HUT74956A - Methodology for the with holding of noxious oxidants and carcinogenic volatile nitrosocompounds from cigarette smoke using biological substances - Google Patents

Abstract

The present invention relates to a filter for filtering tobacco smoke, a filter comprising a fiber matrix enriched with biological material. The biological material is a ferric, copper and / or magnesium porphyrin complex or a stereospecific iron binding material in a protein molecule, or a combination thereof. The invention also relates to the filtering of a cigarette equipped with an enriched filter.

Description

EXTRACT / Removal of harmful oxidants and carcinogenic volatile nitrosating compounds from cigarette smoke using biological agents

The invention relates to the filtering of tobacco smoke. s filter comprising a biological matrix-enriched fiber matrix. The biological material is a porphyrin ring complex of iron, copper and / or magnesium, or a substance having a stereospecific iron bond in a protein molecule, or a combination thereof.

The invention also relates to a cigarette with a fortified filter for filtering a j-s-filter. - [κο ^ πέ ι η- .............

EXAMPLE, Π ·? (Removal of harmful oxidants and carcinogenic volatile nitrosatable compounds from cigarette smoke using biological agents

It is an object of the present invention to provide a method for preventing the inhalation of harmful compounds such as nitrogen oxides, free radicals, aldehydes, hydrogen peroxide, carbon monoxide, trace elements and carcinogenic volatile nitrosatable compounds, which are still not required by conventional smoke filters. .

Many publications in the international literature suggest that cigarette smoke be separated into two phases: a) solid phase (tar); and b) a gas phase. This separation occurs using a typical Cambrige glass fiber filter which filters 99.9% of particles larger than 0.1 µm. Cigarette tar contains extremely high concentrations of very stable free radicals, which can be classified into at least four different categories. Semiquinones in equilibrium with quinone and hydroxyquinones are considered to be the free radicals with the most interesting chemical properties. The chinone system reduces the molecular oxygen content during the formation of superoxide (O ₂ ), which is then spontaneously converted

11532 forms hydrogen peroxide (H ₂ O ₂ ). In the gas phase, there is an organic radical with a half-life of less than 10 seconds per puff which is inhaled. The paradox is that these radicals can maintain their high activity in the gas phase for more than 15 minutes, in contrast to their instant half-lives. In fact, the concentration of these radicals increases considerably as we approach the end of the cigarette smoke filter. We find an explanation for this paradox by maintaining a steady state; caused by the continuous production of free radicals. (Pryor, WA, Stone, K., Ann. NY Acad. Sci. 686: 12-28, 1993).

The most important free radical in the gas phase of cigarette smoke during smoking is nitric oxide (NO), a series of reactions that produce nitrogen dioxide, isoprene radicals, peroxyl radicals and alkoxyl radicals. Cigarette smoke also contains a significant amount of aldehyde compounds, which are also responsible for the harmful, toxic effects. Small amounts of aldehydes extracted from cigarette smoke have been shown to cause protein catabolism and oxidation of thiol groups of plasma proteins.

These properties of aldehydes are the result of reactions between their carbonyl group and the -SH and -NH ₂ groups of plasma proteins (Alving, K., Forhem, C., Lundberg, J.M., Br. J. Pharmacol. 110: 739-746, 193). Trace elements in cigarette smoke tar, such as iron, copper, manganese and cadmium, are involved in many free radical reactions and lead to the formation of very active secondary radicals (such as peroxy radicals, alkoxy radicals, superoxides, cytotoxic aldehydes, etc.). The introduction of these trace elements into the lungs during smoking leads to a series of redox reactions in the lung fluid and alveolar macrophages, resulting in highly active hydroxyl radicals (OH). These hydroxyl radicals are formed mainly in the presence of iron in the Fenton reaction. Copper can react with hydrogen peroxide to form hydroxyl radicals in the lungs. Low concentrations of manganese (10 M) stimulate the activity of soluble guanylate cyclase compounds in endothelial cells of the lungs and produce nitric oxides and zuperoxides via positive feedback (Youn, TK, Lalonde, C., and

Demling, R., Free Rád. Biol. Med. 12: 409-415 (1992).

Carbon dioxide is released during the burning of tobacco. A certain amount of carbon dioxide remains in the lungs after exhalation due to the stimulation of soluble guanylate cyclase. Stimulation is the result of the interaction of endothelial cells with the enzymes of other cells of the lung tissue, the heme. Elevated cyclic GMP levels increase the production of nitric oxides and superoxides by positive feedback between cells (Wattson,

A., Joyce, H., Hopper, L., and Pride, N.B. Thorax 48: 119124,1993) NO gas, which can be produced by many cell types such as vascular endothelial and reticular endothelial cells, causes smooth muscle relaxation (Lowenstein, C.J., Dinerman, J.L., Snyder, S.S. Ann. Intern. Med. 120: 227 to 237.1994). There are also exogenous sources of NO, which are also responsible for damaging blood vessels and other tissues. It has been convincingly shown that secondary and tertiary amines can react with nitrites and other nitrosating agents to form N-nitrosamines (Lowenstein, C.J., Dinerman, J.L., Snyder, S.H. Ann. Intern. Med. 120: 227-237, 1994). Since 1974, many researchers have shown that during harvesting, processing and smoking, alkaloids are nitrosated to tobacco-specific Nitrosamines (TSNA). N-nitrosonornicotine (NNN), 4- (methylnitrosamino) -1- (3-pyridyl) -2-butanone (NNK) and 4- (methylnitrosamino) -1- (3pyridyl) have been identified as TSNAs in tobacco and / or smoke. -1-Butanol (NNAL), which is a potent carcinogen in animals. NNN induces tumors in the lungs of mice, in the trachea of hamsters, and in the oral cavity and esophagus of rats. NNK induces tumors in the lungs of mice, hamsters and rats and in the liver, oral cavity and pancreas of rats. Rinsing of the oral cavity in rats with NNN and NNK mixtures caused tumors in the oral cavity and lungs. Typical amounts of NNK and NNN are cigarettes

200 ng / cigarette (Hecht,

T.E., and Trushin, N.

Carcinogenesis,

9: 161165 (1988).

Our present study on the effects of cigarette smoke on lung tissues has shown that NO reacts with superoxide to produce strong oxidation peroxynitrite radical (ONOO), inducing adverse reactions with major biomolecules.

NO gas. practice on cells! its metabolic and deleterious effects have been investigated in laboratories both in vivo and in vitro.

In the presence of oxygen, NO is oxidized to nitrogen dioxide (NO ₂ ) - The rate of this oxidation reaction depends on the oxygen concentration and the NO concentration on nitrogen dioxide is clearly cytotoxic and is converted to water and nitrate. In addition, NO forms a complex with trace elements and / or meta-protein1, such as hemoglobin (Wink, DA, Darbyshire,

J.F., Nims, R.W.

Saavedra,

J.E.and Ford, P.E. Chem. Toxicol. 6: 23-27,

1993).

NO ONOO can form a harmful compound to prove the toxicity of some superoxides. The ONOO compound is unusually stable when considered. Upon decomposition, highly oxidative compounds are formed, including hydroxyl radicals, nitrogen dioxide and the nitronium ion. Consequently, any modification of NO and superoxide production can lead to the formation of highly oxidative secondary compounds in tissues (Deliconstantinos, G., Villiotou, V., Stavrides, J.C., Cancer. Mol. Biol. 1: 77-86, 1994). Finally, the compound 0N00 and its esters (RO-ONO or RO-ONO ₂ ) tend to deactivate the alpha-1 proteinase inhibitor (alPI). This is evidenced by the following: a) hydrogen peroxide alone does not cause rapid deactivation of alPI, but in the presence of NO, when 0N00 is formed, rapid deactivation of alPI occurs (b-tert-butyl peroxynitrite (R0-0-0- alone N0 ₂₎ or ONOO- cause deactivation of Alpin and c / amines and amino acids protect the Alpin proteinase rapid deactivation (Moreno, JJ, and Pryor, WA, Chem Res Toxicol 5:... 425-431, 1992). Aside from free radicals in cigarette smoke, activated alveolar macrophages are another important source of free radical formation during smoking. Alveolar macrophages activated by cigarette smoke increase the production of free oxygen radicals by respiratory burst (mainly 0 ₂ ,

NO and H ₂ O ₂ ).

Alveolar macrophages and circulating neutrophils are likely to be elevated in smokers. The free oxygen radicals of ci garnet smoke also play a role in the development of lung cancer. Inhaled cigarette smoke in lung tissues increases oxidative stress and thus reduces the concentration of intracellular antioxidants. H ₂ O ₂ reacts with the cellular DNA compound to form hydroxyl radicals and causes the double helix to break. This rupture can be prevented by the addition of catalase and indirectly confirms the damaging effect of H ₂ O ₂ and hydroxyl radicals on cellular DNA (Leanderson, P., Ann. NY Acad. Sci. 686: 249-261, 1993). Furthermore, H ₂ O ₂ causes lesions in the lung tracheal epithelium and plays a role in the development of lung cancer. Thus, the lung tissue damage and role of H ₂ O _{2 in} cigarette smoke and its role in the development of lung cancer are highly probable. Cigarette tar contains semiquinone radicals and iron, thus providing a system for the formation of hydroxyl radicals. The various micronutrients (Fe, Cu, Mn, Cd) in the cigarette resin act intracellularly and extracellularly. And Fe in the well-known Fenton reaction:

Fe ² * + H ₂ O ₂ - + Fe ³⁺ + OH '+ OH ~ undergoes many oxidation reactions via hydroxyl radicals. Similar hydroxyl radical production can be achieved with 2+ 2 + el Cd, Mn has soluble guamate cyclase activity ή

stimulates. Cd in cigarette smoke is particularly toxic to the lungs. It is twice that in smokers' lungs

ι high Cd 'concentration than normal. Cd 24 is found to exchange Zn ions in the lung endothelium (Kostial, K., In: Trace Elements in Human and Animal Nutrition. Ed. W. Mertz) Fifth edit. Vol. 2: 31-345, Academic. Press, Inc. Orlando, FI., 1986). The aldehydes present in the cigarette smoke eventually become inert by reacting with the -SH and -NH ₂ groups of the proteins. Crotonaldehyde (α, β unsaturated aldehyde) in cigarette smoke reduces the -SH groups and increases the concentration of carbonyl proteins (Stadtman, ER, Science, 257: 1220-1224, 1991).

It is highly desirable today to fit a smoke filter on cigarettes. The basic purpose of fitting a smoke filter is to maximally filter out harmful compounds in the gas and solid phase of cigarette smoke. Epidemiological studies in smokers have found that the dose dependence was different for gas, solid, or combined cigarette smoke (Surgeon Generals of the US Public Health Service. The Health Consequence of Using Smokeless Tobacco, NH Publ. No. 86-2874, Bethesda). , MD, 1986). It has been proven that modifying a cigarette in itself is a good way to reduce harmful compounds in cigarette smoke. This was initially achieved by common filters and then by chemical modification of the tobacco composition. They have also changed the technology of cigarette production8 using porous paper or paper made from tobacco leaves. Over the past fifteen years, many measures have been taken to reduce the harmful effects of smoking: per cigarette! reducing the amount of smoke; changing the diameter of the cigarette; and using perforated smoke filters. Perforated smoke filters allow cigarette smoke to be diluted with air up to 50%. Perforated smoke filters are also combined with activated carbon. This resulted in a significant reduction in the tar and nicotine content in the smoke. Such solutions are partially applied in developed countries such as Austria, Canada, France, Germany, Sweden, England and the USA. The average tar and nicotine content of American cigarettes was reduced from 38 mg and 2.7 mg in 1955 to 13 mg and 1 mg in 1991, respectively. Efforts to reduce tar and nicotine emissions from cigarettes in the European Community are continuing. The limit of 15 mg for tar in January 1993 is reduced to 12 mg in January 1998. However, in other countries, the smoke emission limit for cigarette smoke is 22 mg (Mitacek,

E.J., Bruneman, K.D., Pollednak, A.P., Hoffman, D., and Suttajit, M., Pren. Med. 20: 764-773, 1991). Changes in cigarette manufacturing have resulted in specific reductions of some toxic compounds in cigarette smoke; Thus, the introduction of cellulose acetate filters resulted in the partial removal of semi-volatile phenols and volatile N-nitrosamines (Brunnemann, K.D., Hoffman, D., recent. Adv. Tobacco Rev. 17: 71-112, 1989). The perforated filters achieved a selective reduction of carbon monoxide. Nitrite-enriched tobacco has been used to selectively reduce the concentration of carcinogenic polynuclear aromatic hydrocarbons (PAHs). However, reducing PAH content with high concentrations of nitrite has led to an undesirable increase in the concentration of carcinogenic N-nitrosamines, and an alternative method has to be sought (Hoffman, D., Hoffman, I., Wynder, EI, Lung Cancer and the Changing Cigarette). in Relavance to Human Cancer of Nitroso Compounds, Tobacco Smoke and Mycotoxins (eds. 0'Neil, IK, chen, J., and Bartsch, H.) Vol. 105: 449459, 1991).

From the foregoing, it is obvious that a smoke filter must be constructed which is capable of retaining nitrogen oxides, free radicals, hydrogen peroxides, aldehydes and carcinogenic nitrosating compounds which are harmful to the respiratory and cardiovascular systems of cigarette smoke. Chemical and biological experiments have been conducted to identify harmful compounds in cigarette smoke. The chemical experiments were carried out as follows:

0

a) Identification and quantification of NO and NO _x compounds by new chemical and biological methods (developed in our laboratory).

b) Identification of free radicals by lucigenin-dependent chemoluminescent methods.

c) Identification of aldehydes and quinones by stimulation of the luciferin-luciferase enzyme system (also developed in our laboratory).

d) Identification and quantification of trace elements by oxidation of luciferin with luciferase in the presence of ATP (this method was developed in our laboratory).

(e) Identification and quantification of H ₂ O ₂ by the isoluminol microperoxidase-dependent chemiluminescent method.

(f) Identification and quantification of 0N00 by spectrophotometric and chemiluminescent enhanced luminol.

(g) Identification of carcinogenic nitrosating compounds by luminol enhanced chemoluminescence.

A _/ biological experiments were carried out as follows:

(a) Identification of NO by functional guanylate cyclase activity.

b) Identification of 0N00 ~ based on oxidative stress exerted by 0N00 on human erythrocytes.

Ί 1 «· a« * · · «·· ···« ·· ·

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

The following in vitro experiments were performed:

(a) Isolation of alveolar macrophages from rat lungs.

b) Determination of oxidative stress of alveolar macrophage induced by tertiary butyl hyperoxides (t-BHP).

(c) Determination of NO / NO ₂ / 0N00 produced by alveolar macrophages.

(d) Determination of H ₂ O ₂ produced by alveolar macrophages.

e) Effect of exogenous H ₂ O ₂ on NO production of alveolar macrophages.

The following compounds have been identified in in vivo experiments in volunteers:

(a) Determination of NO in exhaled non-smokers.

(b) Determination of NO in exhaled smokers.

(c) Determination of NO in exhaled tobacco smoke.

(d) Determination of ONOO in exhaled tobacco smoke.

(e) Determination of free radicals in exhaled tobacco smoke.

(f) Determination of aldehydes in effected tobacco smoke.

To determine NO, N0 _{x in} (a) tobacco smoke, (b) a test chamber of 2.5 eh diameter, normally emitted from alveolar macrophages exposed to cigarette smoke and (c) tobacco smoke exhaled by volunteers

2 • «pure plexiglas rods drilled from one side on a lathe to create conical cavities of the same size. Further formed at their open ends, a matt inclined mantle was provided, which provided a narrow transition between two conical cavities. A thin Teflon strip was placed between the threaded assemblies. The two tubular sections on both sides of the membrane allowed the injection of biologically active samples and reactive compounds extracted or modified during biological reactions (Figure 1).

A. Determination of NO by chemoluminescence

The standard NO solution was prepared as described in the literature (Deliconstantinos, G., Villiotou, V., Fassitsas, C. (1992) J. Cardiovasc. Pharmacol. 12, S63S65) and (Deliconstantinos, G., Villotu, V.). , Stavrides, JC (1994) In: Biology of Nitric Oxide eds.

M., Busse, R., Moncanda, S., Portland Press, in press). The reaction solution was prepared from Hank's equilibrated saline (HBSS) at pH 7.4; a H ₂ O ₂ (500 μΜ); luminol (30 μΜ) and total volume was 500 μΐ. The contents of the vial were mixed and the emission was measured with a Berthold AutoLumat LB953 luminometer.

B. Chemical determination of NO / NO ₂

Chemical determination of NO by diazotization of sulfonantide with acidic acid at NO and subsequent scopoletin

3 · · ♦ · oxidation was performed by the fluorometric method described earlier (Deliconstantinos, G., Villiotou, V.,

Fassitsas, C., J. Cardiovasc. Pharmacol. 12: S63-S65, 1992). In alveolar macrophages in the HBDD (10 ° cells / ml) in 100 μΐ 20% H ₃ PO dissolved in a reagent containing 20% sulfanilamide in 25 pM szkopoletint stirred totality. Fluorescence decomposition was monitored at room temperature (22 ° C) with an Aminco SPF-500 fluorescence spectrophotometer. Fluorescence was measured continuously until the slope of the line was measurable (about 8 min). The measured slopes were converted to the concentration of NO in nanomolar using a calibration curve of different concentrations of NO containing only solutions. The final product of NO synthesis was determined by the accumulation of nitrite (NO ₂ ) in the surface-floating part of cell cultures with Griess reagent.

C. Spectroscopic Determination of Peroxynitrite (ONOO) The ONOO compound was synthesized, titrated and stored as described previously (Deliconstantinos, G., Villiotou, V., Stavrides, J. C., Ib. Biology of nitric oxide, eds. Feelish, Μ. Busse, R., and Moncada, S.) Portland Press (in press) Due to the decomposition of ONOO at pH 7.4, the UV spectrum was taken immediately after mixing the H ₂ O ₂ and NO solutions. The concentration of the compound was determined from the ε302 nm value of 1670 M ¹ cm ¹ UV spectrum was obtained after subtraction of the H ₂ O ₂ baseline.

D. Definition of free radicals

Free radicals were determined by chemiluminescence-induced lucigerin / DAMCO (1,4 diazabicyclo [2,2,2] octane) as described previously (Deliconstantinos, G., Krueger, GRF, J. Viral. Dis. 1:22 -27, 1993). The reaction mixture was HBSS pH 7.4;

lucigerin (30 μΜ); It consisted of DAMCO (100 μΜ). The contents of the cuvette were mixed and the emission was measured with a Berthold AutoLumat LB953 luminometer. Free radicals (SOD, mannitol, histidine, methionine) were used as oxygen filters.

E. Determination of trace elements and aldehydes

The assay was based on luciferase-catalyzed oxidation of D-luciferin in the presence of an ATP magnesium salt by the following reaction:

LH ₂ + ATPMg ²⁺ -> oxylucifine + ATP + O ₂ + PPi + Mg ²⁺ + light

Trace elements Cd ² , Cu ²⁺ , Fe ²⁺ increase luciferase activity and the maximal chemiluminescence response increased proportionally with the trace element concentration up to the μg limit /. The reaction was carried out in a total volume of 0.5 ml HBSS pH 7.4.

5 • ·

Aldehydes were determined using the same luciferin / luciferase enzyme system but without ATP. Aldahydes react with the enzymatic system in the absence of ATP and produce chemiluminescence. The reagents were used with an ATP assay kit (Calbiochem-Novabiochem CA, U.S.A.).

F. Isolation of alveolar macrophages

Rats were sacrificed by intravenous injection of sodium pentabarbital and, after opening the chest, the lungs were exposed to calcium-free cold (4 ° C) phosphate-buffered saline (PBS;

pH 7.4) and was removed from the thoracic cavity. The lungs were homogenized by repeated syringing and then passaged through a series of 32, 62, and 68 pores / inch stainless steel mesh sieves under constant flow of Finkelstein's stabilized saline (FBSS; pH 7.4). The alveolar macrophage suspension was collected, filtered, and centrifuged at 300Xg for 10 minutes to granulate the cells. Cell pellets containing more than 98% macrophage were washed and resuspended in Ringer's solution. The procedure was then repeated twice. Approximately 10x10 macrophages per rat were isolated. Viability was ensured by precipitation with trypan blue.

F. Identification of nitrating compounds €

• · · · · · · · · · · · · · · · · · · · · · · ···

The nitrosating compounds were identified as slowly released NO after treatment with H ₂ O ₂ . The reaction mixture is dimethyl nitrosamine and / or diethyl nitrosamine (1 μΜ); H ₂ O ₂ -t (500 μΜ); lumitol (30 μΜ) in 0.5 ml HBSS pH 7.4. The contents of the cuvette were mixed and the emission was measured with a Berthold AutoLumat LB953 luminometer. Mannitol (100 mM) was used to identify ONOO; DMSO (100 mM) and cysteine (3.0 mM) were used.

G. Isolation of alveolar macrophages

The rats were sacrificed by intravenous injection of sodium pentabarbital, and after opening the chest, the lungs were anesthetized with calcium-free cold (4 ° C) phosphate-buffered saline (PBS; pH 7.4) and removed from the thoracic cavity. The lungs were homogenized by repeated syringing and then passaged through a series of 32, 62, and 68 pores / inch stainless steel mesh sieves under constant flow of Finkelstein's stabilized saline (FBSS; pH 7.4). The alveolar macrophage suspension was collected, filtered, and centrifuged at 300Xg for 10 minutes to granulate the cells. Cell pellets containing greater than 98% macrophage were washed and resuspended in Ringer's solution. The procedure was then repeated twice. About • 9 · ··· * ·······················································•

10x10 macrophages per rat were isolated. Viability was ensured by precipitation with trypan blue.

H. Oxidation treatment of alveolar macrophages by t-butyl-1-hydroperoxide (tBHP)

The production of free oxygen radicals generated in alveolar macrophages by t-BHP (2.5 mM) was determined by luminol chemiluminescence. Chemiluminescence was determined with a Berthold AutoLumat LB 953 luminometer as described previously (Deliconstantinos, G. _z Krueger, GRF, J. Viral. Dis. 1: 22-27, 1993).

I. Determination of Hydrogen Peroxide An isoluminol / microperoxidase mixture (100 mM sodium borate, ImM isoluminol, 0.01 mM microperoxidase in 70% water and 30% methanol at pH 8) was prepared. 50 μΐ reagent was mixed with the isolated alveolar macrophages (10 ⁶ cells) 0.5 ml of HBSS solution. The chemiluminescent signal is calculated from the calibration curve obtained for pure H ₂ 0 _2, expressed sequence nM concentration.

J. Preparation and Purification of Soluble Guanylate Cyclase (sGC) for CO Determination.

SGC from human endothelial cells was purified by GTPagarose chromatography. Cytosol (10 mg protein) was added to the GTP-agarose column (1.8x9cm) and 250mM in 25mM Tris HCl buffer pH 7.6.

8 • V · · · · · · · · · · · · ·· • • * · * «« · ··· 4 <4 * ·· · · · 'Lommel sucrose and MgCl ₂ were treated with the formation of the charge balance . The sGC was then eluted with 5 ml of this equilibration buffer with the addition of 10 mM GTP.

K. Determination of Cyclic GMP

The concentration of cGMP after acetylation with acetic anhydride was determined by radioimmunoassay (Deliconstantinos, G., and Kopelkina, L., Anticancer et al., 9: 753-760, 1989). The reaction mixture was triethanolamine / HCl (50 mM); creatine phosphate (5 mM); MgCl ₂ (3 mM); isobutylmethyl xanthine (1 mM); creatine kinase (0.6 units); GTP (1 mM); soluble guanylate cyclase (1 pg protein) in a total volume of 150μ1. The reaction was started by the addition of GTP and incubated for 10 minutes at 37 ° C. The incubation medium was aspirated and the cGMP compound was extracted with ice cold HCl (0.1 M). After 10 minutes, it was transferred to a new drying plate and dissolved in 5 mM sodium acetate (pH 4.75) for cGMP determination. The resulting cGMP was determined using the Amersham cGMP kit.

Description of the Invention

It is an object of the present invention to provide and apply a method in which biological agents are used to specifically react and screen for:

(a) NO and NO _X ,

b) CO • »♦ · · 0 * é ·« · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

(c) H ₂ O ₂ ,

d) free radicals,

(e) aldehyde quinones,

(f) carcinogenic nitroso compounds,

(g) cadmium, copper, manganese, iron, etc. retention of trace elements that we inhale during smoking.

The present invention is based on the idea that:

(a) Suitable substances, such as hemoglobin or lysates of erythrocytes or any substance containing stereospecific iron bonds, have a filtering effect.

b) Filters containing iron-containing porphyrin ring (eg protoporphyrin) have a cleaning effect

c) Filters that contain a porphyrin ring that is not necessarily iron have a cleaning effect.

d) Filters that have porphyrin rings with other metals, e.g. They contain complexes with Mg ²⁺ , Cu ² *.

e) A biotechnological process for enriching conventional materials currently used in cigarette smoke filters will be described, which will result in the smoke filters containing the above biological materials.

2Ö • * «···· · <· ·· * ·« »« ♦ * a ·· ·· * »♦» * • · · · · · · · · · · · ·

The basic idea of the present invention is based on the fact that conventional cigarette smoke filters and / or smoke filters containing activated charcoal can be enriched with biological substances. These biological materials are characterized by the presence of Fe ²⁺ , Cu ²⁺ , Mg ² metal ions in the porphyrin ring.

They contain a complex of 2-1, or a stereospecific bond of Fe with a protein molecule, and as a result, the harmful substances in the cigarette can be filtered out before the smoker smokes the cigarette smoke. The present invention, which is undoubtedly new and industrially applicable, is characterized by the aforementioned enriched filter.

Methods for industrial application:

The present invention has been carried out in order to demonstrate its applicability in the industry as follows:

1. A solution containing 1 mg / ml hemoglobin and / or erythrocyte lysate was prepared in phosphate buffered saline (PBS) pH 7.4 and added to 100 mg of activated carbon. The mixture was kept at room temperature for 30 minutes and filtered through S&S Cari Schleicher & Schuell Co USA filter paper. The amount of unabsorbed hemoglobin in the filtrate was determined spectrophotometrically. The hemoglobin-enriched activated carbon was dried at room temperature. 200mg Hemoglobin-rich21 • ····· · ··· · · · · · · · · · · · ··· · Activated carbon was placed between two conventional smoke filters so that the passing cigarette smoke came into contact with the active groups of the molecules (Fe ²⁺ , Fe ³⁺ , -SH, -NH ₂ ) (Fig. ₂ ).

figure) . These compatible materials can now be used to make new cigarette smoke filters, which will be known as biological smoke filters.

Hemoglobin can be replaced by other biological materials that have a complex of Fe ²⁺ , Cu ² \ Mg ²⁺ metal ions with a porphyrin ring, or a Fe ²⁺ stereospecific bond attached to a protein molecule such as transferrin, catalase, protoporphyrin, cytochrome C, chlorophyll.

2. A solution containing 5 mg / ml of hemoglobin and / or erythrocyte lysate was prepared in pH 7.4 phosphate buffered saline (PBS) and analyzed at 25 ° C with an Acta Beckman recording spectrophotometer. Absorbance peaks were consistently observed at 540 nm and 575 nm (Smith, R.P., Kruszyma, H.J. Pharmacol. Exper. Ther. 91, 557-563, 1974). Conventional smoke filters were impregnated with these solutions, and the impregnated smoke filters were air-dried at 25-35 ° C. These compatible materials can now be used to make new cigarette smoke filters, which will be known as biological smoke filters. These new biological smoke filters allow you to smoke the air that you inject into the air. 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • For aesthetic reasons, a small (3mm) conventional smoke filter can be attached to the visible end of the biological smoke filter.

3. A solution containing 5 mg / ml protoporphyrin was prepared in pH 7.4 buffer (PBS) and analyzed at 25 ° C with an Acta Beckman recording spectrophotometer. Excitation of protoporphyrin with ultraviolet light (498-408) resulted in an orange-red fluorescence at 620-630 nm. The conventional smoke filters were then impregnated (soaked) with the above-mentioned solutions and then dried with warm air at 25-35 ° C.

Totten. 4. A solution containing 5 mg / ml transferrin was prepared. we have PH 7.4 in PBS and Acta Beckman recording spectrophotometer. THE

iron-transferrin shows a characteristic spectrum at 470 nm. The method mentioned above was used to impregnate conventional filters currently used.

5. A solution containing 5 mg / ml catalase was prepared in PBS pH 7.4. They followed the above-mentioned method for the production of biological smoke filters.

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6. A solution containing 5 mg / ml chlorophyll was prepared in PBS pH 7.4. The above-mentioned method for the production of biological smoke filters was followed.

7. The aforementioned biological agents are inserted between two conventional smoke filters in solid form so that all cigarette smoke passing through the filter is in contact with

2+ 3+ yen with the active groups of the molecules (Fe, Fe, -SH, NH ₂ ).

Evaluation of results

The results show that the various biological materials with which conventional smoke filters have been enriched retain, to varying degrees, the toxic components of cigarette smoke (NO, CO, free radicals, H ₂ O ₂ , aldehydes, trace elements and nitroso components).

filters WOMAN % CO % Free radicals % ^ 2θ2 % aldehydes % Nitrating Compounds% Trace elements% hemoglobin 90 90 90 80 90 90 95 transferrin 85 90 60 60 60 75 50 catalase 85 90 90 90 80 80 80 protoporphyrin 85 90 70 80 70 75 80 cytochrome C 85 80 70 80 60 60 70 chlorophyll 15 10 40 15 10 10 80

We obtained the extent to which the most destructive substances of cigarette smoke were filtered out and compared the cigarette smoke (20 ml) filtered through the biological smoke filter with the cigarette smoke filtered through a conventional filter. Only 1 ml of cigarette smoke passed through a conventional filter was compared to 40 ml of cigarette smoke passed through a biological filter. It can be seen that the retention capacity of the biological smoke trace element is 40 times that of a conventional smoke filter.

The following experimental results are presented in order to better understand the activity of these biological agents.

a) To measure the NO content of cigarette smoke, the chemiluminescence method was used for:

NO was identified by a luminol-enhanced chemiluminescence method as described in the experimental section. Figures 3 and 4 show a typical experiment for identifying and estimating NO and also how it filters out after cigarette smoke has passed through the biological smoke filter. It is seen that hemoglobin retains more than 90% of NO. The effectiveness of the biological smoke filter can be seen in its ability to retain and neutralize NO, which is a toxic reaction in the lung tissues and lung fluid. cause of the formation of highly oxidant 0N00.

(b) Identification of free radicals in cigarette smoke by chemiluminescence;

Free radicals in cigarette smoke were determined from a chemiluminescence signal following the reaction of the lucigenin / DAMCO system with free radicals. Figure 5 shows the characteristic chemiluminescent response peak recorded within 2 seconds which was 100% inhibited by passage of cigarette smoke through a biological filter. Free radicals remaining in the biological filter prove that oxidative stress induced by cigarette smoke is reduced in alveolar macrophages.

(c) Identification of H ₂ O ₂ in cigarette smoke by chemiluminescence.

H ₂ O ₂ was identified by a chemiluminescence response in the isoluminol / microperoxidase system. THE

Figure 6 shows the chemiluminescence peak due to the presence of H ₂ O ₂ . In the presence of catalase (100 Units / ml), the chemiluminescent response was about 90%

was inhibited. If the cigarette smoke on a biological filter we drove through the 80% chemiluminescence response was inhibited. The isoluminol / microperoxidase system specific for H ₂ O ₂ identify. It's in cigarette smoke free radicals one minute chemiluminescence

• · induce a response after interaction with isoluminol. This minute chemiluminescence signal is about 10% of the chemiluminescence signal induced in the presence of H ₂ O ₂ free radicals, since catalase inhibits 90% of the maximal chemiluminescence signal. The retention of H ₂ O ₂ reduces both the oxidative stress effect and the amount of NO produced by alveolar macrophages.

(d) Determination of trace elements and aldehydes in cigarette smoke by the lysiferin / luciferase enzyme system.

Trace elements in cigarette smoke were identified by their luciferase stimulating capacity. Figure 7 shows:

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

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

3) increased luminescence response in the presence of Cu ions (0.5 mg),

4) Increased chemiluminescence response in the presence of cigarette smoke (Imi) and

5) inhibition of the chemiluminescent response signal after passing through a biological filter of 40 ml of cigarette smoke (relative to the signal produced by cigarette smoke). The chemiluminescence response triggered by trace elements is greater. ₉ * .. * · * · was 40 times greater in conventional cigarette smoke than in biological filter smoke. The trace element retention effect of the biological filter is manifested in short and long term effects. Short-term effects inhibit lung redox reactions (Fe, Μη) and long-term effects inhibit damage to blood components and components (Cd).

Identification and determination of aldehydes in cigarette smoke were performed using the same luciferin / luciferase enzymatic system in the presence of ATP. Aldehydes are prone to oxidation of luciferin. Figure 8 shows a chemiluminescent response signal for more than one hour. This response signal was 100% inhibited after passage of cigarette smoke through the biological filter, demonstrating that the biological filter's binding to toxic aldehyde was significant.

(e) Identification of nitrosating compounds in cigarette smoke;

The identification of nitrosating compounds in cigarette smoke was performed by determining the slow NO emission from H ₂ O ₂ treatment. As shown in Figure 9, a chemiluminescence response was obtained at ca. 900 seconds from now. After passage of the cigarette vapor through a biological filter, the chemiluminescence response showed 90% inhibition and the peak was about. Detected after 1200 seconds. Sodium nitroprusside treated with H ₂ O _{2 was} detected

slow NO emissions. Figure 10 illustrates the slow NO release of diethylnitrosamine and dimethylnitrosamine nitrosating compounds and hemoglobin enriched with nitrous compounds derived from H ₂ O ₂ treated cigarette smoke. It can be seen that the NO emission of the nitrosating compounds of cigarette smoke, which forms an adduct with hemoglobin, has the same type of NO release as the diethyl nitrosamine and dimethyl nitrosamine nitrosating compounds. Figure 11 shows NO emission of the nitrosating compounds of cigarette smoke, which forms an adduct with hemoglobin after one minute of UVB (100 mJ / cm) irradiation with hemoglobin. The NO emission in the presence of H ₂ O ₂ was determined from the chemiluminescence response at 1 second. The gradual increase in Figure 11 can be explained by the effect of H ₂ O _{2 on} hemoglobin.

(Fenton reaction)

(f) NO emission of lung macrophages;

In our laboratory, we performed in vitro experiments using the special chamber we created, which is shown in Figure 1. The Teflon membrane separating the two chamber cavities is permeable to NO gas while impermeable to NO ₂ and ONOO. Lung macrophages not yet treated with smoke as described in the experimental section were placed in HBSS buffer (1x10 ⁶ cells / ml) into chamber A. 2.5 ml of Griess reagent or sulfanyl amide / scopoletin reagent was poured into well B. NO released by macrophages in cavity A, when diffused through the separation membrane into cavity B, bound to Griess reagent or sulfamide / scopoletin reagents and fixed. This process is indicated by the fact that lung macrophages do not release NO gas. The amount of NO gas thus contained in Cavity B was determined spectrophotometrically or fluorophotometrically. The amount of ONOO- and N0 ₂ in the cavity Griess and / or sulfamide / scopoletin reagent was also determined. The above experiments were also repeated with tobacco smoke-treated macrophages, which were also placed in well A. The results shown in Figure 12 show that cigarette smoke reduces the amount of NO produced while lung macrophages stimulate the production of ONOO and thus indirectly show high levels of NO and O ₂ , which then interact to form ONOO.

According to the biological tests repeated (i.e., cigarette smoke transferring biological filter) in the case of biological agents used in the same well of the N0 ₂ and compound ONOO, cavity B is generated similar amounts of NO compounds than without treatment with the macrophages. In this regard, the aqueous solution, physio30

the nitrosation kinetics of intermediates resulting from the N0 / N0 ₂ reaction were also investigated.

Griess using reaction components. Addition of 50 ml cigarette smoke to a solution containing 100 mM phosphate (pH 7.4) containing 25 mM sulfanylamine and 2.5 mM N- (1-naphthyl) ethylenediamine dihydrochloride (NEDD) showed an X _max = 4 at 96 nm, indicating that during the nitration, a catacetic azo product is formed. It is worth mentioning that the observations of the present studies contradict the expected reactivity of NO under physiological conditions, where the maximum concentration of NO in the cellular microenvironment is assumed to be 0.5-1 μΜ. During cigarette smoking, NO levels increase dramatically, damaging lung cells.

g) Oxidation stress of lung macrophages;

Figure 13 shows the effect of cigarette smoke on the oxidative stress of lung macrophages. Oxidation stress as determined by t-BHP was twice as high for phage exposed to cigarette smoke as for phage in non-smokable media. When the cigarette smoke was passed through a biological filter, the oxidative stress was similar to that of a smokeless case. This clearly demonstrated the elimination of oxidative stress induced by cigarette smoke on macrophages. so the cigarette smoke

purified from compounds causing oxidative stress on lung macrophages.

(h) H ₂ O ₂ produced by lung macrophages;

The production rate of H ₂ O ₂ produced by macrophages in contact with cigarette smoke was more than ten times higher than that produced under smoke-free conditions. Using a biological filter, H ₂ O ₂ production was reduced by 90% (Figure 14) compared to conventional filters. It is clear that cigarette smoke promotes oxidative stress in macrophages and increases the toxic production of H ₂ O _{2 by} these cells.

j) Reconstruction attempts;

The alveolar NO released by macrophages her cyclic GMP in FIG. closed chamber where A

cavity-soluble guanylate cyclase and cavity B were alveolar macrophages. The amount of NO produced by macrophages was measured over a period of 50 minutes for smoke-treated and non-smoked phages. The phage treated with cigarette smoke (10 ml) contained ca. 10 times less NO was released than untreated phages, so cyclic GMP production was 10 times lower. The above procedure was repeated with a cigarette smoke filter passed through a biological filter. There was no statistically significant difference between the results of the biological filter experiment and the smokeless experiment (control) (Figure 15). There was more than 5-fold NO accumulation in the AB cavity when the alveolar macrophages were treated with H ₂ O ₂ (5 mM) (Fig. 16). This indicates that H ₂ O ₂ increases NO production by a positive feedback mechanism. The direction of the L-arginine / NO reaction of macrophages is consistent with the theory that cigarette smoke causes NO / NOOO emissions.

(k) Identification of carbon monoxide in cigarette smoke;

The presence of carbon monoxide in cigarette smoke was determined by a biological method based on CO stimulation of soluble guanylate cyclase. HBDD saturated with cigarette smoke (in the presence of superoxide to neutralize NO) was introduced into chamber A of the chamber. By inserting soluble guanylate cyclase in Cavity B, the CO diffusing from Cavity A to Cavity B increased cyclic GMP production. Passing the cigarette filter through a biological filter is approx. Cyclic GMP production was reduced by 80% (Figure 117). These data demonstrated that in cigarette smoke and toxic CO _x N0 compounds retained and neutralized by the biological filters.

IN VIEW EXPERIMENTS

a) First we confirmed the presence of NO and 0N00 'in exhaled cigarette smoke. Volunteers smoked cigarettes with conventional filters. Amount of NO in Exhaled Cigarette Smoke on an Acidic Solution • · * · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Õ Õ Õ Õ Õ Õ "" · · (50 ml) after passage through pH 4 was determined by a luminol-assisted chemiluminescence method (as described in the experimental section using a conventional calibration curve with NO). A concentration of 0.0.45 mM NO was determined. In repeated experiments with biological filters, the amount of NO in the exhaust smoke is approx. It was 70% lower (Figure 18). The ONOO concentration was performed using 1.2 M NaOH and an increase in absorbance was observed at 303 nm (ε _{303 nm} = 1670 M ¹ , Figure 19). In our experiments, during smoking, exhaled smoke contains a large amount of ONOO (50 ml of exhaled cigarette smoke was passed through 5 ml of 1.2 M NaOH solution to give 0.9 mM ONOO). Exhaled smoke gave a NO / ONOO ratio of 1:20.

NO _{X in} the lungs, when it reacted with superoxide, seemed to

ONOO converts. Superoxide is released by the macrophages in the lungs during smoking and may also be formed during redox reactions. There is no ONOO in the pumped cigarette smoke, although some NO _X reacts with superoxide or oxygen to form nitrite ion (NO ₂ ~). ONOO is produced when cigarette smoke enters the lungs. Applying a biological filter reduces NO and NO by 70%

ONOO ejected volume.

• · · · · · · · · · · · · · · · · · · · · · · · ··· ·

(b) ONOO 'reacts with bicarbonate ions of human erythrocytes as follows:

onoo ”+ hco ₃ ” -> hco ₃ + no ₂ + oh ·

The bicarbonate radical oxidizes luminol as well as heterocyclic molecules. Alternatively, ONOO is capable of oxidizing bicarbonate to peroxybicarbonate or other strongly oxidizing agent. On the other hand, superoxide dismutase (SOD) catalyzes the nitration of ONOO 'and several phenolic types, including tyrosine in proteins.

Thus, there are a number of potential mechanisms by which bicarbonate and SOD can influence the resulting reactivity of ONOO in cells. The presence of ONOO in the lungs caused by inhaled smoke causes a dramatic increase in oxidative stress in erythrocytes, which is detected as a chemiluminescent response measured within 5 seconds. Repeating the same experiment with a biological filter, in most cases 100% inhibition was found in the oxidative stress of human erythrocytes (Figure 20). Hemoglobin or lysates of erythrocytes treated with ONOO in exhaled cigarette smoke disappeared the two peaks at 540 nm and 575 nm otherwise observed with hemoglobin. Representative experiments performed as previously described included 12 volunteers and the results are shown in Figure 21. Hemoglobin and / or lysate precipitated with 5 ml and 575 nm peaks shifted to 525 and 555 nm with ¹ (10 ml) of expelled tobacco smoke, respectively. according to hemoglobin formation. The experiments were repeated with biological filters. The observed peaks occurred at typical wavelengths.

d) Aldehydes in cigarette smoke exhaled by volunteers were identified by their characteristic chemiluminescence peaks. The experiments were repeated with biological filters and showed a 90% reduction in the maximum chemiluminescence signal obtained with conventional filters (Figure 22). Obviously, biological filters retain and neutralize aldehydes and oxidants in cigarette smoke, thereby inhibiting redox reactions in the lungs and avoiding the formation of endogenous aldehydes.

e) Free radicals were determined by their characteristic chemiluminescence peaks in cigarette smoke expelled by human volunteers. Volunteers used conventional and biological filter cigarettes. Cigarette smoke (50 mL) was blown into acidic solution (0.01 N HCl) (50 mL) at pH 6 and the chemiluminescent response was measured after 5 minutes and 60 minutes. At pH 6, the released ONOO decomposed spontaneously. Within five minutes, the smokeless tobacco smoke passing through the conventional filter resulted in a 160% increase in chemiluminescence signal compared to the results of the experiments with the biological filter (). Figure 23). If the acidic solution saturated with exhaled tobacco smoke was allowed to stand for one hour, the difference in chemiluminescence signals increased from 160% to 250% (Figure 24). This is consistent with the idea that in cigarette smoke the redox reactions are constantly occurring through the root of the pain and a series of activated oxygen compounds are produced which cause biological damage.

COMMENTARY

Our research has shown that alveolar macrophages, like other cells, have endogenous NO synthase and are able to release NO / ONOO compounds over a longer period of time under the influence of cigarette smoke. Furthermore, once these cells have started to release NO, the release becomes self-sustaining even after removal of the stimulant. The NO content of cigarette smoke can trigger this reaction, which triggers alveolar macrophages to release NO and 0N00 for several hours after removal of the stimulant. Such a reaction is triggered by the production of H ₂ O ₂ by alveolar macrophages produced by cigarette smoke in the lungs. H ₂ O _{2 can} stimulate lung cell NO synthase activity, resulting in NO and ONOO formation for more than one hour after removal of the stimulant. In our experiments, cigarette smoke passes through a biological filter to oxidize the alveolar macrophage lobe to the oxidative * 9 «, 9 9 9 9 9 9 · <·· ············ 90% reduction in stress compared to values observed in conventional filter experiments. The ONOO residue in the lungs is likely to attack and deactivate the alpha proteinase inhibitor (alPI). Inhibition of alPI in human lungs often causes emphysema, which results in a decrease in lung capacity. Smoking has been shown to predispose to the development of emphysema (Southon, PA, Pwis, G., Free Radicals in Medicine. Involvement in Human Disease. Mayo Clin. Proc. 63: 390-408, 1988). In 12 volunteer in vivo experiments, the amount of inhaled NO / ONOO was reduced by 90% when the inhaled cigarette smoke was passed through a biological filter.

Oxygen free radicals have also been implicated in the pathogenesis of alveolitis induced by the IgA immune complex. The animals are pretreated with superoxide dismutase catalase, the iron chelator desfarooxamine, or the hydroxyl radical filter DMSO inhibits the development of lung damage. In contrast, untreated, positive control animals had an increased number of alveolar macrophages in the lungs. Interstitial edema and haemorrhage have been observed. In this model of lung injury, L-arginine also has a potent protective effect, as demonstrated by the reduction in vascular permaebility, vascular hemorrhage, and vascular endothelial and alveolar epithelial cell damage. These results confirm that macrophages are sources of the damaging compounds NO, O ₂ , H ₂ O ₂ and OH (Mullingan, MS, Jonhson, KJ, Ward, PA, In: Biological Oxidants: Generation and Injurious Consequences (eds. Cohrane, CG, and Gilbrone, MA, Jr. Academic Press 157-172, 1992).

The retention and neutralization of oxidants in cigarette smoke by biological filters can be significant in reducing the activity of redox enzymes, which are directly responsible for the oxidative stress of lung cells. Biological filters dramatically reduce the oxidative stress caused by cigarette smoke. Oxidative stress in lung macrophages and endothelial cells may be induced by NO, NO _x , oxygen radicals and / or aldehydes in cigarette smoke. Furthermore, retention of aldehydes and trace elements (especially Cd) by biological filters can have a significant long-term effect on preventing plasma antioxidants and inhibiting the development of arteriosclerosis. Hemoglobin has several neurophilic centers that covalently react with electrophiles. These centers induce the N-terminal valine residues of the α- and β-chains, the residual NI and N3 atoms of histidine and the residual sulfhydryl group of cysteine. The carcinogenic nitrosating compound is a

4- (methylnitrosamino) -1- (3-pyridyl) -1-buhahöT) (JSINK), which is present in tobacco, passes into the smoke during the burning of the cigarette and amounts to 41700 ng per cigarette in the mainstream smoke. can change. NNK can form an adduct with hemoglobin (Hecht, S.S., Kárán, S., and Carmella,

S.G., in:: Human Carcinogen Expose eds. 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 avoid smoking-related harm is to stop smoking and chewing tobacco. However, current smokers' stats suggest that smoking can significantly reduce the dose of carcinogens and modify the deleterious effects of these substances. Principles underlying this goal: 1) Modification of tobacco products, 2) Inhibition of the metabolic activity of tobacco carcinogens and their endogenous formations by micro and macro additives and chemical preventive substances, 3) Retention of tobacco carcinogens by special filters attached to cigarette tobacco. Our invention uses biological materials for the biological filter, discovering nitrosating compounds in cigarette smoke that are retained by the biological materials and thus protect the health of not only smokers but also non-smokers.

···, ·>

· · · • * * <· <<•

Claims (14)

CLAIMS 1. A filter for filtering tobacco smoke comprising a biological matrix enriched in fiber, wherein the biological material is a porphyrin cyclic complex of iron, copper and / or magnesium, or a substance having a stereospecific iron bond in a protein molecule, or a combination thereof. A filter according to claim 1, characterized in that it contains activated carbon enriched with biological material. A filter according to claim 1 or 2, characterized in that the enriched fiber matrix is layered in a non-biological material enriched fiber matrix. 4. A filter according to any one of claims 1 to 3, characterized in that the biological material contains hemoglobin and / or lysate of erythrocytes. 5. Filter according to any one of claims 1 to 3, characterized in that the biological materials are selected from Fe ions stereospecifically bound to one or more of transferrin, catalase, protoporphyrin, cytochrome C and chlorophyll. 6. A filter according to any one of claims 1 to 5, characterized in that the biological material is solid. 7. A cigarette according to claims 1-6. A filter according to any one of the preceding claims. 8. Procedure 1-6. A cigarette smoke filter according to any one of claims 1 to 6, characterized in that a conventional smoke filter is impregnated with one or more of the above biological materials. 9. The method of claim 8, wherein the filter comprises activated carbon. 10. The method of claim 8 or 9, wherein the biological agent comprises hemoglobin and / or lysate of erythrocytes. 11. A process according to any one of claims 1 to 4, wherein the biological material is a solution of 1-10 mg / ml in pH 7.4 phosphate buffered saline. 12. A method for filtering cigarette smoke comprising the steps of 1-6. A filter according to any one of claims 1 to 3, which passes through the cigarette smoke. 13. The process of claim 12 wherein the filter comprises 15-90% NO, 10-90% CO, 40-90% free radicals, 10-90% aldehyde, 10-90% carcinogenic nitrosating compounds , 15-90% H ₂ O ₂ and 50-95% trace elements that were in the cigarette smoke before the filter. 14. The process of claim 13, wherein the filter comprises 85-90% NO, 80-90% CO, 60-90% free radicals, 60-90% H ₂ O ₂ , 60-90% NO. It filters out 90% aldehydes, 60-90% carcinogenic nitroso compounds, and 70-90% trace elements that were in the cigarette smoke before the filter. ADVCPATEN '. PATENT WRITING PATH 42nd t • * e V _vk THE ' SIPZZETÉTEU ARTICLES 0'1 ALVEOLAR MACROPHAGES Patent Office Patent Attorney. 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