Tobacco Documents Online

r INTEROFFICE MEMORANDUM TO: J. D. deBethizy DATE: Dec. 20, 1990 RE: Proposed experiments for 1991 FROM: Jim Walker on nicotine perception ~' I would propose the following experiments as a way to begin to understand the possible effect of different compounds on the sensory perception of vapor phase nicotine: I. Psychophysical and respiratory responses to mixtures of levulinic acid and nicotine in normal and anosmic subjects Rationale: This experiment will determine the quantitative relationship between levulinic acid concentration and degree of effect on nicotine perception. Comparison of the results for normal and anosmic subjects will indicate the relative magnitude of the effect for the human olfactory and trigeminal nerves. Support needed: We will need to have analytical chemistry assistance with the calibration of the olfactometer for levulinic acid. If we use custom-fitted masks, we will also need PT&D support. There is a good possibility that the generic mask used by Dr. Warren at UNC-CH will be sufficient. Description of experiment: Five normal and five anosmic subjects will be given short tracking sessions in which sensitivity (odor and nasal irritation and respiratory behavior) to levulinic acid will be estimated. This will be used to equate the subjective intensity of levulinic acid to be used in mixtures with nicotine. This will be followed by longer sessions in which mixtures will be presented and psychophysical and respiratory responses will be recorded. Resources needed: Following 35 hours of preparation by Jim Walker and Roger Jennings, 60 hours of testing (supervised by Tom Guy) will be required. Data review by Jim Walker will require 25 hours. Alternatively, some or all of this research could be done, on a contract basis, in Dr. Don Warren's laboratory at UNC-Chapel Hill, requiring slightly more time due to the calibration of the olfactometer for levulinic acid. Cost: Timing: Subject fees: $4500 Lab supplies: $2000 Calibration, programming prep. - Jan. 8-19, 1991 Subject testing Data analysis, reporting of results - Jan. 22 - Feb. 22 Ln m - Feb. 25 - Mar. 1 co ~ w Ln I--

IL Psychophysical and respiratory responses to nicotine following pre-exposure to levulinic_acid in normal and anosmic subjects ~ Rationale: We predict that there will be some decrease in sensitivity to nicotine resulting from addition of levulinic acid observed in L If this is due to events occurring in the olfactory and/or trigeminal epithelium, we would also expect a decline in nicotine sensitivity with levulinic acid pre- exposure. A weak or non-existent effect in the pre-exposure study would suggest chemical interaction between the two compounds prior to deposition in the nasal epithelium. As in L, comparison of the results from normal and anosmic testing will demonstrate the relative magnitude of the effect for the human olfactory and trigeminal nerves. Support needed: If we use custom-fitted masks, we will need PT&D support. Description of experiment: With the same subjects used in I., sensitivity to nicotine will be determined as a function of the concentration of levulinic acid used in the pre-exposure period. Resources needed: Following 20 hours of preparation by Jim Walker and Roger Jennings, 50 hours of testing (supervised by Tom Guy) will be required. This will be followed by 20 hours of data review by Jim Walker. Alternatively, some or all of this research could be done, on a contract basis, in Dr. Don Warren's laboratory at UNC-Chapel Hill, requiring approximately the same amount of time. Cost: Subject fees: $3500 Lab supplies: $1000 Timing: Programming preparation/olfactometer setup - Mar. 4-8 Subject testing - Mar. 11 - April 2 Data analysis, reporting of results - April 3-5 III. Psychophysical and respiratory responses to levulinic acid alone in normal and anosmic subjects Rationale: If I. and II. above show a clear effect of levulinic acid on nicotine perception, we would need to collect data on the sensory properties of levulinic acid when presented alone. In this way we can relate, for each levulinic acid concentration, degree of effect on nicotine perception to intensity of odor and irritation. Support needed: If we use custom-fitted masks, we will need PT&D support. 2

Description of experiment: With the same subjects used in I. and IL, psychophysical and respiratory responses to levulinic acid will be determined. Resources needed: Following 20 hours of preparation by Jim Walker and Roger Jennings, 30 hours ^ of testing (supervised by Tom Guy) will be required. This would be followed by 20 hours of data review by Jim Walker. Alternatively, some or all of this research could be done, on a contract basis, in Dr. Don Warren's laboratory at UNC-Chapel Hill, requiring approximately the same amount of time. Cost: Timing: Subject fees: $3500 Lab supplies: $1000 Subject testing - April 8-19, 1991 Data analysis, reporting of results - Apri122-24, 1991 IV. Effect of levulinic acid on perception of acetic acid, amyl acetate and propionic acid Rationale: Assuming that levulinic acid is shown to alter nicotine perception, it will be important to determine the degree to which the effect is specific to nicotine. To address this we will examine, using both the pre-exposure and the mixture approaches, the effect of levulinic acid on the perception of acetic acid, amyl acetate and propionic acid. We have already collected a great deal of data on the psychophysical and respiratory responses of normal and anosmic humans to these three additional compounds. Support needed: If we use custom-fitted masks, we will need PT&D support. Description of experiment: Using the same subjects and approaches (both mixture and pre-exposure) used in I. and II., we will examine the effectiveness of levulinic acid in decreasing sensitivity to each of these three additional compounds. Resources needed: Following 5 hours of preparation by Jim Walker and Roger Jennings, 100 hours of testing supervised by Tom Guy in our automated odor sensitivity testing lab will be required. This would be followed by 35 hours of data review by Jim Walker. Alternatively, some or all of this research could be done, on a contract basis, in Dr. Don Warren's laboratory at UNC-Chapel Hill, requiring approximately the same amount of time. Cost: Subject fees: $7500 Lab supplies: $1000 Timing: Subject testing - April 29 - May 31 Data analysis, reporting of results - June 3 - 7 ~ 3 Ln w

V. Neurophysiological measurement of effect of levulinic acid on perception of a ~___--_---------- -. _ . _: _ _ Rationale: Assuming that levulinic acid is shown to alter nicotine perception, and that the effect is seen in both normal and anosmic human subjects, investigation of the underlying events in the olfactory and trigeminal epithelia will require an animal model. Recordings of the electrical activity in the olfactory and trigeminal nerves, in response to manipulations like those in IL and IL, can be used to demonstrate an effect of levulinic acid on the peripheral sensory response to nicotine. Following this demonstration, binding studies could be used to determine whether the effect is due to an interaction of levulinic acid with receptor molecules for nicotine. Support needed: We will not require outside support. Description of experiment: The olfactory nerve (pigeon) or a branch of the maxillary division of the trigeminal nerve (rat or pigeon) will be cut and the proximal (brain) end will be placed on electrodes for multi-fiber recording. An existing automated olfactometer, similar to that we now use for human experiments, will be used to deliver odor stimuli so that we can measure the response to: i. nicotine following pre-exposure with levulinic acid; and ii. mixtures of this compound and nicotine. Resources needed: Following 50 hours of preparation by Jim Walker and Roger Jennings, 150-250 hours of laboratory experiments conducted mostly by Roger Jennings will be required, followed by 80-100 hours of data review by Jim Walker and Roger Jennings. Cost: Lab supplies: $3000 Timing: Setup - February 4 - 22 Testing of animal subjects - February 25 - April 12 Data analysis, reporting of results - April 15-30 VI. Respiratory physiology in dogs Rationale: Subjective reports of the effects of the addition of levulinic acid to cigarettes indicate that overall sensitivity of the upper respiratory airway from the oropharynx down into the trachea is decreased. To determine if sensory receptors in these areas are stimulated less with levulinic acid addition, physiological experiments, using dogs, on these portions of the respiratory tract could be conducted. Experiments with hexamethonium aerosol suggest that the respiratory reflexes seen when smoke is delivered directly to the larynx and trachea are due in large part to nicotine. This work has been conducted by Dr. L. Lee and his colleagues at the University of Kentucky. The same paradigm could be used to examine the possible effect of levulinic acid vapor or aerosol on respiratory reflexes and or afferent vagal activity in response to smoke andlor nicotine alone. 4

Support needed: This experiment would be conducted on a contract basis. Description of experiment: As described in publications from Lee and colleagues, two related approaches would be used: 1. Respiratory reflexes such as bronchoconstriction would be measured in conscious tracheotomized dogs in response to smoke and nicotine alone; the effect of both pre- exposure to and mixture with levulinic acid on these responses would be quantified. 2. In anesthetized dogs in which vagal afferents were exposed and placed on recording electrodes, the effect of levulinic acid on the sensory nerve responses to nicotine and to smoke would be measured. Resources needed: Contract funds. Cost: est. $100,000 Timing: to be determined VIL Measurement of permeability of nicotine through epithelia in the presence or absence of levulinic acid Rationale: Levulinic acid could act to decUP ay chemoreceptor stimulation by decreasing the ability of nicotine to perm~ate through thelia in which some of these receptors are found. One way to determine this is~to directly measure nicotine's permeability through epithelia in the presence or absence of levulinic acid~ Lloyd Beidler, an 1RT grantee, has developed a technique for making such measurements and.has used it to meas nicotine permeability through the epithelium on the dorsal surface of the tong~u s a function _ H. This approach could be used to measure the permeability of nicotine or other smoke cotnponents through various kinds of epithelia (including that found in the nose or larynx) as a function of levulinic acid concentration. Support needed: This experiment would be conducted on a c'ontract basis. Description of experiment: As described recently by Beidler ` a seminar presented at BGTC, the epithelium of interest would be removed from the animal and placed between two chambers. On one side of the tissue mixtures containing radio-labelled nicotine (ana possibly cigarette smoke condensate) and various concentrations of levulinic acid would be\placed. Samples taken from the other side of the tissue would be used to estimate the quantity of r~cotine per unit area permeating through the tissue in a given length of time. This information is used to calculate a permeability coefficient that would indicate relative degrees of access to chemoreceptors embedded in epithelia of the upper respiratory airway. Resources needed: Contract funds. Cost: est. $50,000 Ln m CO Timing: to be determined \V I ~ %D w Ln 5 cn