Patent Publication Number: US-5250283-A

Title: Organic contrast agent analog and method of making same

Description:
This is a continuation of U.S. patent application Ser. No. 07/500,471, filed Mar. 28, 1990, now U.S. Pat. No. 5,143,715. 
    
    
     TECHNICAL FIELD 
     The present invention relates to organic contrast agent analogs and methods of making the analogs. More particularly, the present invention relates to a method of decreasing the toxicity of such agents through chemical modification. 
     BACKGROUND ART 
     In general, current cholangiographic agents used as biliary X-ray contrast agents are now considered too toxic to meet risk/benefit criteria for routine use as contrast agents to visualize the biliary system. Because of the relative toxicity of these agents, these agents are being replaced in use by alternative imaging methodologies, such as ultrasound and magnetic resonance imaging (MRI). On the other hand, the resolution achievable with X-ray radiology techniques is superb. Accordingly, biliary contrast agents can still be quite useful. 
     A second issue with regard to these agents is the low rate of choleresis associated with biliary excretion of the agent. The low rate of choleresis increases the biliary concentration and thus increases the toxicity. 
     Current biliary X-ray contrast agents have amphophilic properties. These agents have a hydrophilic head portion; that is, the --CO 2  H terminus, and a hydrophobic tail; that is, triiodo aniline moiety with an unsubstituted 5-position. Because of this property, they are excreted nearly exclusively by the biliary route. Typical of most amphilphilic molecules, these biliary contrast agents tend to self associate. This property is reflected by the low critical micellar concentration and low osmolalities of their aqueous salt solution. 
     It is therefore desirable to produce a less toxic and more choleretic contrast agent. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, there is provided an organic contrast agent analog wherein the analog is an oral or intravenous media having the formula I: ##STR1## 
     
         __________________________________________________________________________
          R.sub.1          R.sub.2                                        
__________________________________________________________________________
Iopanoic acid                                                             
           ##STR2##        NH.sub.2                                       
Ipodatic acid                                                             
          CH.sub.2 CH.sub.2 COOH                                          
                           NCHN(CH.sub.3).sub.2                           
Tyropanoic acid                                                           
           ##STR3##        NHCOCH.sub.2 CH.sub.2 CH.sub.3                 
Iocetamic acid                                                            
           ##STR4##        NH.sub.2                                       
Iopronic acid                                                             
           ##STR5##        NHCOCH.sub.3                                   
Iosumetic acid                                                            
           ##STR6##        NHCH.sub.3                                     
__________________________________________________________________________
 
    
     or wherein said analog is an intravenous media having formula II; ##STR7## and R 3  is 
     
         ______________________________________                                    
          R.sub.3                                                         
______________________________________                                    
Iodipamide  NHCO(CH.sub.2).sub.4 CONH                                     
Ioglycamide NHCOCH.sub.2 OCH.sub.2 CONH                                   
Iodoxamate  NHCO(CH.sub.2 CH.sub.2 O).sub.4 CH.sub.2 CH.sub.2 CONH        
Iotroxamide NHCO(CH.sub.2 OCH.sub.2).sub.3 CONH                           
Iosulamide* NHCOCH.sub.2 CH.sub.2 SO.sub.2 CH.sub.2 CH.sub.2 CONH         
______________________________________                                    
 ##STR8##                                                                 
-  or wherein said analog is an urographic media having formula III;      
 ##STR9##
 wherein the analog includes one or more fluorine substitutions or
 additions.
 
    
     The present invention further provides a method of making the organic contrast agent analog including the steps of forming the organic contrast agent analog having formulas I, II, or III and substituting or adding at least one flourine moiety to the analog. 
    
    
     FIGURES IN THE DRAWINGS 
     Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: 
     FIGS. 1 and 2 are high resolution NMR proton spectrums; 
     FIG. 3 is a high resolution NMR fluorine spectrum; 
     FIGS. 4 and 5 are mass spectrum analyses performed on TFA-IOP made in accordance with the present invention; 
     FIG. 6 is a plotted curve comparing biliary concentration over time of IOP versus TFA-IOP; 
     FIG. 7 is comparative data of TFA-IOP and IOP plasma concentration versus time; and 
     FIG. 8 is a comparative date of TFA-IOP and IOP biliary excretion concentration versus time. 
    
    
     DESCRIPTION OF THE INVENTION 
     Generally, an organic contrast agent analog made in accordance with the present invention has the following general formula I: ##STR10## and R 1  and R 2  are 
     
         __________________________________________________________________________
          R.sub.1          R.sub.2                                        
__________________________________________________________________________
Iopanoic acid                                                             
           ##STR11##       NH.sub.2                                       
Ipodatic acid                                                             
          CH.sub.2 CH.sub.2 COOH                                          
                           NCHN(CH.sub.3).sub.2                           
Tyropanoic acid                                                           
           ##STR12##       NHCOCH.sub.2 CH.sub.2 CH.sub.3                 
Iocetamic acid                                                            
           ##STR13##       NH.sub.2                                       
Iopronic acid                                                             
           ##STR14##       NHCOCH.sub.3                                   
Iosumetic acid                                                            
           ##STR15##       NHCH.sub.3                                     
__________________________________________________________________________
 
    
     or wherein said analog is an intravenous media having the formula II; ##STR16## and R 3  is 
     
         ______________________________________                                    
          R.sub.3                                                         
______________________________________                                    
Iodipamide  NHCO(CH.sub.2).sub.4 CONH                                     
Ioglycamide NHCOCH.sub.2 OCH.sub.2 CONH                                   
Iodoxamate  NHCO(CH.sub.2 CH.sub.2 O).sub.4 CH.sub.2 CH.sub.2 CONH        
Iotroxamide NHCO(CH.sub.2 OCH.sub.2).sub.3 CONH                           
Iosulamide* NHCOCH.sub.2 CH.sub.2 SO.sub.2 CH.sub.2 CH.sub.2 CONH         
______________________________________                                    
 ##STR17##                                                                
-  or wherein said analog is an urographic media having the formula III;  
 ##STR18##
 
    
     The agent includes a hydrophilic head (side chain) and a hydrophilic tail (benzine ring). The fluorine addition or substitution provides means for increasing the acidity of the agent and increasing the choleretic activity of the agent to render the agent less toxic. 
     Examples of oral cholecystographic contrast media which can be modified in accordance with the present invention are 
     
                                           TABLE 1                                 
__________________________________________________________________________
          proprietary     Molecular weight(m.w.)                          
                                      Iodine                              
Common name                                                               
          name     Manufacturer                                           
                          Acid form                                       
                                Salt form                                 
                                      (%/m.w.)                            
__________________________________________________________________________
Iopanoic acid                                                             
          Telepaque                                                       
                   Winthrop                                               
                          571         66.7                                
Ipodate.calcium                                                           
          Oragratin.calcium                                               
                   Squibb 598   1.234 63.7                                
Ipodate.sodium                                                            
          Oragratin.sodium                                                
                   Squibb 598   620   63.7                                
Tyropanoate.sodium                                                        
          Bilopaque                                                       
                   Winthrop                                               
                          641   663   59.4                                
Iocetamic acid                                                            
          Cholebrine                                                      
                   Mallinckrodt                                           
                          614         62.0                                
Iopronic acid                                                             
          Oravue   Squibb 673         56.6                                
Iosumetic acid     Schering                                               
                          628         60.6                                
__________________________________________________________________________
 
    
     Examples of intravenous cholangiographic media modifiable in accordance with the present invention are 
     
                                           TABLE 2                                 
__________________________________________________________________________
N-Methylglucamine salt                                                    
            Proprietary   Molecular weight(m.w.)                          
                                      Iodine                              
or contrast media                                                         
            name   Manufacturer                                           
                          Acid form                                       
                                Salt form                                 
                                      (%/m.w.)                            
__________________________________________________________________________
Iodipamide  Chologratin                                                   
                   Squibb 1.140 1.530 66.8                                
Ioglycamide Biligram                                                      
                   Schering                                               
                          1.128 1.518 67.5                                
Iodoxamate  Cholevue                                                      
                   Winthrop                                               
                          1.288 1.678 59.1                                
Iotroxamide        Schering                                               
                          1.316 1.706 57.9                                
Iosulamide         Winthrop                                               
                          1.376 1.764 55.3                                
__________________________________________________________________________
 
    
     More specifically, the starting agent can be a cholangiographic or cholecystographic agent as stated above. The agent can be of the oral or intravenous types. That is, the agent is iodinated such that it can be used as a biliary X-ray contrast agent for routine use to visualize hepatocytes gallbalder and the the biliary system. Unlike previously used cholecystographic agents, such as underivatized iopanoic acid, the present invention is a chemically modified organic contrast agent analog which is rendered less toxic and more choleretic by the modification of the agent including additional fluorine functionalities on the agent, the fluorine functionalities rendering the agent more acidic and more choleretic. 
     It has been hypothesized that if an amphophilic molecule, such as a common organic contrast agent analog is rendered a stronger acid and is fully or at least more disassociated at physiological pH, it will most likely be excreted unconjugated, without causing choloresis and without being trapped in the intrahepatic shunt pathway (Sawkat, MS. et al; Influence of side-chain charge on hepatic transport of bile acids and bile acid analogues. American Journal Physiology 249:G479-G488, 1985; Gurantz D., et al; Influence of bile acid structure on bile flow and biliary lipid secretion in the hamster. American Journal Physiology 247:G738-G748, 1984; Yoon, YB, et al, Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary sectetion of 23-nor-ursodeoxycholate in rodents. Gastroenterology 90:837-852, 1986). Even in the presence of choleresis, if the hepatocyte and canalicular transport steps are rapid, the biliary contrast agent is expected to move as a bolus, and anatomical details, such as the intrahepatic ducts, can be visualized in patients with good hepatocyte function. 
     For example, the agent can be N-trifluoroacetyl-iopanoic acid or alpha-fluoro-N-trifluoroacidic-iopanoic acid. 
     The present invention further provides a method of making the organic contrast agent analog. The method generally includes the steps of forming the organic contrast agent analog having the formulas I, II, or III and then increasing the acidity of the agent while increasing the choleretic activity of agent to render the agent less toxic by adding or subtituting flourine on the agent. 
     EXPERIMENTS 
     1. Synthesis of N-trifluoro Derivative 
     of Iopanoic Acid 
     Three fluorine atoms were attached to the cholecystographic agent iopanoic acid (IOP) so that it could be detected using 19-F magnetic resonance techniques. 
     IOP was reacted with trifluoroacetic anhydride at 0° C. in tetrahydrofuran. After the reaction was complete, the organic solvents were removed. The residue was dissolved in ethyl acetate and washed twice with water. The organic layer was dried over magnesium sulphate and the solvent was removed to obtain the final product. The product, N-trifluorcacetyl iopanoic acid, had a melting point of 193° C. and an extension coefficient of approximately 37,000 at 232 nm. The assigned structure was verified using high resolution proton and fluorine NMR spectroscopy, elemental analysis and mass spectrum analysis. The isolated yield of the product was 97.5%. 
     The following summarizes the reaction. ##STR19## 2. Synthesis of Alpha-fluoro-N-trifluoroacetyl-Iopanoic Acid 
     Functional groups were placed on the carbon atom alpha to the carboxyl group of the organic contrast agent, iopanoic acid. A fluorine atom was placed on the carbon atom alpha to the carboxyl group of N-trifluoroacetyl-iopanoic acid to significantly effect its pKa and thus, effect its biodistribution and metabolism. The following summarizes the synthesis of the alpha-fluoro-N-trifluoroacetyliopanoic acid. ##STR20## 
     Results of elemental analysis of TFA-IOP provided by Galbraith Laboratories, Inc., Knoxville, Tenn. are as follows: 
     
         ______________________________________                                    
Galbraith        Theoretical                                              
                           Error                                          
______________________________________                                    
C      24.33%        24.03%    +0.30%                                     
H      1.93%         2.15%     -0.22%                                     
N      2.04%         2.00%     +0.04%                                     
F      8.50%         8.15%     -0.35%                                     
I      55.24%        54.51%    +0.73%                                     
O                    9.16%                                                
                     100.0%                                               
______________________________________                                    
 
    
     The calculations are based on the inclusion of a molecule of methanol with each molecule of TFA-IOP. 
     High resolution NMR proton spectrums (FIGS. 1 and 2), high-resolution NMR fluorine spectrum (FIG. 3) and mass spectrum analyses (FIGS. 4 and 5) were performed on TFA-IOP. The results of each confirm the expected chemical structure of the new compound. Mass spectral analysis was performed. 
     3. Comparative Data of Biliary Excretion of IOP and Trifluoroacetyl IOP 
     Experiments were conducted to compare the biodistribution and excretion of TFA-IOP to that of the parent compound IOP. TFA-IOP and IOP were each administered intravenously to rats (100 umol/kg) in order to compare the biodistribution and excretion of the compound made in accordance with the present invention and the parent compound. Tissues examined included liver, spleen, upper GI, lower GI, kidney, lung and heart. The length of each study was 90  minutes, during which time bile, urine and plasma samples were taken to monitor uptake and excretion. NMR relaxation rates for liver and plasma were also examined to note whether uptake of either compound might possible alter these parameters. 
     4. Results 
     In general, liver concentrations 90 minutes post injection were 512 uM and 202 uM after IOP and TFA-IOP injection, respectively, while kidney concentrations were 105 uM and 5 uM. Lesser concentrations were found in the other tissues. Excretion of the two compounds were found only in bile with no compound detected in urine. In bile, TFA-IOP reached a maximum concentration of 0.5 uM with the recovery of 40% of the injection dose during a 90 minute time period (see FIG. 6). For IOP, the biliary concentration reached 10 mM with 18% of the injected does recovered during the 90 minute collection period as shown in FIG. 6. Plasma disappearance curves were similar in form, as shown in FIG. 7. Thin layer chromatoqraphy of bile indicated that a vast majority of the injected TFA-IOP was metabolized to a single metabolite. No nonmetabolized compound was found. 
     These results indicate that TFA and IOP are similar to their biodisposition except the rate of biliary excretion of TFA-IOP appears to be approximately twice that of IOP, as shown in FIG. 8. 
     Tables 1 through 5 summarizes the results of each experiment conducted. Table 6 shows a more detailed analysis of the tissue concentration results with values such as total micromoles in tissue, percent of dose/gm, etc. 
     Tables 7 and 8 contain analysis of the bile excretion results. 
     
                       TABLE 1                                                     
______________________________________                                    
TISSUES:                                                                  
         -IOP CONCENTRATION (uM)                                          
         90 MIN POST INJECTION                                            
         I-6   % OF      I-8       % OF                                   
         (IOP) DOSE      (TFA-IOP) DOSE                                   
______________________________________                                    
LIVER      511.90  14.53     201.73  6.04                                 
KIDNEY     105.40  0.69      57.84   0.34                                 
*UPPER GI  307.81  1.31      140.23  0.41                                 
*LOWER GI  135.23  0.59      178.09  0.63                                 
*SPLEEN    255.54  0.45      164.06  0.24                                 
*LUNG      324.91  1.21      277.64  0.83                                 
*HEART     259.38  0.72      242.85  0.72                                 
______________________________________                                    
 No standard curves were compiled for these tissues. The values given     
 represent concentrations generated using the LIVER analysis routine and  
 standard curve. Because this routine utilizes a control LIVER spectra for
 background modeling, erroneous Iodine concentration values for these     
 tissues may result due to inappropriate background removal.              
 
    
     
                       TABLE 2                                                     
______________________________________                                    
PLASMA:                                                                   
          -IOP CONCENTRATION (uM)                                         
SAMPLING    I-6         I-8                                               
TIME (min)  (IOP)       (TFA-IOP)                                         
______________________________________                                    
 0          ****         0.00                                             
10          ****        863.19                                            
20          ****        603.93                                            
30          556.68      573.38                                            
40          ****        510.22                                            
50          507.76      469.19                                            
60          462.07      414.69                                            
70          462.46      373.79                                            
80          403.95      403.05                                            
90          372.89      359.94                                            
______________________________________                                    
 ****Indicates that no samples were obtained for these time periods.      
 
    
     
                       TABLE 3                                                     
______________________________________                                    
BILE:                                                                     
          -IOP CONCENTRATION (uM)                                         
SAMPLING    I-6         I-8                                               
TIME (min)  (IOP)       (TFA-IOP)                                         
______________________________________                                    
 0          ****         635.0                                            
 5          1605.8      3010.0                                            
15          8659.4      5777.6                                            
25          10504.9     6436.1                                            
35          9854.3      7802.9                                            
45          9667.6      7641.1                                            
55          7316.4      8461.4                                            
65          7529.1      5372.5                                            
75          7758.8      5305.7                                            
85          9564.6      4663.1                                            
______________________________________                                    
 ****Indicates that no sample was obtained for this time period.          
 
    
     
                       TABLE 4                                                     
______________________________________                                    
URINE:                                                                    
          -IOP CONCENTRATION (uM)                                         
SAMPLING    I-6         I-8                                               
TIME (min)  (IOP)       (TFA-IOP)                                         
______________________________________                                    
 0-20       0           0                                                 
20-40       0           0                                                 
40-60       0           ***                                               
60-80       0           ***                                               
80-90       0           ***                                               
______________________________________                                    
 ***Indicates that no samples were obtained for these time periods.       
 
    
     
                       TABLE 5                                                     
______________________________________                                    
NMR DATA:                                                                 
         CONTROL       I-6      I-8                                       
TISSUE   VALUE         (IOP)    (TFA-IOP)                                 
______________________________________                                    
       T1 VALUES (msec)                                                   
Liver    214.96        228.26   243.07                                    
Plasma   1332.65       1406.19  1455.34                                   
       T2 VALUES (msec)                                                   
Liver     38.27         28.65    28.94                                    
Plasma   232.18        257.87   221.56                                    
______________________________________                                    
 As expected, the values obtained do not significantly differ from control
 values                                                                   
 
    
     
                                           TABLE 6                                 
__________________________________________________________________________
                 uMOLES           PERCENTAGE OF DOSE                      
       TISSUE                                                             
             % OF      PER              PER                               
       WEIGHT                                                             
             B. WT.                                                       
                 IN TISS.                                                 
                       gram kg    IN TISS.                                
                                        gram kg b.wt.                     
__________________________________________________________________________
INTRAVENOUS IOP RAT STUDY                                                 
RAT NUMBER: 6                                                             
DOSE: 104. umoles/kg                                                      
TIME: 90. minutes                                                         
BODY WEIGHT: 406. grams                                                   
LIVER  11.98 2.95                                                         
                 6.13  .51  511.90                                        
                                  14.52 1.21 35.77                        
SPLEEN .75   .18 .19   .26  255.54                                        
                                  .45   .61  1.12                         
UPPER GI                                                                  
       1.79  .44 .55   .31  307.91                                        
                                  1.31  .73  3.22                         
LOWER GI                                                                  
       1.83  .45 .25   .14  135.23                                        
                                  .59   .32  1.44                         
KIDNEY 2.76  .68 .29   .11  105.40                                        
                                  .69   .25  1.70                         
LUNG   1.57  .39 .51   .32  324.91                                        
                                  1.21  .77  2.98                         
HEART  1.18  .29 .31   .26  259.38                                        
                                  .72   .61  1.79                         
INTRAVENOUS TFA-IOP RAT STUDY                                             
RAT NUMBER: 8.                                                            
DOSE: 100. umoles/kg                                                      
TIME: 90. minutes                                                         
BODY WEIGHT: 435. grams                                                   
LIVER  13.02 2.99                                                         
                 2.63  .20  201.73                                        
                                  6.04  .46  13.88                        
SPLEEN .63   .14 .10   .16  164.06                                        
                                  .24   .38  .55                          
UPPER GI                                                                  
       1.27  .29 .18   .14  140.23                                        
                                  .41   .32  .94                          
LOWER GI                                                                  
       1.54  .35 .27   .18  178.09                                        
                                  .63   .41  1.45                         
KIDNEY 2.56  .59 .15   .06   57.84                                        
                                  .34   .13  .78                          
LUNG   1.30  .30 .36   .28  277.64                                        
                                  .83   .64  1.91                         
HEART  1.29  .30 .31   .24  242.85                                        
                                  .72   .56  1.66                         
__________________________________________________________________________
 
    
     
                                           TABLE 7                                 
__________________________________________________________________________
INTRAVENOUS TFA-IOP RAT STUDY                                             
RAT I.D.: I-8                                                             
COMPOUND: TFA-IOP                                                         
DOSE: 100. umoles/kg                                                      
BODY WEIGHT: 435. grams                                                   
UMOLES INJECTED: 43.50 umoles                                             
LIVER WEIGHT: 13.02 grams                                                 
EXP. LENGTH: 90 minutes                                                   
__________________________________________________________________________
SAMPLE                                                                    
      BILE FLOW   COMPOUND                                                
                          EXCRETION     SAMPLE                            
PERIOD                                                                    
      ml/ ml/min PER                                                      
                  CONC.   umoles/                                         
                               umoles/min PER                             
                                        WEIGHT                            
(min) min kg b.w.                                                         
              kg l.w.                                                     
                  (umolar)                                                
                          min  kg b.w.                                    
                                   kg l.w.                                
                                        (grams)                           
__________________________________________________________________________
10-0  .02093                                                              
          .04811                                                          
              1.60753                                                     
                   635.0  .013 .031                                       
                                    1.021                                 
                                        .20930                            
 0-10 .04071                                                              
          .09359                                                          
              3.12673                                                     
                  3010.0  .123 .282                                       
                                    9.411                                 
                                        .40710                            
10-20 .03675                                                              
          .08448                                                          
              2.82258                                                     
                  5777.6  .212 .488                                       
                                   16.308                                 
                                        .36750                            
20-30 .04050                                                              
          .09310                                                          
              3.11060                                                     
                  6436.1  .261 .599                                       
                                   20.020                                 
                                        .40500                            
30-40 .02992                                                              
          .06878                                                          
              2.29800                                                     
                  7802.9  .233 .537                                       
                                   17.931                                 
                                        .29928                            
40-50 .03047                                                              
          .07005                                                          
              2.34025                                                     
                  7641.1  .233 .535                                       
                                   17.882                                 
                                        .30470                            
50-60 .02817                                                              
          .06476                                                          
              2.16359                                                     
                  8461.4  .238 .548                                       
                                   18.307                                 
                                        .28170                            
60-70 .02721                                                              
          .06255                                                          
              2.08986                                                     
                  5372.5  .146 .336                                       
                                   11.228                                 
                                        .27210                            
70-80 .02653                                                              
          .06099                                                          
              2.03763                                                     
                  5305.7  .141 .324                                       
                                   10.811                                 
                                        .26530                            
80-90 .02586                                                              
          .05945                                                          
              1.98618                                                     
                  4663.1  .121 .277                                       
                                    9.262                                 
                                        .25860                            
__________________________________________________________________________
SAMPLE UMOLES       PERCENTAGE OF DOSE                                    
PERIOD IN    RUNNING                                                      
                    IN    RUNNING                                         
                                 PER gm.                                  
                                       PER kg.                            
(min)  SAMPLE                                                             
             TOTAL  SAMPLE                                                
                          TOTAL  SAMPLE                                   
                                       BODY WT.                           
__________________________________________________________________________
10-0    .13   .13    .31   .31    1.46 .70                                
 0-10  1.23   1.36  2.82   3.12   6.92 6.48                               
10-20  2.12   3.48  4.88   8.00  13.28 11.22                              
20-30  2.61   6.09  5.99  14.00  14.80 13.78                              
30-40  2.33   8.42  5.37  19.36  17.94 12.34                              
40-50  2.33  10.75  5.35  24.71  17.57 12.30                              
50-60  2.38  13.13  5.48  30.19  19.45 12.60                              
60-70  1.46  14.60  3.36  33.56  12.35 7.73                               
70-80  1.41  16.00  3.24  36.79  12.20 7.44                               
80-90  1.21  17.21  2.77  39.56  10.72 6.37                               
__________________________________________________________________________
 
    
     
                                           TABLE 8                                 
__________________________________________________________________________
INTRAVENOUS IOP RAT STUDY                                                 
RAT I.D.: I-6                                                             
COMPOUND: IOP                                                             
DOSE: 104. umoles/kg                                                      
BODY WEIGHT: 406. grams                                                   
UMOLES INJECTED: 42.22 umoles                                             
LIVER WEIGHT: 11.98 grams                                                 
EXP. LENGTH: 90 minutes                                                   
__________________________________________________________________________
SAMPLE BILE FLOW       COMPOUND  EXCRETION                                
PERIOD ml/  ml/min PER CONC.     umoles/                                  
                                       umoles/min PER                     
(min)  min  kg b.w.                                                       
                  kg l.w.                                                 
                       (umolar)  min   kg b.w.                            
                                             kg l.w.                      
__________________________________________________________________________
-10-0  no sample weight                                                   
                       no sample conc.                                    
  0-10 .01375                                                             
            .03386                                                        
                  1.14750                                                 
                       1605.8    .022  .054  1.843                        
 10-20 .00867                                                             
            .02136                                                        
                  .72404                                                  
                       8659.4    .075  .185  6.270                        
 20-30 .01077                                                             
            .02652                                                        
                  .89866                                                  
                       10504.9   .113  .279  9.440                        
 30-40 .00977                                                             
            .02406                                                        
                  .81528                                                  
                       9854.3    .096  .237  8.034                        
 40-50 .01161                                                             
            .02861                                                        
                  .96953                                                  
                       9667.6    .112  .277  9.373                        
 50-60 .00904                                                             
            .02225                                                        
                  .75417                                                  
                       7316.4    .066  .163  5.518                        
 60-70 .01072                                                             
            .02640                                                        
                  .89457                                                  
                       7529.1    .081  .199  6.735                        
 70-80 .01018                                                             
            .02506                                                        
                  .84942                                                  
                       7758.8    .079  .194  6.590                        
 80-90 .00977                                                             
            .02407                                                        
                  .81586                                                  
                       9564.6    .093  .230  7.803                        
__________________________________________________________________________
SAMPLE                                                                    
      SAMPLE                                                              
             UMOLES      PERCENTAGE OF DOSE                               
PERIOD                                                                    
      WEIGHT                                                              
            IN    RUNNING                                                 
                         IN    RUNNING                                    
                                      PER gm.                             
                                            PER kg.                       
(min) (grams)                                                             
            SAMPLE                                                        
                  TOTAL  SAMPLE                                           
                               TOTAL  SAMPLE                              
                                            BODY WT.                      
__________________________________________________________________________
-10-0 no sample concentration, thus cannot calculate these values         
  0-10                                                                    
      .13747                                                              
            .22   .22    .52   .52    3.80  1.29                          
 10-20                                                                    
      .08674                                                              
            .75   .97    1.78  2.30   20.51 4.38                          
 20-30                                                                    
      .10766                                                              
            1.13  2.10   2.68  4.98   24.88 6.60                          
 30-40                                                                    
      .09767                                                              
            .96   3.07   2.28  7.26   23.34 5.61                          
 40-50                                                                    
      .11615                                                              
            1.12  4.19   2.66  9.92   22.98 6.55                          
 50-60                                                                    
      .09035                                                              
            .66   4.85   1.57  11.48  17.33 3.86                          
 60-70                                                                    
      .10717                                                              
            .81   5.66   1.91  13.40  17.83 4.71                          
 70-80                                                                    
      .10176                                                              
            .79   6.45   1.87  15.27  18.38 4.61                          
 80-90                                                                    
      .09774                                                              
            .93   7.38   2.21  17.48  22.65 5.45                          
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