- T-4c: Cholestyramine Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Mean of 5 Rats Cho 0 0 5 10 15 20 100- Figure 6 Cumulative Excretion (Urine + Feces) of Ti@al Carbon-14 After a Sinyle Intravenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine Treated, 13.3 mg/kg; Control, 13.5 mg/kg) Mean of 5 Rats 75 x LW 0 50 @4 (U 84.3t 4.3 @Cho3lestyramine Treated 71.88.9 ontrol 25 0 0 4' 10 is Days Post Dose -26- APPENDIX 1 Determination of Carbon-14 Content of FC-95- 14 C Dosing Solution Just prior to dosing of rat the FC-95- 14 C dosing solution was swnpled with calibrated micropipett:rsa directly into counting vials. Six 10 ul and six go ul aliquots were pipetted, and 1 ml of water and 15 ml of AquasolO- were added to three of each of the 10 ul and 50 ul aliquats. The remaiging three vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSe@c-'-.corrections were made for background and counting efficiency and, using the specific activity already determinede-, the uCi/2.0 ml was calculated. The data are shown in Appendix Table 1. The_dos T4 administered each rat is 2.0 M14 of solution containing 0.519 uCi of FC 95- C which is 1.13 mg of FC-95- C. Sbis is a mean dose of 3.4 mg/kg for the cholestyramine-treated rats and 3.5 mg/kg for the control rats. 2L/I Micropipettor, Lab Industries, Berkeley, California. -@New England Nuclear, Boston, Massachusetts. EModified TSS: 25.2 g PPO, 1.01 q I)imethylPOPOP 2nd 3.8 Liters Toluene. iaqua:ol a and MTSS were found to be suitable solvent-scintillents for 4 FC-9 - C during specific activity determination (4). .sspecific activity = 0.459 uCi/mg. has been reported (4). Specific activity determination -27APPENDIX TABLE 1 Carbon-14 Content of FC-95- 14 C Dosing Solution 10 ul Aliquot uCi/2.0 ml 0.5084-S 0.5012@A C).5284-S 0.516@-b 5064-h O.Sogeb 50 ul Aliquot uCi/2.0 ml o.siii.! 0.5375a 0 -5299-0.5299-b 0.5383b 0.5155b overall average = 0.5194+0.0128 0.519 0.459 uCi/mg 1.133 mg/2.0 ml Sample prepared with Aquasol b Sample prepared with MTSS. -28APPENDIX 2 Determination of Carbon-14 Content of FC-143- 14 C Dosing Solution Just prior to dosing of rats, the PC-143- 14 C dosing solution was sampled with calibrated micropipettors a directly into counting vials. Six 10 ul and six 50 ul aliquots were pipetted, and 1 ml of water and 15 ml of AquasolOb were added to three of each of the 10 ul and 50 ul aliquots. !rhqremaining vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSSS,-a. Corrections were made for background and counting efficiency and, using the specific activity already determined-t, the uCi/2.0 ml was calculated. The data are shown In Appendix Table 2. The dose fiministered each rat is 2.0 ml of Y21ution containing 2.134 uCi of FC-143- C which is 4.21 mg of FC-143- C. This is a mean dose of 13.3 mg/kg for the cholestyraminetreated rats and 13.5 mg/kg for the control rats. !L/I Micropipettor, Lab Industries, Berkeley, California. !tnew England Nuclear, Boston, Massachusetts. -EModified 7SS: 25.2 g PPO, 1.01 g DimethylPOPOP 2nd 3.8 Liters Toluene. OD -AAqua ol 14 and MTSS were found to be suitable solvent-scintillents for Fc-1843- C during specific activity determination (3). ispecific activity = 0.507 uCi/mg. Specific activity determination has been reported (3). -29APPENDIX TABLE 2 14 Carbon-14 Content of FC-143- C Dosing Solution 10 ul Aliquot uC!/2.0 ml 2.131A 2.06932 2.1588-S 2.0884k 2. 08 38-t 2. 0985-t overall average = 2.1344+0.0495 2.134 0.507 uCi/mg 4.21 mg/2.0 ml 50 ul Aliquot uCi/2.0 ml 2.121A 2.1867' 2.1509@@2.1012-b 2.2108b 2. 211 9-b- -S Sample prepared with Aquasol 22 Sample prepared with MTSS.