Tobacco Documents Online

Tlll: O/t'\('li. F(/1i T/)ltA('C'(1 h'I:Sh.1RC1I-j.'.S.1,., INC. August 28,• 1978 MEMx1RANDUNI TO: Drs. Gardner, Sammers and Jacbbscn FROb't: R. C. Hockett SUBJrECT: Application No. 88bB Albert Castro, Ph.D., University of Miami School of Medicine, Miami, Florida "Nicotine and its Metabolites in Blood and Other Tissues: Studies by Immunoassays (Radio-, Enzyme and Fluorescence Polarization) and on Brain Binding Sites". This proposal which is classed as a continuation for competitive consideration de novo contains four parts: Proposal #1. Continustion and rounding out of work on the nicotine . radio immunoassay in blood and plasma: The report of the past year's work is very poorly written . Though there were extenuating circumstances in (a) his usual difficulty in expressing himself in English, and (b) the recent very serious illness of his wife, the problen goes beyond these difficulties. He has worked with several synthetic nicotine antigens, nearly all ably prep'ared and well characterized by Herbert McKennis: Several have produced anti=• bodies in goats and more recently in rabbits so that Castro has had more nicotine- active antibodies to work with than any other investigator we know about. He also has an as'say, bu} it is not very satisfactory. I would gather from his experience that all these antibodies have a rather weak affinity for nicotine and low stability. A very strong]y radimactive label is required for decent results. A nicotine derivative containing a large amount of 1251 had to be used to get a reasonably good assay. It was aa prioprietary substance furnished by Union Carbide and Castro did not even know its structure. It is costly and has a short half-life, hence has to be purified and assayed before each use. The strong radio-activity requires special facilities for handling and imprses a hazard besides. The best assays were obtained with the automated "Centria" apparatus, also supplied by Union Carbide. This method is based upon competitive reaction rates of labelled and unlabelled nicotine (as determined by relative concentrations) so that it does not have to be run to equilibrium and this saves time. To complicate matters, Union Carbide seems to have lost interest in furnishing the iodinated nicotine derivative, and highly triteated nicotine of reliable quality seems no longer to be available commercially, reputedly because it did not keep well enough to be profitable. More than a year ago when the assay seemed to be going pretty well, I urged Castro to do a series of replicability tests in the conventional manner to see how well ptce nicotine added to freshly taken dog and human blood could be recovered. He visited New York last May with his new (able) assistant Dr. Monji and Dr. McKennis to Ahow me the results. They appeared quite chaotic to me. In some experiments the nicotine seemed to be disappearing from serum on storage (metabolism?) and in others it seemed to increase.

Page 2. WEMORANDUid-Re: Castsa 8/28/7C3 An ___deL~ndent method seemed necessary to determine whether nicotine really is metabolized in drawn blood,(a question which nobody seems to have studied heretofore) before the capabilities of this assay could be appraised. McKennis supplied pure samples of a dozen or more metabolites of nicotine and nicotine analogs for study of cross reactions with the nicotine anti- body. As of May 1978, none of these appeared to have been studied. Perhaps confidence in the assay did not justify the work, or else the reagents were not available. (Now, I am told they have all been run but wonder whether the results er1•ll be meaningful ) . Considering the quite limited success other investigators have had with nicotine radioinpnunoassay, (Vesell, the Japanese group and Armitage for example) I am inclined to think that there are basic problems. I have urged Castro to do all he cgn between now and December 31, 1978 to study replicability of the assay and the extent of cross-reactions with nicotine relatives. Unless there is some kindcf break-through, I would counsel discontinuing the study of nicotine radioiimnunoassarj at that time. This conclusion is strongly reinforced by the fact that improvements have recently been made on the chrematographic methods of nicotine and cotinine assay of both nicotine and cctinine reliably, in a single plasma sample as small as 1 to 3 ml: (See Hengen and Hengen, ^1inical Chemistry 24, 50 (1978) copy attached). This method is being explored in Dr. Vesell's laboratory with very good success. I spent a day there and was well impressed. It would appear to be adaptable to tacking a number of the practical problems on our docket that have, been awaiting a suitable assay method. At my suggestion, they agreed to begin at once to look for any indication of nicotine degradation in fresh plasma or serum in vitro. (McKehnis is studying the Hengens' paper for any pitfalls he my recognize). Proposals #2 and 3. Castro and his assistants have surveyed the rather ab%%adant literature in search of other physico-chemical methods thst might be substituted for radio-active labelling in assaying the extend of bin" nicotine with antibody. They have selected two that they regard as the most promSsing-- an enzyme assay, and a fluorescence polarization method. Apparently considerable preliminary work has been done on these. The Board will have to decide whether these ventures are s±Lt`ficiently ptanising to justify support from CTR. Here, some outside consultati:jn may be needed. Proposal #4. This presents the matter of adapting the nicotine antibody for labelling of nicotine binding sites. This proposal will have to be considered in relation to the present restrictions on CNS studies as well as the competitive values of the now numerous other approaches that have already shown promising progress. R .C .H. Note: McKennis' current continuation is largely geared to Castro's proposals. It will obviously have to be revised in the light of what decisions are made about Castro. I can see the need for McKennis' consultation and assistance in many projects. His status will need to be considered carefully. Meanwhile his current grant needs b be pro-rated from September 1 to December 31, 1978.