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fiK rd 11 0 ") 6361 'Titc f'c~t'~c td. Fc)tr'1'OI;.ic'CtS Bc•. A;emorandum To: Dr. W. U. Gardner and Staff From: D. H. Ford and D. Stone I7ovember 7, 1080 Re: Site visit with Dr. A. Janoff and Staff on October 22, at the State University of New York at Stoney Brook, N. Y. Grant Title: "Further Studies on Suppression of Protease Inhibition by Cigarette Smoke", Grant # 1143A; and "Immunological Assay of Lung Elastin Degradation", Grant ~ 1259. During our visit, Dr. Janoff presented'hi~ usual well organized discussion of the progress accomplished by him and his staff. There have been some per- sonnel changes since our last visit. Dr. Harel has returned to Israel and been replaced by Drs. Laurent and Sachewitz. Both are experienced in pulmonary medicine. Mr. Carp has finished his studies and been awarded his Ph.D. and will be continuing with Dr. Janoff. One paper has been published recently (att- achment #1) and one manuscript accepted for publication (02). A third attache- ment (; 3) represents a diagram of Dr. Janoff's hypothesis for the protease; antiprotease imbalance which occurs in the lungs of cigarette smokers. In view of the divergent views of various investigators,on the respective roles of :N1-Pi and a2-MG in preventing protease degradation of elastin, we asked Dr. Janoff if he would enlarge on this schemaAig and review his own current opinion on this in- teraction. Briefly: his view is that elastase becomes.bound to of 1-Pi at the active methionine site and is completely inactivated. A small peptide appears to be split off from GK1-Pi at this time. The -a'1-Pi/protease then enters the blood and immediately breaks down. The rel'eased protease is immediately picked up by -N, 2-MG so rapidly that no protease can be detected free in the blood. However, the ot 2-MG association with the protease is a loose one, permiting some substrate to interact with the protease and be degraded. Finally, the t~, 2-tZ/ protease complex enter the liver and is degraded. Grant .1143A: This program breaks down into 6 separate projects. 1. This project is described in the renewal application and deals with the effect of cigarette smoke on the low molecular weight acid stable bronchial mucous inhibitor (BM Pi). This project has been completed and published (see attachment ##1). The BM Pi was obtained from sputum, partially purified and shown to be inactivated by smoke, chemical oxidants or a myeloperoxidase + H2O2 + C1' system. Phenolic antioxidants prevented the suppression of BM Pi activity. Further, the BNi Pi activity of tracheal aspirants from smokers was found to be 204 lower than that obtained from non amokerp. (Activity was measured against P:MI elastase ) . 2. This program was also described in the renewal and deals with the effect of pretreatment with anitoxidants on the loss of a 1-Pi activity which occurs after exposure to 6 puffs of smoke ( carases a 30°Jo decrease in act ivity in rats ). Can such pretreatment blunt the loss ok'o~ 1-Pi activity? Study undertaken by Dr. Carp. (over)

li E! 11036362 Four studies are to be t>erformed: :,. :,'ix to- week pretreatment with Vitamin ?, a fat saluble vitzmir.. Had no effect: B. Six to 8 week pretreatment with the water soluble vitamin C. Results currently being calculated, but not yet available. C. Short pretreatinent by aerosol wit'h an improved form of N.ucota$st (a reducing agent acting at disulfide bonds). They are workinr on this with a Dr. Drew and a Mr. Norman from the Brookhaven ilatiomal Laboratories. Both of these individuals are knowledgable in ae'osol engineering. Will proceed with the Mucbmitt exposure as soan as an adequate aerosol system is developea which :aill deliver the drug pnst the turbinals into the pulnonary tract system. D. This is also a short term aerosol drug exposure study, but will utilize Schneblis' drug (components unknown; however, Dr. T;rxvis has observed that it protects a 1-Pi against oxidation). Still waiting to receive drug from Schneblis. 3. ThAs project is also described in the renewal: Is there a corre- lation between the degree of depression of oi 1-Pi activity in the bronchial pulmonary lavage fluid of smokers and the degree of airflow obstruction. (which could vary between subjects). Has been undertaken by Dr. Carp. A. a(1-Pi in lavage fluid to be measured and correlated .6-ith various physiologic measures of lung function (ie., FEV1). This part is almost completed and so far there appears to beN„gorrelation between degree of depressed o(1-Pi activity and degree of pulmonary obst- ruction. However, since the physiologic measures did not different- iate between individuals with emphysema and those with chronic brbnchitis, amlysis of these factors may alter tlieir interrretaticn of' the,data. '!"his aspect will be followed up. B. a: 1-Pi in lavage fluid will be compared with morphometric indices of l::r:e fupction, using lung samples obtained at biopsy. This aspect, which will measure size of alveolar sacs, etc. awaits clinical collaboration and ma,}P be slow in developing. Suggested they contact Dr. Eugene Bleecker, chief of i.ulmonary research at Johns Hopkins. k. This project conceives that within the MM a 1-Pi genotype there may be subtle subtype groupingtsuch that one subtype within the population might be more susceptible to pulmonary damage by oxidants than another. This program is just beginning and will be superyised by Dr. Laurent. ModelT Will collect serum from the patients used in project #3, using sera from patients repres- enting a broad range of _Mls. Dr. Laurent has developed an assay for this project wherein the serum is exposed to a chemical oxidant and ther the rate of loss of o(1-Pi activity over a 30 minute period is measured. Will use this assay to compare o(1-Pi activity loss rate in sera from smokers with severe pulmonary disease, no disease and from a non smoker (himself) as a control. (Obviously needs more not ~smoking control subjects).

HKa~~0363b3 Preliva aarv Resuits Subject FEV1 Diagnosis Inactivation 5ate Healthy Smoker 1 3 liters - 109 Sick Smoker 2 1 liters ? >>> lco Sick Smoker 3 1 liters ? 101) Sick Smoker 4 1 liters ? 10~1 Control (Laurent) 5 3 liters - 14C If sick smbker (?Jr12) with greater than 1001~, inacti atiorr shouil ha,re emphysema (once diagnosis is made), assay could prove to be of value for identifying subjects at risk for developing emphysema as a result of a greater than normal rate of inactivation of o~I-Pi. Plan to test 10 more subjects using sick and healthy smokers and will-correlate with clinical evaluation of subject. 5. This is also a new project and is based on the observation that there is still no clear proof that inactivation of a 1-ri is c.-used by o>:i3ation of methionine to ret ;:ar,ine sul_°oxide. To "e under*.eken by Dr. Sicot^t: end Teno.'f. MAy plan to analyze the o( 1-Pi obtained by lavage from smokers and non smokers. The x 1-?i will be isolated and purified and then cleaved, partly by cyandgen bromide. Will then determine the amount of methionine and meth- ionine sulfoxide,present, expressed as a ratio. This will be done by amino acid arialysis. They then plan to use this ratio as an index of the degree to which x 1-Pi inactivation is related to oxidation at the methi.onine site. They 'will also of course be determining the degree to which ,,•1-Pi obtained from these individuals inactivates protease. 6. ;,r. Carp is undertaking to develop en antibody which will recognice oxidized ac 1--Pi. This then might be used as a tool which coul.d also serve to determine the ratio of oxidized to native ;N1-Pi. Grant # 1259. This study has received its terminal support and then been extended for 6 months to provide time for Dr. Laurent to learn the desmosine RIA assay devised by Dr. H:rel. 1. The RIA,assay project for desomosine undertaken by rr. Harel has been completed (see attachment -v"2) and is being published in the American Rev. of Respiratory Disease. The assay.works and can be readily apriied to urinerAr. Yu in St. Louis has applied it already to patients success'-a11y. ^.9r ^ssry tu:•::F out to be :pe'cifi^ Po+_• desmosine from ery elrs`,in source r}•st.c . rac:° ?^cr not tlcn to continue thi: arogrr.m. as a clinical stucb• due to the difi'iculty in obtaining a sufficiently large human populaticn to study. Will instead apply it to two animal -todels (see below). A. A study with sheep will be undertalen at the Brookhaven National Laboratory. This will utilize unanesthetized animals from which lung lymph will be collected via indwelling catheters. Urine will also be collected by catheter from the urinary bladder. Graded doses of pancreatic elastase (PE) will be applied by pulmonPry entheters. Urine and pulmonary lymph will then be (over)

H K 27 10 36364 assayed over a= week period for desmosine (as an index of elastin degradation). They will also employ a radioactive scanning procedure of the lung to evaluv`.e pulmonary damage at vFrious timescfter applying the FE and correle:e this :ota krith the levels of desmosine found in urine or lymph. Also plan to obtain morphological and physiologic evelpations of lung function. 0b;ect of study :s to determine if there are levels of desmosine (indicating damage to elastin) which occur in urine or ],ymph prior to any appearance of physiolooic or morp- hologic indices of lung damage. This sttldy will be conducted by Dr. Laurent. B. The final study will utilize rats which :,ill be exposed to ci,:rxette smoke and PE. Janoff predicts that such animals will demonstrate more puJmonery disease than animals exposed to only one agent. The PE will be appliea i:: -r:.aea doses after smoke exvosure and - , aill, then measure l+,mg damsge (mean linear inter - cepts with the alveolar walls) in relation to levels of urinary desmosine detected. Predict the followine data: Desmosine level or n, # alveolar A intercepts ! ?E ; SA!)YE .-a Dose Pancreatic elasts5e Evaluation: This program continues to be actiti•e snd imaginative w-ith a Z high degree of organization and direction. It merits our continued support. The presentation of progress and future plans were suscinct and illustrate the con- siderable insight possessed by Dr. Janoff concerning the tremendous number of inter-relationships which are associated with the maintenance and turnover of lung elastin as associated with emphysema. Dr. Janoff plans at this point to incorporate his continuing objectives for Grant 7#125o into Grant #11b3A, since the two programs are so closely related. (See Janoff letter of Oct. 28, 1980- attached). D. H. Ford D. Stone