Document 8RrvrvMGkDaDvxZ0wY4byEyDK

f\RM >-o\G\ SUMMARY OF THE EFFECTS OF PFC's ON MITOCHONDRIAL BIOENERGETICS IN VITRO The attached diagram describes the metabolic relationship for the various PFC's that were selected for inclusion in this study. The metabolic chart is adapted from the original as provided by Dr. Steve Gordon. Included in the diagram is a summary description of the biological activity of each metabolite, the details of which are described in the appended report and supplement. The identities of all test compounds were withheld from the person who performed the bioenergetic analyses (Dr. Anatoli Starkov) and the following legend reveals the code to the test compounds as referred to in the research report. Code PF10 PF10H PF12L PF12M PF95 PF95M PF143 SAL 3M identification______________________ FC10 the acetic acid of FC10 FX12-linear FX12-mixed linear and branched FC95 the sulfonamide of FC95 (N-de-ethylated FX12) FC143 salicylic acidincluded as a positive control To briefly summarize, we found that FC95 and FC143 were weak inhibitors of m itochondrial bioenergetics in vitro. They would have to bioaccumulate to substantial levels to pose a significant concern in vivo. On the other hand, FX12 (both linear and mixed) and FC10 and its carboxylic acid metabolite (PF10H) were far more potent. The IC50 for the protonophoric uncoupling of m itochondrial respiration by FX12 (ca., 6 (iM) compares to that of the classic uncoupler 2,4dinitrophenol (DNP). W hether safety concerns are warranted depends on if these metabolites are stable and if they bioaccumulate to sufficient concentrations during the course of chronic exposures. 004191 Our greatest concern is for the sulfonamide (PF95M), which is 5-fold m ore potent than DNP as an uncoupler of mitochondrial oxidative phosphorylation. The IC50 for the sulfonamide is approximately 1.5 jxM, which compares with that of the m ost potent mitochondrial uncouplers known. It is highly reasonable to suspect that exposures to the sulfonamide, or its generation in vivo from an N-substituted precursor metabolite, may be associated with demonstrable symptoms attributable to the uncoupling of mitochondrial respiration. This concern is compounded by the rapid N-de-ethylation of FX12 in vivo and bioconcentration of the sulfonamide in liv er12. Since this metabolite has been detected in sera from both rats and h u m a n s3, an aggressive kinetic characterization seems warranted. W e suggest that future research focus on the sulfonamide. `Johnson, J.D. and Ober, R.D. (1979) Study No. 137-093, International Research and Development Corporation, Mattawan, MI 2Manning, R.O., Bruckner, J.V ., Mispagel, M .E., and Bowen, J.M . (1990) Drug Metab. Dispos. 19: 205-211. 3Anon (1979). Tech. Report No., 723Q, 3M Central Analytical Laboratory, St. Paul, MN. 004192 May 1, 1998 C H 2C H 3 CQF17 -SO0-N \ 8 17 L c h 2c h 2o h FC10 weak, increases passive proton leak @ 60 f/M c h 2c h 3 CsFi7-S02-N ^ c h 2c o o h c h 2c h 3 C8Fi7-S02-N ^ H FX12 strong, protonophore uncoupler @ 6 (mixed and linear are comparable) PF10-H strong, increased permeability to ion conductance @ 6 /iM H c 8f 17 -s o 2-n < CH2COOH CgF 1 7-S 02-NH 2 PF95-M extremely strong, prontonophore uncoupler @ 0.5 fiM Cmpd VIII did not receive for testing C8Fl7-S03- FC95 weak increase in membrane fluidity @ 10 fiM c 7f 15-c o o h FC143 weak detergent @ 100 juM 004193