Document pBdB7ayjRQaEzXxNXEm3bj5qB

16^ BESTG t* ^ p-6 -i$ RECEIVED f u : M AR 1 9 1930 Enhanced Elimination of\FC-95- - 3M TOXICOLOGY and 14 FC-143- C in Rats with Cholestyramine Treatment March 5, 1980 Conducted at : During: Conducted by: Report b y : Riker Laboratories, Inc. Subsidiary of 3M St. Paul, Minnesota 55101 November, 1979 to January, 1980 S. J. Gibson and J. D. Johnson Reviewed by: 002913 -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 i n c r e a s e ^ the eliminjljion of carbon-14 via feces after iv dosing with ejtjher FC-95or FC-143- C. Groups of five raj| were dosed iv with FC-95- C (mean dose, 3.4 mg/kg) or FC-143- C (mean dose, 13.3 m g / k g ) . Groups of five control rats were dosjg similarly but were not treated with cholestyramine. The FC-95- C rats were sacrificed at 21 days and the FC-143- C rats at 14 days post dose. Hie mean liver, plasma, and red blood cell concentration as well as fecal and urinary excretion of carbon-14 for cholesl^ramine-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 (75.9J^with cholestyramine treatment) was increased 9.5 fold. For FC-143- C, mean cholestyraminetreated 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; fecal elimination (43.2%^^ith cholestyrjrjine 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 cholestyramine-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 Cholestyramine, 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 o n ^ a r b o n - 1 4 elimination and tissue retejtjion after iv dosing with FC-95- C {FC-Experiment 8) and with FC-143- C (FC-Experiment 9). C02314 Methods -3- Radiolabeled FC-143-^4C *-+ C_F _ COO NH 7 15 4 FC-143 Denotes postion of carbon-14 The carbon-14 label is at the carbonyl carboy^atom (see above structure). 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 ter! zation,^|nd radiochemical purity have been reported separately (3); the FC-143- C was found suitable for metabolism studies. 14. Radiolabeled FC-95- F J+ C F c-so, K 7 15 I3 F Denotes postion of carbon-14 The carbon-14 label is at the carbon atom e< to the sulfur a t o m ^ s e e above structure). Ihe specific activity of this lot of FC-95- ^C (Riker Isotope Inventory Number 442) is 0.46 uCi/mg. Details of specific activity, chemical characterization^and radiochemical purity have been reported separately (4); the FC-95- C was found suitable for metabolism studies. Animals 14 FC-95- C (FC-Experiment 8) Ten male Charles River-- CD rats, twelve weeks old, were randomly divided into a control group of five (weight 304 to 331 g, mean 320 g) and a group of five to be treated with cholestyramine (weight 322 to 342 g, mean 330 g ) . For 24 hours prior to dosing, the rats were conditioned to individual metal metabolism cages and fagted with free access to water. Hie rats were allowed free access to Purina-- Ground Chow (with or without added cholestyramine) and water immediately after dosing. a ^Charles River Breeding Laboratories, Wilmington, Massachusetts. -- Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. 002915 4- 14. FC-143- (FC-Experiment 9) I Ten male Charles River^ C D 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. The rats were allowed free access to Purina-- Ground Chow (with or without added cholestyramine) and water immediately after dosing. Dosing 14. FC-95- (FC-Experiment 8) Each rat was weighed, anesthetized lightly with dj|thyl ether, and then given a single iv dose (via tail vein) of FC-95- C^4 The dose was 2.0 ml of a 0.9% NaCl solution containing 1.13 mg FC-95- 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. ^he dose was delivered with a 3.0 cc disposable plastic syringe (Monoject -- ) fitted with a 26 gauge J^2" needle. The dosing solution,was prepared by adding *150 m g of FC-95- C to 0.9% NaCl, shaking for one 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). 14 FC-143- C (FC-Experiment 9) Each rat was weighed, anesthetized lightly with ^ e t h y l ether, and then given a single iv dose (via tail vein) of FC-143 C. jlje 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 control rats was 13.5 mg/J^g. The dose was delivered with a 3.0 cc disposable plastic syringe (Monoject -- ) fitted with a 26 gauge l/^ needle. The dosing solution was prepared by dissolving 225 m g of FC-143- C in 0.9% NaCl. The carbon-14 content of the dosing solution was determined by direct counting (see Appendix 2). a j-Charles River Breeding Laboratories, Wilmington, Massachusetts. -- Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. -- Sherwood Medical Industries, Inc., Deland, Florida 32720. 002916 -5- 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 couj|e of the studies. The actual mean dose of cholestyramine for the FC-95 C treated rats was 2.7+0.2 (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 centri fugation. The rats were sacrificed by exsanguination, and liver was collected as the whole organ. 14 FC-143- C (FC-Experiment 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 centri fugation. 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 homogenizing and ali quoting a sample of the homogenate into combustion cones-- . Homogenizing was done in Waring blenders by adding nine parts of water (w/w) 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. a gMead Johnson Research Center, Evansville, Indiana 47721. -- Packard Instrument Co., Inc., 220 Warrenville Road, Downers Grove, Illinois. C02917 -6- Homogenates and red blood cells were combusted with a Packard Model 306 Oxidizer-- . Recovery of carbon-14 from biological samples was determined by combusting suitable blank homogenates (feces and liver) spiked with dilutions of the appropriate dosing solution at the beginning, middle, and end of the experimental sample sets. All mean recj^eries were >J|%; thus, no correction for recovery was made for either FC-95- c or FC-143- u samples. Good recovery from cyijbustion analysis has previously been reported for FC-95- 4C (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 wgs added. It has previously been^Reported that direct counting in^j|quasol 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 -- . Counting efficiency for each sample was determined by use of the AES (Automatic External Standardization) ratio method. Ib 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 blj^d cell analyses for carbon-14 content after intravenous dosing with FC-95- are shown in Table 1; the results after intravenous dosing with FC-143- C are shown in Table 2. Cholestyramine treatment caused a decrease iy^mean liver, p^|sma, and red blood cell carbonj^4 content for both FC-95- C and FC-143- C dosed rats. For FC-95- 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^concentrations 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. 14 The results of carbon-14 fecal analyses for FC-95- 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 fold^ljhe mean percent of dose eliminated via feces by control rats. For FC-143- C a -- Packard Instrument Co., Inc., 220 Warrenville Road, Dcwners Grove, Illinois. C02918 -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 <jybon-14 concentrations 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. 14 The results of the urine analyses for FC-95- 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 elim^rjated 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 the percent 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 eliminatijij Total elimination of carbon-14 (urine plus fej|s) is shown for FC-95- C dosed rats Figure 5 and for FC-143- C dosed rats in Figure 6. For FC-95- 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 percentages 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, J!j.6 u g / g ) . The total carbon-14 eliminated (urine plus feces) by FC-143- C dosed rats is 84% of the dose for cholestyramine-treated rats and 72% of the dose for control rats; the percentages of dose rjrjaining 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 |)pwn, along with corresponding contrj^ groups, in Table 7 for FC-95- C dosed rats and in Table 8 for FC-143- C dosed rats. Cholestyramine does not appear to have caused a change in weight gain or liver weight in either experiment. 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. 002919 8 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, Boylan JJ, Blanke RV, Fgriss MW, Howell JR, Guzelian PS: Treatment of Chlordecone (Kepone ) Toxicity with Cholestyramine. New Engl J Med 298: 243-278, 1978. (3) Behr FE, Johnson JP: Synthesis and Characterization of FC-143- 14 C (Report) December 28, 1979. 14 (4) Johnson JD, Behr FE: Synthesis and Characterization of FC-95- C (Report) November 2, 1979. (5) Johnson JD: Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Rats after a Single IV Dose of FC-95- C (Report) December 28, 1979. (6) Johnson JD: Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Male and Female Rats after a Single IV Dose of FC-143- C (Report) January 30, 1979. C02920 -9' Acknowledgement Ohe preparation of the 4% w/w cholestyramine in Purina Ground Chow by L. J. Sibinski of Pathology-Toxicology is gratefully aknowledged. 092921 -10- List of Tables and Figures Table 1 liver, Plasma, and Red Blood Cell Concentration of Carbon-14 in Rats at 21 Days a|er a Single IV Dose of FC-95- 4C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 25-27. Table 2: Liver, Plasma, and Red Blood Cell Concentration of Carbon-14 in Rats at 14 Days afj|r a Single IV Dose of FC-143- X (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 38,39. Table 3: Excretion of Total Carbon-J| in Feces in Rats a f t e ^ a Single Intravenous Dose of FC-95- C (Mean Dose of FC-95- X : Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) NB 53102 P 20,21. Tabie 4 : Excretion of Total Carbon-14 in Feces in Rats after a Single Intravenous Dose of FC-143- X (Mean Dose of FC-143- X: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 30,32. Table 5 : Excretion of Total Carbon-J^ in Urine in Rats a f t e ^ a Single 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-l|4in Urine in Rats after j^Single 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 Liver Weights Sacrifice for Rats a f t e r S i n g l e 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 26 Table 8: Body Weights at Dosing and Body and Liver Weights a ^ S a c r i f i c e for Rats after j^Single Intravenous Dose of FC-143- C (Mean Dose of FC-143- X : Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg) NB 53102 P 38. Figure 1 Cumulative Excretion of Total Car^n-14 in Feces after a Single^Jntravenous 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. 002922 -11- List of Tables and Figures (continued) Flgure 2: Cumulative Excretion of Total Carbjg-14 in Feces after a Single Jgtravenous Dose of FC-143- (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 3: Cumulative Excretion of Total Car^n-14 in Urine after a Single^ntravenous 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 4: Cumulative Excretion of Total Carb^ij-14 in Urine after a Single Jgtravenous 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. Fi gure 5 : Cumulative Excretion (Urine + Feces) of Tj^al Carbon-14 after a S^rjgle 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. Fi gure 6 : Cumulative Excretion (Urine + Feces) of ToJ|l Carbon-14 after a Single 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. 14 Appendix 1 ; Determination of Carbon-14 Content of FC-95- C Dosing Solution NB 51807 P 41,44. Appendix Table 1 14, ; Carbon-14 Content of FC-95- Dosing Solution NB 51807 P 44. Appendix 2 14 Determination of Carbon-14 Content of FC-143- C Dosing Solution NB 51807 P 41-45. Appendi x Table 2 14 Carbon-14 Content of FC-143- C Dosing Solution NB 51807 P 45. C02923 -12- Table 1 Liver, Plasma, and Red Blood Cell Concentration-- of. Carbon-14 in Rats at 21 Days a|tjer a Single IV Dose of FC-95- 4C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg Liver Plasma Red Blood Cells Cholestyrami ne-Treated Rat Number Mean + SD 1 234 5 9.8 11.5 9.7 7.1 8.8 9.4 + 1.6 0.9 1.1 0.8 0.7 0.9 0.9 + 0.1 0.2 0.3 0.3 0.2 0.3 0.3 + 0.1 Liver Plasma Red Blood Cells 1 30.8 7.0 2.1 Control Rat Number 234 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 1.8 + 0.4 a -- Total carbon-14 concentration for liver a n ^ r e d blood cells is expressed as ug equivalents of FC-95- C/g; plj|ma carbon-14 concentration is expressed as ug FC-95- C equivalents/ml. 002924 -13- Table 2 Liver, Plasma, and Red Blood Cell Concentration-- of Jarbon-14 in Rats at 21 Days afi^r a Single IV Dose of FC-143- c (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 mg/kg Liver Plasma Red Blood Cells 1 14.6 7.1 2.7 Choiestyrami ne-Treated Rat Number Mean + SD 2345 9.4 13.5 11.9 10.9 12.1 + 2.1 3.0 6.1 5.5 3.9 5.1 + 1.7 1.0 2.4 2.0 1.2 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 34 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 a -- Total carbon-14 concentration for liver a ^ red blood cells is expressed as ug equivalents of FC-143- C/g; plj|ma carbon-14 concentration is expressed as ug FC-143- C equivalents/ml. C02925 -14- Table 3 Excretion of Total C ^ b o n - 1 4 in Feces-- in Rats after a Single Intravenous Dose of F C - 9 5 - ^ C (Mean Dose 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 18-21 Total Cholestyrami ne-Treated_____________________ Rat Number Mean+SD 1234 5 0.08 2.90 1.97 2.07 0.0^ 1.40+1.30 1.49 7.94 11.30 7.58 3.97 6.46+3.80 6.75 14.27 15.63 17.59 16.47 14.14+4.31 15.34 11.03 12.68 12.03 14.19 13.05+1.72 11.85 7.24 8.42 9.96 8.71 9.24+1.75 8.54 5.67 5.77 6.02 8.20 6.84+1.41 5.26 3.81 4.42 4.61 5.12 4.64+0.58 4.54 2.69 3.28 3.67 5.13 3.86+0.98 5.07 4.48 4.17 4.80 5.30 4.76+0.45 3.65 3.38 3.37 3.96 3.90 3.65+0.28 4.36 4.57 4.08 4.76 5.27 4.61+0.45 2.48 2.50 2.40 2.63 2.54 2.51+0.08 0.48 0.58 0.64 0.77 0.98 0.69+0.19 69.89 71.06 78.13 80 .45 79.78 75.86+5.01 __________ ______ Control_________________________ Rat Number Mean+SD 12 345 0.35 0.60 0.40 0.52 0.0-- 0.37+0.23 0.64 0.59 0.46 0.86 0.72 0.53 0.58 0.70 0.54 0.92 0.59+0.10 0.72+0.17 0.57 0.41 1.11 0.55 0.50 0.48 0.65 0.45 0.62 0.51 0.69+0.24 0.48+0.05 0.40 0.35 0.35 0.37 0.39 0.43 0.45 0.52 0.29 0.36 0.38+0.06 0.41+0.07 0.32 0.40 0.31 0.36 0.44 0.37+0.05 0.58 0.69 0.69 0.77 1.01 0.75+0.16 0.56 0.64 0.69 0.80 0.75 0.69+0.09 1.01 1.25 0.95 1.17 1.18 1.11+0.13 0.64 0.54 0.65 0.91 1.00 0.75+0.20 0.48 0.58 0.64 0.77 0.98 0.69+0.19 6.90 8.40 7.38 8.65 8.60 7.99+0.80 a -- Data cure expressed as percent of dose excreted during collection period, -- No feces excreted. 002926 -15Table 4 Excretion of Total Ca^ljon-14 in Feces-- in Rats after a Single Intravenous Dose of FC-143 - ^ C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kgj Control, 13.5 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 11-14 Total _______ Cholestyrami ne-Treated_____________________ Rat Number Mean+SD 1234 5 0.04 0.13 0.01 3.15 1.78 1.02+1.40 5.06 3.72 1.73 5.68 4.63 4.16+1.54 5.50 7.21 7.20 8.11 7.81 7.17+1.01 6.85 7.21 7.74 7.52 5.50 6.96+0.88 4.96 4.45 5.91 5.15 3.84 4.86+0.78 4.54 2.79 5.14 3.57 3.22 ... 3.85+0.97 3.58 2.34 4.09 3.35 2.41 3.15+0.76 2.54 2.04 3.26 2.56 1.98 2.48+0.52 4.87 2.73 5.04 3.74 2.91 3.86+1.07 3.66 1.83 3.87 2.61 2.21 2.84+0.89 4.01 1.94 3.61 2.82 2.04 2.88+0.92 45.61 36.39 47.60 48.26 38.33 43.23+5.50 _________________ Control_________________________ Rat Number Mean+SD 12 34 5 0.0^ 0.09 0.21 0.37 0.07 0.15+0.15 0.44 0.54 0.44 0.33 0.0-- 0.35+0.21 0.32 0.51 0.69 0.74 0.71 0.59+0.18 0.34 0.54 0.52 0.44 0.62 0.49+0.11 0.28 0.33 0.51 0.75 0.48 0.47+0.18 0.31 0.33 0.42 0.22 0.26 0.31+0.08 0.21 0.54 0.65 0.20 0.25 0.37+0.21 0.16 0.25 0.22 0.25 0.32 0.24+0.06 0.33 0.97 0.69 0.40 0.73 0.62+0.26 0.41 0.48 0.54 0.35 0.38 0.43+0.08 0.27 0.59 0.61 0.27 0.33 0.41+0.17 3.07 5.17 5.50 4.32 4.15 4.43+0.95 002927 -- Data are expressed as percent of dose excreted durinq collection period. No feces excreted. -16- Table 5 Excretion of Total C ^ b o n - 1 4 in Urine-- in Rats after a Single Intravenous Dose of FC-95-^^C (Mean Dose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kgj 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 18-21 Total ______ Cholestyrami ne-Treated_____________________ Rat Number Mean+SD 12 34 5 1.58 1.53 1.75 1.91 1.53 1.66+0.17 0.49 0.48 0.50 0.30 0.26 0.41+0.12 0.69 0.61 0.58 0.83 0.61 0.66+0.10 0.67 0.61 0.52 0.50 0.62 0.58+0.07 0.59 0.33 0.39 0.48 0.42 0.44+0.10 0.26 0.20 0.22 0.23 0.32 0.25+0.05 0.23 0.16 0.19 0.21 0.25 0.21+0.03 0.22 0.13 0.11 0.15 0.16 0.15+0.04 0.25 0.19 0.18 0.21 0.24 0.21+0.03 0.20 0.17 0.18 0.21 0.19 0.19+0.02 0.28 0.23 0.25 0.26 0.35 0.27+0.05 0.22 0.17 0.23 0.22 0.22 0.21+0.02 0.15 0.11 0.14 0.14 0.12 0.13+0.02 5.83 4.92 5.24 5.65 5.29 5.37+0.36 _________ Control Rat Number 12 34 5 1.98 1.61 1.83 1.02 1.60 1.06 1.00 0.90 0.87 0.92 1.14 1.30 0.94 1.07 1.41 1.45 1.31 1.11 1.19 1.55 1.13 1.17 0.97 1.09 1.30 0.84 0.87 0.88 0.89 0.88 0.96 0.92 0.75 0.91 1.23 0.76 0.68 0.71 0.72 1.08 1.55 1.62 1.26 1.40 1.94 1.51 1.41 1.30 0.97 1.56 2.62 2.51 2.18 2.25 2.69 1.79 1.76 1.63 1.62 2.21 1.72 1.59 1.50 1.34 2.04 18.51 17.75 15.96 15.34 20.41 Mean+SD 1.61+0.37 0.95+0.08 1.17+0.19 1.32+0.18 1.13+0.12 0.87+0.02 0.95+0.17 0.79+0.16 1.55+0.26 1.35+0.23 2.45+0.23 1.80+0.24 1.64+0.26 17.59+2.03 -- Data are expressed as percent of dose excreted during collection period. 002928 17Table 6 Excretion of Total Ca^^on-14 in Urine-- in Rats after a Single Intravenous Dose of FC-143 -*^C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kg; Control, 13.5 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 11-14 Total _______ Cholestyrami ne-Treated_____________________ ___ __ Rat Number ___ Mean+SD 1234 5 4.72 8.60 5.21 8.79 12.17 7.90+3.04 2.46 5.11 2.23 2.97 5.00 3.55+1.40 4.15 9.05 4.09 5.00 8.09 6.08+2.33 3.50 8.68 3.95 4.73 5.75 5.32+2.06 2.87 5.74 4.19 3.18 4.18 4.03+1.12 2.27 3.34 2.65 2.57 2.90 2.75+0.40 2.44 2.99 2.67 1.88 2.36 2.47+0.41 1.87 2.15 2.11 1.41 1.66 1.84+0.31 3.55 3.22 3.48 2.21 3.23 3.14+0.54 2.38 2.19 2.05 1.54 1.98 2.03+0.31 2.69 1.98 1.85 1.48 1.78 1.96+0.45 32.90 53.05 34.48 35.76 49.10 41.07+9.31 _________________ Control________________________ ________ Rat Number Mean+SD 12 3 45 3.85 5.06 4.11 15.09 4.37 6.50+4.83 3.48 4.15 7.88 5.49 5.07 5.21+1.68 6.07 7.21 8.72 11.64 10.66 8.86+2.32 6.35 5.54 6.68 9.00 9.60 7.43+1.77 6.10 4.76 6.67 7.14 9.48 6.83+1.73 5.22 4.20 6.32 4.88 7.35 5.59+1.25 4.83 3.69 5.08 4.45 5.37 4.68+0.65 4.34 3.40 4.14 3.99 5.58 4.29+0.80 7.21 6.37 6.37 6.24 6.69 6.58+0.39 6.09 5.55 5.62 5.24 5.37 5.57+0.32 6.95 6.98 5.57 4.48 5.21 5.84+1.10 60 .49 56.91 67.16 77.64 74.75 67.38+8.90 002929 -- Data are expressed as percent of dose excreted during collection period. -18- Table 7 Body Weights a ^ D o s i n g and Body and Liy^r at Sacrifice for Rats after a Single Intravenous Dose of FC-95- C (Mean nose of FC-95- C: Cholestyramine-Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Cholestyrami ne-Treated Rat Number 12345 Body Weight at Dosing (g) 324 329 322 342 331 Body Wei ght at Sacrifice (g) 400 422 403 410 402 Percent Change in Body Weight 23.5 28.3 25.2 19.9 21.5 Liver Wei ght (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 MeantSD 330+8 407+9 23.7+3.3 13.6+1.2 3.3+0.3 12 Control Rat Number 34 5 Mean+SD 316 304 331 321 328 320+11 393 409 394 440 388 405+21 24.4 34.5 19.0 37.1 18.3 26.7+8.7 14.6 13.3 11.7 14.9 10.8 13.1+1.8 3.7 3.3 3.0 3.4 2.8 3.2+0.4 002930 19- Table 8 Body Weights at^^losing and Body and Livej^Weights at Sacrifice for Rats after a Single Intravenous Dose of FC-143- C (Mean Dose of FC-143- C: Cholestyramine-Treated, 13.3 mg/kgj Control, 13.5 mg/kg) Cholestyrami ne-Treated Rat Number 12 345 Body Wei ght 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 Liver Wei ght (at Sacrifice) (g) 17.0 14.1 11.2 11.8 14.9 Percent Liver Weight of Body Weight (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 12 Control Rat Number 34 5 Mean+SD 341 312 301 300 310 313+17 384 356 383 358 381 372+14 12.6 14.1 27.2 19.3 22.9 19.2+6.1 15.5 14.7 15.0 10.6 14.9 14.1+2.0 4.0 4.1 3.9 3.0 3.9 3.8+0.4 C02931 Figure 1 Cumulative Excretion of To||l Carbon-14 in Feces after a Single Intravenous Dose of FC-95- c (Mean Dose of FC-95- 4C: Cholestyramine Treated, 3.4 mg/kg; Control, 3.5 mg/kg) Mean of 5 Rats 75.85.0 Figure 2 Cumulative Excretion of Total Carbon-14 in Feces after a SingJi Intravenous Dose of FC-143- 4C (Mean Dose of FC-143- C: Cholestyramine Treated, 13.3 mg/kg: Control, 13.5 mg/kg) Mean of 5 Rats 100 75 50 43.2+5.5 25 0 002933 Cumulatibe Percent of Dose in Urine 17.6+2.0 Figure 4 Cumulative Excretion of Total Carbon-l^in Urine after 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 Cumulative Percent of Dose in Urine Figure 5 Figure 6 Cumulative Excretion (Urine + Feces) of Total Carbon-14 After a Sinjle Intravenous Dose of FC-143- C (Mean Dose of FC-143- u: Cholestyramine Treated, 13,3 m g A g ; Control, 13.5 m g A g ) Mean of 5 Rats 100 84.3 4.3 Cholestyramine Treated 75 50 25 0 Days Post Dose -26APPENDIX 1 14 Determination of Carbon-14 Content of FC-95- C Dosing Solution Just prior to dosing of rats, the FC-95- C dosing solution was sampled with calibrated micropipettors-- directly into counting vials. Six 10 ul and six ^0 ul aliquots were pipetted, and 1 ml of water and 15 ml of Aquasol-- were added to three of each of the 10 ul and 50 ul aliquots. The remaining three vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSS-- '-- . Corrections were made for background and counting efficiency and, using the specific activity already determined-- , the uCi/2.0 ml was calculated. The data are shown in Appendix Table 1. Hie dos^adm inistered each rat is 2.0 m ^ o f solution containing 0.519 uCi of FC-95- C which is 1.13 mg of FC-95- C. This is a mean dose of 3.4 mg/kg for the cholesty ramine-treated rats and 3.5 mg/kg for the control rats. -- h / I Micropipettor, Lab Industries, Berkeley, California. -- New England Nuclear, Boston, Massachusetts. -Modified TSS: 25.2 g PPO, 1.01 g DimethylPOPOP 2nd 3.8 Liters Toluene. -Aquaso|~ and MTSS were found to be suitable solvent-scintillents for FC-95- C during specific activity determination (4). -Specific activity = 0.459 uCi/mg. Specific activity determination has been reported (4). 002938 -27- APPENDIX TABLE Carbon-14 Content of FC-95- Dosing Solution 10 ul Aliquot uCi/2.0 ml 0.50840.5012^ 0.52840.5162^ 0.50640.5098^ 50 ul Aliquot uCi/2.0 ml 0.51110.53750.52990.5299-- 0.53830.5155- Overall average = 0.5194+0.0128 0.519 = 1.133 mg/2.0 ml 0.459 uCi/mg -- Sample prepared with Aquasol' -- Sample prepared with MTSS. C02939 28 APPENDIX 2 14 Determination of Carbon-14 Content of FC-143- C Dosing Soluti on 14 JUst prior to dosing of rats, the FC-143- C dosing solution was sampled with calibrated micropipettors-- directly into counting vials. Six 10 ul and six 50 ul aliquots were pipetted, and 1 ml of water and 15 ml of Aquasol --b were added to three of each of the 10 ul and 50 ul aliquots. Thg remaining vials were prepared with 2.5 ml of methanol and 7.5 ml of MTSS-- . Corrections were made for background and counting efficiency and, using the specific activity already determined-- , the uCi/2.0 ml was calculated. The data are shown in Appendix Table 2. The dose j^ministered each rat is 2.0 ml of -solution 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. -- I/I Micropipettor, Lab Industries, Berkeley, California. -- New England Nuclear, Boston, Massachusetts. -- Modified TSS: 25.2 g PPO, 1.01 g DimethylPOPOP 2nd 3.8 Liters Toluene. d -- Aquasol 4*nd MTSS were found to be suitable solvent-scintillents for FC-143- C during specific activity determination (3). -- Specific activity = 0.507 uCi/mg has been reported (3) . Specific activity determination 002940 / -29- APPENDIX TABLE 2 14, Carbon-14 Content of FC-143- Dosing Solution 10 ul Aliquot 50 ul Aliquot uCi/2.0 ml____________________uCi/2.0 ml 2.1312-- 2.06932.15882.08842.08382.0985-- 2.1212-- 2.18672.1509-- 2 .1012^ 2.21082.2119-- Overall average = 2.1344+0.0495 2.134 = 4.21 mg/2.0 ml 0.507 uCi/mg a -- Sample prepared with Aquasol . b -- Sample prepared with MTSS. C02941