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f E" =_Li0R 05C T NESULFQNATE. PE.' FLl G RG FEXANES'JL-O NATE a n o p E R F^U O R O O C TA N E S U LFO N A TE ON SOiV.E / S E C T OF CHOLESTEROL METABOLISM IK VITRO.
jTNO-report of study 3M#Q
TNO-report
Final version
18-04-2005)
Study of the effect of PERFLUOROBUTANESULFONATE, PERFLUOROHEXANESULFONATE AND PERFLUOROOCTANESULFONATE ON SOME ASPECTS OF CHOLESTEROL METABOLISM IN VITRO.
Study / project number: 3 M # 0 1 / 011.2 5 1 7 7
TNO Quality of Life TNO Pharma
P.O. Box 2215 2301 CE Leiden The Netherlands P h o n e +31 71 518 1469 Fax +31 71 518 1904
Author: L.H. Cohen E. Hoegee-de Nobel
In assignment of: 3M, Medical department
Study monitor.
John L. Butenhoff, Ph.D., CIH, DABT
Corporate Scientist
Medical Department
3M Center, Building 0 2 2 0 -0 6 -W -0 2
St.Paul, Minnesota 55144-1000
USA
Phone:
+ 1 .6 5 1 .7 3 3 .1 9 6 2
Fax:
+1.651.733.9066
E-mail:
jlbu ten ho ff@ m m m .com
Study director Dr. Louis H. Cohen
Status and Date:
Final, 18 April 2005
Previous version:
Draft 1:
16-02-2005
Draft 2,
29 March 2005
Number of pages: 34
Authorization by Dr. H.M.G Princen,
Head of the Department of Vascular and Metabolic Diseases
Signature and date: %
t.Q . ,?
n
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fTNO-report of study 3M#Q1
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Contents Contents Testing facility Contributors 1. Agreement and protocol 2. Objective of the study 3. Methods
3.1. Compounds 3.2 Assays 3.2.1. Cholesterol synthesis in primary rathepatocytes 3.2.2. Bile acid synthesis in primary rat hepatocytes 3.2.3. Bile acid uptake with hamster ileum pieces 3.3 Approval Animal ExperimentCommittee TN O
4. Results 4.1. Cholesterol synthesis in primary rathepatocytes
4.2. Bile acid synthesis in primary rat hepatocytes
4.3. Bile acid uptake with hamster ileum pieces
5. Conclusions 5.1. Cholesterol synthesis in primary rat hepatocytes
5.2. Bile acid synthesis in primary rat hepatocytes
5.3. Bile acid uptake with hamster ileum pieces
6. References
Appendix 1, Cholesterol synthesis in primary rat hepatocytes 1.1 Appendix: primary data experiment 1 1.2. Appendix: primary data experiment 2 1,3V Appendix: primary data experiment 3 1.4 Appendix: composite data
IM
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Appendix 2, Bile acid synthesis in primary rat hepatocytes 2.1 Appendix: primary data experiment 1 2.2. Appendix: primary data experiment 2 2.3. Appendix: primary data experiment 3 2.4 Appendix: composite data
Appendix 3, Bile acid uptake with hamster ileum pieces 3.1 Appendix: primary data experiment 1 3.2. Appendix: primary data experiment 2 3.3. Appendix: primary data experiment 3 3.4. Appendix: composite data
Testing facility
TN O Quality of Life
Business Unit Biomedical Research
Department of Vascular and Metabolic Diseases
Gaubius Laboratory
P.O. Box 2215
2301 CE Leiden
The Netherlands
phone: +31 71 5181479
fax: +31 71 5181904
Contributors Study director:
Dr. L.H. Cohen Phone: +31 71 5181469 E-mail: LH.Cohen@pg.tno.nl
Experimental work:
Mr. W . van Duyvenvoorde Mrs. E. Hoegee-de Nobel Mrs. E. Pieterman Mrs. C. van Thiel
18-04-20051
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page 25 page 28 page 31 page 34
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[TNO-report of study 3M#01
* Final version
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1. Agreement and protocol
The research agreement for the present project between TN O and 3M, Medical Department has been entered on 16-01-2003. The project has been performed according to the protocol of 20-9-2004, which has been attached to the research agreement as Annex i.
2. Objective of the study
3M has observed that certain perfluoroalkylsulfonates have cholesterol lowering effects in animal studies. It is the purpose of the present study to gain insight into the possible mechanism of action. Cholesterol lowering can be caused in several ways: inhibition of hepatic cholesterol synthesis, increase in hepatic bile acid synthesis or inhibition of the intestinal uptake of cholesterol or bile acids will all lead to reduction of circulating cholesterol.
In the present study the effects of the compounds on cholesterol and bile acid synthesis in primary culture of rat hepatocytes and on bile acid uptake in hamster ileum pieces in vitro is investigated
3. Methods
3.1 Compounds The three test compounds, potassium perfluorobutanesulfonate (PFBS; MTDID-206; L7038), potassium perfluorohexanesulfonate (PFHS; M TDID-207; L-9051) and potassium perfluorooctanesulfonate (PFOS; MTDID-208; FC -S000-0256-8) have been supplied by 3M, Medical Department. Since the delivery of the compounds was delayed, the start of the project was on 1-12-2004. During the performance of the project and correspondingly, in the appendices of this report these compounds have been designated as test compound 206, 207 and 208, respectively.
The test compounds were dissolved in DM SO at a concentration of 100 mM. In each experiment freshly made solutions of the test compounds have been used.
3.2 Assays
3.2.1.
Cholesterol synthesis in primary rat hepatocytes
Hepatocytes were isolated from freshly obtained rat liver and brought into culture. After
about 20h the cells were refreshed and incubated with 3 different concentrations (up to
100pM) of the 3 test compounds in duplicate for 3.5 h. After a 0.5h-preincubation, [14C]-
acetate was present for 3h for labeling of newly synthesized cholesterol. As a positive
control and reference a HM G-CoA reductase inhibitor (simvastatin) was incorporated in
the experiment. [14C]-labeled lipids were extracted, saponified and the non-saponifiable
lipid fraction, mainly containing sterols, analyzed for incorporated radioactivity [1,2],
These experiments were repeated three times with separately isolated hepatocytes.
3.2.2
Bile acid synthesis in primary rat hepatocytes
Hepatocytes were isolated from freshly obtains rat liver and brought into culture. After
about 28h the cells were refreshed and incubated with 3 different concentrations of the 3
test compounds in duplicate for 24h. Collected cells and media were analyzed on
synthesized bile acids by g.I.c. [3],
These experiments were repeated three times with separately isolated hepatocytes.
t R IIIIIb
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3.2.3
Bile acid uptake with hamster ileum pieces
The uptake measurements were performed according to a modification of the procedure
as described by Kurata et al. [4], It is based on the specific association of [3H]-
taurocholate (TCA) with freshly obtained hamster ileum pieces. The association is
corrected for nonspecific uptake by association in the presence of an excess of non-
labeled TCA. Three concentrations of one test compound in triplicate {the intestinal bile
acid uptake-gradient has to be taken into account) per one ileum were tested.
Experiments were repeated three times.
3.3 Approval Animal Experiment Committee TNO The Animal Experiment Committee (DEC) of TN O has approved the use of animals for this study on 19 -11-2004 under number DEC1779.
4. Results
4.1. Cholesterol synthesis in primary rat hepatocytes The three test compounds have been tested in three independent performed experiments, using three different isolations of rat liver microsomes. The primary data obtained in these experiments are added to this report in Appendix 1. The results of the three independent experiments are shown in figure 4.1.1 to 4.1.3, respectively. The composite curves of the three experiments are given in figure 4.1.4.
test compounds 3M on cholesterol synthesis
experiment 1
-- -- PFBS -- -- PFHS
' * pros
-- V -- Simva
1
concentration (pM)
Fig. 4.1.1. Effect of PFBS, PFHS and PFOS on cholesterol synthesis in experiment 1; mean values range are given.
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test compounds 3M on cholesterol synthesis
18-04-20051 experiment 2
PFBS PFHS PFOS Simva
concentration (pM)
Fig. 4.1.2. Effect of PFBS, PFHS and PFOS on cholesterol synthesis in experiment 2; mean values range are given.
test compounds 3M on cholesterol synthesis
experiment 3
- o - PFBS --ffl -- PFHS - i - PFOS
-- V -- Simva
concentration (pM)
Fig. 4.1.3. Effect of PFBS, PFHS and PFOS on cholesterol synthesis in experiment 3; mean values range are given.
UNO-report of study 3M#Q1
Final version
18-04-20051
composite curve
test compounds 3M on cholesterol synthesis
PFBS PFHS PFOS Simva
0.01 0.1 1 10 100 1000
concentration (pM)
Fig. 4.1.4. Composite curves of the effect of PFBS, PFHS and PFOS on cholesterol synthesis from three independently performed determinations. The values are the mean of the individual experimental values + SEM.
The reference inhibitor of cholesterol synthesis, simvastatin, inhibited in all three experiments as expected with an IC50-value < 1 0 nM. The test compounds showed no clear inhibition of cholesterol synthesis at the concentrations up to 100 pM and under test conditions used. Incubation with the different concentrations of compound PFOS resulted in values for the incorporation of [14C]-acetate into sterols which were similar to the control values (set as 100% in the figures). Remarkable were the higher values obtained with compounds PFBS and PFHS. The explanation is not easy to give since the effect seems not to be dependent on the concentration of the compounds. It may not be an increase of cholesterol synthesis, but can be an effect on the substrate pool as well, since [14C]-acetate is used for this measurement. It could be that the presence of the compounds in the incubation medium increased the influx of label, or, alternatively, reduced the generation of endogenous acetyl-CoA; the latter can be the availability and/or degradation of endogenous or exogenous fatty acids. Additional experiments would have to be performed to give more insight in this matter.
4.2 Bile acid synthesis in primary rat hepatocytes The three test compounds have been tested in three independent performed experiments, using three different isolations of rat liver microsomes. The primary data in these experiments obtained are added to this report in Appendix 2. The results of the three independent experiments are shown in figure 4.2.1 to 4.2.3, respectively. The composite curves of the three experiments are given in figure 4.2.4.
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test compounds 3M on Bile acid synthesis
experiment 1
PFBS PFHS PFOS
concentration (pM)
Fig. 4.2.1. Effect of PFBS, PFHS and PFOS on bile acid synthesis in experiment 1; mean values range are given.
experiment 2
test compounds 3M on Bile acid synthesis
-- PFBS -- PFHS -- PFOS
% of control on Bile acid synthesis
concentration (pM)
Fig. 4.2.2 Effect of PFBS, PFHS and PFOS on bile acid synthesis in experiment 2; mean values range are given. 8 of 34
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test compounds 3M on Bile acid synthesis
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experrnent3
-- Q -- PFBS -- PFHS -- -- PFOS
% of control on Bile acid synthesis
^
0.1 1 10 100 1000
concentration ((jM)
.2.3. Effect of PFBS, PFHS and PFOS on bile acid synthesis in experiment 3; mean values range are given.
composite curve
test compounds 3M on Bile acid synthesis
-- PFBS -- PFHS -- PFOS
% of control on Bile acid synthesis
concentration (pM)
Fig. 4.2.4. Composite curves of the effect of PFBS, PFHS and PFOS on bile acid synthesis from three independently performed determinations. The values are the mean of the individual experimental values SEM. 9 of 34
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In the three experiments there is a concentration dependent trend towards higher biie acid synthesis with all three compounds. This is most pronounced with the highest concentration of compound PFOS (100 pM). The change in the bile acid synthesis is sustained by a shift in the composition of the individual bile acids: p-muricholate (the major bile acid in mouse) is relatively more increased compared to cholate and muricholate (see Appendix 2). The cause of the increase in bile acid synthesis may be changes in activity of rate limiting enzymes, which are members of the cytochrome P450 family, as well as changes in substrate availability. E.g., the latter is the case when cholesterol esterification is inhibited.
4.3 Bile acid uptake with hamster iieum pieces In one ileum three concentrations of one compound each with three different ileum pieces were tested. Since the uptake of taurocholate increases linearly from the jejunum side to the cecum side, the combination of triplicate ileum pieces are chosen in such a way that the average control uptake is more or less the same. The primary data obtained in these experiments are added to this report in Appendix 3. The results of the three independent experiments are shown in figure 4.3.1 to 4.3.3, respectively. The composite curves of the three experiments are given in figure 4.3.4.
test compounds 3M on TCA uptake
experiment 1
concentration (pM)
Fig. 4.3.1. Effect of PFBS, PFHS and PFOS on TCA uptake with hamster ileum pieces in experiment 1; mean values of incubations with three pieces are given
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test compounds 3M on TCA uptake
18-04-2009
experiment 2
PFBS pms pros
concentration (pM)
Fig. 4.3.2. Effect of PFBS, PFHS and PFOS on TCA uptake with hamster ileum pieces in experiment 2; mean values of incubations with three pieces are given
test compounds 3M on TCA uptake
experiment 3
- O -- PFBS -- PFHS
- a -- pros
concentration (iM )
Fig.4.3.3. Effect of PFBS, PFHS and PFOS on TCA uptake with hamster ileum pieces in experiment 3; mean values of incubations with three pieces are given 11 of 34
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iTNO-report of study 3M#Q1
* Final version
*
test compounds 3M on TC uptake
16-04-20051 j composite curve
-- PFBS
j* - PFHS
3a.
Z3
<
a - pros
.u
Fig. 4.3.4. Composite curves of the effect of PFBS, PFHS and PFOS on TCA uptake with hamster ileum pieces from three independently performed determinations. The values are the mean of the three different experiments SEM
Under the experimental conditions used, the test compounds show no or hardly any effect on the specific association of taurocholate with hamster ileum pieces. PFBS showed a minor decrease at the highest concentration tested, but this may be due to the variation in this system.
5. Conclusions
5.1 Cholesterol synthesis in primary rat hepatocytes The test compounds showed no clear inhibition of the cholesterol synthesis at the concentrations and conditions used.
In the presence of PFBS and PFHS, a higher incorporation of [14C]-acetate into sterols was observed. Additional experiments would have to be performed to give more insight in this matter.
5.2 Bile acid synthesis in primary rat hepatocytes There is a concentration dependent trend towards higher bile acid synthesis with the three compounds. This is most pronounced with the highest concentration of PFOS (100 pM). At this concentration there is additionally a small change in the composition of the individual bile acids. Changes in activity of rate limiting enzymes, which are members of the cytochrome P-450 family, as well as changes in substrate availability may be the cause of the increase in bile acid synthesis. Increased bile acid synthesis is often accompanied by increased fecal bile acid excretion. Since cholesterol is the substrate for bile acid synthesis this leads to decreased cholesterol levels. In order to prove this mechanism of action, animal (e.g. mouse) studies can be performed in which bile acid synthesis and flow can be monitored via bile canulation and fecal bile acids (mass and composition) can be analyzed.
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Changes in liver cytochrome P450 enzymes can be analyzed as well.
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5.3 Bile acid uptake with hamster ileum pieces The test compounds show no or hardly any effect on the uptake of taurocholate in hamster ileum pieces under the experimental conditions used.
6. References
[1] Cohen LH, vanVIiet A, Roodenburg L, Jansen LMC, Griffioen M. Pravastatin inhibited the cholesterol synthesis in human hepatoma cell line Hep G2 less than simvastatin and lovastatin, which is reflected in the upregulation of 3-hydroxy-3- methylglutaryl coenzyme A reductase and squalene synthase. Biochem Pharmacol 1993; 45: 22032208.
[2] Van Vliet AK, van Thiel GCF, Huisman RH, Moshage H, Yap SH, Cohen LH. Different effects of 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors on sterol synthesis in various human cell types. Biochimica et Biophysica Acta 1995; 1254: 105111.
[3] Princen HM, Meijer P, Hofstee B. Dexamethasone regulates bile acid synthesis in monolayer cultures of rat hepatocytes by induction of cholesterol 7 alpha-hydroxylase. Biochem J 1989; 262: 341-348.
[4] Kurata.H., Suzuki,S., Ohhata.Y., Ikeda,T., Hasegawa.T., Kitayama.K., Inaba.T., Kono,K., and Kohama.T., A novel class of apical sodium-dependent bile acid transporter inhibitors: the amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinoiine derivatives, Bioorg. Med Chem. Lett., 14 (2004) 1183-1186.
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APPENDIX 1 CHOLESTEROL SYNTHESIS IN PRIMARY RAT HEPATOCYTES
Appendix 1.1 experiment 1
mean
14C dpm per m ean 14C dpm
mean
test compounds control D M SO
14C dpm protein in mg mg protein per mg protein % o f control % o f control range
16520
0.27
61186
control D M SO
13405
0.31
43242
48247
100
control DM SO
13236
0.33
40312
control ethanol control ethanol
12995 9835
0.23 0.24
56913 40980
45980
100
control ethanol test compound 206 100 pM test compound 206 100 pM test compound 206 10 pM test compound 206 10 pM test compound 206 1 pM test compound 206 1 pM test compound 207 100 pM test compound 207 100 pM
12214 33488 35485 42327 52246 47582 58677 31169 36074
0.31 0.19 0.30 0.25 0.33 0.26 0.33 0.19 0.29
40047 179401 116985 170443 159125 185385 179623 161221 123682
148193 164784 182504 142452
372 242 353 330 384 372 334 256
307 342 378 295
64.7 11.7 6.0 38.9
test compound 207 10 pM test compound 207 10 pM
36312 43388
0.20 0.29
186216 147915
167066
386 307
346 39.7
test compound 207 1 pM test compound 207 1 pM test compound 208 100 pM test compound 208 100 pM test compound 208 10 pM test compound 208 10 pM test compound 208 1 pM test compound 208 1 pM Simva 1 pM Simva 1 pM Simva 0.1 pM Simva 0.1 pM Simva 0.01 pM Simva 0.01 pM
45991 36902 25343 19272 8647 12947 14762 11888
109 251 899 564 3499 3438
0.24 0.21 0.25 0.33 0.25 0.26 0.26 0.18 0.21 0.20 0.19 0.21 0.22 0.26
194327 178560 102742 57816 35293 50773 57513 65439
513 1274 4650 2684 16025 13395
186443 80279 43033 61476
894 3667 14710
403 370 213 120 73 105 119 136
1 3 10 6 35 29
386 16.3 166 46.6 89 16.0 127 8.2
2 0.8 8 2.1 32 2.9
[TNO-report of study 3M#01 Appendix 1.2 experiment 2
Final version
18-04-2005)
mean
14C dpm per mean 14C dpm
mean
test compounds
14C dpm protein in mq mg protein per mq protein % of control % of control ranqe
control D M S O
7630
0.26
29728
control DM SO
8472
0.23
36835
33157
100
control DM SO
12725
0.39
32909
control ethanol
8772
0.30
29735
30869
100
control ethanol
8986
0.28
32480
control ethanol
8358
0.28
30394
test compound 206 100 pM 7413
0.22
33193
34103
100
103 2.7
test compound 206 100 pM 9745
0.28
35013
106
test compound 206 10 pM 10698
0.33
32419
34248
98
103 5.5
test compound 206 10 pM 13228
0.37
36076
109
test compound 206 1 pM
10193
0.30
33604
34935
101
105 4.0
test compound 206 1 pM
12391
0.34
36267
109
test compound 207 100 pM 9779
0.34
28482
30416
86
92 5.8
test compound 207 100 pM 9651
0.30
32351
98
test compound 207 10 pM
5459
0.22
24444
28376
74
86 11.9
test compound 207 10 pM 11739
0.36
32309
97
test compound 207 1 pM
13766
0.32
42574
39779
128
120 8.4
test compound 207 1 pM
14917
0.40
36984
112
test compound 208 100 pM 8472
0.27
31187
27503
94
83 11.1
test compound 208 100 pM 8416
0.35
23820
72
test compound 208 10 pM
6943
0.29
24221
27494
73
83 9.9
test compound 208 10 pM 10205
0.33
30768
93
test compound 208 1 pM
8591
0.30
28321
26013
85
78 7.0
test compound 208 1 pM
8138
0.34
23704
71
Simva 1 pM
256 0.29
893
989
3
3 0.3
Simva 1 pM
378 0.35
1085
4
Simva 0.1 pM
606 0.33
1854
1880
6
6 0.1
Simva 0.1 pM
705 0.37
1906
6
Simva 0.01 pM
2407
0.29
8299
7291
27 24 3.3
Simva 0.01 pM
1990
0.32
6284
20
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Appendix 1.3 experiment 3
Final version
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mean
|4C dpm per mean 14C dpm
mean
test compounds
14C dpm protein in mc$ mg protein per mg protein % o f control % o f control ra n a e
control D M SO
9820
0.19
53083
control D M SO
19068
0.32
59588
52303
100
control DM SO
11207
0.25
44237
control ethanol
16024
0.29
56225
61191
100
control ethanol
25059
0.36
69287
control ethanol
19064
0.33
58062
test compound 206 100 pM 3535
0.16
22808
35976
44
69 25.2
test compound 206 100 pM 12532
0.26
49144
94
test compound 206 10 pM 16066
0.25
64695
71017
124
136 12.1
test compound 206 10 pM 22815
0.30
77339
148
test compound 206 1 pM
23573
0.28
83691
70022
160
134 26.1
test compound 206 1 pM
16530
0.29
56352
108
test compound 207 100 pM 21840
0.31
71217
77918
136
149 12.8
test compound 207 100 pM 22706
0.27
84620
162
test compound 207 10 pM 27486
0.33
82458
81235
158
155 2.3
test compound 207 10 pM 22937
0.29
80012
153
test compound 207 1 pM
21833
0.29
74855
80811
143 155 11.4
test compound 207 1 pM
26898
0.31
86767
166
test compound 208 100 pM 12637
0.23
55344
58972
106
113 6.9
test compound 208 100 pM 14607
0.23
62601
120
test compound 208 10 pM 15725
0.24
66444
57850
127
111 16.4
test compound 208 10 pM 10015
0.20
49255
94
test compound 208 1 pM
18330
0.31
59773
57749
114
110 3.9
test compound 208 1 pM
12074
0.22
55725
107
Simva 1 pM
322 0.30
1093
855
2
1 0.4
Simva 1 pM
129 0.21
618
1
Simva 0.1 pM Sim va 0.1 pM
1021 748
0.32 0.31
3208 2440
2824
5 4
5 0.6
Simva 0.01 pM Simva 0.01 pM
5276 3756
0.35 0.27
15294 13826
14560
25 23
24 1.2
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Final version
test compound 206 fyMl
1 10 100
test compound 207 fuMl
1 10 100
test compound 208 [pM]
1 10 100
Simva im 0.01 0.1
1
% of control
exp.1
exp. 2
378 105
342 103
307 103
% of control
exp.1
exp. 2
386 120
346 86
295 92
% of control
exp.1
exp. 2
127 78
89 83
166 83
% of control
exp.1
exp. 2
32 24
86
23
exp. 3 134 136 69
exp. 3 155 155 149
exp. 3 110 111 113
exp. 3 24 5 1
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mean % of control
206 194 160
mean % of control
220 196 179
mean % of control
105 94 121
mean % of control
27 6 2
SEM 86.6 74.8 74.4
SEM 83.5 77.8 60.5
SEM 14.4 8.5 24.3
SEM 2.7 0.6 0.9
fTNO-report of study 3M #01
Final version
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APPENDIX 2 BILE ACID SYNTHESIS IN PRIMARY RAT HEPATOCYTES
Appendix 2.1
experiment '
1 ni injected
Control 1+2 Control 3+4 Control 5+6 mean stdev 3M206 100 7+8 3M206 100 9+10 mean range 3 M 2 G 0 :it'U i11 + 1 2 M 2 0 6 1 0 13+14 m e flfllf range'" 3M206 1 15+16 3M206 1 17+18 Mean Range 3M207 100 19+20 3M207 100 21+22 Mean Range 3M 207 10 23+24 3M 207 10 25+26 Mean Range 3M207 1 27+28 3M207 1 29+30 Mean Range
nor-hyodeoxy ini. standard
area 21136 22079 18448
22799 15957
17016 19559
16706 6079
18616 13236
15566 10261
14490 20442
etiolate
area 4776 5130 5693
8muricholate
area 13882 14339 14491
wmurichoiate
area 1359 1270 1239
cholate
u bile acid 2.3 2.3 3.1
8murlcholate
pg bile acid
6.6
6.5
7.9
W-
muricholate
pg bile acid 0.6 0,6 0.7
7181 4928
18533 12423
1458 980
4.6 11.9 3.1 7,8
0.9 0.6
5182
14090
1207
3.0
8.3
5029
12629
1052
2.6
6.5
0.7 0.5
4633
12547
1143
2.8
7.5
2021
4856
509
3.3 8.0
0.7 0.8
7604 6306
20071 17879
1226 972
4.1 10.8 4.8 13.5
0.7 0.7
3827
11236
1052
2.5
7,2
2708
6750
624
2.6 6,6
0.7 0.6
3001
7170
684
2.1 4.9
5554
13280
1669
2.7
6.5
0.5 0.8
protein mg 5.9 5.8 5.8
5.8 5.6
6.8 6.7
6.6 8.1
7.6 7.1
6.0 6.9
7.2 7.5
cholate nmol /mg
protein 0.94 0.98 1.29 1.07 0.19 1.33 1.35 1.34 0.01 1.09 0.94 1.02 0.08 1.02 1.00 1.01 0.01 1.31 1.63 1.47 0,16 1.00 0.94 0.97 0.03 0.71 0.89 0.80 0.09
8muricholate
nmol /mg protein 2.74 2.75 3.29 2.93 0.31 3.43 3.39 3.41 0.02 2.96 2.37 2.67 0.30 2.77 2.41 2.59 0.18 3.46 4.63 4.05 0.59 2.93 2.34 2.64 0.30 1.69 2.13 1.91 0.22
wmuricholate
nmol /mg protein 0.27 0.24 0.28 0.26 0.02 0.27 0.27 0.27 0.00 0.25 0.20 0.23 0.03 0.26 0.25 0.26 0.01 0.21 0.25 0.23 0.02 0.27 0.22 0.25 0.03 0.16 0.27 0.22 0.06
total nmol /mg
protein 3.95 3.98 4.87 4.27 0.52 5.04 5.01 5.03 0.01 4.31 3.51 3.91 0.40 4.05 3.67 3.86 0.19 4.99 6.51 5.75 0,76 4,20 3.50 3.85 0.35 2.56 3.29 2.93 0.37
%of control
100.0
118.1 117.4 117.8
0.4 101.0 82.3 91.6
9.4 94.9 86.0 90.5 4.5 117.0 152.6 134.8 17.8 98.4 82.0 90.2 8.2 60.0 77.1 68.6 8.6 18 of 34
TN -report of study 3M #01
Final version
18-04-2005\
3M208 100 31+32 3M208 100 33+34 Mean Range 3M208 10 35+36 3M208 10 37+38 Mean Range 3M 2081 39+40 3M208 1 41+42 Mean range
nor-hyodeoxy int. standard
area
19032
21118
cholate
area 6553 9890
limuricholate
area 22371 34108
w~ muricholate
area 1426 1603
cholate
MO bile acid 3.4 4.7
timuricholate
Mg bile acid
11.8
16.2
wrnuricholate
mo bile acid 0.7 0.8
protein
mg 7.4 6.5
21213
5369
16393
1464
2.5
7.7
12880
3952
12571
776
3.1 9.8
0.7 7.6 0.6 6.7
17605
4500
11600
1309
2.6
6.6
22507
5642
15956
1617
2.5
7.1
0.7 7.0 0.7 5.4
cholate nmol /mg
protein 1.14 1.76 1.45 0.31
0.81 1.12 0.97 0.16 0.90 1.13 1.02 0.12
6muricholate
nmol /mg protein 3.89 6.06 4.98 1.09
2.48 3.55 3.02 0.54
2.32 3.19 2.76 0.44
wmuricholate
nmol /mg protein
0.25 0.28 0.27 0.02
0.22 0.22
0.22 0.00
0.26 0.34 0.30 0,04
total nmol /mg
protein 5.28 8.10 6.69 1.41
3.51 4.89 4.20 0.69 3.48 4.66 4.07 0.59
%of control 123.8 189.8 156.8 33,0 82.3 114.6 98.4 16.2 81.6 109.2 95.4 13.8
19 of 34
t f N O -report o f study 3M#01
Final version
18-04-2005\
Appendix 2.2
experiment 2
1 ul iniected
taurodeoxvoholate
Control 1+2 Control 3+4 Control 5+6 mean stdev 3M206 100 7+8 3M206 100 9+10 mean range 3 M 2 0 6 10 11+12 3M 206 10 13+14 mean rande 3M 2061 15+16 3M 2061 17+18 mean ranqe 3M207 100 19+20 3M207 100 21+22 Mean Ranqe 3M207 10 23+24 3M207 10 25+26 Mean Ranqe 3M2071 27+28 3M207 1 29+30 Mean Ranqe
int. standard area 22200 6745 11811
12488 14051
13722 10945
13205 11976
9974 13465
15405 13323
17985 12961
choiate area 4459 2478 3666
4728 5186
3313 3125
3857 3178
4190 4950
3696 3170
3770 4110
ftmuricholati
area 13878 4719 10767
W-
muricholate choiate
area
M9 bile acid
1541
2.0
578 3.7
1100
3.1
limuricholate
Md bile acid 6.3 7.0 9.1
wmurichoiate
Mg bile acid 0.7 0.9 0.9
12328 13118
1689 1116
3.8 3.7
9.9 9.3
1.4 0.8
8985 10122
821 1117
2.4 2.9
6.5 9.2
0.6 1.0
13573 10902
1212 798
2.9 2.7
10.3 9.1
0.9 0.7
11034 17225
600 1305
4.2 3.7
11.1 12.8
0.6 1.0
12772 9662
1193 1085
2.4 2.4
8.3 7.3
0.8 0.8
11771 12978
1271 1287
2.1 3.2
6.5 10.0
0.7 1.0
protein choiate nmol /mg
mg protein
6,07
0.81
5.47
1.64
5.26
1.44
1.30
0.44
6.38
1.45
6.85
1.32
1.39
0.07
6.65
0.89
6.21
1.13
1.01
0.12
7.58
0.94
6.00
1.08
1.01
0.07
7.49
1.37
6.77
1.33
1.35
0.02
6.65
0.88
6.62
0.88
0.88
0.00
7.96
0.64
5.90
1.32
0.98
0.34
amuricholate
nmol /mg protein 2.52 3.13 4.24 3.30 0.87 3.79 3.33 3.56 0.23 2.41 3,65 3.03 0.62 3.32 3.71 3.51 0.20 3.61 4,63 4.12 0.51 3.05 2.68 2.87 0.18 2.01 4.16 3.08 1.07
w-
murichoiate total
nmol /mg
nmol /mg
protein
protein
0.28
3,61
0.38
5.16
0.43
6.12
0.37
4.96
0.08
1.27
0.52
5.76
0.28
4.94
0.40
5.35
0.12
0.41
0.22
3.52
0.40
5.17
0.31
4.35
0.09
0.83
0.30
4.56
0.27
5.07
0.28
4.81
0.01
0.25
0.20
5.18
0.35
6.31
0.27
5.75
0.08
0.56
0,29
4.22
0.30
3.86
0.29
4.04
0.01
0.18
0.22
2.88
0.41
5.88
0.31
4.38
0.10
1.50
%of control
100.0
116.1 99.5 107.8 8,29 70.9 104.3 87.6 16.68 91.8 102.1 96.9 5,13 104.5 127.1 115.8 11,31 85.0 77.9 81.4 3,58 57.9 118.6 88.3 30.32
20 of 34
fTNO -report o f study 3 M # 0 1 _______ ________ Final version
18-04-200S\
3M208 100 31+32 3M208 100 33+34 mean range 3M208 10 35+36 3M208 10 37+38 mean range 3M208 1 39+40 3M2081 41+42 mean range
taurodeoxycholate int. standard area
13182 17106
15959 11623
18140 11498
a- wetiolate muricholate muricholate
area 5170 6730
area 15851 22483
area 858 1307
4037 3362
12134 10846
1044 1034
4808 2753
14972 7667
1357 801
acholate muricholate MS M9 bile acid bile acid
3.9 12.0
3.9 13.1
wmuricholate
99 bile acid
0.7
0.8
2.5 7.6 2.9 9.3
0.7 0.9
2.7 8.3 2.4 6.7
0.7 0.7
protein cholate nmol /mg
mg protein
6.46
1.49
6.75
1.43
1.46
0.03
7.23
0.86
5.02
1.41
1.13
0.28
7.78
0.83
7.02
0.83
0.83
0.00
emuricholate
nmol /mg protein 4.55 4.76 4.66
0.11 2.57 4.55 3.56 0.99
2.60 2.32 2.46 0.14
W-
muricholate nmol /mg protein 0.25 0.28 0.26 0.02 0.22 0.43 0.33 0.11 0.24 0.24 0.24 0.00
total nmol /mg protein 6.29 6.47 6,38 0.09 3.65 6.40 5.02 1.37 3.67 3.40 3.53 0.13
%of control
126.6 130.3 128.5 1.84 73.5 128.9 101.2 27.68 73.9 68.5 71.2 2.7
21 of 34
fTN O -report of study 3M #01
Final version
-IB-04-200S
A ppendix 2.3
experiment 3
2 Ml injected
Taurodeoxycholate cholate
Control 1+2 Control 3+4 Control 5+6 mean stdev 3M206 100 7+8 3M206 100 9+10 mean range
JM 2D6 1D 11+12 3M206 1 13+14 mean ranqe
3M 2061 15+16 3M206 1 17+18 mean range 3M 207100 19+20 3M207 100 21+22 mean range 3M207 10 23+24 3M207 10 25+26 mean range 3M2071 27+28 3M207 1 29+30 mean range
int, standard area 21374 26929 29502
14202 27875
29566 24438
23377 46358
33096 31989
37853 28904
36982 34786
area 2697 3453 3744
5700 4797
5141 3938
3799 5952
5892 6266
5063 4643
4836 4565
6muricholate
area 8034 11392 11405
11499 14110
17073 12243
12273 18727
20208 19379
18253 12269
15795 14419
wmuricholate
area 1611 2282 2470
2565 3241
3332 2620
2733 3769
3073 2605
3214 2504
3245 2646
cholate M9 bile
acid 1.30 1.30 1.30
4.00 1.70
1.70 1.60
1.60 1.30
1.80 2.00
1.30 1.60
1.30 1.30
limuricholate MS bile acid
3.80 4.20 3.90
8.10 5.10
5.80 5.00
5.30 3.90
6.10 6.10
4.80 4.20
4.30 4.10
wmuricholate M9 bile acid
0.80 0.80 0.80
1.80 1.20
1.10 1.10
1.20 0.80
0.90 0.80
0.80 0.90
0.90 0.80
total Mg bile
acid
5.80
6.40
6.00
mg
protein 8.17 7.04 6.42
13,90 7.90
8.14 7.12
8.60 7.70
7.21 6.74
8.00 6.00
7.45 7.71
8.80 8.80
7.27 7.53
7.00 6.70
7.14 6.95
6.50 6.20
7.72 8.44
cholate nmol /mg
protein 0.38 0.45 0.48 1.47 0.05 1.21 0.59 0.90 0.31 0.59 0.59 0.59 0.00 0.53 0.41 0.47 0.06 0.60 0.64 0.62 0.02 0,46 0.57 0.52 0.06 0.41 0.38 0.40 0.02
6muricholate
nmol /mg protein 1.13 1.47 1.47 1.36 0.20 2.43 1.74 2.09 0.35 1.96 1.82 1.89 0.07 1.72 1.25 1.49 0.24 2.05 1.97 2.01 0.04
1.65 1.50 1.58 0.08 1,35 1.20 1.28 0,08
wmurichofate
nmol /mg protein 0.23 0.29 0.32 0.28 0.05 0.54 0.40 0.47 0.07 0.38 0.39 0.39 0.01 0.38 0.26 0.32 0.06 0.31 0.26 0.29 0.03 0.29 0.31 0.30 0.01 0.28 0.22 0.25 0.03
total nmol /mg protein
1.73 2,21 2,28 2.07 0.30 4.18 2,73 3.46 0.73 2.93 2.79 2.86 0.07 2,64 1.92 2.28 0.36 2,97 2.87 2.92 0.05 2.40 2.37 2.39 0.02 2.05 1.80 1.93 0,13
% of control
100.0
201.6 131,7 166.6 35.0 141.3 134.6 137.9
3.4 127.3 92.6 110.0 17.4 143.2 138.4 140.8
2.4 115.8 114.3 115.0
0.7 98.9 86.8 92.8 6,0
22 of 34
f f N O -report o f study 3M#01
Final version
-\S-Q4-2QOS,
BTaurodeoxycbolate cholate muricholate
3M208 100 31+32 3M208 100 33+34
mean range 3M208 10 35+36 3M208 10 37+38 mean range 3M208 1 39+40 3M208 1 41+42 mean range
int. standard area 31073 32836
41038 31137
27688 35363
area 6986 6730
5566 4092
3520 4522
area 23247 22483
20405 14644
12477 15421
wmuricholate
area 3240 1307
3608 2600
2633 3420
cholate gg bile
acid 2.20 2.10
1.40 1.30
1.30 1.28
I5muricholate
MS bile acid 7.50 7.00
5.00 4.70
4.50 4.36
wmurichofate
MP bile acid 1.00 0.90
0.90 0.80
1.00 0.97
total pg bile
acid 10.80 10.00
7.20 6.90
6.70 6.60
mg protein 7.36 7.59
7.11 8.40
7.56 7.53
cholate nmol /mg
protein 0.75 0.67 0.71 0.04 0.47 0.38 0.43 0.05 0.41 0.42 0.42 0.01
6muricholate
nmol /mg protein 2.49 2.25 2.37 0.12
1.71 1.37 1.54 0.17 1.46 1.42 1.44 0.02
w* muricholate
nmol /mg protein 0.35 0.30 0.33 0.03 0.30 0.24 0.27 0.03
0.31 0.31 0.31 0.00
total nmol /mg
protein 3.58 3.23 3.41 0.18 2.48 2.00 2.24 0.24 2.18 2.15 2.17 0.02
% of control 172.7 155.8 164.2
8.4 119.6 96,5 108.0 11.6 105.1 103.7 104.4
0.7
23 of 34
mm-
iTNO-report o f study 3M#01 Appendix 2.4
Final version
8-04-2009
Composite data experiments 1 - 3
test compound 206 iuMl 1 10 100
test compound 207 [pM] 1 10 100
test compound 208 fuM] 1 10 100
% of control exp.1 91 92 118
% of control exp.1 69 90 135
% of control exp.1 95 98 157
exp. 2 97 88 108
exp. 2 88 81 116
exp. 2 71 101 129
exp. 3 110 138 167
mean % of control
99 106 131
SEM 5.6 16.1 18.3
exp. 3 93 115 141
mean % of control
83 95 131
SEM 7.3 10.2 7.5
exp. 3 104 108 164
mean % of control
90 102 150
SEM 9.9 3.0 10.7
24 o f 34
** *
*
ITNO-report of study 3M#Q1_______________ Final version_____________________________ 18-04-2005
APPENDIX 3 BILE ACID UPTAKE WITH HAMSTER ILEUM PIECES
Appendix 3.1 experiment 1 compound 206
Ham ster 1
Condition
# dpm
excess cold TCA 1 10607
1 pM 206
2 70831
10 pM 206
3 67826
100 pM 206 4 97967
control
5 66568
me? dpm /m g # 36 295 10 33 2146 9 33 2055 8 49 1999 7 22 3026 6
dpm 7946 47219 44795 54314 59281
mg dpm /mg # dpm mq dDm/mq 38 209 11 10779 36 299 26 1816 12 35724 36 992 32 1400 13 21943 44 499 36 1509 14 22022 40 551 31 1912 15 14060 40 351
Mean Condition excess cold TCA 1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 268 1652 1318 1353 1763
specific TCA uptake ( TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mq
1 pM 206
2 1879 9 1548 12 725
10 pM 206 3 1788 8 1132 13 231
100 pM 206 4
1732 7 1241 14 283
control
5 2758 6 1645 15 84
Mean specific TCA uptake (and % of control)
1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 1384 1050 1085 1495
% of control 92.54 70.23 72.56 100.00
25 of 34
FINO-report of study 3M#Q1
Final version
18-04-20051
experiment 1 compound 207
Hamster 2
Condition
# dpm
excess cold TCA 1 7748
1 pM 206
2 53168
10 pM 206
3 69209
100 pM 206 4 66290
control
5 75331
mg dpm/mg 31 250 27 1969 33 2097 32 2072 33 2283
# 10 9 8 7 6
dpm 7579 57797 47287 58656 66781
dpm/mg # 39 194 11 43 1344 12 31 1525 13 31 1892 14 36 1855 15
dpm 6230 41456 32380 30414 29882
mg dpm/mg 29 215 30 1382 34 952 30 1014 28 1067
mean Condition excess cold TCA 1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 220 1565 1525 1659 1735
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 207
2 1749 9
1124 12 1162
10 pM 207
3 1878
8
1306 13 733
100 pM 207 4 1852
7
1672 14 794
control
5 2063 6
1635 15 848
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 207
1345
88.79
10 pM 207
1305
86.14
100 pM 207
1439
95.00
control
1515
100.00
[TNO-report o f study 3M#Q1______ experiment 1 compound 208
Final version
18-04-2005)
Hamster 3
Condition excess cold TCA
1 pM 206 10 pM 206 100 pM 206
control
# dpm 1 7216 2 60278 3 83982 4 67831 5 64502
mg dpm/mg # dpm mg dpm/mg # dpm mg dpm/mo 22 328 10 6178 27 229 11 4197 23 182 29 2079 9 44413 30 1480 12 29949 37 809 31 2709 8 45901 31 1481 13 17089 23 743 26 2609 7 50892 29 1755 14 18576 32 581 31 2081 6 75284 31 2429 15 15288 20 764
mean Condition excess cold TCA 1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 246 1456 1644 1648 1758
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 206
2 1832 9 1234 12 563
10 pM 206
3 2463 8 1234 13 497
100 pM 206
4 2362 7 1508 14 334
control
5 1834 6 2182 15 518
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 208 10 pM 208 100 pM 208 control (0.1 %
DMSO)
1210 1398 1402
1511
80.04 92.48 92.74
100.00
27 of 34
fTNO-report of study 3M#Q1 A p pen d ix 3.2 experim ent 2 com pound 206 Hamster 4
Final version
18-04-2005
Condition
# dpm
excess cold TCA 1 6433
1 pM 206
2 40568
10 pM 206
3 40299
100 pM 206
4 33543
control
5 51429
dpm/mg # dpm mg dpm/mg # dpm mg dpm/mq 34 189 10 4660 27 173 11 6560 39 168 25 1623 9 23576 25 943 12 8342 23 363 32 1259 8 33540 24 1397 13 9554 28 341 41 818 7 27052 28 966 14 9159 34 269 29 1773 6 38743 29 1336 15 10911 30 364
Mean Condition excess cold TCA 1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 177 976 999 685 1158
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 206
2 1446 9
766 12 186
10 pM 206
3 1083 8 1221 13 165
100 pM 206
4 641
7
789 14 93
control
5 1597 6 1159 15 187
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 206 10 pM 206 100 pM 206
control
799 81.49 823 83.86 508 51.77 981 100.00
*
*
m
m
fTNO-report of study 3M#Q1_______________ Final version ___________________________18-Q4-2Q05|
experiment 2 compound 207
Hamster 5
Condition excess cold TCA
1 pM 207 10 (JM 207 100 pM 207
control
# dpm mg dpm/mg # 1 5877 15 392 10 2 58425 19 3075 9 3 81952 25 3278 8 4 100229 25 4009 7 5 75039 21 3573 6
dpm 6549 58898 50220 66507 61523
mg dpm/mg # dpm mg dpm/mg 21 312 11 6059 32 189 23 2561 12 36635 21 1745 19 2643 13 31296 31 1010 24 2771 14 25690 22 1168 20 3076 15 32726 23 1423
Mean Condition excess cold TCA 1 pM 207 10 pM 207 100 pM 207
control
dpm/mg 298 2460 2310 2649 2691
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 207
2 2777 9 2263 12 1447
10 pM 207
3 2980 8 2346 13 712
100 pM 207
4 3712 7 2473 14 870
control
5 3276 6 2778 15 1125
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 207
2162
90.36
10 pM 207
2013
84.10
100 pM 207
2352
98.27
control
2393
100.00
29 of 34
rrNO-report of study 3M#01______ experiment 2 compound 208
Hamster 6
Final version
18-04-2005)
Condition excess cold TCA
1 (JM 208 10 (jM 208 100 pM 208
control
# 1 2 3 4 5
dpm 12998 62775 58113 64153 65801
mg dpm/mg # 35 371 10 31 2025 9 21 2767 8 28 2291 7 30 2193 6
dpm 11123 48383 73080 56772 59322
mg dpm/mg # dpm mg dpm/mq 36 309 11 8869 28 317 35 1382 12 23302 25 932 39 1874 13 25201 33 764 28 2028 14 27248 35 779 24 2472 15 29784 38 784
m ean Condition excess cold TCA 1 pM 208 10 pM 208 100 pM 208
control
dpm/mg 332 1446 1802 1699 1816
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 208
2 1693 9 1050 12 600
10 pM 208
3 2435 8
1541 13 431
100 pM 208 4 1959 7 1695 14 446
control
5 1861 6 2139 15 451
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 208
1114
75.08
10 pM 208
1469
99.01
100 pM 208
1367
92.10
control
1484
100.00
mum
30 of 34
iTNQ-report of study 3M#01
Appendix 3.3 experiment 3 compound 206
Final version
18-04-2005)
Hamster 7
Condition excess cold TCA
1 pM 206 10 pM 206 100 pM 206
control
# dpm 1 7991 2 73584 3 73951 4 84905 5 84047
mg dpm/mg 22 363 23 3199 20 3698 21 4043 24 3502
# 10 9 8 7 6
dpm 5509 75435 81803 78671 80739
mg dpm/mg # dpm mg 22 250 11 6805 25 29 2601 12 39200 19 29 2821 13 32999 22 28 2810 14 43312 31 25 3230 15 44087 23
mean Condition excess cold TCA 1 pM 206 10 pM 206 100 pM 206
control
dpm/mg 295 2621 2673 2750 2883
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mq excess cold TCA)
Condition
# dpm/mq # dpm/mq # dpm/mq
1 pM 206
2 2904 9 2306 12 1768
10 pM 206
3 3402 8 2526 13 1205
100 pM 206
4 3748 7 2514 14 1102
control
5 3207 6 2934 15 1622
Mean specific TCA uptake (and % of control)
dpm/mq % of control
1 pM 206
2326
89.89
10 pM 206
2377
91.88
100 pM 206
2455
94.87
control
2587
100.00
31 of 34
TN O-report of study 3M#01______ experiment 3 compound 207
Final version
18-04-2005)
Hamster 8
Condition
# dpm mg dpm/mg #
excess cold TCA 1 5082 17 299 10
1 pM 207
2 38506 15 2567 9
10 pM 207
3 49547 18 2753 8
100 pM 207
4 76185 27 2822 7
control
5 63313 21 3015 6
dpm 7890 49873 46363 55509 72551
mg dpm/mg 22 359 24 2078 23 2016 30 1850 28 2591
# dpm mg dpm/mg 11 6016 21 286 12 34022 20 1701 13 28487 15 1899 14 38643 21 1840 15 48843 28 1744
mean Condition excess cold TCA 1 pM 207 10 pM 207 100 pM 207
control
dpm/mg 315 2115 2223 2171 2450
specific TCA uptake (= TCA uptake indpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 207
2 2252 9 1763 12 1386
10 pM 207
3 2438 8 1701 13 1584
100 pM 207
4 2507 7 1536 14 1525
control
5 2700 6 2276 15 1430
Mean specific TCA uptake (and % of control)
dpm/mg % of control
1 pM 207
1801
84.32
10 pM 207
1908
89.34
100 pM 207 control
1856 2135
86.91 100.00
1 32 of 34
[TNO-repori of study 3M#01 experim ent 3 compound 208
Final version
18-04-2005)
Hamster 8
Condition excess cold TCA
1 pM 208 10 pM 208 100 pM 208
control
# dpm 1 4711 2 45213 3 61953 4 62903 5 70113
mg dpm/mg # dpm JQL dpm/mg # dpm mq dom/mq 17 277 10 6826 30 228 11 6344 19 334 18 2512 9 65417 32 2044 12 44266 23 1925 30 2065 8 74003 31 2387 13 30260 20 1513 21 2995 7 75323 28 2690 14 24664 18 1370 24 2921 6 76474 30 2549 15 33794 27 1252
mean Condition excess cold TCA 1 pM 208 10 pM 208 100 pM 208
control
dpm/mg 280 2160 1988 2352 2241
specific TCA uptake (= TCA uptake in dpm/mg - mean dpm/mg excess cold TCA)
Condition
# dpm/mg # dpm/mg # dpm/mg
1 pM 208 10 pM 208
2 2232 9 1765 12 1645 3 1786 8 2108 13 1233
100 pM 208
4 2716 7 2411 14 1091
control
5 2642 6 2270 15 972
Mean specific TCA uptake (and % of control)__________________________
dpm/mg % of control
1 pM 208 10 pM 208 100 pM 208
1881 1709 2072
95.90 87.14 105.67
control
1961
100.00
33 o f 34
fiN O -report o f study 3M#01 Appendix 3.4 Composite data experiments 1-3
Final version
test compound 206 fuMT
1 10 100
hamster 1 92.54 70.23 72.56
% of control hamster 4
81.49 83.86 51.77
hamster 7 89.89 91.88 94.87
18-04-2009
mean % of control
87.97 81.99 73.07
SEM
3.3 6 12.4
test compound 207 fuMl
1 10 100
hamster 2 88.79 86.14 95.00
% of control hamster 5
90.36 84.10 98.27
hamster 8 84.32 89.34 86.91
mean % of control
87.82 86.53 93.39
SEM
1.8 1.5 3.4
test compound
JQgJpML........
1
10
100
hamster 3 80.04 92.48 92.74
% of control hamster 6
75.08 99.01 92.10
hamster 9 95.90 87.14 105.67
mean % of control
83.67 92.88 96.84
SEM
6.3 3.4 4.4
