Title:
TOBACCO SMOKE FILTERS
United States Patent 3658070
Abstract:
A tobacco smoke filter filled with a smoke permeable composition capable of reducing the toxic effect of tobacco smoke on alveolar (lung) macrophages, comprising N,N'-diphenyl-p-phenylenediamine (DPPD) which may be mixed with flutathione, active charcoal or mixed with or impregnated into known filter material such as cellulose acetate.
US Patent References:
Toracco mixture
Hicks - January 1932 - 1842266
Food product
Ott - March 1939 - 2152602
Stabilization of crops
Chenicek - June 1950 - 2513002
Tobacco smoke filter capable of selective removal of aldehydes
Tovey et al. - January 1961 - 2968306
Absorbing composition for tobacco smoke
Whitefield et al. - April 1964 - 3127901
Toracco mixture
Hicks - January 1932 - 1842266
Food product
Ott - March 1939 - 2152602
Stabilization of crops
Chenicek - June 1950 - 2513002
Tobacco smoke filter capable of selective removal of aldehydes
Tovey et al. - January 1961 - 2968306
Absorbing composition for tobacco smoke
Whitefield et al. - April 1964 - 3127901
Application Number:
05/077247
Publication Date:
04/25/1972
Filing Date:
10/01/1970
View Patent Images:
Export Citation:
Primary Class:
Other Classes:
131/342
International Classes:
A24D3/14; A24D3/00; A24B15/02
Field of Search:
131/1R,266,267,269,262A,261
US Patent References:
| 3340879 | Cigarette filters | September 1967 | Horsewell et al. | |
| 3429318 | SELECTIVE FILTER MEDIUM | February 1969 | Walker et al. | |
| 3550600 | CIGARETTE FILTERS | December 1970 | Horsewell et al. | |
| 3410282 | Filter medium for removing hydrogen cyanide from tobacco smoke | November 1968 | Walker et al. | |
| 3426765 | TOBACCO SMOKE FILTERS | February 1969 | Misenheimer et al. | |
| 2856293 | Stabilization of fatty materials | October 1958 | Chenicek |
Primary Examiner:
Koran, Samuel
Assistant Examiner:
Yahwak G. M.
Claims:
I claim
1. A tobacco smoke filter comprising a cylindrical container having a cross-sectional dimension conforming to that of a tobacco smoke passage and defining a space having an inlet and outlet, said space being filled with a smoke permeable composition comprising N,N'-diphenyl-p-phenylenediamine in an amount from about 0.025 to 0.500 grams to reduce the toxic effect of tobacco smoke passing through the container on alveolar macrophages, the amount used not being toxic to the smoker.
2. A tobacco smoke filter according to claim 1 in which the N,N'-diphenyl-p-phenylenediamine is mixed with glutathione.
3. A tobacco smoke filter according to claim 1 wherein the N,N'-diphenyl-p-phenylenediamine is impregnated into a cellulosic smoke filtering material.
4. A tobacco smoke filter according to claim 1 wherein the N,N'-diphenyl-p-phenylenediamine is incorporated with activated charcoal.
5. A tobacco smoke filter according to claim 2 wherein the mixture is impregnated into a cellulose acetate filtering material.
6. A tobacco smoke filter adapted to remove undesirable components from tobacco smoke, said filter having a cross-sectional dimension conforming to that of a tobacco smoke passage and comprising a carrier material and an amount of from 5 per cent to 70 per cent by weight based on the weight of said carrier of N,N'-diphenyl-p-phenylenediamine to reduce the toxic effect of tobacco smoke passing through said passage, the amount used not being toxic to the smoker.
1. A tobacco smoke filter comprising a cylindrical container having a cross-sectional dimension conforming to that of a tobacco smoke passage and defining a space having an inlet and outlet, said space being filled with a smoke permeable composition comprising N,N'-diphenyl-p-phenylenediamine in an amount from about 0.025 to 0.500 grams to reduce the toxic effect of tobacco smoke passing through the container on alveolar macrophages, the amount used not being toxic to the smoker.
2. A tobacco smoke filter according to claim 1 in which the N,N'-diphenyl-p-phenylenediamine is mixed with glutathione.
3. A tobacco smoke filter according to claim 1 wherein the N,N'-diphenyl-p-phenylenediamine is impregnated into a cellulosic smoke filtering material.
4. A tobacco smoke filter according to claim 1 wherein the N,N'-diphenyl-p-phenylenediamine is incorporated with activated charcoal.
5. A tobacco smoke filter according to claim 2 wherein the mixture is impregnated into a cellulose acetate filtering material.
6. A tobacco smoke filter adapted to remove undesirable components from tobacco smoke, said filter having a cross-sectional dimension conforming to that of a tobacco smoke passage and comprising a carrier material and an amount of from 5 per cent to 70 per cent by weight based on the weight of said carrier of N,N'-diphenyl-p-phenylenediamine to reduce the toxic effect of tobacco smoke passing through said passage, the amount used not being toxic to the smoker.
Description:
The present invention relates to tobacco smoke filters which are suitable for employment in cigarettes, pipes, cigarette holders or cigar holders.
The present filter, classified as a chemical filter, has profound advantages over other filters known to the art by being able to modify the toxic and/or lethal effects of cigarette smoke extracts on alveolar macrophages, the primary host defense cell of the lung.
The filters currently available are essentially employed to remove particulate and solid components of smoke as well as to dilute the gaseous components of smoke with air. The gaseous phase of the smoke readily passes through such filters resulting in irritation to the lung tissue and, as will be noted later, profound lethality to isolated alveolar macrophages, a cell whose primary function is to maintain a proper lung environment by detoxification and destroying a variety of agents.
The gases derived from burning tobacco smoke contain highly reactive, low molecular weight, volatile components, of which acetaldehyde is characteristic, as well as free radicals and large quantities of gaseous ions induced by the high temperatures, which can approach 1,000° C resulting in pyrolization and distillation.
In addition, free radicals which have been demonstrated to be cytotoxic have also been demonstrated to occur in the gaseous phase of smoke. The chemical filtering material incorporated in the present invention has the ability, in the classical role of antioxidants as free radical stoppers, to lower the cytotoxic substances of cigarette smoke, possibly by reducing the attack of free radicals or other such reactive agents on pulmonary alveolar cells and therefore maintaining cell viability. Since free radicals have been implicated in the genesis of neoplasia, a beneficial effect of the antioxidant filler may be implied. In addition, the chemical filter has the ability to prevent smoke-induced decreases in circulating plasma lipid antioxidants.
It is the object of the present invention to provide a filter which, I have discovered, reduces the disclosed toxicity of cigarette smoke on alveolar macrophages.
I have discovered that the above object can be accomplished by having a tobacco smoke filter such as a cylindrical container filled with a smoke permeable composition comprising the lipid soluble antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD), preferably mixed with the water soluble antioxidant glutathione. The DPPD or the DPPD mixed with glutathione may be used alone or mixed with other ingredients such as with activated charcoal or impregnated into a filter carrier material such as cellulose acetate, paper, wood pulp, cotton, fibrous polyolefins, cellulose esters in fibrous or plastic form, regenerated cellulose, silica gel, alumina, tobacco and the like. Likewise the DPPD may be mixed with other chemical filters such as the amide filter of U.S. Pat. No. 3,426,765.
The amount of DPPD required to be effective in reducing the toxic effect of tobacco smoke on alveolar (lung) macrophages is from 0.025 to 0.500 grams in a filter for use in cigarettes and pipes, irrespective of the type and size of the cigarette or pipe in present day use. Preferably the amount will be from 0.050 to 0.200 grams.
The percentage of DPPD with carrier material is preferably from 5 - 70 per cent by weight based on the weight of carrier material, although higher amounts may, of course, be present since the invention is operative with 100 per cent DPPD.
The proportion of glutathione to DPPD may vary widely up to a weight ratio of 10-1or higher, but preferably the glutathione, in order to give increased effectiveness to the DPPD, should be present in a ratio of at least 1-10.
When active carbon is present the weight ration likewise may vary up to a ratio of activated carbon to DPPD of 10-1 or higher, preferably 0.5-1 to 5-1.
Other chemical filtering material may be used in varying amounts, but preferably the DPPD should be present in major amount compared to other material.
The invention is illustrated by reference to the accompanying drawing in which
FIG. 1, FIG. 2 and FIG. 3 show views in elevation, partly in section, of cigarettes having different arrangements of filters and different types of chemical filter compositions.
In FIG. 1 the numeral 10 represents a cigarette consisting of a paper tube 11 packed, except for tip tip portion, with tobacco 16. One end of the paper tube 11 (or a separate butt tube) consists of a section 12 composed of particles of DPPD with or without glutathione between filter sections 13 and 14 composed of cellulose acetate or other known tobacco smoke filter material. The section 12 may also contain DPPD mixed with active charcoal creating a simultaneous mechanical and chemical filter having dual advantages.
In FIG. 2 the numerals 10, 11 and 16 are the same as in FIG. 1 and the section 12 is composed of a cellulosic filter material impregnated with DPPD, with a plain cellulosic filter 13 at the tip of the cigarette. 1
In FIG. 3 the numerals 10, 11 and 16 are the same as in FIG. 1 and the numeral 12 represents cellulosic filter material or other absorbent filter material impregnated with DPPD with or without glutathione. The numeral 15 represents a recess portion of the tip of the cigarette.
Rearrangements of the various combinations shown in the drawings may, of course, be made, and it is not necessary that there be any filter other than the composition consisting of or comprising DPPD.
By having a plain (nonchemical) filter at the tip of the cigarette (FIGS. 1 and 2) no contact of the smoke with the chemical agent occurs. Likewise, the use of a recess portion of the tip (FIG. 3) will prevent contact of the smoker with the chemical agent.
The DPPD and DPPD with glutathione additives specifically employed to reduce the toxic effects of cigarettes on alveolar (lung) macrophages will also restrict certain biochemical changes which occur following exposure of animals and humans to cigarette smoke. These antioxidant filters will contribute to the reduction of chronic pulmonary disease, which can be credited to impaired macrophage function.
The DPPD antioxidant permits to superimposition of a technique which might be described as "selective chemical filtration" onto the current commonly used process of mechanical filtration. Having in a general way indicated the nature and purpose of my invention, the following are examples and data by which specific illustrations of the practice of the invention and the advantages to be obtained are demonstrated and are not to be construed as limiting the same.
EXAMPLE 1
Toxic Effects of aqueous Extracts of Cigarette Smoke on Isolated Alveolar Macrophages.
Alveolar macrophages were isolated from rat lungs by the technique of lung washing and equal aliquots of obtained cells were added to three flasks containing the following:
Flask A. 3 ml of Krebs-Ringer Phosphate Medium (KRP).
Flask B. 2.9 ml of KRP plus 0.1 ml of KRP smoke solution.
Flask C. 2.5 ml of KRP plus 0.5 ml of KRP smoke solution.
Flask D. 2.0 ml of KRP plus 1.0 ml of KRP smoke solution.
The KRP smoke solution was prepared by bubbling through 10 ml of KRP solution the smoke from one unfiltered cigarette. This solution was used within 10 minutes of its preparation.
The isolated alveolar macrophages (1.7 × 10 6 ) were added to each flask and the flasks were incubated for 30 minutes at 37° C. Viability was determined at 30 minutes after the incubation in the first experiment, and 30 and 60 minutes in the second experiment.
Experiment I Experiment II ____________________________________________________________ ______________ Cell Cell Smoke Survival, Survival, Percent Flask Solution % 30 Min. 30 Min. 60 Min. ____________________________________________________________ ______________ A 0 80 74.2 69.3 B 0.1 ml 72.2 74.5 66.2 C 0.5 ml - 41.9 20.3 D 1.0 ml 16.0 1.6 1.7 ____________________________________________________________ ______________
These studies, as well as those to follow, clearly denote the aqueous extracts of cigarette smoke exert a lethal effect on isolated rat alveolar macrophages which is dose dependent. While no reduction in viability was observed when 0.1 ml of smoke solution was added, a 42 and 71 per cent decrease in viability was observed at 30 and 60 minutes, respectively upon the addition of 0.5 ml of smoke solution. When 1.0 ml of smoke solution was added, or a volume equivalent to 0.1 of the original smoke extract obtained from one cigarette, an almost total loss of viability was observed. Thus, exposure of alveolar macrophages to gaseous, water soluble elements of cigarette smoke is associated with significant cellular mortality.
EXAMPLE 2
Further Studies on the the Toxic Effect of Cigarette Smoke.
Additional studies were conducted on the effects of varying concentrations of cigarette smoke on alveolar macrophage viability. Rat alveolar macrophages, 7.5 × 10 5 macrophages per flask, were incubated in the presence or absence of aqueous extracts of cigarette smoke prepared as Example 1 from one unfiltered cigarette. ------------------------------------------------------------ ---------------
Added Viability, Percent Flask Extract 0 Min. 30 Min. 60 Min. ____________________________________________________________ ______________ A - 75.4 75.8 55.3 B 0.1 74.2 85.7 62.5 C 0.5 70.8 48.5 43.6 D 1.0 76.0 41.5 13.6 A - 77.9 76.0 77.5 B 0.1 84.0 75.3 C 0.5 63.4 58.6 D 1.0 36.3 12.0 ____________________________________________________________ ______________
these studies confirm the dose dependent toxic effect of aqueous extracts of cigarette smoke on isolated lung macrophages.
EXAMPLE 3
Modification of Toxic Effects of Cigarette Smoke by Antioxidant Filter.
In view of the definitive toxic effects of cigarette smoke extract on isolated alveolar macrophages, attempts were made to modify the toxic effects of smoke extract on alveolar macrophages. An antioxidant filter was designed and developed (FIG. 1), consisting of 100 mg of antioxidant filter (50 mg of glutathione and 50 mg of N,N'-diphenyl-p-phenylenediamine (DPPD). Control filters were employed using non-antioxidant chemicals, such as sodium chloride (50 mg), and sodium bicarbonate (50 mg), which have comparable physical appearances of the antioxidants. These filters are designated as "Inert Chemical Filters, having no antioxidant activity." Aqueous extracts were prepared using the following type cigarettes:
A. nonfiltered Cigarettes.
B. antioxidant Filtered Cigarettes.
C. control Inert or Non-antioxidant Containing Chemical Filtered Cigarettes.
D. regular Commercial Cellulose Acetate Filtered Cigarettes.
The aqueous smoke extracts were prepared and added in the amount of 1 ml to incubation flasks containing 1.2 × 10 6 alveolar macrophage cells isolated from rat lungs. Incubations were carried out at 37° C for 0, 60 and 120 minutes and cell viability determined at each period by means of the trypan blue exclusion test. The following data were obtained: ##SPC1##
These studies clearly denote the toxic lethal effects of cigarette smoke extract on cell viability as essentially 100 per cent mortality of alveolar macrophages is observed after 2 hours of incubation (B GROUP). During the initial hour of incubation, the mean 62 per cent decrease in viability, which characterized the cells incubated in aqueous extracts, was not observed when the antioxidant chemical filter consisting of equal parts of DPPD and glutathione was employed. Significant protective effects are observed at the 120 minute period when the antioxidant filter is employed. Likewise, essentially 100 per cent mortality was seen in the cells incubated with aqueous extracts of cigarette smoke derived from filtered cigarettes, chemical control filtered cigarettes, i.e., the non-antioxidant type filter, and the nonfiltered cigarette.
Thus, the beneficial effect of DPPD antioxidant on negating the toxic effect of cigarette smoke on lung macrophages is manifested. These macrophages, an important host defense cell of the body, function in phagocytosis and destruction of bacteria, viruses altered or aged cells, as well as the removal and digestion of toxic macromolecular compounds which are present in their environment.
EXAMPLE 4
The influence of Antioxidant Impregnated Filters on Alveolar Macrophage Viability.
The effect of DPPD antioxidant with glutathione impregnated on cellulose acetate filters (FIGS. 2 and 3), in maintaining alveolar macrophage viability was also tested. Our tests indicated that approximately 1 ml of Krebs-Ringer phosphate solution is taken up by a cellulose acetate filter, which were sectioned to enhance surface area. A Krebs-Ringer phosphate medium was prepared, which had a concentration of 50 mg of glutathione and 50 mg of DPPD/per milliliter. The sectioned filters were immersed in the aqueous suspension for one minute, removed and dried under vacuum over night. The approximate total uptake of antioxidants by each filter was 100 mg. Filters prepared in such a fashion were then inserted back into the end of the cigarette from which the filters were originally removed and aqueous extracts of cigarette smoke prepared from the resulting cigarettes as previously described. Incubation of the aqueous smoke extracts were isolated macropages were conducted at 37° C for 60 and 120 minutes. The results are as follows: ##SPC2##
These studies, in essential agreement with previous observations, clearly demonstrate the toxicity of cigarette smoke (group B) and the inability of currently employed filters (group D) to modify the lethal action of cigarette smoke on alveolar macrophages. In profound contrast, the impregnation of DPPD and glutathione antioxidants into a cellulose acetate filter results in complete inhibition of the lethal effects of cigarette smoke extract (group C).
The protective effect of the DPPD-glutathione filter was due principally to the DPPD, since glutathione did not exert any protective activity on maintaining alveolar macrophage viability in the presence of aqueous smoke extracts. These findings are presented in the following table. As can be observed, the incorporation of glutathione with DPPD resulted in enhancement in survival of the smoke exposed pulmonary macrophages. This suggests a synergistic action between the lipid soluble and water soluble antioxidant on maintaining pulmonary macrophage viability. The DPPD filter was found to be equally effective if the DPPD was suspended either in water to impregnate the filter or dissolved in ethyl alcohol prior to filter impregnation.
INFLUENCE OF DPPD AND GLUTATHIONE, ALONE OR CONJOINTLY ON ALVEOLAR ------------------------------------------------------------ --------------- MACROPHAGE VIABILITY IN THE PRESENCE OF AQUEOUS SMOKE EXTRACT
Cell Viability, % Smoke Incubation Time Filter Extract 0 Min. 60 Min. ____________________________________________________________ ______________ - 0 85 77 - + 11 Cellulose Acetate + 14 DPRD + + 33 DPPD xx + 34 DPPD + Glutathione xxx + 51 Glutathione + 9 ____________________________________________________________ ______________ +-- DPPD (100 mg) prepared as water suspension to impregnate filter. xx -- DPPD (100 mg) dissolved in ethyl alcohol to impregnate filter. xxx -- DPPD (50 mg) and glutathione (50 mg) was used to impregnate filter.
EXAMPLE 5
Evaluation of the Ability of Antioxidants, Added Directly to the Trapping Solution, to Modify Cellular Toxicity of Cigarette Smoke.
Since antioxidants were protective when incorporated into filters, studies were undertaken to determine if the addition of antioxidants directly into the smoke trapping solution would modify the lethal effects of cigarette smoke extracts. To this end, glutathione and/or DPPD were added in the amount of 100 mg/10 ml of the Krebs-Ringer phosphate trapping solution and 1 ml of the resulting smoke extract obtained from unfiltered cigarettes was added to each flask which contained approximately 1.2 × 10 6 alveolar macrophages. ##SPC3##
In agreement with our previous observation, aqueous extracts of cigarette smoke were toxic to alveolar macrophages (Flask B and E vs. Flask A and D, respectively). In contrast to manifested cytotoxicity, the addition of the water soluble antioxidant, glutathione, and the lipid soluble antioxidant DPPD to the smoke trapping solution markedly reduced the lethality of cigarette smoke (Flasks C and H). Glutathione by itself did not appear to exert significant protective effect.
These studies eliminate the possibility of a non-specific mechanism of protection of antioxidants, and indicate that the addition of DPPD, or DPPD and glutathione to the KRP medium in vitro, will modify the toxic effects of cigarette smoke on alveolar macrophages.
EXAMPLE 6
Antioxidants and Photo-activation Studies.
In a further effort to evaluate the role of DPPD antioxidant in reducing alveolar macrophage injury following exposure to cigarette smoke extracts and to evaluate the concept of free radical generation by cigarette smoke, cigarette smoke was trapped in 10 milliliters of KRP solution and its phosphorescene activity determined prior to, and after, the addition of 1 m1 of ml peroxide. The hydrogen peroxide was added to 1 ml of solution prepared from unfiltered cigarette smoke, antioxidant filtered, commercial charcoal filtered, and a combination of charcoal plus glutathione and DPPD antioxidant mixture and counted one minute later by means of a liquid scintillation system. The addition of hydrogen peroxide results in immediate emission of electromagnetic radiation, or light, from as yet an unidentified compound present in the smoke extract.
These studies, and those conducted with toluene trapping solutions, indicate that unknown phosphorescent substances are present in cigarette smoke extracts which produce photons. The excited molecular species, as yet unidentified, product light quanta which can be detected by a phototube of a liquid scintillation counting system. The energy of the excited molecular species is appreciably below that of Tritium, i.e., less than 0.018 MEV.
It can be noted in the table below that the Krebs-Ringer Phosphate buffer had background activity (10-20counts per minute). Similarly, in aqueous solutions, like Krebs-Ringer Phosphate medium, the smoke solutions prepared from the various filters were comparable. However, when hydrogen peroxide was added to various KRP-smoke solutions, an approximate twentyfold increase occurred in the photon producing activity of the sample. This contrasted markedly to but a fourfold increase when DPPD plus glutathione was employed in the filter to prepare the KRP smoke solution. Commercial charcoal-cellulose acetate filter was effective in reducing the increase in photoactivation when compared to the unfiltered cigarette; however, the activity was twice that observed when the chemical antioxidant filter was employed. Employing the activated carbon plus antioxidant filter, prepared as previously described, complete suppression of light energy from the aqueous solution was obtained. ##SPC4##
Footnote to above table:
One ml of aqueous extract was placed in scintillation vial without phosphorous and counted. No photoactivity was manifested initially in all samples. When H 2 0 2 was added, photoactivation occurred to varying degrees in the aqueous smoke extract solution and was dependent upon filter employed.
Additional studies were conducted where smoke was passed through 20 ml of toluene containing 2.5 diphenyloxazole and p-bis-(2-5-phenyloxazolyl)-benzene to enhance detection of light energy emitted by the sample under study. When samples were prepared and counted one minute following completion of preparation, the photoactivity of the toluene sample was increased approximately one thousandfold when smoke from an unfiltered cigarette was used (sample B vs. sample A). This was significantly decreased 68 per cent when the DPPD-glutathionic antioxidant filter was employed. ------------------------------------------------------------ ---------------
Activity in Sample Filter Smoke Counts/Minute ____________________________________________________________ ______________ A - - 40 B - + 38,725 C Cellulose Acetate + 71,502 D DPPD-Glutathione + 15,775 ____________________________________________________________ ______________
EXAMPLE 7
Influence of DPPD Antioxidant Filters on Lipid Soluble Antioxidant Activity of Plasma and Other Tissues of Smoke Exposed Rats.
Studies were also conducted to determine if DPPD antioxidant filters could modify certain specific biochemical events associated with cigarette smoke exposure. Plasma and tissue lipid soluble antioxidant activity was determined in normal, untreated control rats, and in rats exposed to smoke from five unfiltered cigarettes. In addition, groups of rats were also exposed to smoke from five regular filter (cellulose acetate) cigarettes and to smoke derived from five antioxidant filter (FIG. 1) cigarettes. Values below are listed as means ± standard error of mean and are derived from groups of rats number from 6 to 13. Values of DPPD lipid antioxidants are expressed as microequivalents of antioxidant per milliliter of plasma or per gram of tissue. ------------------------------------------------------------ ---------------
DPPD Antioxidant Activity Plasma Lung Liver Group uEq/ml uEq/g uEq/g ____________________________________________________________ ______________ Control ±0.002 0.140 0.329 ± 0.014 ± 0.026 Cigarette Smoke 0.0110 0.126 0.343 (Unfiltered--1 ±0.002 ± 0.018 ± 0.031 Cigarette Smoke 0.0094 0.102 0.255 (Unfiltered--5) ±0.003 ± 0.026 ± 0.027 Cigarette Smoke 0.0092 0.160 0.380 (Regular Filter ±0.0011 ± 0.0197 ± 0.031 Cigarette Smoke 0.0180 0.175 0.337 (Antioxidant Filter) ±0.002 ± 0.160 ± 0.022 ____________________________________________________________ ______________
These studies denote that exposure of rats to alternating environments of cigarette smoke and air, by means of a partial vacuum to draw in alternating amounts of air and smoke into an enclosed chamber in which the rats were housed, induces a profound fall in plasma lipid soluble antioxidant activity. The decrease is observed with as little as one cigarette. A regular commercially available cellulose acetate filter does not modify the 72 per cent decrease in plasma lipid antioxidants. However, the decrease observed when the DPPD lipid soluble antioxidant filter was employed was but 44 per cent. In other words, the animals exposed to smoke obtained from DPPD antioxidant filtered cigarettes had plasma antioxidant levels that were approximately 100 per cent higher than that observed when regular filtered or nonfiltered cigarettes were employed. Thus, the depletion of plasma lipid soluble antioxidant activity which follows cigarette smoke exposure is significantly inhibited when DPPD antioxidants are incorporated directly into the cigarette filter.
Thus, by my invention, I have devised a novel filter comprising the lipid soluble antioxidant DPPD which can be readily adapted to reduce the toxicity of cigarette smoke. This chemical antioxidant additive can readily be incorporated by impregnation into conventional type filters or as inserts into conventional filters. The resulting cigarette, which has been smoked by the investigator and others, is a mild, pleasant smoke without appreciable loss of flavor or aroma.
The present filter, classified as a chemical filter, has profound advantages over other filters known to the art by being able to modify the toxic and/or lethal effects of cigarette smoke extracts on alveolar macrophages, the primary host defense cell of the lung.
The filters currently available are essentially employed to remove particulate and solid components of smoke as well as to dilute the gaseous components of smoke with air. The gaseous phase of the smoke readily passes through such filters resulting in irritation to the lung tissue and, as will be noted later, profound lethality to isolated alveolar macrophages, a cell whose primary function is to maintain a proper lung environment by detoxification and destroying a variety of agents.
The gases derived from burning tobacco smoke contain highly reactive, low molecular weight, volatile components, of which acetaldehyde is characteristic, as well as free radicals and large quantities of gaseous ions induced by the high temperatures, which can approach 1,000° C resulting in pyrolization and distillation.
In addition, free radicals which have been demonstrated to be cytotoxic have also been demonstrated to occur in the gaseous phase of smoke. The chemical filtering material incorporated in the present invention has the ability, in the classical role of antioxidants as free radical stoppers, to lower the cytotoxic substances of cigarette smoke, possibly by reducing the attack of free radicals or other such reactive agents on pulmonary alveolar cells and therefore maintaining cell viability. Since free radicals have been implicated in the genesis of neoplasia, a beneficial effect of the antioxidant filler may be implied. In addition, the chemical filter has the ability to prevent smoke-induced decreases in circulating plasma lipid antioxidants.
It is the object of the present invention to provide a filter which, I have discovered, reduces the disclosed toxicity of cigarette smoke on alveolar macrophages.
I have discovered that the above object can be accomplished by having a tobacco smoke filter such as a cylindrical container filled with a smoke permeable composition comprising the lipid soluble antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD), preferably mixed with the water soluble antioxidant glutathione. The DPPD or the DPPD mixed with glutathione may be used alone or mixed with other ingredients such as with activated charcoal or impregnated into a filter carrier material such as cellulose acetate, paper, wood pulp, cotton, fibrous polyolefins, cellulose esters in fibrous or plastic form, regenerated cellulose, silica gel, alumina, tobacco and the like. Likewise the DPPD may be mixed with other chemical filters such as the amide filter of U.S. Pat. No. 3,426,765.
The amount of DPPD required to be effective in reducing the toxic effect of tobacco smoke on alveolar (lung) macrophages is from 0.025 to 0.500 grams in a filter for use in cigarettes and pipes, irrespective of the type and size of the cigarette or pipe in present day use. Preferably the amount will be from 0.050 to 0.200 grams.
The percentage of DPPD with carrier material is preferably from 5 - 70 per cent by weight based on the weight of carrier material, although higher amounts may, of course, be present since the invention is operative with 100 per cent DPPD.
The proportion of glutathione to DPPD may vary widely up to a weight ratio of 10-1or higher, but preferably the glutathione, in order to give increased effectiveness to the DPPD, should be present in a ratio of at least 1-10.
When active carbon is present the weight ration likewise may vary up to a ratio of activated carbon to DPPD of 10-1 or higher, preferably 0.5-1 to 5-1.
Other chemical filtering material may be used in varying amounts, but preferably the DPPD should be present in major amount compared to other material.
The invention is illustrated by reference to the accompanying drawing in which
FIG. 1, FIG. 2 and FIG. 3 show views in elevation, partly in section, of cigarettes having different arrangements of filters and different types of chemical filter compositions.
In FIG. 1 the numeral 10 represents a cigarette consisting of a paper tube 11 packed, except for tip tip portion, with tobacco 16. One end of the paper tube 11 (or a separate butt tube) consists of a section 12 composed of particles of DPPD with or without glutathione between filter sections 13 and 14 composed of cellulose acetate or other known tobacco smoke filter material. The section 12 may also contain DPPD mixed with active charcoal creating a simultaneous mechanical and chemical filter having dual advantages.
In FIG. 2 the numerals 10, 11 and 16 are the same as in FIG. 1 and the section 12 is composed of a cellulosic filter material impregnated with DPPD, with a plain cellulosic filter 13 at the tip of the cigarette. 1
In FIG. 3 the numerals 10, 11 and 16 are the same as in FIG. 1 and the numeral 12 represents cellulosic filter material or other absorbent filter material impregnated with DPPD with or without glutathione. The numeral 15 represents a recess portion of the tip of the cigarette.
Rearrangements of the various combinations shown in the drawings may, of course, be made, and it is not necessary that there be any filter other than the composition consisting of or comprising DPPD.
By having a plain (nonchemical) filter at the tip of the cigarette (FIGS. 1 and 2) no contact of the smoke with the chemical agent occurs. Likewise, the use of a recess portion of the tip (FIG. 3) will prevent contact of the smoker with the chemical agent.
The DPPD and DPPD with glutathione additives specifically employed to reduce the toxic effects of cigarettes on alveolar (lung) macrophages will also restrict certain biochemical changes which occur following exposure of animals and humans to cigarette smoke. These antioxidant filters will contribute to the reduction of chronic pulmonary disease, which can be credited to impaired macrophage function.
The DPPD antioxidant permits to superimposition of a technique which might be described as "selective chemical filtration" onto the current commonly used process of mechanical filtration. Having in a general way indicated the nature and purpose of my invention, the following are examples and data by which specific illustrations of the practice of the invention and the advantages to be obtained are demonstrated and are not to be construed as limiting the same.
EXAMPLE 1
Toxic Effects of aqueous Extracts of Cigarette Smoke on Isolated Alveolar Macrophages.
Alveolar macrophages were isolated from rat lungs by the technique of lung washing and equal aliquots of obtained cells were added to three flasks containing the following:
Flask A. 3 ml of Krebs-Ringer Phosphate Medium (KRP).
Flask B. 2.9 ml of KRP plus 0.1 ml of KRP smoke solution.
Flask C. 2.5 ml of KRP plus 0.5 ml of KRP smoke solution.
Flask D. 2.0 ml of KRP plus 1.0 ml of KRP smoke solution.
The KRP smoke solution was prepared by bubbling through 10 ml of KRP solution the smoke from one unfiltered cigarette. This solution was used within 10 minutes of its preparation.
The isolated alveolar macrophages (1.7 × 10 6 ) were added to each flask and the flasks were incubated for 30 minutes at 37° C. Viability was determined at 30 minutes after the incubation in the first experiment, and 30 and 60 minutes in the second experiment.
Experiment I Experiment II ____________________________________________________________ ______________ Cell Cell Smoke Survival, Survival, Percent Flask Solution % 30 Min. 30 Min. 60 Min. ____________________________________________________________ ______________ A 0 80 74.2 69.3 B 0.1 ml 72.2 74.5 66.2 C 0.5 ml - 41.9 20.3 D 1.0 ml 16.0 1.6 1.7 ____________________________________________________________ ______________
These studies, as well as those to follow, clearly denote the aqueous extracts of cigarette smoke exert a lethal effect on isolated rat alveolar macrophages which is dose dependent. While no reduction in viability was observed when 0.1 ml of smoke solution was added, a 42 and 71 per cent decrease in viability was observed at 30 and 60 minutes, respectively upon the addition of 0.5 ml of smoke solution. When 1.0 ml of smoke solution was added, or a volume equivalent to 0.1 of the original smoke extract obtained from one cigarette, an almost total loss of viability was observed. Thus, exposure of alveolar macrophages to gaseous, water soluble elements of cigarette smoke is associated with significant cellular mortality.
EXAMPLE 2
Further Studies on the the Toxic Effect of Cigarette Smoke.
Additional studies were conducted on the effects of varying concentrations of cigarette smoke on alveolar macrophage viability. Rat alveolar macrophages, 7.5 × 10 5 macrophages per flask, were incubated in the presence or absence of aqueous extracts of cigarette smoke prepared as Example 1 from one unfiltered cigarette. ------------------------------------------------------------ ---------------
Added Viability, Percent Flask Extract 0 Min. 30 Min. 60 Min. ____________________________________________________________ ______________ A - 75.4 75.8 55.3 B 0.1 74.2 85.7 62.5 C 0.5 70.8 48.5 43.6 D 1.0 76.0 41.5 13.6 A - 77.9 76.0 77.5 B 0.1 84.0 75.3 C 0.5 63.4 58.6 D 1.0 36.3 12.0 ____________________________________________________________ ______________
these studies confirm the dose dependent toxic effect of aqueous extracts of cigarette smoke on isolated lung macrophages.
EXAMPLE 3
Modification of Toxic Effects of Cigarette Smoke by Antioxidant Filter.
In view of the definitive toxic effects of cigarette smoke extract on isolated alveolar macrophages, attempts were made to modify the toxic effects of smoke extract on alveolar macrophages. An antioxidant filter was designed and developed (FIG. 1), consisting of 100 mg of antioxidant filter (50 mg of glutathione and 50 mg of N,N'-diphenyl-p-phenylenediamine (DPPD). Control filters were employed using non-antioxidant chemicals, such as sodium chloride (50 mg), and sodium bicarbonate (50 mg), which have comparable physical appearances of the antioxidants. These filters are designated as "Inert Chemical Filters, having no antioxidant activity." Aqueous extracts were prepared using the following type cigarettes:
A. nonfiltered Cigarettes.
B. antioxidant Filtered Cigarettes.
C. control Inert or Non-antioxidant Containing Chemical Filtered Cigarettes.
D. regular Commercial Cellulose Acetate Filtered Cigarettes.
The aqueous smoke extracts were prepared and added in the amount of 1 ml to incubation flasks containing 1.2 × 10 6 alveolar macrophage cells isolated from rat lungs. Incubations were carried out at 37° C for 0, 60 and 120 minutes and cell viability determined at each period by means of the trypan blue exclusion test. The following data were obtained: ##SPC1##
These studies clearly denote the toxic lethal effects of cigarette smoke extract on cell viability as essentially 100 per cent mortality of alveolar macrophages is observed after 2 hours of incubation (B GROUP). During the initial hour of incubation, the mean 62 per cent decrease in viability, which characterized the cells incubated in aqueous extracts, was not observed when the antioxidant chemical filter consisting of equal parts of DPPD and glutathione was employed. Significant protective effects are observed at the 120 minute period when the antioxidant filter is employed. Likewise, essentially 100 per cent mortality was seen in the cells incubated with aqueous extracts of cigarette smoke derived from filtered cigarettes, chemical control filtered cigarettes, i.e., the non-antioxidant type filter, and the nonfiltered cigarette.
Thus, the beneficial effect of DPPD antioxidant on negating the toxic effect of cigarette smoke on lung macrophages is manifested. These macrophages, an important host defense cell of the body, function in phagocytosis and destruction of bacteria, viruses altered or aged cells, as well as the removal and digestion of toxic macromolecular compounds which are present in their environment.
EXAMPLE 4
The influence of Antioxidant Impregnated Filters on Alveolar Macrophage Viability.
The effect of DPPD antioxidant with glutathione impregnated on cellulose acetate filters (FIGS. 2 and 3), in maintaining alveolar macrophage viability was also tested. Our tests indicated that approximately 1 ml of Krebs-Ringer phosphate solution is taken up by a cellulose acetate filter, which were sectioned to enhance surface area. A Krebs-Ringer phosphate medium was prepared, which had a concentration of 50 mg of glutathione and 50 mg of DPPD/per milliliter. The sectioned filters were immersed in the aqueous suspension for one minute, removed and dried under vacuum over night. The approximate total uptake of antioxidants by each filter was 100 mg. Filters prepared in such a fashion were then inserted back into the end of the cigarette from which the filters were originally removed and aqueous extracts of cigarette smoke prepared from the resulting cigarettes as previously described. Incubation of the aqueous smoke extracts were isolated macropages were conducted at 37° C for 60 and 120 minutes. The results are as follows: ##SPC2##
These studies, in essential agreement with previous observations, clearly demonstrate the toxicity of cigarette smoke (group B) and the inability of currently employed filters (group D) to modify the lethal action of cigarette smoke on alveolar macrophages. In profound contrast, the impregnation of DPPD and glutathione antioxidants into a cellulose acetate filter results in complete inhibition of the lethal effects of cigarette smoke extract (group C).
The protective effect of the DPPD-glutathione filter was due principally to the DPPD, since glutathione did not exert any protective activity on maintaining alveolar macrophage viability in the presence of aqueous smoke extracts. These findings are presented in the following table. As can be observed, the incorporation of glutathione with DPPD resulted in enhancement in survival of the smoke exposed pulmonary macrophages. This suggests a synergistic action between the lipid soluble and water soluble antioxidant on maintaining pulmonary macrophage viability. The DPPD filter was found to be equally effective if the DPPD was suspended either in water to impregnate the filter or dissolved in ethyl alcohol prior to filter impregnation.
INFLUENCE OF DPPD AND GLUTATHIONE, ALONE OR CONJOINTLY ON ALVEOLAR ------------------------------------------------------------ --------------- MACROPHAGE VIABILITY IN THE PRESENCE OF AQUEOUS SMOKE EXTRACT
Cell Viability, % Smoke Incubation Time Filter Extract 0 Min. 60 Min. ____________________________________________________________ ______________ - 0 85 77 - + 11 Cellulose Acetate + 14 DPRD + + 33 DPPD xx + 34 DPPD + Glutathione xxx + 51 Glutathione + 9 ____________________________________________________________ ______________ +-- DPPD (100 mg) prepared as water suspension to impregnate filter. xx -- DPPD (100 mg) dissolved in ethyl alcohol to impregnate filter. xxx -- DPPD (50 mg) and glutathione (50 mg) was used to impregnate filter.
EXAMPLE 5
Evaluation of the Ability of Antioxidants, Added Directly to the Trapping Solution, to Modify Cellular Toxicity of Cigarette Smoke.
Since antioxidants were protective when incorporated into filters, studies were undertaken to determine if the addition of antioxidants directly into the smoke trapping solution would modify the lethal effects of cigarette smoke extracts. To this end, glutathione and/or DPPD were added in the amount of 100 mg/10 ml of the Krebs-Ringer phosphate trapping solution and 1 ml of the resulting smoke extract obtained from unfiltered cigarettes was added to each flask which contained approximately 1.2 × 10 6 alveolar macrophages. ##SPC3##
In agreement with our previous observation, aqueous extracts of cigarette smoke were toxic to alveolar macrophages (Flask B and E vs. Flask A and D, respectively). In contrast to manifested cytotoxicity, the addition of the water soluble antioxidant, glutathione, and the lipid soluble antioxidant DPPD to the smoke trapping solution markedly reduced the lethality of cigarette smoke (Flasks C and H). Glutathione by itself did not appear to exert significant protective effect.
These studies eliminate the possibility of a non-specific mechanism of protection of antioxidants, and indicate that the addition of DPPD, or DPPD and glutathione to the KRP medium in vitro, will modify the toxic effects of cigarette smoke on alveolar macrophages.
EXAMPLE 6
Antioxidants and Photo-activation Studies.
In a further effort to evaluate the role of DPPD antioxidant in reducing alveolar macrophage injury following exposure to cigarette smoke extracts and to evaluate the concept of free radical generation by cigarette smoke, cigarette smoke was trapped in 10 milliliters of KRP solution and its phosphorescene activity determined prior to, and after, the addition of 1 m1 of ml peroxide. The hydrogen peroxide was added to 1 ml of solution prepared from unfiltered cigarette smoke, antioxidant filtered, commercial charcoal filtered, and a combination of charcoal plus glutathione and DPPD antioxidant mixture and counted one minute later by means of a liquid scintillation system. The addition of hydrogen peroxide results in immediate emission of electromagnetic radiation, or light, from as yet an unidentified compound present in the smoke extract.
These studies, and those conducted with toluene trapping solutions, indicate that unknown phosphorescent substances are present in cigarette smoke extracts which produce photons. The excited molecular species, as yet unidentified, product light quanta which can be detected by a phototube of a liquid scintillation counting system. The energy of the excited molecular species is appreciably below that of Tritium, i.e., less than 0.018 MEV.
It can be noted in the table below that the Krebs-Ringer Phosphate buffer had background activity (10-20counts per minute). Similarly, in aqueous solutions, like Krebs-Ringer Phosphate medium, the smoke solutions prepared from the various filters were comparable. However, when hydrogen peroxide was added to various KRP-smoke solutions, an approximate twentyfold increase occurred in the photon producing activity of the sample. This contrasted markedly to but a fourfold increase when DPPD plus glutathione was employed in the filter to prepare the KRP smoke solution. Commercial charcoal-cellulose acetate filter was effective in reducing the increase in photoactivation when compared to the unfiltered cigarette; however, the activity was twice that observed when the chemical antioxidant filter was employed. Employing the activated carbon plus antioxidant filter, prepared as previously described, complete suppression of light energy from the aqueous solution was obtained. ##SPC4##
Footnote to above table:
One ml of aqueous extract was placed in scintillation vial without phosphorous and counted. No photoactivity was manifested initially in all samples. When H 2 0 2 was added, photoactivation occurred to varying degrees in the aqueous smoke extract solution and was dependent upon filter employed.
Additional studies were conducted where smoke was passed through 20 ml of toluene containing 2.5 diphenyloxazole and p-bis-(2-5-phenyloxazolyl)-benzene to enhance detection of light energy emitted by the sample under study. When samples were prepared and counted one minute following completion of preparation, the photoactivity of the toluene sample was increased approximately one thousandfold when smoke from an unfiltered cigarette was used (sample B vs. sample A). This was significantly decreased 68 per cent when the DPPD-glutathionic antioxidant filter was employed. ------------------------------------------------------------ ---------------
Activity in Sample Filter Smoke Counts/Minute ____________________________________________________________ ______________ A - - 40 B - + 38,725 C Cellulose Acetate + 71,502 D DPPD-Glutathione + 15,775 ____________________________________________________________ ______________
EXAMPLE 7
Influence of DPPD Antioxidant Filters on Lipid Soluble Antioxidant Activity of Plasma and Other Tissues of Smoke Exposed Rats.
Studies were also conducted to determine if DPPD antioxidant filters could modify certain specific biochemical events associated with cigarette smoke exposure. Plasma and tissue lipid soluble antioxidant activity was determined in normal, untreated control rats, and in rats exposed to smoke from five unfiltered cigarettes. In addition, groups of rats were also exposed to smoke from five regular filter (cellulose acetate) cigarettes and to smoke derived from five antioxidant filter (FIG. 1) cigarettes. Values below are listed as means ± standard error of mean and are derived from groups of rats number from 6 to 13. Values of DPPD lipid antioxidants are expressed as microequivalents of antioxidant per milliliter of plasma or per gram of tissue. ------------------------------------------------------------ ---------------
DPPD Antioxidant Activity Plasma Lung Liver Group uEq/ml uEq/g uEq/g ____________________________________________________________ ______________ Control ±0.002 0.140 0.329 ± 0.014 ± 0.026 Cigarette Smoke 0.0110 0.126 0.343 (Unfiltered--1 ±0.002 ± 0.018 ± 0.031 Cigarette Smoke 0.0094 0.102 0.255 (Unfiltered--5) ±0.003 ± 0.026 ± 0.027 Cigarette Smoke 0.0092 0.160 0.380 (Regular Filter ±0.0011 ± 0.0197 ± 0.031 Cigarette Smoke 0.0180 0.175 0.337 (Antioxidant Filter) ±0.002 ± 0.160 ± 0.022 ____________________________________________________________ ______________
These studies denote that exposure of rats to alternating environments of cigarette smoke and air, by means of a partial vacuum to draw in alternating amounts of air and smoke into an enclosed chamber in which the rats were housed, induces a profound fall in plasma lipid soluble antioxidant activity. The decrease is observed with as little as one cigarette. A regular commercially available cellulose acetate filter does not modify the 72 per cent decrease in plasma lipid antioxidants. However, the decrease observed when the DPPD lipid soluble antioxidant filter was employed was but 44 per cent. In other words, the animals exposed to smoke obtained from DPPD antioxidant filtered cigarettes had plasma antioxidant levels that were approximately 100 per cent higher than that observed when regular filtered or nonfiltered cigarettes were employed. Thus, the depletion of plasma lipid soluble antioxidant activity which follows cigarette smoke exposure is significantly inhibited when DPPD antioxidants are incorporated directly into the cigarette filter.
Thus, by my invention, I have devised a novel filter comprising the lipid soluble antioxidant DPPD which can be readily adapted to reduce the toxicity of cigarette smoke. This chemical antioxidant additive can readily be incorporated by impregnation into conventional type filters or as inserts into conventional filters. The resulting cigarette, which has been smoked by the investigator and others, is a mild, pleasant smoke without appreciable loss of flavor or aroma.