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Notes on analytical reports prepared by Dr. Renwick, University of Southampton. X;hese reportsnow form a complete record of the analysis of all_ the samples taken during the four phases of the pl~armacokinetic / pi~armacodynamic studies at the General Hospital, Southampton, jointly by the Clinical Pharmacology Group (Professor C F George) an.] BAT. The planned experiment was the largest and most comprehensive of its type although, since beginning the work, several studies covering various aspects of the ;.;ork, have appearred in the literature (e.g. Benowitz's work and the ~",othman sponsore.~ stu~y). The purpose of the study was to compare the phar~aacodynnmic effects of nicotine (e.g. effects on heart rate, skin t~mperature (as an index of peripheral blood flow), systolic and diastolic blood pressure) wi~en the same dose of nicotine ,.-;as taken by inhalation (via cigarette or by synthetic aerosol) an."] by intravenous injection, thus deter~nininq the importance of route of administration to the peak and trough levels of nicotine in the plasma (and, by inference, in the central nervous system) and on the physiological effects of the compound. Subjects ~ere recruited as ~er the arranjed protocol and, during the first phase of t!~e stu',~y, smoke.] a State Express cigarette with nicotine delivery corresponding to th,eir own preferred blend. Durin,j phases 2 and 3, a sub-grou[; of ~ive subjects fron] the main group of fifteen recei:zed ~he noL~in-,-ul dose o~P nicotine of the cigarette smoki~, as a~,, intr..%venous dose either infused over 10 minutes (phase 2) or as a series of boli at :~::I minute intervals (phase 2). Phase 4 involvec~ the sa~e d~e ef nicotine a<]:ninistere~! as a series of 8 boli at I uinute i!~terva!s via an electronic nebuliser. Initially, the bloo:l sam!:~les taken during nicotine dosing were analysed (as plasma) for nicoti:~e and cotinine using an ,~LC metho~ develo[~ed at GR 3 UC, Southampton. This method offered considerable a~]vanta.jes over existing methodology, al!o~:ing determination of both nicotine and cotinine in the same sample, at io~ levels of detection, by a method capable of automation. Analysis of samples fro~] Phase I (own blen~ smokinl) of the stu'.[y >;as undertaken at G2 .~, DC butdata production did not keep up with arrival of samples. ~, the Thus, in smite of the .aqvantages of ...... ~ by metho,], the ma.npo~.:erava ilable prove~ un.~qu~l to t~.~e rate of analysi~ required. Analysis ~.~as therefore contracte./ out to Dr A G BAT Industries document for Province of British Columbia 17 November 2000 BAT INDUSTRIES 00290311

Renwick of Southampton University :.d]ose team had considerable experience of sample purification and HPLC techniques. Dr Renwick's group modified the G:", & DC methodology to includi a 3H nicotine internal standard (allowing accurate correction for losses duri~.~g purification) and optimised the extraction procedure to maximise both recovery and rate of s~laple throughput. : Report number I covers most of this assay optimisation procedure. It was decided that assay of the samples from Phase I should be completed at G~ ,5 DC to avoid the problems associated with change of method/o!?era tot mid sample. Dr Renwick's group therefo~-e comr.~enced assay ~-it!~ the samples from Phase 2. Data from Phase I sa~nples are available from Ian Anderson. Report number 2 concerns the data from sa,nples taken following intravenous infusion of nicotine. Concentrations of nicotine in the samples ;,~ere very io~.: and thus close to the limit of detection of the methodolo,fy. In general, duplicate results were reasonably reliable but there :.;ere also some unaccountably Door matches. Cotinine concentrations ~.;ere, not unexpec t~_dly, higher due to the lon~er half life of the metabolite in plasma. Again, as e::pec~ed, large interindividual " differences in cotinine concentrations were apparent. Ho effects of the nicotine infusion on cotinine levels were obvious, but a~Tain this mig!~thave been predicted given (a) the large starting cotinine values and (b) the relatively short sampling period Vost infusio:~ (2 hours). Reporting of the data from the intravenous infusion studies (Phase 2) i~ complete.~ in Zeuort 3. Given t!~e complete data for intravenous infusion fro:~ all 5 subjects in this phase of the trial, several conclusions can }~,e dr:n:.~n from this part of the study. (a) Poor nicotine value dunlicates often seem to be due to random contamination of the sa!.~pl-_~s rather than ~ipetting or extraction errors since cetinine ,'].u:~ lica te s for the salae samples are alright an'~ the 3[! nicotine soike recoveries are reproducible. (b) Caffeine interference still proves a problem occassionally in spite of atte~p~s to :ninimise pre-e::Deriment c.=~ffeine intake and modification of the UPLC methodolo,/y to ~.~a:,.imise the se[]aration bet:..;ee,~ cotinine and caffeine peaks. (c) Some subjects appear to l,ave c!,eate'~ !)y smohing i~!nnediately prior to the study (pro-dose nicotine levels higher titan pos~-dos9 levels ! ) BAT Industries document for Province of British Columbia 17 November 2000 BAT INDUSTRIES 00290312

Since the data are less than ideal, it is useless to perform any sophisticated phar::lacokinetic analysis, howeve9 an attempt to derive some useful data has been made. U's ing corrected data (i.e. taking pre-dose nicotine levels, into account) the area under the curve has been plotted for each subject, ~.;here possible. [.]aturally, any existing nicotine in plasma is eliminated at the same rate (i.e. with the same tl/2 ) as the dosed nicotine , thus for this calculation it is more correct to use unmodified data (i.e. without correction for initial nicotine levels). Due to the poor quality of the data, little new can be learned about nicotine pharmacokinetics. This poor data quality is not a reflection on the !IPLC assay, nor on the operators, since the data are as good as could be expected given the low plasma nicotine concentrations present. Knowing the levels of plasma nicotine found for the nicotine infusion.study, it comes as no surprise that the concentrations of nicotine in the intravenous bolus study and the aerosol study are, again, lo~;er than hoped. Forewarned, an improvement was attempted by using ~he whole plasma sample for a single estimate of nicotine (thus doubling the concentration but removing the facility of duplicate estimates). Broadly, an overall pattern' can be detected in the sa:~rples which is consistent with the bolus nature of the do~ing, but auain the data require considerable correction and deletion of inappropriate values, where d_~ta do not meet pre-existing quality criteria. Overall, the plasma nicotine concentrations have been too low for accurate d_~termina tion using the current methodology. It is unfortunate that analysis of samples not proceed apace at the outset of the study. It would then have been clear, befor~ the intravenous or aerosol Dhases, that the doses being administered ~;ere too low. Hi~her dose reuimes could then have been instituted ( note that, in the absence of data to the contrary, Professor George/~)r Upwoed insisted lowe~ nicotine doses for the intravenous study than thoes which . . t~_ absence of BAT personnel recommended) Alternatively, in ' ~ higher nicotine doses, fe~-~er but larger samples could have teen taken to allow a larger amount of extractable nicotine in each sample. Despite the problems with the ~3ata, the concentrations of. nicotine found are consistent with ,]ata re~orted by others (notably Feyerabend et al, De~o~itz e~ al, ~inclair et al). BAT Industries document for Province of British Columbia 17 November 2000 BAT INDUSTRIES 00290313

The maj or interindividual differences in plasma nicotine concentrations suggest that the pharmacodynamic effects of the dose may well be different in each individual, if there i~ any relationship bet~.;een concentration and effect. This should be checked. Overail, the study has been disappointing in that no re liable n e~.,; pharmacokinet ic data can be ,.~ er ived. In retrospect, it is easy to see areas ;-;here tl~e study could have been improved, however as a first "toe in the water" for BAT, we all learned a lot. Unfortunately, the underaking of a second study, benefiting from these mistakes, is unlikely. BAT Industries document for Province of British Columbia 17 November 2000 BAT INDUSTRIES 00290314