Tobacco Documents Online

V ; K E 1 2 0 (', :, 'I'm: C'nu.Ncu.Fuie Tom%cco Ri:si:.»:cn -U.S.A.. INc. March 25, 1980 Na7oRAA1DUM M: W. U. Garc3ner and Staff F`laCaN1: D. H. Ford and D. Stone SUE'JFC-T: Grant 1208 A. Carlton K. Frickson, Ph.D., The University of Texas, Austin. "Blood-Brain Nbnitoring of Sustained Nicotine Levels in Rats." SITE VISrI' on March 12, 1980. This study, has proceeded :rore slowly than anticipated, largely L-ecause the type of device with which they had previous experience as recently described for release of morphine (J. Pharmaceut. Sci. 68:622, 1979), using a silicone polymer, released the nicotine base so rapidly that the animals convulsed and died. T`his led then to develop and test a number of other devices (see progress report) which has culminated in a glass cup sealed at one end with an elastMmer. Whet iinplantezi upside down under the flank skin of a rat o these devices have been used to release varying mg amounts of from 1.4 to 4.0 mg of free nicotine base/24 hours. Amount released based on data obtained fran in vitro testing in water with amount of nimtine released dPteraLined by U.V. absor;atio'~ n: Aor b»6b4. Y1I G.+ar.a bA.St t/.,ItI w,c,L Inplants have been left in place for up to 19 days without detectable pathologic change other th2,n the formation of a connective tissue capsule around the cup. Since the formation of such a capsule may cause sane plugging of the elastQmer ^.nd of the cup through which the nicotine diffuses, tests have been un3ertaY,en to determine if this occurs. So far inVlantations of 2 and 5 days have not impaired nicotine release from the cup when it has been removed frcm the rat and tested in water in vitro (see table). Plan to test this after 10 and 20-day implantation periods next. They assxmie that nicotine release is occurring in these tests since hypothermta develops, I,iave done samp prellmir,ary tests with nicotine tartrate in the cup, but so far it fails to be released. Adding alginate or lactose to the solution has not helped. Measurements of nicotine in blood and brain, using GC with N-ettaylnornicotine-bis-oxalate as an internal standard. Has c,orkedl well for nicotine suspended in water and in extracts fran whole blood, assaying nicotine levels as low as 2.5 ng. Extraction procedures which have been developed work well witk: blood; ho4mver, scme contaminants are still appearing in the assay of brain extract. In this test, nicotine was added to whole blood and then extracted. Have not yet lone any tests where nicotine reached blood fram an implanted release device.

Cll)ts, Expect wnrk perfarmed during next 3-6 months to provide data on distribution of nicotine and cotinine in blood qd brain following release fran the implanted glass cup. Also plaviing to measure C-labeled nicotine and cotinine in an at.teavt to quantitate relative levels of the base and its prir,iary metabolite with time after release fron an implant. Will use TIC for this aspect of the work. (Note: one problem with the U.V. assays of nicotine release in vitro into water is the possible presence of the N-oxide of nicotine in the ass'ayed fXuid. This could be particularly true where they were testing the long-term effects of storage of firee nicotine base in the sealer3 glass cups, where there may b6- sane breakdown of nicotine. So far their assays of nicotine in the cups after prolor~ged storage is t2iat there has been a minimrn of breakdown, 1-rut the presence of an N-oxide might be providing a false impression of only nicotine being present.) siol' ic rpasurements of response to nicotine Erickson has focased on h y _ while l9cGinity has rnrked on devel ping the release device and Stavchansky has worked on the assays. Animals injected with 4.0 mg/kg of nicotine twice daily (P7-Ahallenge) . convulse (sane die) after the first exposure. By the 10th day, convulsions are , <<~' markedly reduced, as is the -hypotheimic response to the N-challenge. (9bl.erance?) »~ Animals pre-it:jmlanted with a cup containiiag nicotine for sustained release 3 days before N-challenge show less convulsive response, though the initial hypothex-rai.c ,,..; response is greater than with N-challange alone. By the 10th day, there is a ~! ;,„ipoecrrn_olete lack of hvpothermic or convulsive response to N-challenge, again suggesting A„ tolerance. Animalsinjected with saline and only one N-challenge dose/day show some<ahat less response and also tend to develop tolerance. A new control will be ,y~.. ~.,,. ~. added to these groups which be to implant N-release devices containing only water e ~qrl d th b t h ll en su ~ an ject a o c eng P.c I '~e ,...f: -. ~ ~''° Pleasurements of activity in a open field device with a crossed electric- eve eye circuit showed that 1.0 mg of nicotine challenge increased activity markedly in controls. Animals previously implanted with a continuous nicotine release device showed a significantly less n.arked increase in activity. The dose administered here presunably represents the 1.0 ng/kg challenge dose plus whatever was released frcdn the cup - thus a total of a slightly higher dose. 7.'hus, this data may illust.~ate that low doses of nicotine stimulate activity and that, as doses increase, the degree of stimulation decreases until one would expect to observe the depression known to occur with higher doses. They plan to investigate the possibility. Studies on weight gain or loss after nicotine show that twice daily injections of nicotine or saline injections had no effect on weight. Howev'er, anxmals with the implants lost weight. Pair-fed studies for the control groups as well as on animals with unloaded implants are indicated here and being planned. Review. (1) Tissue extraction procedures apnear to be established for whole blood loaded with nicotine. While obvious nicotine~ peaks were obtained by CC fram ea^tracted brain lxmogenates after nicotine loading, a nunber of other peaks also occurred which Stavchansky did not seem to feel would be difficult to clean up. (2) Creation of a release device for nicotine base appears to have been acconp?.ishhd with primarily t-)e in vivo testing yet to )e done to be sure it does not clog during prolonged inplantation-73) Testing for nicotine and cota.nine in blood and brain folloioing im.plantat.ion still renains to be done also. (4) The develoanent of the bioch®nical assays for Wotine and cotininp in blood and brain appears almost cc.mplete using (aC and PLC (with C-nicotine) . They will be emphasizi.ng the ccnpletion of the

- 3 - HK~1`"iSOh"r extraction and assay procedures during the next months, and predict that these will be completed during the next 3-6 months. They then expect to begin to explore various physiologic or pharmacologic studies: (Curent studies on hypothermia, weight, and activity are performed essentially to manitor that nicotine is escapying fran the release device.) A paper will be presented at the forthoaning Federation meeting by S. A. Stavchansky and J. ASeGinity which aclanaaledges CrR support. Title: Nicotine in Sustained Release Form: In Vitro and In Vivo. C.rnment. Reazmm-d one more year of support as they seem close to oanpletinq the tievelopnent of a sustained release device in oonjunction with the necessary testi,exJ to ensure that a steady rate of release is obtained and can be monitored reliably in blood and tissues. However, any subsequent studies dealing with physiologic or phanmacologic paradigms should be reviewed ccmpet `atively. b. H. Ford D. Stone /ek encl.

NK0 i~.' 7 :SC1U(S ~ Table Pirst Dtperiment UV 74bsorLame Uav for N released Location __.. ~.__. 1 0.407 2 0.432 In water 3 0.405 4 0.407 T}= put in rat for 2 days. In rat 7 0.426 8 0.423 In water 9 0.420 Second fkpesiment 3 0.398 4 0.441 In water 5 0.491 6 0.454 Then rut in rat for 5 days. In rat 12 0.435 13 0.435 In water These data suggent no cloggi.ng of implant when placed in rat.