Tobacco smoke filter
United States Patent 3903898
A tobacco smoke filter body containing dispersed therethrough antibody to carcinogen found in tobacco smoke in an amount effective to remove said carcinogen as the tobacco smoke passes through the filter.
Application Number:
05/472051
Publication Date:
09/09/1975
Filing Date:
05/21/1974
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Export Citation:
Assignee:
Shubik, Phillipe (Omaha, NB)
Primary Class:
Other Classes:
131/334
International Classes:
A24D3/14; C07K16/44; A24D3/00; A24B15/02
Field of Search:
424/85 131/9,264-269,261,262,10-10.9
Primary Examiner:
Rein, Melvin D.
Attorney, Agent or Firm:
Larson, Taylor And Hinds
Claims:
What is claimed
1. A tobacco smoke filter body containing dispersed therethrough a small amount of antibody to a carcinogen found in tobacco smoke, said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
2. A tobacco smoke filter body according to claim 1 wherein the antibody is an antibody to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine and benzo(b)fluoranthene.
3. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to benz(a)pyrene.
4. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to dibenz(a,h)anthracene.
5. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to 7-H-dibenz(c,g)carbazole.
6. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to dibenz(a,h)acridine.
7. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to benzo(b)fluoranthene.
8. A tobacco smoke filter body according to claim 1 wherein the filter body is a cellulose filter.
9. A tobacco smoke filter body according to claim 1 wherein the filter is a glass filter.
10. A tobacco smoke filter body according to claim 1 wherein the amount of said antibody is about 8 to 20 percent by weight and effective to substantially remove said carcinogen from said tobacco smoke as it passes through said filter.
11. The tobacco smoke filter according to claim 1 wherein the antibody is dispersed in the form of serum containing said antibody.
12. A tobacco smoke filter body having dispersed therethrough small amounts of antibodies to each of the following carcinogens: benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine and benzo(b)fluoranthene, said amounts being effective to remove at least substantial quantities of each of said carcinogens from the smoke as it passes through the filter.
13. A tobacco smoke filter body according to claim 12 wherein the antibodies are dispersed in the form of a single serum containing all of the antibodies to said carcinogens.
14. A filter assembly for smoking articles comprising a wrapper and a solid filter body having dispersed therethrough a small amount of antibody to a carcinogen found in tobacco smoke, said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
15. The filter assembly of claim 14 wherein the solid filter has dispersed therethrough antibodies to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine or benzo(b)fluoranthene.
16. A cigarette comprising: a tobacco column, a wrapper and a filter assembly including the filter body of claim 1.
17. The cigarette of claim 16 wherein the filter assembly includes the filter body of claim 2.
18. The cigarette of claim 16 wherein the filter assembly includes the filter body of claim 12.
19. A method for smoking a filter body for adsorbing carcinogen from tobacco smoke which comprises impregnating a tobacco smoke filter with small amounts of antibody to a carcinogen found in tobacco smoke, and said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
20. The method of claim 19 wherein the antibody is an antibody to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c.g)carbazole, dibenz(a,h)acridine or benzo(b)fluoranthene.
1. A tobacco smoke filter body containing dispersed therethrough a small amount of antibody to a carcinogen found in tobacco smoke, said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
2. A tobacco smoke filter body according to claim 1 wherein the antibody is an antibody to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine and benzo(b)fluoranthene.
3. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to benz(a)pyrene.
4. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to dibenz(a,h)anthracene.
5. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to 7-H-dibenz(c,g)carbazole.
6. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to dibenz(a,h)acridine.
7. The tobacco smoke filter body of claim 2 wherein the antibody is an antibody to benzo(b)fluoranthene.
8. A tobacco smoke filter body according to claim 1 wherein the filter body is a cellulose filter.
9. A tobacco smoke filter body according to claim 1 wherein the filter is a glass filter.
10. A tobacco smoke filter body according to claim 1 wherein the amount of said antibody is about 8 to 20 percent by weight and effective to substantially remove said carcinogen from said tobacco smoke as it passes through said filter.
11. The tobacco smoke filter according to claim 1 wherein the antibody is dispersed in the form of serum containing said antibody.
12. A tobacco smoke filter body having dispersed therethrough small amounts of antibodies to each of the following carcinogens: benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine and benzo(b)fluoranthene, said amounts being effective to remove at least substantial quantities of each of said carcinogens from the smoke as it passes through the filter.
13. A tobacco smoke filter body according to claim 12 wherein the antibodies are dispersed in the form of a single serum containing all of the antibodies to said carcinogens.
14. A filter assembly for smoking articles comprising a wrapper and a solid filter body having dispersed therethrough a small amount of antibody to a carcinogen found in tobacco smoke, said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
15. The filter assembly of claim 14 wherein the solid filter has dispersed therethrough antibodies to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)acridine or benzo(b)fluoranthene.
16. A cigarette comprising: a tobacco column, a wrapper and a filter assembly including the filter body of claim 1.
17. The cigarette of claim 16 wherein the filter assembly includes the filter body of claim 2.
18. The cigarette of claim 16 wherein the filter assembly includes the filter body of claim 12.
19. A method for smoking a filter body for adsorbing carcinogen from tobacco smoke which comprises impregnating a tobacco smoke filter with small amounts of antibody to a carcinogen found in tobacco smoke, and said amount being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
20. The method of claim 19 wherein the antibody is an antibody to at least one of benz(a)pyrene, dibenz(a,h)anthracene, 7-H-dibenz(c.g)carbazole, dibenz(a,h)acridine or benzo(b)fluoranthene.
Description:
FIELD OF THE INVENTION
This invention relates to tobacco smoke-filters. More particularly, the invention is directed to improved tobacco smoke filters especially useful in the removal of carcinogens from the smoke stream.
BACKGROUND OF THE INVENTION
In recent years the concern of smokers with the possible presence in tobacco smoke of cancer-inducing components and other substances hazardous to health has led to the development of various filtering materials for use in or with cigarettes, cigars and pipes. These filters, primarily designed to remove nicotine and the tarry components of cigarette smoke from the latter prior to its induction into the buccal cavity, have been incorporated in cigarettes as so-called "filter tips", have been provided for replaceable use in cigarette and cigar holders, have been built into throwaway holders for cigars and the like and have been proposed for incorporation in the stems of pipes.
Unfortunately, it has been found that such prior art filters do not in fact remove or even sufficiently reduce the carcinogens present in tobacco smoke, that is, the components known to induce cancer and other disorders in rats.
It has long been desired, therefore, and the object of many research projects of considerable cost to provide a tobacco smoke filter which is particularly effective against the carcinogens present in tobacco smoke while not eliminating therefrom the essential taste sought by tobacco smokers.
OBJECT OF THE INVENTION
Accordingly, one object of the invention is to provide a filter body which effectively removes known carcinogens from tobacco smoke passing axially therethrough without eliminating or adversely affecting desired taste sensations.
Another object of the invention is to provide a filter body and filter assemblies including same which offer the aforementioned advantages without the necessity of increasing the pressure drop over the filter bodies.
A further object of the invention is to provide a simple and economical method of making filter bodies capable of removing tobacco smoke carcinogens from existing preformed tobacco smoke filters.
SUMMARY OF THE INVENTION
The foregoing and other objects which will be apparent to one having ordinary skill in the art are achieved according to the present invention by a tobacco smoke filter having dispersed therethrough small amounts of antibody to a carcinogen found in tobacco smoke, said amounts being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
The tobacco smoke filter of the invention may contain antibody to at least one of the several carcinogens known to be present both in tobacco and tobacco smoke and advantageously the filter contains a plurality of antibodies each of which is specific to one of the carcinogens of tobacco smoke. Principal carcinogens known to be present in tobacco smoke are benz(a)pyrene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)anthracene, dibenz(a,h)acridine, benzo(b)fluoranthene.
The mechanism by which the antibody removes the respective carcinogens is not completely understood but it is believed that the antibody, being protein, is rehydrated by the water of combustion and while in its rehydrated state binds the carcinogen as it passes through the filter. Whatever the removal mechanism the high efficiency exhibited by the antibody-impregnated filter in removing tobacco smoke carcinogens is indeed surprising. As will be demonstrated in the working examples set forth below, the antibody-containing filter of the invention has been found to remove on the order of 20 times the amount of for instance, benz(a)pyrene removed by the same filter without the antibody.
The antibodies of the invention may be prepared by any suitable method known in the prior art. One appropriate method involves coupling the carcinogen to an immunogenic carrier material to form an antigen which is then injected into host animals from which antibodies specific to the carcinogen are obtained. The tobacco smoke carcinogens to which the invention is directed are small non-protein, molecules (haptens) which by themselves are devoid of antigencity. When the hapten moiety is coupled to an immunogenic carrier such as a protein, however, a antigenic material exhibiting an antibody response is provided. In a preferred embodiment, the carcinogen is covalently bonded to a protein or polypeptide molecule for instance by urea or peptide linkages. The urea linkage may involve for example reaction of an amino group on the protein or polypeptide and an isocyanate derivative of the carcinogen while a peptide linkage may involve either a carboxyl group on the protein or polypeptide chain or an amino group on the carcinogen and a carboxyl group on the polypeptide chain. It should be understood any of the prior methods known to those skilled in the art may be employed for covalently bonding the carcinogenhapten to the immunogenic carrier material.
As used herein, the term "immunogenic carrier material" is meant to include those materials which have the property of independently eliciting an immunogenic response in a host animal when injected therein and which can be coupled with covalent bonding to the aforementioned carcinogen haptens. Suitable carrier materials include, for example, proteins; natural or synthetic polymeric compounds such as polypeptides, e.g. polylysine or polyglutamic acid; polysaccharides; and the like. Particularly preferred carrier materials are proteins and polypeptides, especially proteins.
The identity of the protein material utilized in the preparation of a preferred antigen is not critical. Examples of preferred proteins useful in the practice of the invention include mammalian serum proteins such as, for example, horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and human gamma gobulin. Other suitable protein products will be suggested to one skilled in the art. It is generally preferred that proteins be utilized which are foreign to the animal hosts in which the resulting antigen is injected for antibody formation.
The reaction introducing isocyanate groups into the carcinogens for subsequent coupling with protein or polypeptide is conveniently prepared, for instance, as described in Creech, H. J., J. Amer. Chem. Soc. 63 576 (1941). According to this synthesis the carcinogen as, for example, benzo(a)pyrene is subjected to nitration with nitric acid to form the nitro-derivative of the carcinogen which is reduced with stannous chloride to the amine. Addition of phosgene to the amine produces the isocyanate of the carcinogen.
The coupling of the isocyanate of the carcinogen with protein or polypeptide to form a preferred antigen can be readily accomplished utilizing techniques now well known in protein chemistry as described, for instance, in Creech, H. J., Jones, R. N., J. American Chemical Society, 63 1661 (1941) and Creech, H. J., Jones, R. N., J. Amer. Chem. Soc. 62 1970 (1940). The reaction proceeds under relatively mile reaction conditions and involves the ε-amino groups of the lysine side chains of the protein. The general reaction may be schematic represented as follows:
ArNCO + H 2 N - protein ➝ ArNHCONH-protein
Ar = the aromatic carcinogen moiety.
The present invention also contemplates coupling a plurality of the carcinogens or even all of the carcinogens known to be present in tobacco smoke to an immunogenic carrier such as protein or polypeptide to thereby produce an antigenic substance which will produce upon injection into a host animal a serum containing antibodies to the various carcinogens. Such antigen containing a plurality of covalently bonded tobacco smoke carcinogens can be prepared by employing the general procedure described above for the covalent bonding of isocyanate derivatives of carcinogens with the ε-amino groups of proteins but utilizing in the reaction a mixture of the different carcinogens in their isocyanate form. Also, instead of taking one protein and attaching a plurality of tobacco smoke carcinogens, each carcinogen could be attached to a separate antigen and each of the carcinogen carriers injected into the same animal which will produce a serum containing antibodies to the various carcinogens.
The antibodies used in the filters of the present invention are obtained by injecting the antigens described above into a host animal repeatedly over a period of time and collecting the serum. The serum thus obtained and containing the antibody or antibodies specific to the carcinogen or carcinogens may be used as such in the treatment of the filters. If desired, however, the antibody or antibodies may be precipitated from the serum with a neutral salt solution, purified as by dialysis and/or column chromatography and resuspended in any suitable filter treating or impregnating solution. Illustrative of neutral salts for use in such antibody precipitations are ammonium sulfate, sodium sulfate and the like. It is clear to people skilled in the art that by precipitation of antibodies it is meant that we are precipitating the gamma globulin fraction from the serum. Suitable host animals for inducing formation of the antibodies include mammals such as rabbits, horses, goats, guinea pigs, rats, cows, sheep, etc.
The formation of carcinogen specific antibodies in the host animals can be monitored by taking blood samples from the host animals and adding to it an amount of the carcinogen-protein antigen after absorption of antibodies to the protein antigen. The presence of a precipitate indicates antibody activity. The antigen treatment of the animal can be continued until the antibody titer reaches a maximum titer. In general, the antibody titer is defined as being the maximum concentration of protein precipitated following the addition of varying known concentrations of antigen to fixed volumes of serum, e.g. 0.5 ml.
The filter body through which the antibody of antibodies are dispersed may be any solid filter body, preferably shaped to conform to the smoke passageway of a smoking article. In one embodiment, the invention comprises a filter assembly for a smoking body including the antibody-impregnated filter body which filter body has, in respect to smoke flowing therethrough, a surface facing upstream and a surface facing downstream. The filter body can comprise the sole or multiple parts of a filter assembly and may either be a solid adsorbent or a material capable of reacting with the antibody to chemically bond it thereto. Thus, the filter body impregnated in accordance with the present invention may be incorporated into known filter assembly structures including the pupular "filter tip" structures generally defined by one or more filter plugs or cellulose materials such as cellulose fiber, particularly cellulose acetate fiber; glass fiber and the like; convoluted crepe paper sections, etc., with or without other adsorbent filter material, secured in coaxial alignment by suitable means such as a paper wrapper.
The term "filter body" as used in here includes as well the various adsorbents commonly used in tobacco smoke filter assemblies either as fillers in filter assembly chambers enclosed by plugs and a wrapper or as surface coatings on confoluted crepe paper filters. Thus, the antibodies of the invention may be impregnated into adsorbent aggregate materials commonly included in tobacco filter assembiles amongst which can be included, for example, activated carbon or charcoal, silica gel, manganese greensand, activated bentonite, cellulose powder, fullers earth, diatomaceous earth, alumina, molecular sieves, asbestos fiber, and the like.
Although the filter body of the invention has use in combination with any tobacco smoking article, it perhaps will find its greatest utility as a cigarette comprising a tobacco column wrapped in cigarette paper having attached integral thereto by means of a paper wrapper a filter assembly that includes the antibody-impregnated filter body of the invention. Illustrative of such filter-cigarette structures are those described, for example, in U.S. Pat Nos. 2,881,770, 2,915,069, 3,251,365 and 3,251,365.
Impregnation of the filter bodies of the invention can be effected in any desired manner, the only important criteria being that the antibody be dispersed throughout substantially the entire surface area of the filter body through which the tobacco smoke passes and that the antibody be present in amounts effective to effect removal of the tobacco smoke carcinogen. Impregnation may be conducted by immersing the filter body into a serum containing the antibody or other suitable impregnating solutions containing the antibody but ordinarily application of a drop or two of antiserum, that is, the serum containing the antibody, of sufficient strength, for instance, having a titer for a given antibody of at least about 1:1000 is usually effective to remove substantially all of the carcinogen. In cases where a filter assembly comprised of a plurality of filter bodies is employed, the application or impregnation of the antibody may be to any one or more of the filter bodies.
Where reduction or elimination of the various carcinogens contained in tobacco smoke is desired either multiple impregnations of antibody solutions or antiserums specific to the various carcinogens will be required or a single impregnation with a mixture of the different antibodies can be employed.
The optimum effective amount of antibody in the filter body will vary depending on various factors such as the size and concentration of the smoke stream, the particular filter material the filter body is fabricated from, the filter assembly employed, the surface area of the filter body utilized, etc. In general, however, the antibody will be present in an amount which falls in the range of 8 to 20 percent by weight based on the combined weight of the filter body and antiserum. If instead of antiserum, purified gamma globulin containing the carcinogen-specific antibodies is used, the gamma globulin will be present in an amount which falls in the range of 0.5 to 2.0 percent by weight.
The present invention will be further described below in conjunction with the following examples which are set forth for illustrative purposes and which are not to be construed as a limitation on the scope of the invention.
EXAMPLE I
Conjugation of Horse Serum Albumin with the Isocyanate
Dioxane was added to an aqueous solution of 2 percent horse serum albumin having a pH of 10 after which 3,4-benzo(a)pyrene-5-isocyanate in dioxane was gradually added. The final concentration of dioxane was brought to 45 percent and the temperature throughout the reaction was maintained at 0°-2°C. The solution was then diluted with water, stirred, and dialysed against water. The preparation was then centrifuged and filtered to remove particulate matter.
The resulting benzo(a)pyrene-horse serum albumin conjugate was then purified by a series of 2.8 M ammonium sulfate precipitations. The protein content of the resulting solution was determined and further purification of the conjugate was accomplished by -15°C acetone extraction of adsorbed amine. The precipitated conjugate was dissolved in cold water, dialysed and reprecipitated with ammonium sulfate. The benzo(a)pyrene-horse serum conjugate was then stored in a 2.5 M ammonium sulfate solution at 5°.
EXAMPLE II
Antibody Preparation
The benzo(a)pyrene-horse serum conjugate (17 mg/ml) of Example I was mixed with an equal volume of Freund's Complete Adjuvant. Injections were given to several young adult male rabbits consisting of multiple (5-6) intramuscular (I.M.) injections of 0.2 ml of the conjugate-adjuvant mixtures. Three weeks later a second intramuscular injection was given to each rabbit consisting of several multiple injections (0.2 ml) of the conjugate.
One week after the second immunization injection, the rabbits were bled by cardiac puncture and the blood was allowed to clot overnight in a refrigerator. The serum was removed by centrifugation from the clotted blood and analyzed to determine if antibodies specific for the benzo(a)pyrene portion of the conjugate were present.
To initially demonstrate that there were antibodies specific for benzo(a)pyrene present in the collected rabbit antisera, the antibodies specific for horse serum albumin (HSA) were adsorbed from the rabbit antisera. To remove the HSA antibodies, the antisera (0.7 ml) were incubated at 37° for 30 minutes with 0.1 ml of various dilutions of horse serum albumin (HSA). The adsorbed antisera was then centrifuged at 10,000 RPM (12,000 X G) for 30 minutes and the precipitated horse serum albumin-antibody complexes discarded. The resultant adsorbed (antibodies for HSA removed) antisera was then tested on Ouchterlony gel diffusion plates to demonstrate that the adsorbed antisera no longer reacted with HSA. This same adsorbed antisera was then tested on Ouchterlony plates against the original benzo(a)pyrene-HSA conjugate and found to give precipitin bands indicating that antibodies specific for benzo(a)pyrene were present.
The following example demonstrates preparation of cigarette filters contemplated by the present invention and the advantage offered by same.
EXAMPLE III
The cellulose acetate fiber filter of 500 cigarettes received a drop of the rabbit antiserum prepared as in Example II above and the cigarettes were allowed to dry. The cigarettes were then lit and smoked using the set up and procedure described in Hoffman, D., Wynder, E. L., A Study of Tobacco Carcinogenesis Cancer, 27, 848, 864 (1971), hereby incorporated by reference. The cigarette smoke was collected into a water wash bottle containing 500 ml of acetone through a plastic tubing into one end of which was placed the cigarette. The water wash bottle was placed under vacuum to effect the smoking. Collected water was removed by distillation and the acetone was evaporated leaving a residual "tar." The residual tar was subjected to the various separations and chromatographic steps as described in the aforementioned article by D. Hoffman and E. L. Wynder to obtain a relatively purified benzo(a)pyrene for quantitative analyses. In addition, to determine the % recovery of the benzo(a)pyrene during the separation procedures, the isotope dilution technique of adding 10 ug of 14 C-labeled benzo(a)pyrene was used.
The separation involved partitioning the sample between cyclohexane and methanol-water (4:1), followed by nitromethane. The nitromethane layers were evaporated leaving a residual material. This material was passed through an aluminum oxide column in n-hexane. Assays of the various fractions eluted from the column were by both gas chromatography with flame ionization detection and U.V. spectrometry. The percentage recovery was calculated by liquid scintillation of the benzo(a)pyrene fraction as described in ED. Wydner, E. L., Hoffman, D., Tobacco and Tobacco Smoke, Chapter 8, Certain Constituents of Tobacco Products.
A control group of 500 cigarettes was treated in the same manner except that the filter of the cigarette did not receive a drop of the antisera.
The results of the tests showed that the control smoke had on the average 2.0 ug of benzo(a)pyrene per 100 cigarettes and the smoke from the cigarettes treated with the antisera of the invention had an average of 0-0.1 ug of benzo(a)pyrene per 100 cigarettes.
EXAMPLES IV - VII
Antibodies to the following carcinogens:
Example IV dibenz(a,h)anthracene
Example V 7-H-dibenz(c,g)carbazole
Example VI dibenz(a,h)acridine
Example VII benzo(b)fluoranthene
are prepared using the general procedures of Examples I and II by substituting the above carcinogen for the benzo(a)pyrene. Impregnation of the cellulose acetate filter of cigarettes containing same produces essentially the same results as obtained in Example III.
This invention relates to tobacco smoke-filters. More particularly, the invention is directed to improved tobacco smoke filters especially useful in the removal of carcinogens from the smoke stream.
BACKGROUND OF THE INVENTION
In recent years the concern of smokers with the possible presence in tobacco smoke of cancer-inducing components and other substances hazardous to health has led to the development of various filtering materials for use in or with cigarettes, cigars and pipes. These filters, primarily designed to remove nicotine and the tarry components of cigarette smoke from the latter prior to its induction into the buccal cavity, have been incorporated in cigarettes as so-called "filter tips", have been provided for replaceable use in cigarette and cigar holders, have been built into throwaway holders for cigars and the like and have been proposed for incorporation in the stems of pipes.
Unfortunately, it has been found that such prior art filters do not in fact remove or even sufficiently reduce the carcinogens present in tobacco smoke, that is, the components known to induce cancer and other disorders in rats.
It has long been desired, therefore, and the object of many research projects of considerable cost to provide a tobacco smoke filter which is particularly effective against the carcinogens present in tobacco smoke while not eliminating therefrom the essential taste sought by tobacco smokers.
OBJECT OF THE INVENTION
Accordingly, one object of the invention is to provide a filter body which effectively removes known carcinogens from tobacco smoke passing axially therethrough without eliminating or adversely affecting desired taste sensations.
Another object of the invention is to provide a filter body and filter assemblies including same which offer the aforementioned advantages without the necessity of increasing the pressure drop over the filter bodies.
A further object of the invention is to provide a simple and economical method of making filter bodies capable of removing tobacco smoke carcinogens from existing preformed tobacco smoke filters.
SUMMARY OF THE INVENTION
The foregoing and other objects which will be apparent to one having ordinary skill in the art are achieved according to the present invention by a tobacco smoke filter having dispersed therethrough small amounts of antibody to a carcinogen found in tobacco smoke, said amounts being effective to remove at least substantial quantities of said carcinogen from said tobacco smoke as it passes through said filter.
The tobacco smoke filter of the invention may contain antibody to at least one of the several carcinogens known to be present both in tobacco and tobacco smoke and advantageously the filter contains a plurality of antibodies each of which is specific to one of the carcinogens of tobacco smoke. Principal carcinogens known to be present in tobacco smoke are benz(a)pyrene, 7-H-dibenz(c,g)carbazole, dibenz(a,h)anthracene, dibenz(a,h)acridine, benzo(b)fluoranthene.
The mechanism by which the antibody removes the respective carcinogens is not completely understood but it is believed that the antibody, being protein, is rehydrated by the water of combustion and while in its rehydrated state binds the carcinogen as it passes through the filter. Whatever the removal mechanism the high efficiency exhibited by the antibody-impregnated filter in removing tobacco smoke carcinogens is indeed surprising. As will be demonstrated in the working examples set forth below, the antibody-containing filter of the invention has been found to remove on the order of 20 times the amount of for instance, benz(a)pyrene removed by the same filter without the antibody.
The antibodies of the invention may be prepared by any suitable method known in the prior art. One appropriate method involves coupling the carcinogen to an immunogenic carrier material to form an antigen which is then injected into host animals from which antibodies specific to the carcinogen are obtained. The tobacco smoke carcinogens to which the invention is directed are small non-protein, molecules (haptens) which by themselves are devoid of antigencity. When the hapten moiety is coupled to an immunogenic carrier such as a protein, however, a antigenic material exhibiting an antibody response is provided. In a preferred embodiment, the carcinogen is covalently bonded to a protein or polypeptide molecule for instance by urea or peptide linkages. The urea linkage may involve for example reaction of an amino group on the protein or polypeptide and an isocyanate derivative of the carcinogen while a peptide linkage may involve either a carboxyl group on the protein or polypeptide chain or an amino group on the carcinogen and a carboxyl group on the polypeptide chain. It should be understood any of the prior methods known to those skilled in the art may be employed for covalently bonding the carcinogenhapten to the immunogenic carrier material.
As used herein, the term "immunogenic carrier material" is meant to include those materials which have the property of independently eliciting an immunogenic response in a host animal when injected therein and which can be coupled with covalent bonding to the aforementioned carcinogen haptens. Suitable carrier materials include, for example, proteins; natural or synthetic polymeric compounds such as polypeptides, e.g. polylysine or polyglutamic acid; polysaccharides; and the like. Particularly preferred carrier materials are proteins and polypeptides, especially proteins.
The identity of the protein material utilized in the preparation of a preferred antigen is not critical. Examples of preferred proteins useful in the practice of the invention include mammalian serum proteins such as, for example, horse serum albumin, bovine serum albumin, rabbit serum albumin, human serum albumin, and human gamma gobulin. Other suitable protein products will be suggested to one skilled in the art. It is generally preferred that proteins be utilized which are foreign to the animal hosts in which the resulting antigen is injected for antibody formation.
The reaction introducing isocyanate groups into the carcinogens for subsequent coupling with protein or polypeptide is conveniently prepared, for instance, as described in Creech, H. J., J. Amer. Chem. Soc. 63 576 (1941). According to this synthesis the carcinogen as, for example, benzo(a)pyrene is subjected to nitration with nitric acid to form the nitro-derivative of the carcinogen which is reduced with stannous chloride to the amine. Addition of phosgene to the amine produces the isocyanate of the carcinogen.
The coupling of the isocyanate of the carcinogen with protein or polypeptide to form a preferred antigen can be readily accomplished utilizing techniques now well known in protein chemistry as described, for instance, in Creech, H. J., Jones, R. N., J. American Chemical Society, 63 1661 (1941) and Creech, H. J., Jones, R. N., J. Amer. Chem. Soc. 62 1970 (1940). The reaction proceeds under relatively mile reaction conditions and involves the ε-amino groups of the lysine side chains of the protein. The general reaction may be schematic represented as follows:
ArNCO + H 2 N - protein ➝ ArNHCONH-protein
Ar = the aromatic carcinogen moiety.
The present invention also contemplates coupling a plurality of the carcinogens or even all of the carcinogens known to be present in tobacco smoke to an immunogenic carrier such as protein or polypeptide to thereby produce an antigenic substance which will produce upon injection into a host animal a serum containing antibodies to the various carcinogens. Such antigen containing a plurality of covalently bonded tobacco smoke carcinogens can be prepared by employing the general procedure described above for the covalent bonding of isocyanate derivatives of carcinogens with the ε-amino groups of proteins but utilizing in the reaction a mixture of the different carcinogens in their isocyanate form. Also, instead of taking one protein and attaching a plurality of tobacco smoke carcinogens, each carcinogen could be attached to a separate antigen and each of the carcinogen carriers injected into the same animal which will produce a serum containing antibodies to the various carcinogens.
The antibodies used in the filters of the present invention are obtained by injecting the antigens described above into a host animal repeatedly over a period of time and collecting the serum. The serum thus obtained and containing the antibody or antibodies specific to the carcinogen or carcinogens may be used as such in the treatment of the filters. If desired, however, the antibody or antibodies may be precipitated from the serum with a neutral salt solution, purified as by dialysis and/or column chromatography and resuspended in any suitable filter treating or impregnating solution. Illustrative of neutral salts for use in such antibody precipitations are ammonium sulfate, sodium sulfate and the like. It is clear to people skilled in the art that by precipitation of antibodies it is meant that we are precipitating the gamma globulin fraction from the serum. Suitable host animals for inducing formation of the antibodies include mammals such as rabbits, horses, goats, guinea pigs, rats, cows, sheep, etc.
The formation of carcinogen specific antibodies in the host animals can be monitored by taking blood samples from the host animals and adding to it an amount of the carcinogen-protein antigen after absorption of antibodies to the protein antigen. The presence of a precipitate indicates antibody activity. The antigen treatment of the animal can be continued until the antibody titer reaches a maximum titer. In general, the antibody titer is defined as being the maximum concentration of protein precipitated following the addition of varying known concentrations of antigen to fixed volumes of serum, e.g. 0.5 ml.
The filter body through which the antibody of antibodies are dispersed may be any solid filter body, preferably shaped to conform to the smoke passageway of a smoking article. In one embodiment, the invention comprises a filter assembly for a smoking body including the antibody-impregnated filter body which filter body has, in respect to smoke flowing therethrough, a surface facing upstream and a surface facing downstream. The filter body can comprise the sole or multiple parts of a filter assembly and may either be a solid adsorbent or a material capable of reacting with the antibody to chemically bond it thereto. Thus, the filter body impregnated in accordance with the present invention may be incorporated into known filter assembly structures including the pupular "filter tip" structures generally defined by one or more filter plugs or cellulose materials such as cellulose fiber, particularly cellulose acetate fiber; glass fiber and the like; convoluted crepe paper sections, etc., with or without other adsorbent filter material, secured in coaxial alignment by suitable means such as a paper wrapper.
The term "filter body" as used in here includes as well the various adsorbents commonly used in tobacco smoke filter assemblies either as fillers in filter assembly chambers enclosed by plugs and a wrapper or as surface coatings on confoluted crepe paper filters. Thus, the antibodies of the invention may be impregnated into adsorbent aggregate materials commonly included in tobacco filter assembiles amongst which can be included, for example, activated carbon or charcoal, silica gel, manganese greensand, activated bentonite, cellulose powder, fullers earth, diatomaceous earth, alumina, molecular sieves, asbestos fiber, and the like.
Although the filter body of the invention has use in combination with any tobacco smoking article, it perhaps will find its greatest utility as a cigarette comprising a tobacco column wrapped in cigarette paper having attached integral thereto by means of a paper wrapper a filter assembly that includes the antibody-impregnated filter body of the invention. Illustrative of such filter-cigarette structures are those described, for example, in U.S. Pat Nos. 2,881,770, 2,915,069, 3,251,365 and 3,251,365.
Impregnation of the filter bodies of the invention can be effected in any desired manner, the only important criteria being that the antibody be dispersed throughout substantially the entire surface area of the filter body through which the tobacco smoke passes and that the antibody be present in amounts effective to effect removal of the tobacco smoke carcinogen. Impregnation may be conducted by immersing the filter body into a serum containing the antibody or other suitable impregnating solutions containing the antibody but ordinarily application of a drop or two of antiserum, that is, the serum containing the antibody, of sufficient strength, for instance, having a titer for a given antibody of at least about 1:1000 is usually effective to remove substantially all of the carcinogen. In cases where a filter assembly comprised of a plurality of filter bodies is employed, the application or impregnation of the antibody may be to any one or more of the filter bodies.
Where reduction or elimination of the various carcinogens contained in tobacco smoke is desired either multiple impregnations of antibody solutions or antiserums specific to the various carcinogens will be required or a single impregnation with a mixture of the different antibodies can be employed.
The optimum effective amount of antibody in the filter body will vary depending on various factors such as the size and concentration of the smoke stream, the particular filter material the filter body is fabricated from, the filter assembly employed, the surface area of the filter body utilized, etc. In general, however, the antibody will be present in an amount which falls in the range of 8 to 20 percent by weight based on the combined weight of the filter body and antiserum. If instead of antiserum, purified gamma globulin containing the carcinogen-specific antibodies is used, the gamma globulin will be present in an amount which falls in the range of 0.5 to 2.0 percent by weight.
The present invention will be further described below in conjunction with the following examples which are set forth for illustrative purposes and which are not to be construed as a limitation on the scope of the invention.
EXAMPLE I
Conjugation of Horse Serum Albumin with the Isocyanate
Dioxane was added to an aqueous solution of 2 percent horse serum albumin having a pH of 10 after which 3,4-benzo(a)pyrene-5-isocyanate in dioxane was gradually added. The final concentration of dioxane was brought to 45 percent and the temperature throughout the reaction was maintained at 0°-2°C. The solution was then diluted with water, stirred, and dialysed against water. The preparation was then centrifuged and filtered to remove particulate matter.
The resulting benzo(a)pyrene-horse serum albumin conjugate was then purified by a series of 2.8 M ammonium sulfate precipitations. The protein content of the resulting solution was determined and further purification of the conjugate was accomplished by -15°C acetone extraction of adsorbed amine. The precipitated conjugate was dissolved in cold water, dialysed and reprecipitated with ammonium sulfate. The benzo(a)pyrene-horse serum conjugate was then stored in a 2.5 M ammonium sulfate solution at 5°.
EXAMPLE II
Antibody Preparation
The benzo(a)pyrene-horse serum conjugate (17 mg/ml) of Example I was mixed with an equal volume of Freund's Complete Adjuvant. Injections were given to several young adult male rabbits consisting of multiple (5-6) intramuscular (I.M.) injections of 0.2 ml of the conjugate-adjuvant mixtures. Three weeks later a second intramuscular injection was given to each rabbit consisting of several multiple injections (0.2 ml) of the conjugate.
One week after the second immunization injection, the rabbits were bled by cardiac puncture and the blood was allowed to clot overnight in a refrigerator. The serum was removed by centrifugation from the clotted blood and analyzed to determine if antibodies specific for the benzo(a)pyrene portion of the conjugate were present.
To initially demonstrate that there were antibodies specific for benzo(a)pyrene present in the collected rabbit antisera, the antibodies specific for horse serum albumin (HSA) were adsorbed from the rabbit antisera. To remove the HSA antibodies, the antisera (0.7 ml) were incubated at 37° for 30 minutes with 0.1 ml of various dilutions of horse serum albumin (HSA). The adsorbed antisera was then centrifuged at 10,000 RPM (12,000 X G) for 30 minutes and the precipitated horse serum albumin-antibody complexes discarded. The resultant adsorbed (antibodies for HSA removed) antisera was then tested on Ouchterlony gel diffusion plates to demonstrate that the adsorbed antisera no longer reacted with HSA. This same adsorbed antisera was then tested on Ouchterlony plates against the original benzo(a)pyrene-HSA conjugate and found to give precipitin bands indicating that antibodies specific for benzo(a)pyrene were present.
The following example demonstrates preparation of cigarette filters contemplated by the present invention and the advantage offered by same.
EXAMPLE III
The cellulose acetate fiber filter of 500 cigarettes received a drop of the rabbit antiserum prepared as in Example II above and the cigarettes were allowed to dry. The cigarettes were then lit and smoked using the set up and procedure described in Hoffman, D., Wynder, E. L., A Study of Tobacco Carcinogenesis Cancer, 27, 848, 864 (1971), hereby incorporated by reference. The cigarette smoke was collected into a water wash bottle containing 500 ml of acetone through a plastic tubing into one end of which was placed the cigarette. The water wash bottle was placed under vacuum to effect the smoking. Collected water was removed by distillation and the acetone was evaporated leaving a residual "tar." The residual tar was subjected to the various separations and chromatographic steps as described in the aforementioned article by D. Hoffman and E. L. Wynder to obtain a relatively purified benzo(a)pyrene for quantitative analyses. In addition, to determine the % recovery of the benzo(a)pyrene during the separation procedures, the isotope dilution technique of adding 10 ug of 14 C-labeled benzo(a)pyrene was used.
The separation involved partitioning the sample between cyclohexane and methanol-water (4:1), followed by nitromethane. The nitromethane layers were evaporated leaving a residual material. This material was passed through an aluminum oxide column in n-hexane. Assays of the various fractions eluted from the column were by both gas chromatography with flame ionization detection and U.V. spectrometry. The percentage recovery was calculated by liquid scintillation of the benzo(a)pyrene fraction as described in ED. Wydner, E. L., Hoffman, D., Tobacco and Tobacco Smoke, Chapter 8, Certain Constituents of Tobacco Products.
A control group of 500 cigarettes was treated in the same manner except that the filter of the cigarette did not receive a drop of the antisera.
The results of the tests showed that the control smoke had on the average 2.0 ug of benzo(a)pyrene per 100 cigarettes and the smoke from the cigarettes treated with the antisera of the invention had an average of 0-0.1 ug of benzo(a)pyrene per 100 cigarettes.
EXAMPLES IV - VII
Antibodies to the following carcinogens:
Example IV dibenz(a,h)anthracene
Example V 7-H-dibenz(c,g)carbazole
Example VI dibenz(a,h)acridine
Example VII benzo(b)fluoranthene
are prepared using the general procedures of Examples I and II by substituting the above carcinogen for the benzo(a)pyrene. Impregnation of the cellulose acetate filter of cigarettes containing same produces essentially the same results as obtained in Example III.