CZ58996A3 - Process for removing harmful oxidative and carcinogenic volatile nitrosocompounds from cigarette smoke by making use of biological substances - Google Patents

Abstract

The invention relates to a method for removing harmful compounds contained in cigarette smoke (NO, NOX, carcinogenic nitro compounds, free radicals, H2O2, CO, aldehydes and trace elements), which have not yet been sufficient retained by conventional cigarette filters. Specifically includes the enrichment of current current filters biological substances of metal ions (Fe2 +, Cu2 +, Mg 2+) complexed with the porphyrin ring as well as Fe2 + ions stereospecifically bound to protein molecules either separately or in combination. Enriching these conventional filters with the above biological substances does not change the physical characteristics of cigarette smoke (smell, taste and appearance) or the physical properties of the filter itself.

Description

Method for removing harmful oxidative and carcinogenic volatile nitroso compounds from cigarette smoke using biological substances

Technical field

The present invention relates to a method for the removal of harmful compounds, i.e., nitrogen oxides, free radicals, aldehydes, hydrogen peroxide, carbon monoxide, trace elements and carcinogenic volatile nitroso compounds which are inhaled when smoking cigarettes, substances which have not yet been sufficiently retained using conventional cigarette filters.

BACKGROUND OF THE INVENTION

A set of publications in international journals confirms that cigarette smoke is divided into two phases: a) solid phase (tar) and b) gas phase. This separation occurs using a typical Cambridge glass fiber filter which removes 99.9% of particles larger than 0.1 µm. Cigarette tar contains significantly high concentrations of very stable free radicals that can be classified into at least four different categories. Semichinones in equilibrium with quinone and hydroxyquinones are considered to be free radicals with the most interesting chemical properties. The quinone system reduces the molecular oxygen to form superoxide (O 2), which then spontaneously dismounts to form hydrogen peroxide (H 2 O 2). In the gas phase, there are more than 10 ¹⁵ retractable organic radicals with a half-life of less than 1 second that are inhaled. Paradoxically, however, despite their flash half-life, these radicals can maintain high levels of activity for more than 10 minutes in the gas phase. In fact, the concentration of these radicals is correspondingly increased as they approach the end of the cigarette filter. An explanation of this paradox can be found in maintaining a lasting state; due to the continued production of free radicals (Pryor WA, Stone K., Ann.NYAcad.Sci.686: 12-28, 1993).

Nitric oxide (NO) is the most important free radical in the gas phase of cigarette smoke, which during the smoking process participates in the reaction sequence which generates nitrogen dioxide, isoprene radicals, peroxyl radicals and alkoxy radicals. Cigarette smoke also contains an adequate number of aldehydes that contribute to its toxic effects. It has been found that instantaneous amounts of aldehydes extracted from cigarette smoke cause both protein catabolism and oxidation of the thiol groups of plasma proteins. These properties are attributed to the aldehydes and are the result of reactions between the carbonyl group of the aldehydes and the -SH and -NH2 groups of the plasma proteins. For example, acrolein from cigarette smoke reacts rapidly with -SH groups to form carbonyl compounds (Alving K., Forhem C. and Lundberg J.M.,

Br.J.Pharmacol. 110: 739-746,1993). Trace elements such as iron, copper, manganese and cadmium are present in the tar of cigarette smoke, which are involved in many free radicals producing reactions and lead to the formation of very active secondary radicals (e.g., peroxy radicals, alkoxy radicals, superoxide, cytotoxic aldehydes, etc.). The introduction of trace elements into the lungs during cigarette smoking results in a series of redox reactions in both lung fluids and alveolar macrophages, resulting in the formation of very active hydroxyl radicals (OH *). These hydroxyl radicals are mostly formed in the presence of iron by a Fenton reaction. Copper can also form hydroxyl radicals by reaction with hydrogen peroxide in the lungs. Manganese in low concentrations (IQ Μ ^-7), stimulates the soluble guanylate cyclase of the endothelial cells, lung inducing the production of nitric oxide and superoxide through a positive feedback mechanism (Youn YK _t Lalonde, C. and Demling, R., Free Rad.Biol.Med.12: 409 (1992). Carbon monoxide is produced during the combustion of tobacco. The amount of CO is retained in the lungs even after exhalation, resulting in stimulation of soluble guanylate cyclase after its interaction with the herae family of endothelial cell enzymes and other cells in lung tissue. Increased levels of cyclic GMP in cells associated with a positive feedback mechanism increase nitric oxide and superoxide production (Watson A., Joyce, Hopper L., and Pride)

N.B., Thorax 48: 119-124,1993). NO gas, which can be produced by many cell types, including vascular endothelial cells and reticular endothelial cells, causes smooth muscle relaxation (Lowenstein C.J., Dinerman J.L., Snyder S.H.

Ann.Intern.Med.120: 227-237, 1994). There are also exogenous sources of NO, which are considered similarly responsible for causing damage to blood vessels and other tissues. It is well known that secondary and tertiary amines can react with nitrites and other nitrosating agents to form N-nitrosoamine (Lowenstein C.J., Dinerman J.L., Snynder S. H. Ann. Intern. Med. 120: 227-237, 1994). Since 1974, many studies have demonstrated that during harvesting, tobacco pressing and smoking, alkaloids are nitrosated to tobacco-specific N-nitrosoamines (TSNA). Among the TSNAs identified in tobacco and / or its smoke are N-nitrosonornicotine (NNN),

4- (methylnitrosoamino) -1- (3-pyridyl) -1-butanone (NNK) and 4- (methylnitrosoamino) -1- (3-pyridyl) -1-butano1 (NNAL) by strong animal carcenogens. NNN induces tumor in the lungs of mice, tumors of the trachea in hamsters and tumors of the nasal cavity and esophagus in rats. NNK induces lung tumors in mice, hamsters and rats as well as liver, nasal cavity, and pancreatic tumors in rats. Oral administration of a mixture of NNN and NNK elicits tumors in the oral cavity and rat lungs. A typical amount of both NNK and NNN in a mainstream cigarette is 200 ng / cigarette (Hecht S.S., Spratt T.E. and Trushing N. Carcinogenesis, 9: 161-165, 1988).

Our recent research on the effect of cigarette smoke on lung tissue has found that NO reacts with superoxide to produce a strong oxidative radical peroxynitrite (ONOO-), which causes secondary damaging reactions in key biomolecules. Both the metabolic and harmful effects of NO in cells have been studied in our laboratory in vitro and in yivo experiments.

NO is oxidized, in the presence of oxygen, to nitrogen dioxide (NO2). The rate of this oxidation depends on the oxygen concentration and the square of the NO concentration. Nitrogen dioxide is clearly cytotoxic and is transformed into nitrite and nitrate in aqueous solutions. In addition, NO forms complexes with trace elements and / or metalloproteins, such as hemoglobin (Wink, D.A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. E., Chem. Res. Oxicol. 6: 23-27, 1993).

NO, which reacts with superoxide to produce the harmful compound ONOO, may cause certain types of superoxide toxicity. ONOO 'is unusually stable, consistent with its strong oxidation potential (+1.4 V). During decomposition, it strongly forms oxidizing derivatives, including the hydroxyl radical, nitrogen dioxide and nitronium ion. Consequently, any modification in NO and superoxide tissue production may result in creatively strong secondary oxidation radicals (Deliconstantinos G., Villiotou V., Stavrides JC, Cancer M.Biol. 1: 77-86, 1994). Finally, ONOO 'and its esters (RO-ONO or RP-ONO2) tend to inactivate the alpha-1-proteinase inhibitor (alPI). This can be attributed to the fact that: (a) hydrogen peroxide alone does not cause rapid inactivation of aPlI, but acts only in the presence of NO, producing ONOO * and rapid inactivation of alPI; ONOO * acts to inactivate 1PI alone and c) amines and amino acids protect 1PI protenase from rapid inactivation (Mořen JJ and Pryor WA, Chem.Res.Toxicol. 5: 425-431, 1992). In addition to the free radicals contained in cigarette smoke, activated alveolar macrophages are another important source of free radical production by smokers. Alveolar macrophages activated by cigarette smoke burst when breathing, leading to increased production of * oxygen free radicals (mainly 0 * 2, NO and H2O2). Increased levels of both alveolar macrophages and circulating neutrophils appear in smokers. Oxygen free radicals of cigarette smoke have also been involved in the development of lung cancer. Inhaled cigarette smoke causes increased oxidative stress in lung cells, leading to a reduction in the concentration of intracellular antioxidants, H2O2 reacts, through the production of hydroxyl radicals, to the DNA of the cells and causes double strand disruption. Since this interruption can be prevented by the addition of catalase, the harmful effects of H2O2 and hydroxyl radicals on cellular DNA are thus indirectly confirmed (Leanderson P., Ann.NY Acad. Sci. 686: 249-261, 1993). In addition, H2O2 can cause transformation in the tracheal epithelium of the lung and have been linked in the development of bronchogenic cancer in smokers. This harmful role of H2O2 (contained in cigarette smoke) in lung cells and the development of lung cancer is thus strongly indicated. The cigarette tar tar contains both semichinone radicals and iron and a system for producing hydroxyl radicals. The various trace elements contained in the tar of cigarette smoke (Fe, Cu, Mn, Cd) can act both intracellularly and extracellularly. Fe ^{2 +} well-known Fenton reaction:

Fe ²⁺ + H ² O 2> Fe ³⁺ + OH 'and OH * cause the oxidation reaction to the hydroxyl radical. Similarly, production of hydroxyl radicals can be achieved in Cd ² *. Mn ² * (|

is a characteristic stimulator of soluble guanylate cyclase activity. Cd ² * contained in cigarette smoke is exceptionally [toxic in the lungs. Smokers experience twice the normal E concentration of Cd ² * in their lungs. This suggests that Cd ² * (replaces Zn ² * in the presence of a normal state in the endothelium of the lung vessels (Kostial K., in: Trace Elements in Human and. J

Animal Nutrition (ed. W.Mertz) fifth ed., SV.2: 319-345, j

Academic Press, Inc.Orlando, Fl. 1986). The aldehydes present in cigarette smoke react immediately with the -SH and -NH 2 groups of proteins and become inert. Crotonaldehyde (a, 0 unsaturated aldehyde contained in cigarette smoke decreases the concentration of -SH groups and increases the concentration of carbonylproteins (Stadtman E.R.,

Science 257: 1220-1224 (1991).

Currently, cigarette filters are highly recommended.

The immediate effect of adding filters to cigarettes is to achieve maximum retention of harmful compounds present in both the gaseous and solid phase in cigarette smoke. Epidemiological studies in smokers have shown that it is dose-dependent here whether cigarette smoke is administered in the gas phase, the solid phase or the combined phase (Surgeon General of U.S.Public

Health Service. The health consequences of using smokeless (tobacco, N.H.Publ. No 86-2874, Bethesda, MD, 1986). It has been found that modification of the cigarette is itself a practical approximation to the reduction of harmful compounds contained in cigarette smoke. This was achieved first by using conventional filters and then by changing the tobacco composition by chemical treatment.

Changes in cigarette production were also made using porous paper or paper made from tobacco leaves. Much effort has been made over the last 15 years to make smoking less harmful: by reducing the amount of smoke to

Ί cigarette; changing the cigarette diameter; and using perforated filters. Perforated filters allow the cigarette smoke to be diluted with air up to 50%. Activated carbon was also used in combination with perforated filters. This was followed by a significant reduction in tar and nicotine yields in the smoke. Such techniques are used in particular for developed countries such as Austria, Canada, France, Germany, Sweden, England and the USA. The average yield of tar and nicotine in an American cigarette was reduced from 39 mg and 2.7 mg in 1955 to 13 mg and 1 mg in 1991. In the European Community, the trend of tar and nicotine reduction in cigarette smoke is still ongoing. The maximum permitted tar threshold in January 1993 is 15 mg and is to be reduced to 12 mg in early January 1998. However, in other countries the tar content of cigarette smoke is 22 mg (Mitacek EJ, Brunneman KD, Pollednak AP, Hoffman D. and Suttajit M 20: 764-773 (1991). The changes made in cigarette production have led to the specific removal of certain toxic substances from cigarette smoke; more specifically, cellulose acetate filters have been introduced that allow partial removal of semi-volatile phenols and volatile N-nitrosoamines (Brunnermann K.D., Hoffman D., Recent

Adv.Tobacco Res. 17: 71-112 (1989)]. Carbon monoxide is selectively reduced using perforated filters. The concentration of carcinogenic polynuclear aromatic hydrocarbons (PAH) was selectively reduced using nitrite-enriched tobacco. However, the reduction of PAH in tobacco using high nitrite concentrations leads to an undesirable increase in carcinogenic N-nitrosoamines, so it was necessary to reduce PAH by alternative means (Hoffman D., Hoffman I., Wynder E. 1., Lung Cancer and the Changing Cigarette in Relevance to Human Cancer of N-Nitroso Compound, Tobacco Smoke and Mycotoxins.

O'Neill I.K., Chen J. and Bartsch H.) vol.105: 449-459, 1991).

From the above it is clear that it is necessary to produce a filter

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c capable of removing harmful nitrogen oxides, free radicals, _________ hydrogen peroxide, aldehydes and carcinogenic nitroso compounds, all of which are responsible for the harmful effects of cigarette smoke on the respiratory and cardiovascular systems. To identify harmful compounds contained in a cigarette smoke, chemical, biological experiments were performed. j

The chemical experiments performed are as follows: i

(a) Identification and quantitative determination of NO and NOx using a new chemical and biological method (developed in our laboratory).

b) Identification of free radicals using lucigen-dependent chemiluminescence methods.

c) Identification of aldehydes and quinone by stimulation of the enzymatic system 5 luciferin-luciferase (this method was also developed in our laboratory).

(d) Identification and quantification of trace elements using the luciferin oxidation method in the presence of luciferin

ATP (this method was developed in our laboratory).

(e) Identification and quantitative determination of H2O2 using isoluminol microperoxidase-dependent chemiluminescence method.

f) Identification and quantitative determination of 0N00 by spectrophotometric and luminol enhanced chemiluminescence methods.

g) Identification of carcinogenic nitroso compound by luminol enhanced chemiluminescence.

Biological experiments were performed as follows:

a) Identification of NO using isolated soluble guanylate cyclase activity as a functional parameter.

b) Identification of 0N00 using 0N00 induced oxidative stress evaluation of human erythrocytes.

c) Identification of CO using isolated soluble guanylate cyclase activity as a functional parameter.

Furthermore, the following in vitro experiments were performed:

a) Isolation of alveolar macrophages from rat lung.

b) Evaluation of tert-butyl hydroperoxide-induced oxidative stress of alveolar macrophages (t-BHP).

c) Determination of NO / NO2 / 0N00 ~ produced by alveolar macrophages.

d) Determination of H2O2 produced by alveolar macrophages.

e) Effect of exogenous H2O2 on NO production by alveolar macrophages

In vivo experiments on human volunteers were performed to determine the following compounds:

(a) Determination of NO in non-smoker's exhaled air.

b) Determination of NO in the exhaled air of smokers.

c) Determination of NO in exhaled cigarette smoke.

d) Determination of 0N00 ~ in the exhaled cigarette smoke.

e) Determination of free radical in exhaled cigarette smoke

f) Determination of aldehydes in exhaled cigarette smoke.

To determine the NO, NOx contained in a) cigarette smoke, b) released by alveolar macrophages upon stimulation by cigarette smoke and c) in exhaled cigarette smoke of human volunteers, a 2.5 cm clear Plexiglass clear rod chamber was designed and manufactured. One end drilled by a lathe to create an identical conical cavity in each of the Plexiglas rods. These were further processed and polished at the open ends to form a joined tapered area, forming a very tight connection between the two conical cavities. A thin square Teflon layer (0.0015 inch thick polytetrafluoroethylene) was placed between the assemblies, which were re-compressed by screws tightened with fingers. The two tubular portions on both sides of the membrane allow the biologically active sample and reactive substance to inject, remove or modify on either side of the membrane during the biological reaction (Fig. 1).

A. Determination of NO by chemiluminescence

A standard NO solution was prepared according to literature (Deliconstantinos G., Villiotou V., Fassitsas C., (1992),

J.Cardiovasc.Pharmacol. 12 pp.63-65) and (Deliconstantinos G., Villitou V., Stavrides JC, (1994) in: Biology of Nitric Oxide, ed. Feelish M., Busse R., Moncanda S., Portland Press, in press) . Reaction solution composed of Hank's Balanced Salt Solution (HBSS) pH 7.4; H2O2 (500 μΜ); luminol (30 μΜ and total volume was 500 μΐ) The vial was vigorously stirred and emission was recorded in a Bedrthold AutoLumat LB953 luminometer.

B. Chemical determination of NO / NO2

The chemical determination of NO was based on diazotization of sulfanolamide NO at acidic pH and subsequent oxidation of scopoletin, which can be detected fluorometrically as previously described by Deliconstantinos G., Villitou V., Fassitsas C.,

J. Cardiovasc. Pharmacol 12: 63-65, 1992). Alveolar macrophages (HBSS ¢ 10 ⁶ cells / ral) were mixed with 100 μΐ reagent consisting of: 20% sulfanilamide in 20% H3PO4 and 25 μΜ scopoletin. Fluorescence decomposition was monitored at room temperature (22 ^° C) using an Aminco SPF-500 Fluorescence Spectrophotometer. Fluorescence was monitored continuously until the slope of the line could be measured (approx. 8 min). The slope measurements were converted to nmol NO using a standard curve constructed with different concentrations of pure NO. The nitrite (NO2) end product of NO synthesis was measured based on its accumulation in the supernatants of cells cultured by its reaction with Griess reagent. _______

C. Spectroscopic determination of peroxynitrite (ONOO ')

ONOO 'was synthesized, titrated, and stored as previously described (Deliconstantinos G., Villitou V., Stavrides JC, in: Biology of nitric oxide (ed. Feelisch,., Busse R. and Moncada S.) Protland Press (in press) . Due to the instability of ONOO "at pH 7.4, UV stektra were recorded directly after mixing of H2O2 and NO solution. the concentration of ONOO" was determined based on E302 nm value of 1670 M ^"1 cm ^-1. UV spectra were reported after subtraction of the basal UV H2O2 to the corresponding concentrations.

D. Evaluation of free radicals

Free radical scoring was performed using lucigenin / DAMCO (1,4-diazabicyclo [2.2.2] octane) -induced chemiluminescence as previously described (Deliconstantinos G., Villitou V., Krueger GRF, J. Viral Dis. 1 : 22-27 (1993). The reaction mixture contains HBSS pH 7.4; lucigenin (30 μΜ); DAMCO (100 μΜ). The vial was vigorously mixed and emission was recorded on a Bedrthold AutoLumat LB953 luminometer. Oxygen free radical acceptors (SOD, mannitol, histidine, methionine) were used.

E. Evaluation of trace elements and aldehydes

The assays were based on luciferase catalyzed oxidation of D-luciferin in the presence of ATP-magnesium salt according to the reaction:

luci ferasa

LH2 + ATPMG ² * + 02 ------> 0xylucifine + ATP + O2aPPi + Mg ^{2 +} + light

The trace elements Cd ²⁺ , Cu ² * Fe * - * increase the α-t-vvituluciferase and the maximum cherailuminescence response is proportionally increased according to the trace element concentration up to 10 µg. The reaction was carried out in HBSS pH 7.4 in a total volume of 0.5 ml.

The same luciferin / luciferase enzyme system was used for the evaluation of aldehydes, but in the absence of ATP. The aldehydes react with the enzyme system to produce chemiluminescence in the absence of ATP. The reagents that were used were obtained from the ATP assay kit (Calbiochem-Novabiochem CA, USA).

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F. Isolation of alveolar macrophages

Briefly, rats were sacrificed by intravenous injection of sodium pentobarbital, thorax was opened, lung perfused with blood Ca ²⁺ free cold (4 ° C) phosphate buffered saline (PBS; pH 7.4) and removed whole from the thoracic cavity. Rat lung homogenate was obtained by repeatedly passing tissue through a syringe and then gradually passing it through finer 32, 62 and 68 pore mesh sieves and under a constant flow of Finkelstein's balanced salt solution (FBSS; pH 7.4). The final suspension of alveolar macrophages was collected, filtered, and centrifuged at 300 xg for 10 minutes per cell pellet. The cell pellet containing more than 98% macrophages was washed and resuspended in Ringer's solution. The procedure was repeated twice. Approximately 10 x 10 ⁸ macrophages were obtained per rat. Viability was assessed using trypan blue.

F. Identification of nitroso compounds

The nitroso compounds were identified by slow release. - The solution contains dimethylnitrosamine and / or diethylnitrosamine (1 μΜ); H2O2 (500 μΜ); Lurainol (30 μΜ) in HBSS pH 7.4, total volume 0.5 ml. The violet was vigorously stirred and emission was recorded using a Bedrthold AutoLumat LB953 luminometer. Mannitol (100 mM), DMSO (100 mM) and cysteine (3.0 mM) were used to identify ONOO formation.

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G. Isolation of alveolar macrophages

Briefly, rats were sacrificed by intravenous injection of sodium pentobarbital, thorax was opened, lung perfused with blood Ca ²⁺ free cold (4 ° C) phosphate buffered saline (PBS; pH 7.4) and removed completely from the thoracic cavity. Rat lung homogenate was obtained by repeatedly passing the tissue through a syringe and then passing it gradually through finer steel screens of 32, 62, and 68 per inch mesh, and under a constant flow of Finkelstein's balanced salt solution (FBSS; pH 7.4). The final aplveolar macrophage suspension was collected, filtered, and centrifuged at 300 xg for 10 minutes onto the cell pellet. The cell pellet containing more than 98% macrophages was washed and resuspended in Ringer's solution. The procedure was repeated twice. Approximately 10 x 10 ⁸ macrophages were obtained per rat. Viability was assessed using trypan blue.

H. Tert-butyl 1-hydroperoxide-induced oxidative stress of alveolar macrophages (t-BHP)

Al-free macrophage-induced oxygen-free radical generation by t-BHP (2.5 mM) was determined using the luminol chemiluminescence method.

The chemiluminescence response was recorded as previously stated and

with the help of Bedrthold ΑυΐοΒυΓη3ΐ (ϋΤ1ίοοη5ΐ3ηζίηο5θ., ______________ „__________ ^r

Krueger G. R. F., J. Viral Dis. 1, 22-27, 1993). , p

I. Determination of hydrogen peroxide (H2O2) i

AND

An isoluminol / microperoxidase cocktail (100 mM sodium borate, 1 mM isoluminol, 0.01 mM microperoxidase in 70% water and 30% methanol at pH 8) was prepared. 50 μΐ of this reagent was mixed with isolated alvelolar macrophages (10 ⁶ cells) in.

HBSS in a total volume of 0.5 ml. The chemilurainescent response was | converted to nmol H2O2 using a standard curve generated by j

with different concentrations of pure H2O2. !

J. Preparation and purification of soluble guanylate cyclase (sGC) for CO 2

rating!

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sGC from human endothelial cells was purified

GTP-agarose chromatography. Cytosols (10 mg protein) were loaded onto a GTP-agarose column (1.8 x 9 µm), equilibrated with 25 mM Tris-HCl buffer pH 7.6 containing 250 mM.

sucrose and 10 mM MnCl 2. The sGC was then eluted from the column with 5 ml of equilibration buffer plus 10 mM GTP.

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and

K. Determination of cyclic GMP s

CGMP concentrations were determined by radioimmunoassay following acetylation of the samples with acettanhydride (Delikonstantinos G., and Kopeikina L., Anticancer Res: 9: 753-760, 1989). The reaction mixture contains triethanolamine / HCl (50 mM); ceratine phosphate (5 mM);

MgCl 2 (3 mM), isobutylmethylxanthine (1 mM), creatine kinase (0.6 µ units), GTP (1 mM); soluble guanylate cyclase (1 pg protein) in a total volume of 150 μΐ. Reactions were initiated by the addition of GTP and incubated for 10 min at 37 ° C. The incubation medium was aspirated and cGMP was extracted by addition of ice

Cold HCl - (Or fM). After 10 min, samples were transferred to a new plate, dried and reconstituted in 5 mM sodium acetate (pH 4.75) for the determined cGMP. The generated cGMP was determined using a cGMP assay kit (Amersham).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide and apply methods using biological substances that specifically react and capture the following:

(a) NO and NOx;

(b) CO,

(c) H2O2;

d) free radicals,

(e) aldehyde-quinones;

(f) carcinogenic nitroso compounds;

(g) remove trace elements cadmium, copper, manganese, iron, etc., which are inhaled during smoking.

The present invention is based on the assumption that:

(a) There is a choice of suitable capture agents, hemoglobin-like or erythrocyte-lysate, or any substance that contains stereospecifically bound iron

b) There is a choice of acceptors that contain a porphyrin ring with iron (eg protoporphyrin)

c) There is a selection of acceptors that contain a porphyrin ring that does not necessarily contain iron

d) There is a choice of acceptors that contain a porphyrin ring complexed with other metals, eg Mg ² *, Cu ² *

e) The biotechnological process will be set up to enrich the materials currently used for the manufacture of cigarette filters containing the above-mentioned biological substances - acceptors.

The basic idea of the present invention consists in the concept that the impregnation of conventional cigarette filters and / or activated carbon filters can be enriched with biological substances characterized by the presence of metal ions Fe ² *, Cu ²⁺ , Mg ² * complexed with a porphyrin ring, as well as Fe ²⁺ specifically bound to protein molecules, allowing the harmful compounds contained in the cigarette to be removed before the smoker inhales cigarette smoke. This is a major characteristic of the present invention and constitutes an indisputable innovation with very easy industrial applicability.

DETAILED DESCRIPTION OF THE INVENTION

Ways of industrial use

The present invention has been practiced as follows in terms of its industrial applicability:

A solution of 1 mg / ml hemoglobin and / or erythrocyte lysate in phosphate buffered saline (PBS) pH 7.4 was prepared and added to 100 mg activated charcoal. The mixture was incubated for 30 min at room temperature and filtered through S&S Carl Schleier & Schuell Co USA filter paper. The amount of unabsorbed hemoglobin in the filtrate was determined spectrophotometrically. Hemoglobin-enriched activated carbon was dried at room temperature. An amount of 200 mg of dry activated carbon enriched with hemoglobin was placed between two conventional filters so that all sucked cigarette smoke comes into contact with the active groups of molecules (Fe ² *, Fe ³ *, -SH,

-NH 2) (Fig. 2). These compatible materials are now able to be used for the production of new cigarette filters, which will now be referred to as biological filters.

- Alternatively, hemoglobin may be replaced by biological ________s characterized by the presence of Fe ² *, Cu ² *, Mg ² * metal ions complexed with the porphyrin ring, as well as Fe ² * bound stereospecifically to protein molecules such as transferrin, catalase, protoporphyrin, cytochrome C, chlorophyll.

Alternatively, a solution of 5 mg / ml hemoglobin and / or erythrocyte lysate in phosphate buffered saline (PBS) pH 7.4 was prepared and scanned at 25 ° C using an Acta Beckman recording spectrophotometer. The absorbance peak was consistently observed at 540 nm and 575 nm (Smith R.P., Kruzsyma H., J.Pharmacol.Exper.Ther. 191, 557-563, 1974).

Commercially available cigarette filters were impregnated with these solutions and dried at 25-35 ° C. These compatible materials are now ready for use in the manufacture of new cigarette filters, which will now be referred to as biological filters. These new biological filters ensure that the smoke that is inhaled comes into full contact with the active groups of hemoglobin molecules and / or the lysate in the filter without altering the physical properties or taste of cigarette smoke. For aesthetic reasons, a small portion (3 mm) of a conventional filter may be adapted to the visible eye of the biological filter.

Alternative industrial methods include the following:

A solution of 5 mg / ml protoporphyrin in buffer solution (PBS) pH 7.3 was prepared and scanned at 25 ° C using an Acta Beckman recording spectrophotometer. The excitation of protoporphyrin by ultraviolet radiation (498-408) produces orange-red fluorescence between 620-630 nm. Conventional filters were then impregnated with the above solution and dried

----- by hot-air- (25-35 ^α C) .----------- Alternatively, a solution of 5 mg / ml transferrin in PBS pH 7.4 is scanned using an Acta Beckman recording spectrophotometer The ferric transferrin shows a characteristic spectrum at 470 nm. Currently used methods for impregnation of conventional filters were used.

Alternatively, a solution of 5 mg / ml catalase in PBS pH is prepared

7.4. The method described above for preparing the biological filter is then followed.

Alternatively, a solution of 5 mg / ml cytochrome C in PBS pH 7.4 is prepared. The method described above for preparing the biological filter is then followed.

Alternatively, the above biological substances are placed between two conventional filters in solid form such that all cigarette smoke passing through the filter comes into contact with active groups of molecules (Fe ²⁺ , Fe ³⁺ , -SH, -NH 2).

Analysis of results

The various biological substances used to enrich conventional filters have been found to contain toxic compounds (NO, CO, free radicals, H2O2, aldehydes and trace elements and nitroso compounds) from cigarette smoke at varying degrees, as shown in the table below:

Akre.D-tnr ----- NG .... ΟΏ --- vtrbné H-rG-> ------- alrtehvd.v_ race-stop thread - compounds niny % % radi-% kály % % % % hemoglo- bin 90 90 90 80 90 90 95 transferin 85 90 60 60 60 75 50 katalasa 85 90 90 90 80 80 80 protopor- f yrin 85 90 70 80 70 75 80 cytoch- rom C 85 80 70 80 60 60 70 chlorophyll 15 Dec 10 40 15 Dec 10 10 80 It was achieved a high degree detention highly harmful cigarette components smoke and smoke from cigarettes (20 ml) was filter over biological filter a compared with smoke that

was filtered through a conventional filter (20 mL). Only 1 ml of cigarette smoke collected through a conventional filter was compared to 40 ml of cigarette smoke collected through a biological filter. Obviously, biological filters have 40 times the ability to retain trace elements compared to conventional filters.

In the following detailed description of the examples, representative results are presented to better understand the activity of these biological substances.

(a) Identification of NO contained in cigarette smoke using the cemiluminescence method:

NO was identified using the luminol-enhanced chemiluminescence method as described in the Examples section. Figures 3 and 4 illustrate a typical experiment for identifying and assessing NO as well as its capture after the passage of a cigarette.

and smoke through the biological filter. More than 90% of NO appears to be trapped by hemoglobin. The efficacy of the biological filter is apparent from the capture and neutralization of NO, which has been implicated in the toxic reactions of both lung cells and lung fluids, especially when |

I formed by strongly oxidizing ONOO '

(b) Identification of free radicals contained in cigarette smoke using the chemiluminescence method:?

Free radicals have been identified by chemiluminescence!

the response induced by the lucigenin / DAMCO system after its reaction with!

| free radicals. Fig. 5 shows a characteristic peak in 2 seconds of the chemi-luminescence response that was inhibited 100% after passing cigarette smoke through the biological filter. 1

The capture of free radicals by biological filters suggests that and

here will reduce oxidative stress in alveolar!

| macrophages, which is caused by normal cigarette smoke. | and

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(c) Identification of the H2O2 contained in cigarette smoke using the b chemiluminescence method

H2O2 was evaluated by the chemiluminescence response produced by the isoluminol / microperoxidase system. Giant. 6 shows a characteristic peak of chemiluminescence caused by the presence of H2O2 in cigarette smoke. In the presence of catalase (100 units / ml), the chemiluminescence response was inhibited by approximately 90%. If cigarette smoke passed through the biological filter, an 80% reduction in chemiluminescence was observed. The isoluminol / microperoxidase system is specific for the identification of H2O2. The free radicals contained in cigarette smoke evoke immediate chemiluminescence upon reaction with isoluminol. This instant chemiluminescence appears to be 10% of the total C Hum iscencevyvoTáně HE m '' ~ r H2O2 ^- i-- vetnýc píftomno st hr adr Kahu since catalase inhibits the maximum chemiluminesent response up to about 90%. Apparently, H2O2 retention reduces both oxidative stress and NO production by alveolar macrophages.

(d) Identification of trace elements and aldehydes contained in cigarette smoke using the luciferrin / luciferase enzyme system.

The trace elements contained in cigarette smoke have been identified by their ability to stimulate luciferase activity. Giant. 7 presents:

1) chemiluminescence response induced by oxidation of luciferin in the presence of ATP,

2) increased chemiluniniscence response in the presence of Cd ²⁺ ions (0.5 mg),

3) increased chemi-luminescence response in the presence of Cu ² · * · ions (0.5 mg),

Increased chemiluminescence response caused by cigarette smoke (1 ml); and

5) inhibition of the chemiluminescence response (with respect to the response caused by cigarette smoke) induced by 40 ml of cigarette smoke after passing it through the biological cigarette filter. Obviously, the chemiluminescence responses caused by the trace elements contained in conventional cigarette smoke are more than 40 times higher than for the smoke passing through the biological filter. Removal of trace elements by biological filters can have both short and long term effects. Short-term effects may cause inhibition of blood components (Cd) damage.

The aldehydes contained in cigarette smoke were identified and evaluated using the same luciferin / luciferase enzyme system in the absence of ATP. The aldehydes are capable of effecting oxidation of acylcifurine. The compositions also melt a characteristic chemiluminescence response, which should be at least greater than an hour. The chemiluminescence response was 100% inhibited when the cigarette smoke used passed through the biological filter, i.e.. The effectiveness of a biological filter to remove toxic aldehydes is essential.

(e) Identification of nitrites of legumes in cigarette smoke.

The identification of nitroso compounds contained in cigarette smoke was achieved by assessing the slow release of NO from nitroso compounds after treatment with H2O2. As shown in Figure 9, the peak of the chemiluminescence response was obtained in approximately 900 seconds. The passage of cigarette smoke through the biological filter shows 90% inhibition in the observed chemiluminescence response and its peak occurs in approximately 1200 seconds. The slow release of NO by sodium nitroprusside (SNP) after its treatment with H2O2 is also shown. Giant. 10 shows the slow release of NO from both the nitro compounds diethylnitrosamine and dimethylnitrosamine and the hemoglobin-enriched nitroso compounds from cigarette smoke treated with H2O2. Obviously, the NO released by the nitroso compounds from cigarette smoke, which formed the adducts with hemoglobin, is similar to the NO release of the nitroso compounds diethylnitrosamine and dimethylnitrosamine. Giant. 11 represents the NO release of nitroso compounds of cigarette smoke that formed adducts with hemoglobin after the adducts of the hemoglobin nitroso compound were irradiated with UVB (100 mJ / cm ² ) for one minute. The release of no was evaluated in the presence of H 2 O 2 and gave a chemiluminescence response in 1 second. The gradual increase observed in Figure 11 is due to the effect of H2O2 on hemoglobin.

(E ent on re action) i ----------------------------------- (f) NO production by pulmonary macrophages :

In vitro experiments were performed using a special chamber created in our laboratory as shown in Figure 1. The Teflon membrane separating the two compartments in the chamber is permeable to NO gas and impermeable to NO2 and ONOO.Kidium pulmonary macrophages isolated as described in the Example section, were suspended in HBSS buffer solution (1 x 1% ⁶ cells / ml) and placed in chamber A of the chamber. 2.5 ml Griess reagent or sulfaniamide / scopoletin reagent are placed in chamber B of the chamber. The NO released by macrophages in space A diffuses through the Teflon membrane into space B and binds with Griess and / or sulfamide / scopoletin by the agent in which they are trapped. This means that pulmonary macrophages produce NO gas. The amount of NO actually present in space B is then determined spectrophotometrically or fluorophotometrically. Quantity ONOO ^- and NO2 contained in compartment A of the chamber was also determined using the Griess and / or sulfamide / scopoletin reagents. The above experiments were repeated after challenge the macrophages with cigarette smoke before placing them in compartment A. The results, as shown in Figure 12, show that cigarette smoke decreases the amount of NO produced whilst increasing production of ONOO ^- in lung macrophages, indirectly indicating which huge production of both NO and O 2, which interact to form ONOO.

Repetition of the above experiments using biological filters (i.e., in which cigarette smoke was drawn through the biological filter) shows that when biological substances are used, the same amounts of NO2 and 0N00 ~ are produced in space A and similar amounts of NO in space B, as if macrophages were not ignited by cigarette smoke. In this context, the components of the invention were evaluated for the evaluation of nitrosation kinetics of the intermediate (s) generated during the NO / Oz reaction in aqueous solution at physiological pH. Add cigarette smoke (50 mL) to a 100 mM phosphate pH solution

7.4 containing 25 mM sulfanilamide and 2.5 mM

N- (1-naphthylethylenediamine dihydrochloride (NEDD) generates absorption at lambdaaax = 496, indicating the characteristics of the azo product resulting from nitration. microenvironment is expected to be in the range of 0.5 to 10 μΜ.NO concentrations are significantly increased during cigarette smoking with harmful effects on lung cells.

g) Oxidative stress of lung macrophages:

The results of the effects of cigarette smoke on the oxidative stress of pulmonary macrophages are illustrated in Figure 13. The evaluation of oxidative stress using t-BHP, shows that cigarette smoke causes twice the oxidative stress than occurs with unexposed macrophages. When cigarette smoke passed through the biological filter, oxidative stress similar to that of unexposed pulmonary macrophages was observed. Thus, the elimination of cigarette smoke-induced oxidative stress in macrophages is clearly indicated. Cigarette smoke is now devoid of a substance that causes oxidative stress in lung macrophages.

(h) H2O2 produced by pulmonary macrophages:

H2O2 induced by macrophages exposed to cigarette smoke exhibits more than 10-fold production than untreated macrophages. The use of a biological filter shows a 90% reduction in H2O2 production (FIG. 14) compared to conventional filters, as the macrophage-producing stress of macrophages is increasing as the blood pressure of the macrophages increases. H2Q2 in these cells.

(i) Reconstruction of bold trials:

The amount of cyclic GMP produced by NO released by alveolar macrophages was determined using the chamber shown in Figure 1, where soluble guanylate cyclase was placed in compartment A and alveolar macrophages were placed in compartment B. Amounts of NO produced by macrophages were determined over 50 minutes with and without cells exposed cigarette smoke. Macrogages exposed to cigarette smoke (10 ml) release approximately 10 times less NO relative to untreated cells, which is 10 times less cyclic GMP production. The above procedure was repeated using cigarette smoke passing through the biological filter. There was a statistically insignificant difference with respect to non-exposed macrophages (control) (Fig. 15). The accumulation of NO in space B was increased more than 5-fold when alveolar macrophages were treated with H2O2 (5 mM) Fig. 6. This confirms that H2O2 increases NO production by a positive feedback mechanism. The L-arginine / NO pathway in macrophages is consistent with the idea that cigarette smoke causes NO / ONOO release.

(k) Identification of carbon monoxide (CO) in cigarette smoke:

The presence of CO in cigarette smoke was determined using a biological method based on stimulation of soluble guanylate cyclase by CO.

Introduction of cigarette smoke saturated HBSS into chamber A, in the presence of superoxide, so as to neutralize NO and

Incorporation of insoluble guanylate thyroids into the phosphorylation of FB The production of cyclic GMP caused by the diffusion of CO from space A to space B is increased. The passage of cigarette smoke through the biological filter 1 reduces the amount of cyclic GMP produced by approximately by 80% (Fig. 17). The above data indicates that they are harmful the NOx and CO components contained in the cigarette smoke are retained and neutralized by biological filters.

A I

In vivo experiments

(a) The presence of NO and ONOO 'in the exhaled substance was first confirmed

cigarette smoke. NO was identified in exhaled I cigarette smoke in human volunteers smoking a cigarette carrying a conventional filter after introducing the exhaled smoke into E acidic solution (50ml) pH 4. The NO concentration was evaluated by the luminol enhanced chemiluminescence method described in the experimental section using standard curves ready with!

and commercial NO. A NO concentration of 0.045 mM was found. The experiments were repeated using biological filters and the NO concentration in the inhaled smoke was approximately 70% lower than the conventional filter (Fig. 18). The ONOO concentration was determined using a NaOH solution of 1.2M, and showed an increase in absorption at 303 nm (Fig. 19) (E3 3 m = 0 M 1670 ^_1 cm ^"1). Our experiments showed that during smoking the exhaled smoke contains large quantities of ONOO-" I (passage of 50ml exhaled smoke into 5ml NaOH 1.2M gives a 0.9 mM ONOO solution - the NO / ONOO ratio in the exhaled smoke was determined 1:20.

Therefore, it appears that NOx in the lungs is transformed into ON00 in response to superoxide in the lungs. Superoxide is released from both macrophages and redox reactions appear in the lungs during smoking. The cigarette smoke collected by the pump does not contain ONOO-, but many NOx react with superoxide or oxygen to form nitrites (NO 2). ONOO is only formed when cigarette smoke enters the lungs. The use of biological filters reduces the exhaled amounts of NO and ONOO 'by 70%

b) ONOO reacts with bicarbonate ions of human erythrocytes according to the equation

ONOO- + HCO3- —---> HCO ₃ + NO2 + OH '

Bicarbonate ions oxidize luminol as well as aromatic and heterocyclic molecules. Alternatively, ONOO 'can peroxide bicarbonate to peroxyhydrogen carbonate another strongly oxidizing species. On the other hand, superoxide dismutase (SOD) catalyzes the nitration of ONOO 'and a wide range of phenolic compounds including tyrosine in proteins.

Thus, there are several potential mechanisms by which bicarbonate and SOD could affect the overall reactivity of ONOO 'in cells. The presence of ONOO formed in the lungs by inhalation of cigarette smoke shows a marked increase in oxidative stress in erythrocytes, which was detected by a chemiluminescent response occurring within 5 seconds. The same experiment performed using a biological filter usually results in 100% inhibition of oxidative stress in human erythrocytes (Fig. 2). Hemoglobin or erythrocyte lysates exposed to ONOO '(contained in exhaled cigarette smoke) induce the removal of two peaks at 540 and 575 nm, normally observed with hemoglobin. Results similar to those described above were obtained in 12 volunteers and are shown in FIG.

21. When hemoglobin and / or lysate were exposed to a small amount of exhaled smoke (10 ml), a shift of peaks from 540 and 575 to 525 and 555 nm was observed consistent with the formation of nitrosylhemoglobin. The experiments were repeated using

-bio4og i-ckých-fil il ... Pozar-Qvané-pi acid t-preserves jT ~ its - - | and

characteristic wavelengths. and

d) Aldehydes were identified in the exhaled smoke of human volunteers by their characteristic chemiluminescent peaks. The experiments were repeated using!

of biological filters and 90 reductions were observed and

the chemiluminescence responses observed using conventional i

filter (fig. 22). Obviously, the biological filters. get off and -!

neutralize the aldehydes in cigarette smoke because they retain oxidants and thus clearly inhibit the initiation of a redox reaction occurring in the lungs that could lead to production!

endogenous aldehydes. í i

i i

e) Free radicals were identified in cigarette smoke from human volunteers using both characteristic and chemiluminescent peaks of these substances. Volunteers used cigarettes with conventional and biological filters. They were asked to blowing smoke (50 ml) into an acidic solution (0.01N HCl) (50 1 ml) pH: 6 and after 5 min and 60 min the chemiluminescence was determined.

response. At pH 6, exhaled ONOO- spontaneously decomposes. |

Within 5 min, a 160% increase in chemiluminescence response is generated in exhaled smoke passed through a conventional filter as compared to I i

cigarette smoke passed through the bilogical filter (Fig. 23). and

If exhaled smoke saturation was left for one hour, the difference in chemiluminescence response increased from 160% to 250% (Fig. 24). This is consistent with the idea that redoc reactions occur continuously in cigarette smoke via quinone radicals and produce a series of activated oxygen species that cause biological damage.

Comment

Our studies have shown that alveolar macrophages allow endogenous NO synthesis to be more likely to result in the release of NO / ONOO for extended periods of time after exposure to cigarette smoke. Further, once NO cells are released by these cells, NO production becomes self-sustaining even after the stimulus is removed. Such a response is attributed to the ability of NO from cigarette smoke to stimulate alveolar macrophages to release NO and ONOO for several hours after stimulus removal. The reaction can be initiated by the production of H2O2 in the lungs upon stimulation of alveolar macrophages by cigarette smoke. H2O2 stimulates NO synthase activity of the lung cells to produce NO and ON00 for more than one hour after stimulus removal.

Indeed, our experiments have shown that the passage of cigarette smoke through a biological filter leads to a 90% reduction (compared to a conventional filter) of oxidative stress in rat alveolar macrophages. The ONOO 'radical formed in the lungs may allow attack and inactivate the α-proteinase inhibitor (α1PI). Inhibition of alPI in human lungs often acts by emphysium, which reduces lung capacity. Statistical data suggest that smoking is a prerequisite for the development of emphysema (Southon P.A., Pwis G., Free Radicals in Medicine, Involment in Human Disease. Mayo Clin. Proc. 63: 390-408, 1988). In vivo experiments conducted in 12 volunteer smokers showed a 90% reduction in exhaled NO / ONOO 'when the inhaled cigarette smoke has passed through a biological filter.

Oxygen free radicals have also been implicated in the pathogenesis of IgA immune complex-induced alveolitis. Pre-treatment of animals with superoxide dismutase, catalase, iron chelator desferioxamine, or an hydroxyl radical acceptor DMSO suppresses the development of lung damage. In contrast, the lungs of untreated positive control animals are characterized by the presence of an increased number of alveolar macrophages.

Interstitial edema and haemorrhage are also present. Furthermore, in this model, lung injury is also highly protective as demonstrated by reduction: vascular.

permeabil1; vascular bleeding; and damage to vascular andothelial and alveolar epithelial cells. These findings suggest that macrophages are a source of the danger of NO, O2, H2O2 and OH compounds (Mullingan M.S., J

Jonhson K.J., Ward P.A., in Biological Oxidants: Generation and Injurious Consequences (eds. Cochrane C.G., and Gilbrone.

(M.A., Jr.Academie Press 157-172, 1992). j

The retention and neutralization of oxidants contained in cigarette smoke by biological filters can play an essential role in reducing the j activity of redox enzymes that are directly related to oxidative stress in lung cells. Biological filters also drastically reduce oxidative stress caused by inhaled cigarette smoke. j

Oxidative stress in the lung macrophages and endothelial cells of the lung vessels can be caused by NO, NOx by oxygen radicals and / or aldehydes contained in cigarette smoke. Furthermore, the retention of aldehydes and trace elements (especially Cd) by biological I

Also filters can have desirable long-term effects in protecting plasma.

• antioxidants and inhibiting the development of atherosclerosis. Hemoglobin | it contains several neutrophil centers that undergo covalent reactions with electrophiles. These centers induce 1

N-terminal valine residues on the α- and O-chain, N1 and n3 atoms of histidine residues and sulfhydryl groups of cysteine residues. Carcinogenic compound

The 4- (methylnitrosoamine) -1- (3-pyridyl) -1-butanone (NNK) present in the tobacco is transferred to the smoke during cigarette burning and its mainstream smoke levels can vary from 4 to 1700 ng per cigarette. NNK may form adducts with hemoglobin (Hecht S.S., Karan S., and Carmella S.G., in Human Carcinogen Expose, edited by Garmer R.C., Farmer P.B., Steel G.I., and Wricht A. S. IRL Press pp. 267-274, 1991). It is clear that the only way to dump tobacco-induced tobacco is to keep chewing tobacco and smoking. However, current smokers statistics suggest that much needs to be done to reduce exposure to tobacco carcinogens and modify their mode of action. The basic approaches to this objective are: 1) modification of tobacco products, 2) inhibition of raetabolic activation of tobacco carcinogens and their endogenous production by certain micro- and macro-nutrients and chemopreventive agents, and 3) retention of tobacco carcinogens using specific filters to be included in cigarette tobacco. Our invention using biological substances for the production of biological filters finally involves the discovery that nitroso compounds present in inhaled cigarette smoke are removed by biological substances, which protects the health of not only smokers but also non-smokers.

'' The changed sheet

Claims (14)

PATENT CLAIMS A tobacco smoke filter comprising a fibrous matrix enriched with a biological substance selected from one or more substances including iron, copper and / or magnesium complexed with a porphyrin ring and iron bound stereospecifically in protein molecules. ia A filter according to claim 1, characterized in that it contains activated carbon enriched with biological substances. 3. The filter of claim 1 or 2 wherein said enriched fibrous matrix is adjacent to a fibrous matrix that is not enriched with said biological substance. Filter according to any one of claims 1 to 3, characterized in that the biological substance comprises hemoglobin and / or erythrocyte lysate. Filter according to any one of claims 1 to 3, characterized in that the biological substances are selected from ferrous Fe ²⁺ ions bound stereospecifically to one or more of transferrin, catalase, protoporphyrin, cytochrome C and chlorophyll. Filter according to any one of claims 1 to 5, characterized in that the biological substance is in solid form. changed sheet _ 7Cigarette, v. Jlz. n and -c u - j — — — c — i ---------- s e --- 1- m m is -----------. provided with a filter according to any one of claims 1 to 6. A method of manufacturing a tobacco smoke filter according to any one of claims 1 to 6, comprising impregnating a conventional tobacco smoke filter with one or more of said biological substances. 9. The method of claim 8 wherein the filter comprises activated carbon. A method according to claim 8 or 9, characterized in that the biological substance comprises hemoglobin and / or erythrocyte cysate. The method of any one of claims 8 to 10, wherein the biological substance is provided as a 1-10 mg / ml solution in a phosphate buffered saline having a pH of 7.4. A method of filtering tobacco smoke, comprising providing a filter according to any one of claims 1 to 6 and passing tobacco smoke through the filter. The method of claim 12, wherein the filter retains from 15 to 90% NO, 10 to 90% CO, 40 to 90% free radicals, 10 to 90% aldehydes, 10 to 90% carcinogenic nitroso compounds, 15 to 90% % H2O2 and 50 to 95% of the trace elements present in the tobacco smoke before passing through the fi ll. 14. The method of claim 13, wherein the filter retains from 85 to 90% NO, 80 to 90% CO, 60 to 90% free radicals, 60 to 90% aldehydes, 60 to 90% altered carcinogenic nitroso compound sheet. , -60 to 90% H2O2 and 70 to 95 trace elements present in the tobacco smoke prior to passing through the film.