Document 6wVX5KRyaq01YQ6xmzEpY6VB3

SUMMARY OF THE EFFECTS OF PFC's ON MITOCHONDRIAL IN VITRO BIOENERGETICS The attacheddiagram describesthe metabolic relationshipfor the various PFC's thatwere selectedforinclusionin thisstudy. The metabolicchartisadapted from the originalasprovidedby Dr. Steve Gordon. Includedin the diagram is a summary descriptionof the biologicaalctivityof each metabolite,the detailsof which aredescribedin the appended reportand supplement. The identitieosf all testcompounds were withheld from the person who performed the bioenergetic analyses(Dr.AnatoliStarkov)and the followinglegend revealsthe code to the test compounds as referredto in the researchreport. Code PF10 PF10H PF12L PF12M PF95 PF95M PF143 SAL 3M identification FC10 the aceticacidof FC10 FX12-linear FX12-mixed linearand branched FC95 the sulfonamideof FC95 (N-de-ethylatedFX12) FC143 salicyliaccidincluded as a positivecontrol To brieflysummarize, we found thatFC95 and FC143 were weak inhibitorsof mitochondrial bioenergeticsin vitro. They would have to bioaccumulate to substantiallevelsto pose a significanctoncern in vivo. On the other hand, FX12 (bothlinearand mixed) and FC10 and itscarboxyliaccidmetabolite(PF10H) were far more potent. The IC50 for the protonophoricuncoupling of mitochondrial respiratiobny FX12 (ca.,6 gM) compares to that of the classicuncoupler 2,4dinitrophenol(DNP). Whether safetyconcernsarewarranted depends on ifthese metaboliteasrestableand iftheybioaccumulateto sufficienctoncentrationdsuring the course of chronicexposures. Our greatestconcern isforthe sulfonamide (PF95M),which is5-foldmore potentthan DNP as an uncoupler of mitochondrialoxidativephosphorylation.The IC50 forthe sulfonamideisapproximately1.5gM, which compares with thatof the most potentmitochondrialuncouplersknown. Itishighly reasonableto suspect thatexposuresto the sulfonamide,or itsgenerationin vivo from an N-substituted precursormetabolite,may be associatedwith demonstrable symptoms attributablteo the uncoupling ofmitochondrialrespirationT.his concerniscompounded by the rapidN-de-ethylationof FX12 in vivo and bioconcentratioonf the sulfonamide in liver". Sincethismetabolitehas been detectedin serafrom both ratsand humanS3, an aggressivekineticcharacterizatiosneems warranted. We suggestthat future researchfocus on the sulfonamide. 'Johnson,J.D.and Ober,R.D. (1979) StudyNo. 137-093,InternationRaelsearchand Development Corporation, Mattawan, @M 'Manning,R.O.,Bruckner,J.V.,Mispagel,M.E.,and Bowen, J.M. (1990) Drug Metab.Dispos.19:205-211. 'Anon (1979).Tech.ReportNo., 723Q, 3M CentralAnalyticaLlaboratoryS,t.Paul,MN. CH2CH3 C8Fl7-SO2-N< CH2CH20H FCIO weak,increaspeasssivperotonleak@ 60tiM C112CH3 C8F17-SO2-N < CH2COOH CH2CH3 C8F17-SO2-N < H FX12 strongp,rotonophoruencouple@r 6 pM (mixedand lineaarrecomparable) PFIO-H strongi,ncreasedpermeabilittyo ionconductance@ 6 liM H C8Fl7-SO2-N< CH2COOH C8Fl7-SO2-NH2 PF--95-A/I extremelystrong,prontonophore uncoupler@ 0.5pM CitzpdVIII didnotreceivefortesting I C8Fl7-SO3- FC95 weak increasienrnet-nbrane fluidit@y 10 liM C7Fl weak