U.S. patent application number 12/711974 was filed with the patent office on 2010-08-19 for reduced risk tobacco products and use thereof.
This patent application is currently assigned to VECTOR TOBACCO, INC.. Invention is credited to Anthony P. Albino, Thomas Jeffrey Clark, Richard L. Coyte, Gene Gillman, Patrick Rainey, Gregory Andrew Sulin.
Application Number | 20100206317 12/711974 |
Document ID | / |
Family ID | 42558823 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100206317 |
Kind Code |
A1 |
Albino; Anthony P. ; et
al. |
August 19, 2010 |
REDUCED RISK TOBACCO PRODUCTS AND USE THEREOF
Abstract
Reduced risk tobacco-related products and methods of use. These
tobacco-related products (e.g., cigarettes or filters) are designed
to reduce the biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of biological insult induced by another cigarette, such as a
conventional or reference cigarette (e.g., 2R4F), in human cells,
and to provide a reduced risk cigarette that meets a cigarette
smoker's sensory/perception needs.
Inventors: |
Albino; Anthony P.; (New
York, NY) ; Sulin; Gregory Andrew; (Chapel Hill,
NC) ; Coyte; Richard L.; (Raleigh, NC) ;
Clark; Thomas Jeffrey; (Elon, NC) ; Rainey;
Patrick; (Raleigh, NC) ; Gillman; Gene;
(Durham, NC) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
VECTOR TOBACCO, INC.
Morrisville
NC
|
Family ID: |
42558823 |
Appl. No.: |
12/711974 |
Filed: |
February 24, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2008/077739 |
Sep 25, 2008 |
|
|
|
12711974 |
|
|
|
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61164301 |
Mar 27, 2009 |
|
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60976291 |
Sep 28, 2007 |
|
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Current U.S.
Class: |
131/88 ; 131/202;
131/360 |
Current CPC
Class: |
A24D 1/00 20130101; A24D
3/12 20130101; A24B 15/10 20130101; A24D 3/163 20130101 |
Class at
Publication: |
131/88 ; 131/360;
131/202 |
International
Class: |
A24C 5/47 20060101
A24C005/47; A24B 1/00 20060101 A24B001/00; A24F 1/20 20060101
A24F001/20 |
Claims
1. A cigarette comprising: a blend of cured tobacco comprising a
Burley tobacco and a Flue-cured tobacco; and a filter comprising at
least 70 mg of carbon and at least 30 mg of resin, wherein the
cigarette is configured to produce a mainstream smoke that
generates fewer DNA double strand breaks (DNA DSBs) in human cells
than the mainstream smoke from a reference cigarette under the same
smoking conditions.
2. The cigarette of claim 1, wherein the Burley tobacco is present
in an amount of about 45-70% by weight based on the combined weight
of the blend, and the Flue-cured tobacco is present in an amount of
about 55-30% by weight based on the combined weight of the
blend.
3. The cigarette of claim 1, wherein the resin is a weak base
amine-containing resin.
4. The cigarette of claim 1, wherein said filter comprises at least
100 mg of carbon and at least 30 mg of resin.
5. The cigarette of claim 2, wherein said filter comprises at least
100 mg of carbon and at least 30 mg of resin.
6. The cigarette of claim 4, wherein the resin is a weak base
amine-containing resin.
7. The cigarette of claim 5, wherein the resin is a weak base
amine-containing resin.
8. The cigarette of claim 1, wherein the reference cigarette is
IM16, 2R4F, or 1R5F.
9. The cigarette of claim 2, wherein the reference cigarette is
IM16, 2R4F, or 1R5F.
10. The cigarette of claim 1, wherein the human cells are human
lung cells.
11. The cigarette of claim 2, wherein the human cells are human
lung cells.
12. A cigarette comprising: a blend of cured tobacco comprising a
Burley tobacco and a Flue-cured tobacco; and a filter comprising at
least 100 mg of carbon, wherein the cigarette is configured to
produce a mainstream smoke that generates fewer DNA double strand
breaks (DNA DSBs) in human cells than the mainstream smoke from a
reference cigarette under the same smoking conditions.
13. The cigarette of claim 12, wherein the Burley tobacco is
present in an amount of about 45-70% by weight based on the
combined weight of the blend, and the Flue-cured tobacco is present
in an amount of about 55-30% by weight based on the combined weight
of the blend.
14. The cigarette of claim 12, wherein the filter further comprises
a weak base amine-containing resin.
15. The cigarette of claim 13, wherein the filter further comprises
a weak base amine-containing resin.
16. The cigarette of claim 12, wherein the reference cigarette is
IM16, 2R4F, or 1R5F.
17. The cigarette of claim 13, wherein the reference cigarette is
IM16, 2R4F, or 1R5F.
18. The cigarette of claim 12, wherein the human cells are human
lung cells.
19. The cigarette of claim 13, wherein the human cells are human
lung cells.
20. A cigarette filter comprising at least 70 mg of carbon and at
least 30 mg of resin, wherein the filter is configured to reduce
DNA double strand breaks (DNA DSBs) in mainstream smoke.
21. The cigarette filter of claim 20, wherein the resin is a weak
base amine-containing resin.
22. The cigarette filter of claim 20, wherein the filter comprises
at least 100 mg of carbon and at least 30 mg of resin.
23. The cigarette filter of claim 20, wherein the resin is a weak
base amine-containing resin.
24. A cigarette filter comprising at least 100 mg of carbon,
wherein the filter is configured to reduce DNA double strand breaks
(DNA DSBs) in mainstream smoke.
25. The cigarette filter of claim 24, further comprising a weak
base amine-containing resin.
26. A method of making a filtered cigarette comprising: preparing a
blend of cured tobacco comprising a Burley tobacco and a Flue-cured
tobacco; incorporating at least 70 mg of carbon and at least 30 mg
of resin into a cigarette filter; and generating a filtered
cigarette that contains said blend of cured tobacco and said
cigarette filter, wherein the cigarette is configured to produce a
mainstream smoke that generates fewer DNA double strand breaks (DNA
DSBs) in lung cells than the mainstream smoke from a reference
cigarette under the same smoking conditions.
27. The method of claim 26, wherein the Burley tobacco is present
in an amount of about 45-70% by weight based on the combined weight
of the blend, and the Flue-cured tobacco is present in an amount of
about 55-30% by weight based on the combined weight of the
blend.
28. The method of claim 26, wherein said filter comprises at least
100 mg of carbon and at least 30 mg of resin.
29. A method of making a filtered cigarette comprising: preparing a
blend of cured tobacco comprising a Burley tobacco and a Flue-cured
tobacco; incorporating at least 100 mg of carbon into a cigarette
filter; and generating a filtered cigarette that contains said
blend of cured tobacco and said cigarette filter, wherein the
cigarette is configured to produce a mainstream smoke that
generates fewer DNA double strand breaks (DNA DSBs) in lung cells
than the mainstream smoke from a reference cigarette under the same
smoking conditions.
30. The method of claim 29, wherein the Burley tobacco is present
in an amount of about 45-70% by weight based on the combined weight
of the blend, and the Flue-cured tobacco is present in an amount of
about 55-30% by weight based on the combined weight of the blend.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of and claims the
benefit of priority to international application number
PCT/US2008/077739, having an international filing date of Sep. 25,
2008, which designated the United States of America and was
published in English and, which claims the benefit of priority to
U.S. Provisional Application No. 60/976,291, filed Sep. 28, 2007.
This application also claims the benefit of priority to U.S.
Provisional Application No. 61/164,301, filed Mar. 27, 2009. The
disclosures of all of the aforementioned applications are hereby
expressly incorporated by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the development of reduced
risk cigarettes and methods of use thereof. More particularly,
aspects of the invention concern cigarettes that are designed to
reduce the induction of biological insult, for example, DNA double
strand breaks (DNA DSBs), cell death or perturbation of RNA
transcriptome or proteome in human. Another aspect concerns
approaches to gradually reduce the presence of a toxicant in
cigarette smoke while adjusting a cigarette smoker's
sensory/perception needs.
[0004] 2. Background
[0005] Toxicants in tobacco smoke induce several biochemical
changes in human cells. Cigarette smoke contains over 4,000
chemicals. At present, the International Agency for Research on
Cancer (IARC) concludes that there are at least 81 possible,
probable, or proven human carcinogens in tobacco smoke
(International Agency for Research on Cancer, Tobacco Smoking and
Involuntary Smoking: Monograph on the Evaluation of Carcinogenic
Risk of Chemicals to Humans. Vol. 83, 2003, Lyon, France). These
include nicotine, tar, and carbon monoxide, as well as
formaldehyde, ammonia, hydrogen cyanide, and arsenic, for example.
Cigarette smoke is also known to cause the induction of biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome in cells that contact the cigarette smoke (e.g.,
cells of the oral cavity and lungs), which may lead to adverse
health consequences. Despite the health risk associate with
smoking, people continue to consume cigarettes.
[0006] Many companies have developed various products that have a
reduced level of toxicants delivered in cigarette smoke but very
few products have had measurable success. Historically, cigarette
smokers have associated "reduced risk" or "reduced nicotine" or
"low tar" cigarettes with a lack of satisfaction and poor taste.
For example, Phillip Morris introduced large carbon length filters
and documented that an increase in the length of the carbon filter
decreased the acceptance based on taste. Gaworski C L. SCoR
Program. Carbon filter technology. Philip Morris USA. Powerpoint
Meeting presentation at Massachusetts Department of Health, Feb. 7,
2004.
[0007] Many products in this class of cigarettes achieve a low
level nicotine delivery by using expanded, puffed tobacco in the
rod accompanied by significant filter ventilation. The response to
these types of products has been one of general dissatisfaction
with taste and, oftentimes, compensatory behavior (e.g., blocking
of ventilation holes or longer, deeper inhalation) is developed in
order to improve the taste and/or delivery of nicotine.
[0008] Accordingly, cigarette smokers do not readily switch from
their favorite brand to reduced risk cigarettes because the reduced
risk cigarettes do not fulfill the tobacco user's
sensory/perception needs. There remains a need for reduced risk
cigarettes, particularly products that fulfill a tobacco user's
sensory perception needs (e.g., those that have an agreeable taste
and/or odor).
SUMMARY OF THE INVENTION
[0009] In one embodiment, a cigarette comprises a blend of cured
tobacco, wherein the blend comprises a non-transgenic Burley
tobacco and a non-transgenic Flue-cured or Bright tobacco, wherein
the non-transgenic Burley tobacco is present in an amount of about
45-70% by weight based on the combined weight of the non-transgenic
Burley tobacco and the non-transgenic Flue-cured or Bright tobacco;
and the non-transgenic Flue-cured or Bright tobacco is present in
an amount of about 55-30% by weight based on the combined weight of
the non-transgenic Burley tobacco and the non-transgenic Flue-cured
or Bright tobacco.
[0010] In another embodiment, a cigarette comprises a blend of
cured tobacco, wherein the blend comprises a non-transgenic Burley
tobacco and a non-transgenic Flue-cured or Bright tobacco, wherein
the non-transgenic Burley tobacco is present in an amount of about
85-92% by weight based on the combined weight of the non-transgenic
Burley tobacco and the non-transgenic Flue-cured or Bright tobacco;
and the non-transgenic Flue-cured or Bright tobacco is present in
an amount of about 8-15% by weight based on the combined weight of
the non-transgenic Burley tobacco and the non-transgenic Flue-cured
or Bright tobacco.
[0011] The non-transgenic Burley tobacco may be a low alkaloid
variety such as LA Burley 21, and/or the non-transgenic Flue-cured
or Bright tobacco may be a low alkaloid variety. In one embodiment,
the cigarette described herein comprises the non-transgenic Burley
tobacco that is present in the amount of about 50% by weight, and
the non-transgenic Flue-cured or Bright tobacco that is present in
the amount of about 50% by weight. In another embodiment, or the
cigarette described herein comprises the non-transgenic Burley
tobacco that is present in the amount of about 90% by weight, and
the non-transgenic Flue-cured or Bright tobacco that is present in
the amount of about 10% by weight.
[0012] The cigarette may further comprise expanded stem tobacco or
Oriental tobacco.
[0013] In another embodiment, the cigarette is configured to
produce a mainstream smoke that generates reduced biological
insult, for example, DNA double strand breaks (DSBs), cell death or
perturbation of RNA transcriptome or proteome in lung cells than
the mainstream smoke from a 2R4F reference cigarette under the same
smoking conditions.
[0014] Another aspect of the invention is directed to a filter, and
the cigarette described above comprising a filter, where the filter
comprises a carbon and/or a weak base amine-containing resin. A
carbon may be used in the filter that has a total pore volume of
from 0.1 mL/g to 0.9 mL/g. In another embodiment, a certain
percentage of the carbon has a pore volume distribution of 0.1 mL/g
to 0.9 mL/g, wherein the percentage of carbon having the pore
volume distribution is least about 50%. In another embodiment, the
carbon has an average pore diameter of 0.6 nm to 1.1 nm.
[0015] In one embodiment, the filter comprises about 30-100 mg of
the carbon. In another embodiment, the filter comprises an
activated carbon having an activity of 50-60. In a further
embodiment, carbon is TA95 activated carbon.
[0016] In another embodiment, the filter comprises about 10-50 mg
of the weak base amine-containing resin. In a further aspect the
weak base amine-containing resin contains at least or equal to
about 1.3-1.5%, such as 1.4% nitrogen atoms (N) in the form of
amine functional groups. In one embodiment, all of the nitrogen
atoms are in the form of primary amine functional groups, and in
other embodiments the nitrogen atoms are in the form of a mixture
that includes primary amine functional groups. In another
embodiment, the ratio of the carbon to the weak base
amine-containing resin in the filter is from about 1:1 to 4:1.
[0017] The filter may also comprise sepiolite. The filter may also
be configured in a tripartite design comprising three compartments
for containing each of the carbon, the weak base amine-containing
resin, and the sepiolite.
[0018] Another aspect is directed to a cigarette filter comprising
a carbon that has a total pore volume of 0.1 mL/g to 0.9 mL/g, or
about 0.3 mL/g to about 0.7 mL/g, or about 0.4 mL/g to about 0.6
mL/g, and/or having a certain percentage of the activated carbon
having a pore volume distribution of 0.1 mL/g to 0.9 mL/g, wherein
the percentage of carbon having the pore volume distribution is
least about 50%; and optionally a carbon having an average pore
diameter of 0.6 nm to 1.1 nm.
[0019] The cigarette filter may further comprise a weak base
amine-containing resin, such as one in which the ratio of the
carbon to the weak base amine-containing resin in the filter is
from about 1:1 to 4:1. The cigarette filter may comprise about
30-100 mg of the carbon. In one embodiment, the carbon is an
activated carbon having an activity of 50-60. In one embodiment,
the activated carbon is TA95.
[0020] The cigarette filter may also comprise about 10-50 mg of
weak base amine-containing resin. The weak base amine-containing
resin may contain at least or equal to about 1.3-1.5%, such as 1.4%
nitrogen atoms (N) in the form of amine functional groups. In one
embodiment, all of the nitrogen atoms are in the form of primary
amine functional groups, and in other embodiments the nitrogen
atoms are in the form of a mixture that includes primary amine
functional groups.
[0021] The cigarette filter described above may also further
comprise sepiolite. The filter described above may have a
tripartite design comprising three compartments for containing each
of the carbon, weak base amine-containing resin, and sepiolite.
Filters described above may be used in a cigarette.
[0022] One aspect is directed to method of making a filtered
cigarette comprising: preparing a blend of cured tobacco, wherein
the blend comprises a non-transgenic Burley tobacco and a
non-transgenic Flue-cured or Bright tobacco, wherein the
non-transgenic Burley tobacco in the blend is present in an amount
of 45-70% by weight based on the combined weight of the
non-transgenic Burley tobacco and the non-transgenic Flue-cured or
Bright tobacco, and the non-transgenic Flue-cured or Bright tobacco
in the blend is present in an amount of 30-55% by weight based on
the combined weight of the non-transgenic Burley tobacco and the
non-transgenic Flue-cured or Bright tobacco; determining at least
one of total pore volume and percentage pore volume distribution of
an activated carbon; selecting an activated carbon having a total
pore volume of 0.1 mL/g to 0.9 mL/g and/or having a certain
percentage of the activated carbon having a pore volume
distribution of 0.1 mL/g to 0.9 mL/g, wherein the percentage of
carbon having the pore volume distribution is least about 50%;
optionally measuring and/or selecting an activated carbon having an
average pore diameter of 0.6 nm to 1.1 nm; incorporating the
selected activated carbon into a cigarette filter; and generating a
filtered cigarette that contains the blend of cured tobacco and the
cigarette filter. In the method, the cigarette filter may further
comprise a weak base amine-containing resin. The ratio of the
activated carbon to the weak base amine-containing resin in the
filter in the method may be from about 1:1 to 4:1. The weak base
amine-containing resin used in the method may contain at least or
equal to 1.3-1.5%, such as 1.4% nitrogen atoms (N) in the form of
amine functional groups. In one embodiment, all of the nitrogen
atoms are in the form of primary amine functional groups, and in
other embodiments the nitrogen atoms are in the form of a mixture
that includes primary amine functional groups. The activated carbon
used in the method may have an activity of 50-60.
[0023] Another aspect is directed to method of making a filtered
cigarette comprising: preparing a blend of cured tobacco, wherein
the blend comprises a non-transgenic Burley tobacco and a
non-transgenic Flue-cured or Bright tobacco, wherein the
non-transgenic Burley tobacco in the blend is present in an amount
of 85-92% by weight based on the combined weight of the
non-transgenic Burley tobacco and the non-transgenic Flue-cured or
Bright tobacco, and the non-transgenic Flue-cured or Bright tobacco
in the blend is present in an amount of 8-15% by weight based on
the combined weight of the non-transgenic Burley tobacco and the
non-transgenic Flue-cured or Bright tobacco; determining at least
one of total pore volume and pore volume distribution of an
activated carbon; selecting an activated carbon having a total pore
volume of 0.5 mL/g, a pore volume distribution of 0.1 mL/g to 0.9
mL/g or a pore diameter of 0.6 nm to 1.1 nm, wherein the percentage
of carbon having the pore volume distribution is least about 50%;
optionally selecting an activated carbon having an average pore
diameter of 0.6 nm to 1.1 nm; incorporating the selected activated
carbon into a cigarette filter; and generating a filtered cigarette
that contains the blend of cured tobacco and the cigarette filter.
In the method, the cigarette filter may further comprise a weak
base amine-containing resin. The ratio of the activated carbon to
the weak base amine-containing resin in the filter in the method
may be from about 1:1 to 4:1. The weak base amine-containing resin
used in the method may contain at least or equal to 1.3-1.5%, such
as 1.4% nitrogen atoms (N) in the form of amine functional groups.
In one embodiment, all of the nitrogen atoms are in the form of
primary amine functional groups, and in other embodiments the
nitrogen atoms are in the form of a mixture that includes primary
amine functional groups. The activated carbon used in the method
may have an activity of 50-60.
[0024] In another embodiment, the method further comprises:
generating mainstream smoke from the filtered cigarette; and
measuring the presence or absence of a toxicant retained in the
filter. In addition, the method may further comprise: generating
mainstream smoke from the filtered cigarette; and measuring the
appearance of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in lung cells
contacted with the mainstream smoke, a fraction of the mainstream
smoke, or a smoke condensate.
[0025] Another aspect of the invention is directed to a kit
comprising: a first cigarette comprising a first cigarette filter
that comprises a carbon or a weak base amine-containing resin, or
both; and a second cigarette comprising a second cigarette filter
that comprises a carbon, a weak base amine-containing resin, or
both, wherein the second cigarette filter is configured to retain a
greater amount of a toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells than the first cigarette filter. In the
kit, the second cigarette filter may comprise a greater amount of
the carbon or the weak base amine-containing resin or both than the
first cigarette filter.
[0026] Another aspect of the invention is directed to a method of
reducing biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells that contact
cigarette smoke comprising: advising a tobacco consumer of the need
to reduce the induction of biological insult, for example, DSBs,
cell death or perturbation of RNA transcriptome or proteome in
cells that contact cigarette smoke; and replacing a cigarette
habitually consumed by the tobacco consumer with any of the
cigarettes described above.
[0027] Another aspect of the invention is directed to a method of
reducing biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of a tobacco
consumer comprising: identifying the tobacco consumer in need of a
reduction in biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of the
tobacco consumer; and replacing a cigarette habitually consumed by
the identified tobacco consumer with any of the cigarettes
described above. The identifying step may comprise analyzing the
presence of DNA DSBs, cell death or perturbation of RNA
transcriptome or proteome in cells of the tobacco consumer. This
method may further comprise measuring the presence of DNA DSBs,
cell death, or perturbation of RNA transcriptome or proteome in the
cells of the tobacco consumer before and after providing any of the
cigarettes described above. The cells may comprise lung cells,
cheek cells, throat cells, or buccal cells.
[0028] Another aspect of the invention is directed to a method of
gradually reducing the exposure of a tobacco user to a toxicant
that caudrd biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome comprising:
identifying the tobacco user to receive a gradual reduction in
exposure to a toxicant that induces biological insults such as DNA
DSBs, cell death, or perturbation of RNA transcriptome or proteome
in human cells; replacing a cigarette habitually consumed by the
identified tobacco user with a first cigarette for a predetermined
length of time, wherein the first cigarette comprises a first
cigarette filter that comprises a carbon, a weak base
amine-containing resin, or both; replacing the first cigarette with
a second cigarette after the predetermined length of time, wherein
the second cigarette comprises a second cigarette filter that
comprises the carbon, the poly-amine containing resin, or both,
wherein the second cigarette filter is configured to retain a
greater amount of the toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells than the first cigarette' filter. The
predetermined length of time may be about 3-6 weeks.
[0029] In another embodiment, this method may further comprise
replacing the second cigarette after a second predetermined length
of time with a third cigarette, wherein the third cigarette
comprises a third cigarette filter that comprises the carbon, the
weak base amine-containing resin, or both, wherein the third
cigarette filter is capable of retaining a greater amount of the
toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the second cigarette filter.
[0030] Another aspect of the invention is directed to a method of
marketing a cigarette that is configured to reduce the induction of
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome comprising: replacing a cigarette
habitually consumed by a tobacco consumer with a first cigarette
comprising a first cigarette filter comprising a carbon and a weak
base amine-containing resin for a predetermined length of time;
replacing the first cigarette after the predetermined period of
time with a second cigarette comprising a second cigarette filter
configured to retain a greater amount of the toxicant that induces
biological insults such as DNA DSBs, cell death, or perturbation of
RNA transcriptome or proteome in human cells than the first
cigarette filter; and marketing the first and second cigarettes,
wherein the first cigarette is introduced to a consumer prior to
the second cigarette and the first cigarette is marketed for a time
sufficient to adjust a tobacco consumer's taste prior to marketing
the second cigarette. The time to adjust the tobacco consumer's
taste may be less than 1 month, 2 months, 3 months, 4 months, 5
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
months, or 12 months.
[0031] In another embodiment, the method of marketing the cigarette
further comprises replacing the second cigarette with a third
cigarette that comprises a third cigarette filter capable of
retaining a greater amount of a toxicant that induces biological
insults such as DNA DSBs, cell death, or perturbation of RNA
transcriptome or proteome in human cells than the second cigarette
filter. In another embodiment, the first cigarette, the second
cigarette and the third cigarette have substantially similar
packaging. In further embodiment, the first cigarette, the second
cigarette, and the third cigarette are sold under the same brand.
In yet another embodiment, the first cigarette, the second
cigarette, and the third cigarette have the same packaging.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 depicts a multiple section filter cigarette.
[0033] FIG. 2 depicts a chart comparing DSBs for cigarette samples
containing 90:10 Flue-cured:Burley tobacco and filters comprising
varying amounts of activated carbon (TA95). The DSB level in
relative fluorescence units compared to sham control are
illustrated for cigarette smoke from cigarette samples having one
(1) conventional cellulose acetate (CA) filter and four (4) filters
containing 40 mg, 60 mg, 80 mg, and 100 mg of TA95 activated
carbon.
[0034] FIG. 3 depicts a chart comparing DSBs that were measured
using an H2AX assay (discussed below in Example 1 in detail) for
cigarette samples containing 10:90 Flue-cured:Burley tobacco and
filters comprising varying amounts of activated carbon (TA95). The
DSB level in relative fluorescence units compared to sham control
are illustrated for cigarette samples having one (1) conventional
cellulose acetate (CA) filter, and four (4) filters containing 40
mg, 60 mg, 80 mg, and 100 mg of TA95 activated carbon.
[0035] FIG. 4 illustrates a comparison of H2AX damage by the blend
type of the cigarette sample. The chart illustrates the amount of
DSBs based on an H2AX assay as a percentage of DSBs induced by
cigarette smoke from a control cigarette of the same blend type
having a cellulose acetate filter. The results are shown for
cigarette samples comprising a 90:10 Flue-cured:Burley tobacco
blend (left/blue) and a 10:90 Flue-cured:Burley tobacco blend
(right/red). The DSB percentages are illustrated for cigarette
samples comprising four (4) filters containing 40 mg, 60 mg, 80 mg,
and 100 mg of TA95 activated carbon. The results show that the H2AX
damage using the 10:90 Flue-cured:Burley tobacco blend is
significantly lower than the 90:10 Flue-cured:Burley tobacco
blend.
[0036] FIG. 5 illustrates a comparison of cloning efficiency by the
blend type of the cigarette sample. The percentage of cell death
using clonogenic assay (discussed below in Example 1 in detail) is
illustrated using a cigarette smoke from a cigarette sample having
a cellulose acetate control filter for 90:10 Flue-cured:Burley
tobacco blend (left/blue) 10:90 Flue-cured:Burley tobacco blend
(right/red). Cell death as a percentage of total cells is
illustrated for cigarette samples having one (1) conventional
cellulose acetate (CA) filter cigarette of the same blend type as
the control and two (2) filters containing 40 mg and 100 mg of TA95
activated carbon. The chart shows that cell death based on a
cigarette sample having the 10:90 Flue-cured:Burley tobacco blend
is significantly lower than the 90:10 Flue-cured:Burley tobacco
blend in the cigarette sample having a filter with 100 mg of
activated carbon (TA95).
[0037] FIG. 6 depicts a chart comparing DSBs that were measured
using an H2AX assay for cigarette samples containing 50:50
Flue-cured:Burley tobacco. The DSB level in relative fluorescence
units compared to sham control are illustrated for cigarette
samples containing three (3) filters containing 50 mg TA95
activated carbon, 50 mg A109 ion exchange resin, and 50 mg each of
TA95 and A109, in comparison to a control conventional cigarette
having a cellulose acetate (CA) filter. The cigarette samples
having filters containing 50 mg TA95 activated carbon or 50 mg A109
ion exchange resin did not result in a reduction in the DSBs in
comparison to the control; however, the cigarette sample containing
a filter combining 50 mg each of TA95 and A109 illustrated an
unexpected synergistic effect.
[0038] FIG. 7 shows a comparison of the percentage of cell death
that was measured using a clonogenic assay for cigarette samples
containing 50:50 Flue-cured:Burley tobacco blends. The results
compare cigarette samples containing three (3) filters containing
50 mg TA95 activated carbon, 50 mg A109 ion exchange resin, and 50
mg each of TA95 and A109 in comparison to a control having
conventional cellulose acetate (CA) filter. The cigarette sample
having a filter comprising 50 mg TA95 activated carbon resulted in
an approximately 10% decrease in cell death in comparison to the
control. The cigarette sample having a filter comprising 50 mg A109
ion exchange resin resulted in a slightly higher percentage of cell
death in comparison to the control. The cigarette sample having a
filter comprising a combination of 50 mg each of TA95 and A109
resulted in a greater than 40% reduction in cell death in
comparison to the control, which is significantly greater than what
would be expected from an additive effect.
[0039] FIG. 8 illustrates the synergistic reduction of the number
of DSBs as measured by H2AX at various efficacies of three
cigarettes having filters comprising 50/50 v/v of TA95 and each of
three resins A109, Duolite and CR20 compared to a 100 mg TA95
control cigarette of the same tobacco blend. A109 is a weak base
primary amine resin containing about 1.4% nitrogen atoms (N) in the
form of primary amines, whereas Duolite and CR20 are weak base
primary amine resins containing about 1.4% nitrogen atoms (N) but
in a mixed composition of primary, secondary and tertiary amine
functional groups. As the percentage of primary amine functional
groups increase within the resin, the amount of DSBs is reduced. At
the same time, however, increasing the percentage of primary amine
functional groups within the resin results in increasingly poor
sensory/taste characteristics of the cigarette.
[0040] FIG. 9 illustrates the synergistic reduction of the number
of DSBs as measured by H2AX of filters containing a mixture of
sepiolite and A109 resin in comparison to a filter containing
sepiolite alone.
[0041] FIG. 10: A549 cells were either mock-exposed (A) or exposed
to whole CS from 2R4F cigarettes for 20 min, replated with fresh
medium and harvested at 1 (B) or 2 (C) hours later. Each dot
represents a single cell. The dotplots are divided into cell cycle
phases based on the DNA index (DI=1 for G.sub.1, DI=2 for G.sub.2M
and the intervening cells in S). The red lines indicate the
boundary of mock-treated cells that includes >97% of unexposed
cells. The mean values of .gamma.H2AX for each cell cycle
compartment, collected at various times up to 2 h after exposure,
are plotted in (D).
[0042] FIG. 11: Panel A. Comparisons of whole CS exposures in terms
of .gamma.H2AX induction using FTC/ISO protocol based on style of
cigarette: IM16 (industry reference cigarette), Full (commercially
available full-flavor king size), Light (commercially available
light king size), and Ultra (commercially available ultra-light
king size). Panel B. A comparison of .gamma.H2AX levels in A549
cells exposed to whole CS generated by the FTC/ISO smoking regimen:
IM16 (16 mg tar), Full (15 mg tar), Light (9.5 mg tar), Ultra (6 mg
tar).
[0043] FIG. 12: Induction of .gamma.H2AX normalized for the
estimated tar delivery per 20 minute whole CS exposure.
[0044] FIG. 13: Panel A: Plot of absorbance at 300 nm versus total
.gamma.H2AX IF of PBS media post exposure to whole CS from two,
three and four 1R5F industry reference ultra-light cigarettes with
35, 55 and 75 ml puff volumes. Panel B: Plot of absorbance at 300
nm versus .gamma.H2AX IF of PBS media post exposure to whole CS
from two and three 2R4F industry reference light cigarettes with 35
and 55 ml puff volumes. Panel C: Combined plot of Panels A and
B.
[0045] FIG. 14: Induction of .gamma.H2AX by the representative
market survey cigarettes: Marlboro Light, Marlboro Ultra-Light and
Camel Ultra-Light. Cigarettes were smoked according to the MDPH
protocol and cells were exposed to whole CS from one, two, and
three cigarettes of each type.
[0046] FIG. 15: Linear regression plot of measured total
particulate matter (TPM) vs. .gamma.H2AX immuno-fluorescence
obtained from each cigarette described in FIG. 5. The y-intercept
of the regression line represents the amount of TPM required from
each cigarette to initiate DSBs detectable by the .gamma.H2AX
assay.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] Creating a cigarette that produces the least amount of
biological insult while retaining taste is desirable in the
cigarette market. Approaches are being developed to analyze
biological insult such as described in WO2006/124448, which is
expressly incorporated by reference herein in its entirety. In the
past, attempts have been made to overcome this problem by using low
alkaloid tobacco (WO2006/124448). In addition, others in the field
have used filters.
[0048] In the past, cigarettes containing tobacco blends having a
large proportion of Burley tobacco also contained a relatively
higher amount of precursors that deliver tobacco specific
nitrosamines (TSNAs), which are carcinogens, to a tobacco consumer.
Cigarettes containing a large proportion of Burley, and having
lengthy carbon filters, although effective for reducing the amount
of TSNAs, resulted in cigarettes that did not satisfy tobacco
users' sensory/perception needs, i.e., such cigarettes had an
unacceptable taste. For example, a prototype cigarette was
developed by Philip Morris having a lengthy filter measuring
approximately 32 mm but the cigarette did not enjoy appreciable
sales due to unacceptable taste.
[0049] An illustration of the effect of the amount a carbon on DSBs
is shown in Example 1. Specifically, DNA double strand breaks
(DSBs) in relative fluorescence units compared to sham control
decreased upon increasing amounts (40 mg, 60 mg, 80 mg, or 100 mg)
of activated carbon present in the filter as shown in FIGS. 2-3 for
cigarette samples containing about 90:10 Flue-cured:Burley tobacco
(FIG. 2) or 10:90 Flue-cured:Burley tobacco (FIG. 3).
[0050] Surprisingly, however, it was found that cigarettes
containing tobacco blends having a lower proportion of
non-transgenic Flue-cured or Bright tobacco to non-transgenic
Burley tobacco resulted in less biological insult (for example,
DSBs, cell death or perturbation of RNA transcriptome or proteome)
in comparison to cigarettes containing tobacco blends having a
relatively higher proportion of non-transgenic Flue-cured or Bright
tobacco to non-transgenic Burley tobacco. It was not expected that
a cigarette containing a higher proportion of Burley tobacco, which
contains a higher amount of carcinogenic TSNAs than Flue-cured or
Bright tobacco, would result in a cigarette that induces relatively
less biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of biological insult induced by a conventional or reference
cigarette (e.g., 2R4F). Thus, in one embodiment, a cigarette is
configured to produce a mainstream smoke that generates less
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome as compared to mainstream smoke from
a 2R4F reference cigarette under the same smoking conditions. In
one embodiment, the mainstream smoke generates 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%
produce a mainstream smoke that generates less biological insult,
for example, DSBs, cell death or perturbation of RNA transcriptome
or proteome in cells, such as lung cells, such as fewer DSBs in
cells, such as A549 cells in a H2AX assay described herein, than
the mainstream smoke from a 2R4F reference cigarette under the same
smoking conditions.
[0051] Cigarettes containing tobacco blends having a lower
proportion of non-transgenic Flue-cured or Bright tobacco to
non-transgenic Burley tobacco result in less biological insult as
shown in Example 1. Specifically, H2AX damage by a carbon filtered
cigarette sample comprising the 90:10 Flue-cured:Burley tobacco
blend (FIG. 4, left/blue) was compared to a cigarette sample
comprising 10:90 Flue-cured:Burley tobacco blend (FIG. 4,
right/red), wherein the filters associated with each blend
contained increasing amounts, namely 40 mg, 60 mg, 80 mg, or 100
mg, of TA95 activated carbon. FIG. 4 illustrates that the H2AX
damage using the 10:90 Flue-cured:Burley tobacco blend was
significantly lower than the 90:10 Flue-cured:Burley tobacco
blend.
[0052] A similar comparison was made using the clonogenic assay
described above. Specifically, the percentage of cell death by a
carbon filtered cigarette sample comprising the 90:10
Flue-cured:Burley tobacco blend (FIG. 5, left/blue) was compared to
a cigarette sample comprising 10:90 Flue-cured:Burley tobacco blend
(FIG. 5, right/red), wherein the filters associated with each blend
contained 40 mg or 100 mg of TA95 activated carbon. Surprisingly,
in the cigarette sample having a filter comprising 100 mg of
activated carbon (TA95), cell death was significantly lower for the
10:90 Flue-cured:Burley tobacco blend in comparison to the 90:10
Flue-cured:Burley tobacco blend as shown in FIG. 5.
[0053] A cigarette filter also plays a role in reducing biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome as compared to the amount of biological
insult induced by a conventional or reference cigarette (e.g.,
2R4F). In general, as the length of a carbon filter increases, the
biological insult conferred to a tobacco user decreases, for
example, the induction of DSBs, cell death or perturbation of RNA
transcriptome or proteome decreases. Surprisingly, it has been
found that carbon filters, such as an activated carbon filter, are
more effective in reducing H2AX damage in cigarettes containing a
higher proportion by weight of Burley tobacco with respect to
Flue-cured tobacco, such as shown in FIG. 4 described above, in
comparison to cigarettes containing a higher proportion by weight
of Flue-cured tobacco with respect to Burley tobacco.
[0054] At the same time, as mentioned above, carbon filters that
are too lengthy are not well accepted due to unacceptable taste. In
general, carbon filters greater than 60 mm have not been well
accepted. Thus, in one embodiment, the amount of a carbon in a
filter must be present in an amount to fulfill the
sensory/perception needs of the consumer. In addition to reduced
biological insult, surprisingly, the change in taste of carbon
filtered cigarettes containing a higher proportion by weight of
Burley tobacco with respect to Flue-cured tobacco is less in
comparison to cigarettes containing a higher proportion by weight
of Flue-cured tobacco with respect to Burley tobacco.
[0055] Accordingly, in some embodiments, cigarettes contain a blend
of at least, greater than, less than or equal to about 8% by weight
of Flue-cured tobacco (such as Virginia Bright leaf), for example
8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight based on the
combined weight of the non-transgenic Flue-cured tobacco and
non-transgenic Burley tobacco. Ranges of Flue-cured tobacco in the
blend may include at least, greater than, less than or equal to
about 8-15%, such as, at least, greater than, less than, equal to,
or any number in between about 8-9%, 8-10%, 8-11%, 8-12%, 8-13%,
8-14%, 9-10%, 9-11%, 9-12%, 9-13%, 9-14%, 9-15%, 10-11%, 10-12%,
10-13%, 10-14%, 10-15%, 11-12%, 11-13%, 11-14%, 11-15%, 12-13%,
12-14%, 12-15%, 13-14%, 13-15%, or 14-15% by weight based on the
combined weight of the non-transgenic Flue-cured tobacco and
non-transgenic Burley tobacco. Therefore, non-transgenic Burley
tobacco may be present in the blend in an amount of at least,
greater than, less than, or equal to about 85%, for example, at
least, greater than, less than, equal to, or any number in between
about 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 92% by weight based on
the combined weight of the non-transgenic Flue-cured tobacco and
non-transgenic Burley tobacco. Ranges of Burley tobacco in the
blend may include at least, greater than, less than or equal to
about 85-92%, such as at least, greater than, less than, equal to,
or any number in between about 85-86%, 85-87%, 85-88%, 85-89%,
85-90%, 85-91%, 85-92%, 86-87%, 86-88%, 86-89%, 86-90%, 86-91%,
86-92%, 87-88%, 87-89%, 87-90%, 87-91%, 87-92%, 88-89%, 88-90%,
88-91%, 88-92%, 89-90%, 89-91%, 89-92%, 90-91%, 90-92%, or 91-92%,
by weight based on the combined weight of the non-transgenic
Flue-cured tobacco and non-transgenic Burley tobacco.
[0056] In another embodiment, a cigarette contains a blend at
least, greater than, less than or equal to about 30% by weight of
Flue-cured tobacco (such as Virginia Bright leaf), for example, at
least, greater than, less than, equal to, or any number in between
about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or
55% by weight based on the combined weight of the non-transgenic
Flue-cured tobacco and non-transgenic Burley tobacco. Ranges of
Flue-cured tobacco in the blend may include at least, greater than,
less than, or equal to about 30-55%, such as, at least, greater
than, less than, equal to, or any number in between about 45-55%,
46-54%, 47-53%, 48-52%, 49-51%, or about 50% by weight based on the
combined weight of the non-transgenic Flue-cured tobacco and
non-transgenic Burley tobacco. Therefore, non-transgenic Burley
tobacco may be present in the blend in an amount of at least,
greater than, less than or equal to about 45%, for example, at
least, greater than, less than, equal to, or any number in between
about 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or
70% by weight based on the combined weight of the non-transgenic
Flue-cured tobacco and non-transgenic Burley tobacco. Ranges of
Burley tobacco in the blend may include at least, greater than,
less than or equal to about 45-70%, such as, at least, greater
than, less than equal to or any number in between about 45-65%,
45-60%, 45-55%, 50-70%, 50-65%, 50-60%, 50-55%, 55-70%, 55-65%,
55-60%, 60-70% or about 50% by weight based on the combined weight
of the non-transgenic Flue-cured tobacco and non-transgenic Burley
tobacco.
[0057] Surprisingly, it has also been discovered that a filter
comprising both a carbon and a weak base amine-containing resin act
synergistically and thus, when used in a cigarette, reduce the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of biological insult expected by a filter containing either
the carbon or the weak base amine-containing resin alone. DSBs
induced by a 50:50 Flue-cured:Burley tobacco cigarette having a
filter containing 50 mg each of TA95 and A109 were significantly
fewer than filters containing either 50 mg TA95 activated carbon or
50 mg A109 ion exchange resin as shown in FIG. 6. A cigarette
having a filter containing either 50 mg TA95 activated carbon or 50
mg A109 ion exchange resin had no effect in comparison to a
reference 2R4F cigarette. However, the combination of 50 mg TA95
activated carbon and 50 mg A109 ion exchange resin showed
significant reduction in DSBs. It is expected that various weak
base amine-containing resins similarly decrease H2AX damage, and
act synergistically when combined with activated carbon as shown in
FIG. 6.
[0058] Synergistic effects were also observed based on a percentage
of cell death using a clonogenic assay for cigarette samples
containing 50:50 Flue-cured:Burley tobacco blends. Filters
containing 50 mg each of TA95 and A109 showed significant reduction
in percentage of cell death in comparison to filters containing
either 50 mg TA95 activated carbon or 50 mg A109 ion exchange
resin. For instance, A109 (50 mg) does not improve cloning
efficiency by itself, but does when mixed with TA95, illustrating a
synergistic effect. It is expected that that various weak base
amine-containing resins similarly reduce cell death in clonogenic
assays when combined with activated carbon, as shown in FIG. 7.
[0059] Thus, another embodiment is directed to a cigarette filter
containing a proportion by weight of either a weak base
amine-containing resin to a carbon or a carbon to weak base
amine-containing resin of individually at least, greater than, less
than or equal to about 50:50, or 40:60 or 30:70, such as, at least,
greater than, less than equal to or any number in between about
31:69, 32:68, 33:67, 34:66, 35:65, 36:64, 37:63, 38:62, 39:61,
40:60, 41:59, 42:58, 43:57, 44:56, 45:55, 46:54, 47:53, 48:52, or
49:51. In addition to the weight of the carbon, it is desired to
describe the total pore volume within the filter regardless of
weight of carbon. For instance, a 100 mg of carbon type A with a
total pore volume of 0.5 mL/g yields a total pore volume in the
filter of 0.05 mL. However the 100 mg of carbon type B with a pore
volume distribution of 1.0 mL/g yields a total pore volume in the
filter is 0.1 mL. Therefore, a filter containing carbon type B will
result in less biological insult, for example, fewer DSBs, cell
death or perturbation of RNA transcriptome or proteome as compared
to the amount of biological insult induced by a conventional or
reference cigarette (e.g., 2R4F) than a filter containing the same
weight of carbon type A. This result is non-obvious because pore
volumes do not necessarily track with surface area measurements,
which is how "high activity" carbons were traditionally selected
for removal efficiency. In one embodiment, as less carbon by weight
is used in the filter, there is less of an impact on a tobacco
consumer's sensory perception and therefore results in a better
tasting cigarette.
[0060] In another embodiment, a cigarette filter contains a carbon
having a total pore volume of at least, greater than, less than or
equal to about 0.1 mL/g to 0.9 mL/g or about 0.5 mL/g, a pore
volume distribution of at least, greater than, less than or equal
to about 0.1 mL/g to 0.9 mL/g, or a pore diameter of at least,
greater than, less than or equal to about 0.6 nm to 1.1 nm, wherein
the percentage of carbon having the pore volume distribution is at
least, greater than, less than or equal to about 50%, and wherein a
carbon is selected for a filter based on a total pore volume, total
pore volume distribution and/or pore diameter. Total pore volume of
the carbon has significantly more effect on removal of a toxicant
from mainstream smoke and reduction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome as compared to the amount of biological insult induced by
a conventional or reference cigarette (e.g., 2R4F), than the
surface area of the carbon. Surprisingly, a carbon having an
increased pore volume but the same surface area as a carbon with a
lower pore volume will allow increased removal efficiency of vapor
phase smoke constituents. The lowering of vapor phase components in
smoke leads to a reduction of biological insult, for example, DSBs,
cell death or perturbation of RNA transcriptome or proteome as
compared to the amount of biological insult induced by a
conventional or reference cigarette (e.g., 2R4F), resulting in
comparatively reduced biological effects. This approach
advantageously allows cigarette designs to provide higher
reductions in unwanted smoke components by using lower quantities
of carbon in the cigarette filter. For example, 50% by weight less
of a carbon with the total pore volume distribution described above
can be used in comparison to a carbon with half of the total pore
volume distribution. In another embodiment, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, less carbon by weight may be used in
comparison to a carbon having a total pore volume of less than 0.1
mL/g to 0.9 mL/g. In addition, the same amount of a carbon by
weight having the total pore volume distribution described above,
will have 75-100% more activity (adsorption) than a carbon having
half of the total pore volume distribution. In another embodiment,
a carbon having the total pore volume distribution described above
may have at least, greater than, less than or equal to about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, more activity
based on the same amount of a carbon by weight. The use of lower
quantities provides a cigarette with increased sensory perception
and more consumer acceptability.
[0061] The toxicants reduced in the cigarettes described herein
include any of those known to be present in cigarette smoke,
including but not limited to, nicotine, tar, and carbon monoxide,
as well as formaldehyde, ammonia, sulfur-containing compounds, and
hydrogen cyanide. The reduced risk cigarettes described herein may
comprise in one embodiment, three components. The first component
is a cut filler composition that comprises a cured tobacco blend
having at least one tobacco, such as, for example, a non-transgenic
Burley tobacco and/or low alkaloid Burley tobacco. In one
embodiment, the Burley tobacco has been cured to have a low or
reduced level of compounds that can cause the induction of
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome as compared to the amount of
biological insult induced by a conventional or reference cigarette
(e.g., 2R4F), e.g., in human cells. In some embodiments, the cut
filler composition comprises a cured tobacco blend having a Burley
or a low alkaloid Burley tobacco that has been cured to have a low
level of tobacco-specific nitrosamines (TSNAs). The second
component is a cigarette wrapper with reduced ignition propensity
that circumscribes the cut filler composition. The third component
is a cigarette filter that comprises carbon and/or an ion-exchange
resin.
[0062] Biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome are generated by a
variety of genotoxic agents, and are among the most critical
lesions that may lead to apoptosis, mutations, translocations or
the loss of significant sections of chromosomal material. Detection
of biological insult, for example, DSBs, cell death or perturbation
of RNA transcriptome or proteome upon cell exposure to a potential
carcinogen, therefore, provides a way to assess the potential
hazard of the exposure to cigarette smoke. In one embodiment, a
sensitive assay of DSBs detection based on analysis of histone H2AX
phosphorylation is used. Histone H2AX, a variant of a family of at
least eight protein species of the nucleosome core histone H2A,
becomes phosphorylated in live cells upon induction of DSBs. The
phosphorylation of H2AX on Ser 139 at sites flanking the DSBs is
carried out by ATM-, ATR-, and/or DNA-dependent protein kinases
(DNA-PKs). The phosphorylated form of H2AX is denoted .gamma.H2AX.
Antibodies and fragments thereof, and related methods for
selectively detecting gamma-H2AX, are known in the art, as
exemplified in U.S. Pat. Nos. 6,362,317 and 6,884,873, both of
which are hereby expressly incorporated by reference in their
entireties. This and additional methods for assessing the level of
compounds that can cause DSBs in human cells are known in the art
as exemplified in WO2006/124448 and WO 2005/113821, both of which
are hereby expressly incorporated by reference in their
entireties.
[0063] In other embodiments, a clonogenic assay or an assay that
detects perturbation of the RNA transcriptome or proteome are used,
which are described below in more detail.
[0064] Low alkaloid tobacco, for example, tobacco that has been
chemically treated or extracted to remove nicotine, or tobacco that
has been selectively bred to have low alkaloid or low nicotine
levels, such as tobacco having one or more mutations in genes
encoding enzymes involved in nicotine biosynthesis, can be employed
in the cigarettes described herein, and the inclusion of these
reduced risk tobaccos with a filter comprising an ion-exchange
resin and/or carbon, results in a cigarette that reduces the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of induction of biological insult induced by a conventional
or reference cigarette (e.g., 2R4F), in cells of a tobacco consumer
or in cells contacted with smoke from said cigarettes in an in
vitro assay, as described herein.
[0065] The level or amount of DNA DSBs induced by the cigarettes
described herein may be determined by several types of DNA DSB
assays, including the one described in Example I. In certain
embodiments, the reduced risk cigarettes described herein induce
less than, equal to, or any number in between about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, or 90% fewer DNA DSBs than a conventional
or reference cigarette (e.g., 2R4F). Similarly, the cigarettes
described herein reduce cell death or perturbation of RNA
transcriptome or proteome of less than, equal to or any number in
between about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% the
amount of cell death or perturbation of RNA transcriptome or
proteome as compared to a conventional or reference cigarette
(e.g., 2R4F).
Reduced Risk Tobacco
[0066] Several approaches to create a reduced risk tobacco product
having a reduced amount of a harmful compound are described. At
least some of the reduced risk tobacco products provided herein
contain modified tobacco. As used herein, "modified tobacco" refers
to a tobacco that contains a mutation in a gene involved in
nicotine biosynthesis or has been subjected to one or more genetic
modifications (e.g., genetic engineering), chemical or processing
steps (e.g., extraction) that is different than the conventional
treatment or processing of traditional and/or "wild-type" tobaccos.
In one example, a tobacco can be selectively bred for reduced risk
properties such as crossing a wild-type tobacco with a tobacco that
has a mutation in a gene involved in nicotine biosynthesis (e.g.,
LA Burley 21 or LA Flue 53). In another example, a tobacco can be
chemically modified, by, for example, extracting or chemically
altering one or more components of tobacco, according to methods
known in the art, as exemplified in U.S. Pat. Nos. 6,789,548,
4,557,280; 4,561,452; 4,848,373; 4,183,364; 4,215,706; 4,257,430;
4,248,251; 4,235,251; 4,216,784; 4,177,822; 4,055,191 (all of which
are herein expressly incorporated by reference in their entireties)
or by adding one or more compounds (e.g., an antioxidant) to a
tobacco or tobacco plant prior to harvesting the tobacco, as known
in the art and exemplified in U.S. Pat. Pub. No. 2005/0072047,
herein expressly incorporated by reference in its entirety.
Additional modified tobaccos contemplated herein include
reconstituted tobacco, extracted tobacco, and expanded or puffed
tobacco. Any one or more of these techniques can be combined with
the modified tobaccos above and/or as otherwise described herein.
In some embodiments, the tobacco is modified to have a reduced
amount of a compound that contributes to a tobacco-related disease,
including, but not limited to, a compound associated with a
tobacco-related disease or a metabolite thereof (e.g., tobacco
sterols, nicotine, a TSNA, and a gene product that is involved in
the production of a compound associated with a tobacco-related
disease or a metabolite thereof). Numerous methods for preparing a
reduced-risk tobacco product are known in the art, including, for
example, those described in WO2006/124448 and US2006/0157072, which
are herein expressly incorporated by reference.
[0067] Researchers have developed several approaches to reduce some
of these harmful compounds, but many of these conventional
techniques result in a product that has poor taste, fragrance, or
smoking properties. Some processes, for example, reduce the
nicotine content of tobacco by selective breeding, microbial
enzymatic degradation, chemical extraction, or high pressure
extraction. (See e.g., U.S. Pat. Nos. 4,557,280; 4,561,452;
4,848,373; 4,183,364; and 4,215,706, all of which are hereby
expressly incorporated by reference in their entireties). More
recently, techniques in genetic engineering and chemically-induced
gene suppression have been employed to make reduced nicotine and/or
reduced tobacco specific nitrosamine (TSNA) tobacco. (See e.g.,
Conkling et al., WO98/56923; U.S. Pat. Nos. 6,586,661; 6,423,520;
and U.S. patent application Ser. Nos. 09/963,340; 10/356,076;
09/941,042; 10/363,069; 10/729,121; 10/943,346; Timko et al., WO
00/67558, which designated the United States and was published in
English, Nakatani et al., U.S. Pat. Nos. 5,684,241; 5,369,023;
5,260,205; and Roberts et al., U.S. Pat. No. 6,700,040, all of
which are hereby expressly incorporated by reference in their
entireties. Any one or more of these techniques can be used to
create a tobacco or tobacco product used with the teachings
herein.
[0068] Also provided herein are modified tobaccos and tobacco
blends that have reduced levels of compounds that induce biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome as compared to the biological insult
induced by a control or reference cigarette such as 2R4F, in human
cells contacted by smoke from said tobacco, while still maintaining
sufficiently desirable consumer taste, so as to receive market
acceptance. Methods for assessing sufficiently desirable consumer
taste of a tobacco product are well-established in the art and are
exemplified elsewhere herein, and include, but are not limited to,
limited geographic introduction into the market and/or focus group
testing, where the results of such assessments are evaluated
according to well-established standards in the art. These methods
are employed to develop a step-wise program for introduction of
cigarettes that gradually (in a step-wise fashion) reduce the
levels or amounts of a toxicant in cigarette smoke while
maintaining or adjusting consumer sensory/perception of taste or
consumer acceptance over time.
[0069] Tobacco products that comprise a modified tobacco described
herein include "full-flavor," "lights," and "ultra light"
cigarettes typically having a reduced level of compounds that
induce biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of biological insult induced by a conventional or reference
cigarette (e.g., 2R4F), in human cells contacted by smoke from said
Burley tobacco. The term "tobacco products" includes, but is not
limited to, smoking materials (e.g., cigarettes, cigars, pipe
tobacco), snuff, snus, chewing tobacco, gum, and lozenges. The term
"reduced risk tobacco product" or "reduced risk tobacco" includes,
but is not limited to, a tobacco product or tobacco comprising a
modified tobacco that has a reduced amount of a compound that
contributes to a tobacco-related disease, such as nicotine,
nornicotine, a sterol, or the metabolites thereof including, but
not limited to, a TSNA, or harmful compounds generated upon
pyrolysis of tobacco, including but not limited to, acrolein,
aldehydes, aromatic amines, aromatic hydrocarbons, carbon monoxide,
carbonyl compounds, free radicals, hydrogen cyanide, nitriles,
oxides of nitrogen, phenolics, polycyclic aromatic hydrocarbons,
volatile hydrocarbons, as compared to the amount of these compounds
in or generated by a reference tobacco or reference tobacco product
(e.g., IM16, 2R4F or 1R5F), a commercially available tobacco
product of the same class (e.g., full-flavor, lights, and
ultra-lights), or a tobacco of the same variety (e.g., Burley,
Virginia Flue-cured, or Oriental) or strain (e.g., LA Burley 21,
K326, Tn90, Djebe1174) as the modified tobacco). In some
embodiments, the "reduced risk tobacco product" or "reduced risk
tobacco" is a tobacco product or tobacco that contains an additive
that reduces the harmful effects of conventional tobacco (e.g.,
reduces the induction of biological insult, for example, DSBs, cell
death or perturbation of RNA transcriptome or proteome as compared
to the amount of biological insult induced by a conventional
tobacco.
[0070] Various tobaccos may be used in accordance with the
embodiments described herein. For example, MB1 is a higher than
normal Burley blend. In addition, MB2 is a low nitrosamine
Burley.
[0071] The modified tobacco described herein is suitable for
conventional growing and harvesting techniques (e.g. topping or no
topping, bagging the flowers or not bagging the flowers,
cultivation in manure rich soil or without manure) and the
harvested leaves and stems are suitable for use in one or more
traditional tobacco products including, but not limited to, pipe,
cigar and cigarette tobacco and chewing tobacco in any form
including leaf tobacco, shredded tobacco or cut tobacco. It is also
contemplated that the modified tobacco described herein can be
processed and blended with conventional tobacco so as to create a
wide range of tobacco products with varying amounts of compounds
that are harmful in the tobacco or smoke generated therefrom.
[0072] In some embodiments, the reduced risk modified tobacco is a
selectively bred low alkaloid tobacco, such as, for example, LA
Burley 21, LA Flue 53, and other known low alkaloid tobacco
strains. This tobacco can be blended with conventional tobacco to
yield a blend of tobacco with varying taste characteristics and/or
toxicants in the tobacco or smoke generated therefrom.
[0073] In some embodiments, the reduced risk modified tobacco is a
tobacco grown under altered conditions that result in a reduced
amount of compounds that cause the induction of biological insult,
for example, DSBs, cell death or perturbation of RNA transcriptome
or proteome as compared to the amount of biological insult induced
by a conventional or reference cigarette (e.g., 2R4F) in human
cells, nicotine and TSNA. Exemplary conditions include, but are not
limited to reduced nitrate soils, increased watering of tobacco
plants, use of clay soils in preference to volcanic soils, and any
other conditions known in the art for reducing the amount of
compounds that cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells, nicotine, TSNA, sterol and/or PAH in
tobacco. The "reduced risk" modified tobacco described herein can
also be processed or cured or otherwise treated so as to reduce the
amount of a harmful compound (e.g., aseptic processing, removal of
microbes, bacteria or fungi or mold, air curing, stalk cutting
wherein the tobacco is not contacted with the soil, and aseptic
packaging).
[0074] In some embodiments, the modified tobacco has reduced levels
of nicotine and/or nornicotine. Alkaloids such as nicotine and
nornicotine are precursors for a number of harmful compounds that
contribute to tobacco-related disease (e.g., the tobacco specific
nitrosamines (TSNAs): N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal
(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),
4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or
4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC)).
Sterols are precursors for a number of harmful compounds, which are
generated by pyrolysis of tobacco, that also contribute to
tobacco-related disease (e.g., polycyclic aromatic hydrocarbons
(PAHs), such as benz[a]pyrene (BAP), heterocyclic hydrocarbons,
terpenes, paraffins, aromatic amines, and aldehydes). Because the
presence of these harmful compounds in tobacco contributes to
tobacco-related disease, a modified tobacco that comprises a
reduced amount of any one of these compounds, as compared to a
reference tobacco (e.g., the industry standard reference tobacco
IM16 (Philip Morris.RTM. USA) or the low tar reference cigarette
2R4F or the ultra low tar cigarette 1R5F, which are Kentucky
reference cigarettes that can be obtained from the Tobacco and
Health Institute at the University of Kentucky, a conventional
tobacco (e.g., a commercially available tobacco of the same class
(e.g., "full-flavor" or "light" or "ultra-light") or a
non-transgenic tobacco (e.g., a tobacco of the same variety, such
as Burley, Virginia Flue-cured, or Oriental, can be assessed, for
example, by measuring a reduced level of compounds that cause the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells
contacted by smoke from the reduced risk tobacco. Tobacco products
comprising the modified tobacco can also be analyzed by various
approaches to confirm that the tobacco is "reduced risk," as
compared to a parental strain or a reference tobacco using a H2AX
analysis, clonogenic analysis or RNA transcriptome or proteome
analysis.
[0075] The modified tobacco can be sterilized or otherwise made
substantially free of microbes, and said tobacco can be
incorporated into tobacco products, preferably, cigarettes,
optionally, by an aseptic approach so as to not introduce microbes
(e.g., bacteria, mold, yeast, and fungi) into the products. Tobacco
products comprising the modified tobacco can then be packaged,
optionally, by an aseptic approach in air-tight or microbe-free
yeast, mold and fungi-free packaging so as to not introduce
microbes yeast, mold or fungi into the products. In this manner,
the conversion of alkaloid to TSNA, which results from microbial
growth on the tobacco when microbes are introduced during
processing, packaging, and storage, is significantly reduced. By
using the embodied tobacco preparative methods, which may include
several aseptic processing, manufacturing, and packaging
procedures, one can maintain an amount of total TSNA (e.g., the
collective content of NNN, NAT, NAB, and NNK) in or delivered by
(e.g., as measured by FTC or ISO methodologies) a commercially
available tobacco product of less than or equal to 0.5 .mu.g/g
(e.g., less than, equal to or any number in between about 0.05
.mu.g/g, 0.1 .mu.g, 0.2 .mu.g/g, 0.3 .mu.g/g, 0.44 g, or 0.5
.mu.g/g) for a period of at least 1 week, 1 month, or 1-5 years
after packaging or incorporation of the tobacco into a tobacco
product (e.g., at least, greater than, less than, equal to, or any
number in between about 1-30 days, 30-90 days, 90-180 days, 180-270
days, 270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0
years-2.5 years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0
years-5.0 years).
[0076] The reduced risk modified tobacco can be made substantially
free of microbes (e.g., an aseptic preparation) by employing
sterilization, heat treatment, pasteurization, steam treatment, gas
treatment, and radiation (e.g., gamma, microwave, and ultraviolet).
The term "substantially free of microbes" in some contexts can mean
an amount of bacteria, mold, fungi, or yeast that is reduced to the
point that the conversion of nicotine or total alkaloid to TSNA is
negligible (e.g., the resultant concentration of or the amount of
delivered or provided total TSNA (e.g., NNN, NNK, NAT, and NAB) in
or delivered by a tobacco or tobacco product is equal to or below
0.5 .mu.g/g (e.g., at least, greater than, less than, equal to, or
any number in between about 0.05 .mu.g/g, 0.1 .mu.g, 0.2 .mu.g/g,
0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .mu.g/g) after prolonged storage
(e.g., at least 1-30 days, 30-90 days, 90-180 days, 180-270 days,
270 days-365 days, 1 year-1.5 years, 1.5-2.0 years, 2.0 years-2.5
years, 2.5 years-3.0 years, 3.0 years-4 years, and 4.0 years-5.0
years)). The term "substantially free of microbes" also includes
the term "substantially free of bacteria," which means in some
contexts that the tobacco or tobacco product is substantially free
of Arthrobacter, Proteus, nicotine oxidizing bacteria, such as
P-34, Psuedomonas, Xantomonas, or Zoogloea strains of bacteria. For
example, a tobacco or tobacco product is substantially free of
bacteria or a particular strain of bacteria when said tobacco or
tobacco product has less than or equal to 20% of the bacteria or a
specific strain of bacteria normally present on the tobacco or
tobacco product in the absence of application of a technique to rid
the tobacco or tobacco product of bacteria (e.g., less than, equal
to, or any number in between about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). With
respect to modified tobacco described herein, the term
"substantially free of bacteria" can refer to tobacco or a tobacco
product containing the modified tobacco that has less than or equal
to 20% of the bacteria normally present on the strain of tobacco
prior to modification and/or application of a technique to rid the
tobacco or tobacco product of bacteria (e.g., less than, equal to,
or any number in between about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%).
[0077] In some embodiments, a modified tobacco comprises a reduced
amount of alkaloid (e.g., a reduced amount of nicotine,
nornicotine, and/or TSNAs), which can then be contacted with an
exogenous nicotine so as to raise the level of nicotine in the
contacted modified tobacco in a controlled fashion. By this
approach, nicotine levels in modified tobacco that comprises a
reduced amount of endogenous nicotine can be selectively raised to
levels that are commensurate with conventional full-flavor
cigarettes, light cigarettes, or ultra-light cigarettes. (See e.g.,
WO 2005/018307, herein expressly incorporated by reference in its
entirety.) For example, modified tobacco comprising a reduced
amount of endogenous nicotine and/or TSNAs can be contacted with an
amount of exogenous nicotine that is at least, equal to, or more
than 0.3 mg/g-20.0 mg/g (nicotine/gram of tobacco). In some of the
aforementioned embodiments, the modified tobacco contacted with the
exogenous nicotine is a tobacco from a plant that has one or more
mutations in a gene involved in nicotine biosynthesis (e.g., LA
Burley 21 or LA Flue 53).
[0078] It should also be appreciated that in some embodiments, the
reduced risk tobacco products comprise an amount of tar similar to
the amount of tar in standard cigarettes. While not intending to be
limited to the following, it has been postulated that consumer
acceptance of a cigarette is related to the amount of tar in the
product, and, accordingly, by delivering a similar amount of tar
but fewer toxicants in the smoke in a reduced risk tobacco product,
the product will likely be accepted by a consumer. In such
embodiments, the tobacco product (e.g. a cigarette) can have about
0.5 mg to about 30 mg of tar. Such a tobacco product comprises
(e.g., on the leaf or tobacco rod) or delivers (e.g., side-stream
or main-stream smoke by the FTC and/or ISO methods), for example,
at least, greater than, less than, equal to, or any number in
between about 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4
mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg,
9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13
mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 m, 17 mg,
17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 20.5 mg, 21 mg,
21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg,
25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg,
29.5 mg, or 30 mg of tar.
[0079] Reducing the Amount of Nicotine, Tunas and Sterols in
Tobacco
[0080] As discussed above, TSNAs, nicotine, nornicotine, and
sterols contribute significantly to tobacco-related disease, most
notably the carcinogenic potential of tobacco and tobacco products.
Thus, tobacco and tobacco products that have or produce reduced
amounts of these compounds are reduced risk compositions (e.g.,
products that have a reduced potential to contribute to a
tobacco-related disease). Without wishing to be bound by any
particular theory, it is contemplated that the creation of tobacco
plants, tobacco and tobacco products that have a reduced amount of
nicotine and/or related alkaloids can also have reduced amounts of
TSNAs. That is, by removing or reducing nicotine in tobacco plants,
tobacco and tobacco products, one effectively removes the most
significant alkaloid substrate for TSNA formation. TSNAs can also
be reduced by employing modified harvesting (e.g., stalk cutting
wherein the tobacco is not contacted with the soil), as well as air
curing and growing in steps to avoid an accumulation of nitrates
(e.g., growing in clay soils with low nitrogen) and steps to avoid
introduction of microbes (e.g., sterilization of green tobacco).
Similarly, it is contemplated that by reducing sterols in tobacco
blends, one can reduce the amount of PAHs generated from pyrolysis
of the tobacco (e.g., using selectively bred tobacco having a
reduced level of sterols).
[0081] It should be emphasized that the phrase "a reduced amount"
as applied herein can refer to an amount of a compound in a
modified tobacco or a tobacco product that is less than what would
be found in a tobacco or a tobacco product from the same variety of
tobacco, processed in the same manner, which has not been treated
or otherwise provided according to the methods provided herein, or
can refer to an amount of a compound in a modified tobacco or a
tobacco product that is less than what would be found in a
reference tobacco or tobacco product. Thus, in some contexts,
wild-type tobacco of the same variety that has been processed in
the same manner is used as a control by which to measure whether a
reduction in a particular compound is present.
[0082] Some embodiments comprise cured tobaccos (e.g., Burley,
Flue-cured, or Oriental) with reduced amounts of nicotine as
compared to control varieties, wherein the amount of nicotine
delivered by the product (e.g., as measured by FTC or ISO
methodologies) is less than about 2 mg nicotine in smoke/g tobacco,
1 mg/g, 0.75 mg/g, 0.5 mg/g or desirably less than about 0.1 mg/g,
and preferably less than 0.08 mg/g, 0.07 mg/g, 0.06 mg/g, 0.05
mg/g, 0.04 mg/g, 0.03 mg/g, 0.02 mg/g, 0.01 mg/g. Tobacco products
made from these reduced nicotine and TSNA tobaccos are also
embodiments. In some embodiments, the cigarettes comprise a low
alkaloid tobacco (e.g., LA Burley 21 or LA Flue 53), wherein the
amount of nicotine in the tobacco (e.g., the tobacco blend) is from
about 0.2 mg/g to about 30 mg/g. In some embodiments, the
cigarettes comprise a low alkaloid tobacco (e.g., LA Burley 21 or
LA Flue 53), wherein the amount of nicotine in the tobacco (e.g.,
the tobacco blend) is from about 0.5 mg/g to about 20 mg/g. In some
embodiments, the cigarettes comprise a low alkaloid tobacco (e.g.,
LA Burley 21 or LA Flue 53), wherein the amount of nicotine in the
tobacco (e.g., the tobacco blend) is from about 1.0 mg/g to about
15 mg/g.
[0083] Alternatively, a cigarette provided herein can have or
deliver, for example, at least, greater than, less than, equal to,
or any number in between about 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg,
0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg,
0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg,
1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg,
1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg,
1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg,
2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg,
2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg,
3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg,
3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg,
4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg,
4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg,
4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0 mg,
6.5 mgmg, 6.7 mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg,
9.0 mg, 9.5 mg, 9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, and 11.0 mg
nicotine and at least, greater than, less than, equal to, or any
number in between about 0.001 .mu.g, 0.002 .mu.g, 0.003 .mu.g,
0.004 .mu.g, 0.005 .mu.g, 0.006 .mu.g, 0.007 .mu.g, 0.008 .mu.g,
0.009 .mu.g, 0.01 .mu.g, 0.02 .mu.g, 0.03 .mu.g, 0.04 .mu.g, 0.05
.mu.g, 0.06 .mu.g, 0.07 .mu.g, 0.08 .mu.g, 0.09 .mu.g, 0.1 .mu.g,
0.15 .mu.g, 0.2 .mu.g, 0.25 .mu.g, 0.3 .mu.g, 0.336 .mu.g, 0.339
.mu.g, 0.345 .mu.g, 0.35 .mu.g, 0.375 .mu.g, 0.4 .mu.g, 0.414
.mu.g, 0.45 .mu.g, 0.5 .mu.g, 0.515 .mu.g, 0.55 .mu.g, 0.555 .mu.g,
0.56 .mu.g, 0.578 .mu.g, 0.58 .mu.g, 0.6 .mu.g, 0.611 .mu.g, 0.624
.mu.g, 0.65 .mu.g, 0.7 .mu.g, 0.75 .mu.g, 0.8 .mu.g, 0.85 .mu.g,
0.9 .mu.g, 0.95 .mu.g, 1.0 .mu.g, 1.1 .mu.g, 1.114 .mu.g, 1.15
.mu.g, 1.2 .mu.g, 1.3 .mu.g, 1.35 .mu.g, 1.4 .mu.g, 1.45 .mu.g, 1.5
.mu.g, 1.55 .mu.g, 1.6 .mu.g, 1.65 .mu.g, 1.7 .mu.g, 1.75 .mu.g,
1.8 .mu.g, 1.85 .mu.g, 1.95 .mu.g, 2.0 .mu.g, 2.1 .mu.g, 2.15
.mu.g, 2.2 .mu.g TSNA (e.g., collective content of NNN, NAT, NAB,
and NNK) per cigarette.
[0084] Curing
[0085] The curing process brings out the flavor and aroma of
tobacco. Several methods for curing tobacco may be used, and indeed
many methods have been previously disclosed. For example, U.S. Pat.
No. 4,499,911 to Johnson; U.S. Pat. No. 5,685,710 to Martinez
Sagrera; U.S. Pat. No. 3,905,123 to Fowler; U.S. Pat. No. 3,840,025
to Fowler; and U.S. Pat. No. 4,192,323 to Horne (all of which are
hereby expressly incorporated by reference in their entireties)
describe aspects of the tobacco curing process, which may be used
for some embodiments provided herein. In some aspects, the curing
process begins with growing techniques and harvesting techniques
that promote reduced risk tobacco. For example, growing the tobacco
in low nitrogen conditions can influence the amount of TSNA
formation. Harvesting the tobacco in a fashion that avoids contact
with the soil can also reduce the formation of TSNAs. Selectively
harvesting leaves from the top of the plant (e.g., at stalk
locations distal to the root mass) can help to produce reduced risk
tobacco. Harvesting the tobacco from plants that have not been
topped or that have been treated with antioxidants or
anti-inflammatory compounds can also help in generating reduced
risk tobacco. Removing the midvein and reducing the stem content in
the tobacco can make for a reduced risk tobacco, as well. Any or
all of the techniques can be employed to generate reduced risk
tobacco.
[0086] Conventionally, "sticks" that are loaded with tobacco are
placed into bulk containers and placed into structures known as a
curing barn. A Flue can be used in some embodiments (thus earning
the term "Flue-cured"). The method of curing will depend, in some
cases, on the type of tobacco product desired, (i.e., snuff, snus,
cigarettes, or pipe tobacco may preferably utilize different curing
methods) and preferred methods may vary from region to region and
in different countries. In some approaches, the stems and midveins
of the leaf, which contain a high proportion of TSNAs, are removed
from the leaves prior to curing to yield a high quality, low TSNA
tobacco product.
[0087] "Flue-curing" is a popular method for curing tobacco in
Virginia, North Carolina, and the Coastal Plains regions of the
United States. Flue-curing requires a closed building equipped with
a system of ventilation and a source of heat. The heating can be
direct or indirect (e.g., radiant heat). When heat and humidity are
controlled, leaf color changes, moisture is quickly removed, and
the leaf and stems dry. Careful monitoring of the heating and
humidity can reduce the accumulation of TSNAs.
[0088] Another curing method is termed "air-curing." In this
method, an open framework is prepared in which sticks of leaves (or
whole plants) are hung so as to be protected from both wind and
sun. Leaf color changes from green to yellow, as leaves and stems
dry. As contemplated herein, in some embodiments, the air curing of
tobaccos such as Burley is performed in an open structure with wide
spacing in a manner that ensures exposure of the tobacco to large
amounts of air during the curing process so as to obtain a very low
TSNA tobacco. Air-curing experiments at a higher temperature have
shown that considerably higher levels of N-nitrosamines are formed
at a curing temperature of 32.degree. C. than at 16.degree. C.,
which is associated with a rise of the nitrite level in the
tobacco, and may also be associated with a rise in microbial
enzymatic activity. Modified curing that involves faster drying
from wider spacing or from more open curing structures has been
shown to reduce TSNA levels in Burley tobacco. The climatic
conditions prevailing during curing exert a major influence on
N-nitrosamine formation, and the relative humidity during
air-curing can be of importance. Stalk curing results in higher
TSNA levels in the smoke than primed-leaf curing. Sun-cured
Oriental tobaccos have lower TSNA levels than Flue- and air-cured
dark tobaccos. Accelerated curing of crude tobaccos, such as
homogenized leaf curing, limits the ability of bacteria to carry
out the nitrosation reactions. However, many of the methods
described above for reducing TSNAs in Burley tobacco can have
undesirable effects on tobacco taste.
[0089] TSNA formation in Flue-cured tobacco also results from
exposure of the tobacco to combustion gases during curing, where
nearly all of the TSNAs in Flue-cured tobacco (e.g., Virginia
Flue-cured) result from a reaction involving NOx and nicotine or
other alkaloids. The predominant source of NOx is the mixture of
combustion gases in direct-fired barns. At present, Flue-cured
tobacco is predominantly cured in commercial bulk barns. As a
result of energy pressures in the U.S. during the 1960's,
farmer-built "stick barns" with heat-exchanged Flue systems were
gradually replaced with more energy efficient bulk barns using
direct-fired liquid propane gas (LPG) burners. These LPG
direct-fired burner systems exhaust combustion gases and combustion
by-products directly into the barn where contact is made with the
curing tobacco. Studies indicate that LPG combustion by-products
react with naturally occurring tobacco alkaloids to form TSNAs.
[0090] In contrast to direct-fired curing, heat-exchange burner
configurations completely vent combustion gases and combustion
by-products to the external atmosphere rather than into the barn.
The heat-exchange process precludes exposure of the tobacco to LPG
combustion by-products, thereby eliminating an important source of
nitrosating agent for TSNA formation, without degrading leaf
quality or smoking quality. The use of heat exchangers can reduce
TSNA levels by about 90%, depending on the type of tobacco.
[0091] "Fire-curing" employs an enclosed barn similar to that used
for Flue-curing. The tobacco is hung over low temperature fire so
that the leaves cure in a smoke-laden atmosphere. This process uses
lower temperatures, so the process may take up to a month, in
contrast to Flue-curing, which takes about 6 to 8 days.
[0092] A further curing method, termed "sun-curing" is the drying
of uncovered sticks or strings of tobacco leaves in the sun. The
best known sun-cured tobaccos are the so-called Oriental tobaccos
of Turkey, Greece, Yugoslavia, and nearby countries.
[0093] Although many of the approaches described in this section
have significant drawbacks, it should be understood that any or all
of these techniques can be used with other techniques, as described
herein, to make tobacco and tobacco products having reduced
TSNAs.
[0094] The cured tobacco may then be blended with other tobaccos or
other materials to create the product to be used herein.
[0095] Additional Tobacco Modifications
[0096] Additional techniques can be used to make the tobacco
products provided herein, including but not limited to, chemical
modification, expansion, extraction, or puffing, and
reconstitution.
[0097] Any of a variety of chemically modified tobaccos can be
included in the methods and tobacco products provided herein. For
example, the chemical modification can include palladium, or can
include an auxin or auxin analog (see e.g., U.S. Pat. No. 6,789,548
and U.S. Pat. App. Pub. No. 20050072047, both of which are hereby
expressly incorporated by reference in their entireties).
[0098] By one approach, a chemically modified tobacco is made as
follows. A tobacco is provided and a casing solution is applied
thereto. Thereafter, a plurality of metallic or carbonaceous
catalytic particles having a mean average or a mode average
particle size of less than about 20 microns is applied to the
tobacco in a form separate from the casing solution. Next, a
nitrate or nitrite source in a form separate from the casing
solution and in a form separate from the plurality of metallic or
carbonaceous catalytic particles is applied to the tobacco, before,
after or simultaneously with applying the plurality of particles
but after applying the casing solution, whereby a smoking
composition is obtained. In some embodiments of this modified
tobacco, a polyaromatic hydrocarbon, azaarene, carbazole, or a
phenolic compound is reduced.
[0099] In another example, the chemically modified tobacco can be
extracted tobacco. By some approaches the chemically modified
tobacco is extracted with an organic solvent and other processes
use super-critical fluid extraction or carbon dioxide. In another
example, the chemical modification can be a biotic modification.
Microbes that ingest nitrates and alkaloids can be applied to
tobacco so as to obtain a reduced nicotine tobacco; for example
such a biotic modification can include bacteria. In another
example, the tobacco is processed to remove the presence of a
microbe. In another example the chemically modified tobacco can be
sterilized, pasteurized, or irradiated.
[0100] Tobacco can also be modified to decrease one or more
toxicants such as metabolites, nicotine-related compounds and
sterols in smoke generated therefrom. In some methods, a tobacco
that has been modified to produce lower levels of one or more
toxicants, such as nicotine or a nicotine metabolite, or a sterol,
can have exogenously added thereto, an antioxidant or radical
scavenger, for example. Tobacco at any stage of its processing can
have added thereto an antioxidant compound or a composition with
radical scavenger properties. Any of a variety of known antioxidant
compounds and additives containing antioxidants or radical
scavengers can be added to the tobacco, including, but not limited
to, lycopene, tocopherol, tocopherol metabolites, ascorbic acid,
unsaturated fatty acids, N-acetyl cysteine, and other antioxidants
known in the art. An additive with antioxidant properties can
include a biological composition or extract that can neutralize
oxidants, such as milk or milk proteins, turmeric or turmeric
extracts, barley or barley extracts, alfalfa or alfalfa extracts.
Other compounds that can be added to the tobacco include
thiol-containing proteins, plant extracts, aromatic compounds
(e.g., caffeine or pentoxyfyllen, which are contemplated to
scavenge radicals).
[0101] Another form of modified tobacco is expanded or puffed
tobacco. Included herein are methods to produce reduced-exposure
tobacco products by utilizing the tobacco provided herein,
deproteinized tobacco fiber, and freeze dried tobacco in any
combination and in conjunction with expanded or puffed tobacco.
More than 150 patents have been issued related to tobacco expansion
(e.g., U.S. Pat. No. 3,991,772, herein expressly incorporated by
reference in its entirety). "Expanded tobacco" is an important part
of tobacco filler, which is processed through expansion of suitable
gases so that the tobacco is "puffed" resulting in reduced density
and greater filling capacity. It reduces the weight of tobacco used
in cigarettes. Advantageously, expanded tobacco reduces tar,
nicotine and carbon monoxide deliveries and finds use, for example,
in making low tar, low nicotine, and low carbon monoxide delivery
cigarettes. Expanded tobacco is particularly useful in making
low-tar delivery cigarettes. Carlton.RTM. cigarettes, which have
had claims of being the lowest tar and nicotine delivery cigarette,
are reportedly made with a very large percentage of expanded
tobacco. Any method for expansion of tobacco known in the art can
be used in the methods provided herein. The most common method used
today incorporates liquid carbon dioxide (U.S. Pat. Nos. 4,340,073
and 4,336,814, herein expressly incorporated by reference in their
entireties). Liquid propane has also been used for making
commercial cigarettes, predominantly in Europe (U.S. Pat. No.
4,531,529, herein expressly incorporated by reference in its
entirety). Liquid propane offers advantages over carbon dioxide
since higher 3Q degrees of expansion are possible, in the range of
200%. Under pressure, the liquid carbon dioxide (or liquid propane)
permeates the tobacco cell structure. When the tobacco is rapidly
heated the carbon dioxide (or liquid propane) expands the cell back
to its pre-cured size.
[0102] Another form of modified tobacco is reconstituted tobacco.
Included herein are methods to produce reduced-exposure tobacco
products by utilizing the tobacco provided herein, deproteinized
tobacco fiber, and freeze dried tobacco in any combination and in
conjunction with reconstituted tobacco. "Reconstituted tobacco"
("Recon") is an important part of tobacco filler made from tobacco
dust and other tobacco scrap material, processed into sheet form
and cut into strips to resemble tobacco. In addition to the cost
savings, reconstituted tobacco is very important for its
contribution to cigarette taste from processing flavor development
using reactions between ammonia and sugars.
[0103] The process to produce sheets of Recon began during the
1950s. U.S. patents that describe such processes include: U.S. Pat.
Nos. 3,499,454, 4,182,349 4,962,774, and 6,761,175, herein
expressly incorporated by reference in their entireties. Recon is
traditionally produced from tobacco stems and/or smaller leaf
particles in a process that closely resembles a typical paper
making process. The tar and nicotine yields of reconstituted
tobacco are lower than those from equivalent quantities of whole
tobacco leaf. This process entails processing the various tobacco
portions that are to be made into Recon. After the Recon sheets are
produced they are cut into a size and shape that resembles cut rag
tobacco made from whole leaf tobacco. This cut Recon then is mixed
with cut-rag tobacco and is ready for cigarette making. Cigarettes
can be manufactured with all Recon, no Recon, or any combination
thereof. Processes of removing proteins from tobacco, thereby
creating "deproteinized tobacco fiber" are known in the art, as
exemplified in U.S. Pat. Nos. 4,289,147 and 4,347,324, herein
expressly incorporated by reference in its entirety. Tobacco fiber
is a major byproduct after removing protein. The fibrous remains
from deproteinized tobacco can be included in any percentage as an
ingredient of Recon. Cigarettes made from deproteinized tobacco
have a different taste than conventional cigarettes. However,
appropriate amounts of additives, including flavorings and
nicotine, can be added to help alleviate this taste deficiency.
Extracting tobacco fiber from modified tobacco effectively
eliminates virtually all carcinogenic TSNAs in such tobacco, since
nitrosamines require relatively high concentrations of nicotine and
other alkaloids to form at detectable levels. Therefore, it can be
advantageous to utilize such a tobacco in reduced-risk cigarettes
or other tobacco products to further reduce TSNAs. In some
embodiments, a low alkaloid (e.g., LA Burley 21 and/or LA
Flue-cured 53) is used in order to manufacture reconstituted
tobacco as a means for manufacturing recon with low TSNA levels.
PAHs are formed from high temperature pyrolysis of amino acids,
sugars, paraffins, terpenes, phytosterols, celluloses and other
components of tobacco. Most of these components are greatly reduced
in tobacco fiber, effectively reducing formation of PAHs. Catechols
and phenols, recognized carcinogenic co-factors in cigarette smoke,
would also be reduced since low levels of soluble sugar are present
in tobacco fiber. Harmful gas phase compounds such as hydrogen
cyanide, nitrogen oxides, and carbon monoxide are also reduced when
a cigarette containing only tobacco fiber is smoked compared to a
cigarette made with whole-leaf tobacco. Hydrogen cyanide is formed
from burning proteins and chlorophyll. Nitrogen oxides are formed
from burning soluble protein, chlorophyll, nitrates, and alkaloids.
These components are not present in significant amounts in
deproteinized tobacco. Tobacco fiber has approximately 85 percent
less starches and cellulosic material thus reducing the major
pyrolytic precursors of carbon monoxide.
[0104] In another embodiment, TSNAs can be virtually eliminated
through processing freshly harvested tobacco using lyophilization.
This is accomplished by processing freshly harvested tobacco
through freeze-drying units located near tobacco farms.
[0105] Tobacco Blending
[0106] It may be desirable to blend tobacco having varying levels
of toxicants so as to create a reduced risk tobacco product with
varying taste and/or toxicant characteristics. This blending
process is typically performed after the curing process, and may be
performed by conventional methods. Preferred tobacco blending
approaches are provided below. A mixture that contains different
types of tobacco is desirably substantially homogeneous throughout
in order to avoid undesirable fluctuations in taste or toxicant
levels. Typically, tobacco to be blended may have a moisture
content from about 10 to about 40%. As an example, the tobacco is
first cut or shredded to a suitable size, then mixed in a mixing
device, such as a rotating drum or a blending box. One such known
mixing device is a tumbling apparatus that typically comprises a
rotating housing enclosing mixing paddles which are attached to
and, therefore, rotate with the housing to stir the tobacco
components together in a tumbling action as the drum turns.
[0107] After the desired tobaccos are thoroughly mixed, the
resulting tobacco blend is removed from the mixing apparatus and
bulked to provide a continuous, generally uniform quantity of the
tobacco blend. The tobacco is then allowed to remain relatively
undisturbed (termed the "bulking step") for the required period of
time before subsequent operations are performed.
[0108] The tobacco blend can be expanded by the application of
steam. After the tobacco blend has been expanded, it is dried. The
dried, expanded tobacco blend is then in a suitable mode to be
processed into a reduced risk product, as described below.
[0109] Some blending approaches begin with tobacco prepared from
one or more tobaccos that have a reduced level of compounds that
cause the induction of biological insult, for example, DSBs, cell
death or perturbation of RNA transcriptome or proteome as compared
to level of compounds that cause the induction of biological insult
induced by a conventional or reference cigarette (e.g., 2R4F) in
human cells contacted by smoke from the tobacco. In some blending
approaches, one or more tobaccos that have extremely low amounts of
nicotine, nornicotine, sterols and/or TSNAs are used (e.g., low
alkaloid tobaccos, such as LA Burley 21 and/or LA Flue 53). In some
embodiments, the reduced-risk tobacco is blended with one or more
conventional tobaccos.
[0110] Typical tobacco blends include a blend of Oriental tobacco,
Burley tobacco and Flue cured tobacco. The tobacco products
provided herein can contain about 0-50%, 5-40%, or 5-25% Oriental
tobacco. The tobacco products provided herein can contain about
0-90%, 5-80%, 10-80%, 10-70%, 10-60%, or 10-50% Burley tobacco
(preferable LA Burley 21). The tobacco products provided herein can
contain about 0-70%, 5-60%, 5-50%, 10-50%, 10-40%, or 10-30%
Flue-cured tobacco (e.g., conventional Flue-cured tobacco or LA
Flue 53). An exemplary tobacco product, e.g., a cigarette, contains
a blend of about 5-25% Oriental tobacco, 10-50% Burley and 10-50%
Flue cured. In particular embodiments provided herein, a blend of
Oriental tobacco, Burley tobacco and Flue cured tobacco is
provided, wherein at least, greater than, less than, equal to, or
any number in between about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or 90%, or all, of the Burley tobacco is a Burley tobacco that
has been grown, harvested, and cured to have a reduced level of
compounds that cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome as compared to the level of compounds that cause
biological insult induced by a conventional or reference cigarette
(e.g., 2R4F), in human cells contacted by smoke from the tobacco
(e.g., LA Burley 21).
[0111] In one embodiment, conventional Flue-cured bright tobacco is
blended with a low alkaloid Burley (e.g., LA Burley 21), and
without conventional Burley, to yield a blended reduced-risk
tobacco. In another embodiment, conventional Flue-cured tobacco and
Oriental tobacco is blended with a low alkaloid Burley (e.g., LA
Burley 21). Optionally, conventional Burley and/or a low alkaloid
Flue tobacco (e.g., LA Flue 53) can be used to yield a blended
reduced-risk tobacco.
[0112] In an embodiment, the tobacco blend comprises Flue-cured
tobacco, Burley tobacco, and Oriental tobacco. In an embodiment,
the Flue-cured tobacco is present in an amount from about 1% to
about 15% by weight of the total blend. In an embodiment, the
Flue-cured tobacco is present in an amount from about 5% to about
10% by weight of the total blend, including, for example, about
6-7% by weight of the total blend. In an embodiment, the Burley
tobacco is present in an amount from about 50% to about 70% by
weight of the total blend. In an embodiment, the Burley tobacco is
present in an amount from about 55% to about 65% by weight of the
total blend, including, for example about 58-60% by weight of the
total blend. In an embodiment, a portion of the Burley tobacco
comprises low-alkaloid Burley tobacco (e.g., LA Burley 21). In an
embodiment, low alkaloid Burley tobacco is present in an amount
from about 5% to about 25% by weight of the total blend. In an
embodiment, low alkaloid Burley tobacco is present in an amount
from about 50% to about 70% by weight of the total blend,
including, for example, about 55-65% by weight of the total blend.
In an embodiment, the Oriental tobacco is present in an amount from
about 5% to about 25% by weight of the total blend. In an
embodiment, the Oriental tobacco is present in an amount from about
10% to about 20% by weight of the total blend, including, for
example about 11-15% by weight of the total blend. In an
embodiment, the tobacco blend further comprises cut rolled expanded
stem. In an embodiment, the cut rolled expanded stem is present in
an amount from about 5% to about 25% by weight of the total blend.
In an embodiment, the cut rolled expanded stem is present in an
amount from about 15% to about 25% by weight of the total blend,
including, for example, about 22% by weight of the total blend. In
an exemplary embodiment, the tobacco blend comprises about 7%
Flue-cured tobacco, about 59% Burley tobacco, about 12% Oriental
tobacco, and about 22% cut rolled expanded stem. In an embodiment,
a portion of the Burley tobacco is low alkaloid Burley tobacco
(e.g., LA Burley 21), which is present in an amount of about 59% by
weight of the total blend.
[0113] It should be appreciated that tobacco products are often a
blend of many different types of tobaccos, which were grown in many
different parts of the world under various growing conditions. As a
result, the amount of nicotine, TSNAs, sterols, PAHs and other
toxicant may differ from crop to crop. Nevertheless, by using
conventional techniques one can easily determine an average amount
of compounds that cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells, and/or an average amount of nicotine,
TSNA, sterol, PAH or any toxicant per crop used to create a desired
blend. It should also be appreciated that reconstituted, expanded,
chemically treated, or microbial treated tobacco can be blended
with the modified tobacco described herein. By adjusting the amount
of each type of tobacco that makes up the blend one of skill can
balance the amount of compounds that cause the induction of
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells, nicotine, TSNA,
sterol and/or PAH with other considerations such as appearance,
flavor, and smokability. In this manner, a variety of types of
tobacco products having varying level of compounds that cause the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells,
nicotine, TSNA, PAH, or other toxicants as well as, appearance,
flavor and smokability can be created.
Filters
[0114] In some embodiments, the cigarette filters used with the
cigarettes described herein comprise a carbon and/or an
ion-exchange resin.
[0115] Activated Carbon in Filters
[0116] Activated carbon in a cigarette filter for selective removal
of vapor phase smoke constituents has been utilized for over almost
four decades. Commercial products have utilized a wide range of
activated carbon types with various properties and significant
effort has been used to identify materials that provide specificity
for removal of toxic constituents while maintaining acceptable
sensory properties during smoking. Selective filtration occurs
through a vapor phase molecule interacting with an active site on
the carbon though a physical adsorption or chemical adsorption
mechanism. During the smoking process this molecule is in
equilibrium with the smoke particle and the gas phase. In addition,
the vapor phase molecule is in equilibrium with the surfaces inside
the cigarette filter. The cellulose acetate filter material has a
weak binding affinity for vapor phase molecules therefore the
equilibrium is shifted toward the gas phase. Carbon, silica gel,
zeolites, resins and other common filter additives, however, have a
stronger affinity for vapor phase molecules and the equilibrium is
shifted toward the molecule residing on the surface of the filter
additive. The vapor phase molecule structure and the surface of the
filter additive have a significant effect on filtration. This
structure and surface affect whether the material is retained
through a physical adsorption process or a chemical process.
[0117] More recent developments have identified that carbons with
higher carbon tetrachloride activity provide increased removal
efficiency for vapor phase constituents including 1,3-butadiene,
isoprene, acetaldehyde, acetone, acrylonitrile, benzene and
butyraldehyde etc. (US 2007/0261706A1 to Banerjea et. al.).
Numerous studies have indicated that carbon loses its efficiency to
remove specific vapor phase components from smoke as the product
ages. This has been attributed to depletion of active sites by
triacetin in the filter. In order to compensate, cigarette designs
have incorporated higher activity carbons and higher levels of
activated carbon in the filter to achieve the same removal
efficiency. Activated beaded carbons have also been proposed (US
2003/0154993A1 to Paine et al.). Beaded activated carbons have been
selected based on their activities and surface area determined
using the BET (Brunauer, Emmet and Teller) method. These beaded
carbons have been manufactured with a well defined micropore and
mesopore distribution of 50 angstrom in diameter and identified as
optimal performance measures.
[0118] The surface structure of carbon has been very well
characterized and consists of an amorphous structure containing a
series of mesopores, micropores and super micropores. These pores
are characterized based on their pore volume and pore diameter.
Surface area as measured by the BET method is a measure of the
amorphous portions of the surface plus the interior volumes
collectively of the series of pore types. Pore volume can be
measured but is generally not utilized with conventional
applications and is not provided by activated carbon manufacturers.
Depending upon the manner in which the carbon is activated, the
pore volume can vary dramatically. Carbons with the same surface
area can have vastly differing pore volumes. Sasaki et al. examined
the effect of pore size and pore volume on adsorption of acetone
and other volatiles in cigarette smoke. (Sasaki, T.; Matsumoto, A.;
Yamashita, Y. "The effects of pore size and volume of activated
carbon on adsorption efficiency of vapor phase compounds in
cigarette smoke" Colloids and Surfaces A 325 (2008) 166-172.) In
general, adsorption tracked surface area. We have discovered a
unique feature not revealed by the prior art, namely, correlation
between pore volume and biological insult. Pore volume has
significantly more effect on reducing the induction of biological
insult on removal than surface area.
[0119] In one embodiment, of the current invention, a carbon is
selected based on pore volume. Higher pore volume carbons having
the same surface area and lower pore volume carbons will allow
increased removal efficiency of vapor phase smoke constituents. The
lowering of vapor phase components in smoke leads to less DNA
damage resulting in reduced biological effects. The usage of this
approach allows cigarette designs to provide higher reductions in
unwanted smoke components by using lower quantities of carbon in
the cigarette filter. The use of lower quantities provides a
cigarette with increased sensory perception and more consumer
acceptability.
[0120] Theoretically, the behavior of the carbon is based on the
equilibrium reaction:
Smoke ParticleVapor PhaseCarbon Surface
[0121] In order to remove the material from smoke and protect the
smoker from exposure to volatile constituents the equilibrium must
be driven toward the right (carbon surface). Selection of a carbon
that retains the vapor phase components and does not allow them to
re-enter the vapor phase provide reduced deliveries of these
constituents in smoke and therefore less biological damage. A
portion of the molecules that are adsorbed onto the outer surface
of the carbon are readily desorbed back into the smoke flow and
delivered to the smoker, shifting the equilibrium to the left.
Pores behave differently. As molecules enter the pore and adsorb to
the walls inside the pore, the probability of desorption and
re-entry into the main flow of smoke is diminished as it is more
likely the molecule contact another location within the pore. As
the pore becomes larger this probability is more likely. During the
smoking process, the pores begin to fill leading to lower level of
vapor phase removal in later puffs. Increase in pore volume allows
better removal efficiency for the later puffs. Pore diameter (as
specified in US 2003/0154993A1 to Paine et al.) becomes more
important for smaller diameter pores due to the potential for
adsorbed molecules to block the opening to the pore thereby
limiting removal in later puffs. In regards to larger diameter
pores, >50 nm as specified in (US 2003/0154993A1 to Paine et
al.) molecules that adsorb closer to the pore entrance have an
increased probability to desorb and return to the mainstream smoke
flow.
[0122] In one embodiment, the pore volume of a carbon is at least,
greater than, less than, equal to, or any number in between about
0.1 mL/g-0.9 mL/g, such as, at least, greater than, less than,
equal to, or any number in between about 0.2 mL/g-0.8 mL/g, 0.3
mL/g-0.7 mL/g, 0.4 mL/g-0.6 mL/g, or about 0.5 mL/g, based on a
nitrogen adsorptive anaylsis. In other embodiments, the pore volume
of a carbon is at least, greater than, less than, equal to, or any
number in between about 0.1 mL/g-0.8 mL/g, 0.1 mL/g-0.7 mL/g, 0.1
mL/g-0.6 mL/g, 0.1 mL/g-0.5 mL/g, 0.1 mL/g-0.4 mL/g, 0.1 mL/g-0.3
mL/g, 0.1 mL/g-0.2 mL/g, 0.2 mL/g-0.9 mL/g, 0.2 mL/g-0.8 mL/g, 0.2
mL/g-0.7 mL/g, 0.2 mL/g-0.6 mL/g, 0.2 mL/g-0.5 mL/g, 0.2 mL/g-0.4
mL/g, 0.2 mL/g-0.3 mL/g, 0.3 mL/g-0.9 mL/g, 0.3 mL/g-0.8 mL/g, 0.1
mL/g-0.7 mL/g, 0.3 mL/g-0.6 mL/g, mL/g, 0.3 mL/g-0.5 mL/g, mL/g,
0.3 mL/g-0.4 mL/g,-0.9 mL/g, 0.4 mL/g,-0.8 mL/g, 0.4 mL/g,-0.7
mL/g, 0.4 mL/g,-0.6 mL/g, 0.4 mL/g,-0.5 mL/g, 0.5 mL/g,-0.9 mL/g,
0.5 mL/g,-0.8 mL/g, 0.5 mL/g,-0.7 mL/g, 0.5 mL/g,-0.6 mL/g, 0.6
mL/g,-0.9 mL/g, 0.6 mL/g,-0.8 mL/g, 0.6 mL/g,-0.7 mL/g, 0.7
mL/g,-0.9 mL/g, 0.7 mL/g,-0.8 mL/g, or 0.8 mL/g,-0.9 mL/g.
[0123] In another embodiment, the total pore volume distribution of
a carbon is at least, greater than, less than, equal to, or any
number in between about 0.1 mL/g-0.9 ml/g such as, at least,
greater than, less than, equal to, or any number in between about
0.2 mL/g-0.8 mL/g, 0.3 ml/g-0.7 mL/g, 0.4 mL/g-0.6 mL/g, wherein
the percentage of a carbon having the total pore volume
distribution is at least 50% in one embodiment, and in other
embodiments the percentage of carbon having the total pore volume
distribution is at least, greater than, less than, equal to, or any
number in between about 50-90%, 50-85%, 50-80%, 50-75%, 50-70%,
50-65%, 50-60%, 50-55%, 55-90%, 55-85%, 55-80%, 55-75%, 55-70%,
55-65%, 55-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%,
65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%,
70-75%, 75-90%, 35-85%, 75-80%, 80-90%, 80-85%, or 85-90%. In other
embodiments, the pore volume distribution of a carbon is at least,
greater than, less than, equal to, or any number in between about
0.1 mL/g-0.8 mL/g, 0.1 mL/g-0.7 mL/g, 0.1 mL/g-0.6 mL/g, 0.1
mL/g-0.5 mL/g, 0.1 mL/g-0.4 mL/g, 0.1 mL/g-0.3 mL/g, 0.1 mL/g-0.2
mL/g, 0.2 mL/g-0.9 mL/g, 0.2 mL/g-0.8 mL/g, 0.2 mL/g-0.7 mL/g, 0.2
mL/g-0.6 mL/g, 0.2 mL/g-0.5 mL/g, 0.2 mL/g-0.4 mL/g, 0.2 mL/g-0.3
mL/g, 0.3 mL/g-0.9 mL/g, 0.3 mL/g-0.8 mL/g, 0.3 mL/g-0.7 mL/g, 0.3
mL/g-0.6 mL/g, mL/g, 0.3 mL/g-0.5 mL/g, mL/g, 0.3 mL/g-0.4 mL/g,
0.4 mL/g-0.9 mL/g, 0.4 mL/g,-0.8 mL/g, 0.4 mL/g,-0.7 mL/g, 0.4
mL/g,-0.6 mL/g, 0.4 mL/g,-0.5 mL/g, 0.5 mL/g,-0.9 mL/g, 0.5
mL/g,-0.8 mL/g, 0.5 mL/g,-0.7 mL/g, 0.5 mL/g,-0.6 mL/g, 0.6
mL/g,-0.9 mL/g, 0.6 mL/g,-0.8 mL/g, 0.6 mL/g,-0.7 mL/g, 0.7
mL/g,-0.9 mL/g, 0.7 mL/g-0.8 mL/g, or 0.8 mL/g,-0.9 mL/g, wherein
the percentage of a carbon having each of the total pore volume
distributions above is at least 50% in one embodiment, and in other
embodiments, the percentage of carbon having each of the total pore
volume distributions above is at least, greater than, less than,
equal to, or any number in between about 50-90%, 50-85%, 50-80%,
50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 55-90%, 55-85%, 55-80%,
55-75%, 55-70%, 55-65%, 55-60%, 60-90%, 60-85%, 60-80%, 60-75%,
60-70%, 60-65%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%,
70-85%, 70-80%, 70-75%, 75-90%, 35-85%, 75-80%, 80-90%, 80-85%, or
85-90%.
[0124] In another embodiment, the pore size measured as the
diameter of the pore is at least, greater than, less than, equal
to, or any number in between about 0.6 nm-1.0 nm, 0.7 nm-0.9 nm or
0.8 nm to 0.9 nm. In another embodiment, a filter comprising carbon
having a combination of the pore size and pore volume is used. For
example, at least, greater than, less than, equal to, or any number
in between about 0.1 mL/g-0.9 mL/g pore volume and 0.5 nm-1.1 nm
pore diameter.
[0125] Activated carbon, such as TA95, which may be used in the
filters described herein may have adsorption capacity for acetone
of at least, greater than, less than, equal to, or any number in
between about 0.3 mL/g-0.9 mL/g, such as 0.4 mL/g-0.8 mL/g, 0.5
mL/g-0.7 mL/g.
[0126] Other materials such as silica gel, zeolites, resins etc.
have slightly different surface structures and less pore structure.
Removal of vapor phase constituents and retention on these
materials relies more heavily on chemical adsorption such as
hydrogen bonding effects or chemical reaction whereas carbon relies
more heavily on London forces and with aromatic molecules,
"p.pi.-p.pi." interactions of graphite like portions of the carbon
surface.
[0127] Forms of Filters
[0128] Filters comprising a carbon, ion-exchange resins, or a
combination thereof can take several forms and the manner in which
the carbon and/or ion-exchange resin is incorporated into the
cigarette filter may vary. In one embodiment, the filter is a
tripartite design. In one tripartite design, the court in its
downstream from the resident in the filter, for example the
cigarette will have a cellulose acetate filter portion, a portion
containing carbon, and a portion containing resin, immediately
before the tobacco. Another tripartite design comprises three
compartments for containing carbon, weak base amine-containing
resin, and sepiolite, or any acceptable adsorbent material capable
of removing volatile and/or semi-volatile constituents from the
vapor phase of cigarette mainstream smoke.
[0129] In one embodiment, it was found that the a mixture of
sepiolite and an ion exchange resin yielded a filter that was
significantly more effective in reducing toxicants causing
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome, than a filter containing the same
amount of the either or sepiolite or resin by weight. Without being
bound by a theory, it is believed that the combination with
sepiolite allows the resin to be more uniformly dispersed
throughout the filter segment as well as assists sepiolite' s
ability to adsorb and subsequently desorb volatile components from
the mainstream cigarette smoke ultimately providing longer
residence time for the volatile components to desorb from the
sepiolite and irreversibly bind to the weak-base primary amine
resin.
[0130] In an embodiment, the cigarette filter comprises a
Dalmatian-type cigarette filter. For example, the filter can
comprise a Dalmatian-type filter, wherein the carbon and/or
ion-exchange resin is distributed throughout the filter in a
uniform manner. In some embodiments, the filter comprises carbon in
a particulate form, wherein the carbon particles are dispersed
throughout the filter uniformly. In some embodiments, the filter
comprises an ion-exchange resin in particulate form, wherein the
ion-exchange resin particles are dispersed throughout the filter
uniformly. In more embodiments, the filter comprises carbon in a
particulate form and ion-exchange resin in a particulate form and
both the carbon particles and the ion-exchange resin particles are
dispersed throughout the filter uniformly.
[0131] The carbon and/or ion-exchange resin can be dispersed within
a material that makes up the filter. The filter can be made from a
variety of materials. For example, the filter may comprise any
filter material known for use in cigarette filters. In some
embodiments, the cigarette filter is manufactured from cellulose
acetate tow, gathered cellulose acetate web, polypropylene tow,
gathered polypropylene web, gathered polyester web, gathered paper,
or combinations thereof.
[0132] In some embodiments, the cigarette filter comprises multiple
sections. A filter section can comprise any portion of the
cigarette filter, for example, the filter section may comprise a
longitudinally extending section within the filter. In some
embodiments, the filter comprises at least a first longitudinally
extending section of filter material positioned at the end of the
filter proximal to the tobacco rod (i.e., tobacco end section) and
at least a second longitudinally extending section of filter
material positioned at the end of the filter element distal to the
tobacco rod (i.e., mouth end section). The number and type of
sections in the filter can vary over a wide range, including one,
two, three, four, five, six, or seven sections, wherein any one
section can be the same or different from any other section.
[0133] One or more sections of a multiple section filter can
comprise a Dalmatian-type section, wherein the carbon and/or
ion-exchange resin is uniformly dispersed within that section.
Additionally, one or more sections of a multiple section filter can
comprise carbon and/or ion-exchange resin in concentrated amounts,
rather than being uniformly dispersed throughout the filter.
[0134] In some embodiments, the cigarette filter comprises one
longitudinally extending section that is a Dalmatian-type section,
wherein the carbon and/or ion-exchange resin is uniformly dispersed
within that section. In some embodiments, the cigarette filter
comprises two longitudinally extending sections that are a
Dalmatian-type section, wherein the carbon and/or ion-exchange
resin is uniformly dispersed within those sections. In some
embodiments, the cigarette filter comprises three longitudinally
extending sections that are a Dalmatian-type section, wherein the
carbon and/or ion-exchange resin is uniformly dispersed within
those sections. In some embodiments, the cigarette filter comprises
four longitudinally extending sections that are a Dalmatian-type
section, wherein the carbon and/or ion-exchange resin is uniformly
dispersed within those sections.
[0135] In some embodiments, the cigarette filter comprises one
longitudinally extending section comprising carbon and/or
ion-exchange resin in a concentrated amount. In some embodiments,
the cigarette filter comprises two longitudinally extending
sections that each comprise carbon and/or ion-exchange resin in a
concentrated amount. In some embodiments, the cigarette filter
comprises three longitudinally extending sections that each
comprise carbon and/or ion-exchange resin in a concentrated amount.
In some embodiments, the cigarette filter comprises four
longitudinally extending sections that each comprise carbon and/or
ion-exchange resin in a concentrated amount.
[0136] FIG. 1 shows an illustrative embodiment of a cigarette
filter 230 comprising three sections 132, 134, 136. The mouth-end
section 132 can comprise any material suitable for manufacturing
cigarette filters, for example, cellulose acetate. In some
embodiments, the mouth-end section comprises cellulose, cellulose
acetate tow, paper, cotton, polypropylene web, polypropylene tow,
polyester web, polyester tow or combinations thereof. In some
embodiments, the mouth-end section includes a plasticizer. In some
embodiments, the mouth-end section comprises carbon, ion-exchange
resin, or a combination thereof. In more embodiments, carbon,
ion-exchange resin, or a combination thereof is absent from the
mouth-end section.
[0137] Cigarette filter sections 134 and 136 can be the same or
different as the mouth-end section 132, and some embodiments
comprise cellulose, cellulose acetate tow, paper, cotton,
polypropylene web, polypropylene tow, polyester web, polyester tow
or combinations thereof. Furthermore, sections 134 and 136 can be
the same or different. In some embodiments, the filter section 134
adjacent the tobacco rod comprises an adsorbent 144. In some
embodiments, the adsorbent 144 is carbon, ion-exchange resin, or a
combination thereof. In some embodiments, the middle filter section
136 comprises an adsorbent 146. In some embodiments, the adsorbent
146 is carbon, ion-exchange resin, or a combination thereof.
[0138] In some embodiments, the cigarette filter comprises two
sections. In some embodiments, the mouth-end section of a
two-section filter comprises cellulose, cellulose acetate tow,
paper, cotton, polypropylene web, polypropylene tow, polyester web,
polyester tow or combinations thereof. In some embodiments, the
mouth-end section of a two-section filter comprises cellulose
acetate. In some embodiments, the tobacco-end section of a
two-section filter comprises cellulose, cellulose acetate tow,
paper, cotton, polypropylene web, polypropylene tow, polyester web,
polyester tow or combinations thereof. In some embodiments, the
tobacco-end section of a two-section filter comprises cellulose
acetate. In some embodiments, the tobacco-end section of a
two-section filter comprises carbon, ion-exchange resin, or a
combination thereof. In some embodiments, the tobacco-end section
of a two-section filter is a Dalmatian-type section and comprises
carbon, ion-exchange resin, or a combination thereof.
[0139] The length of the cigarette filter can vary over a wide
range. In some embodiments, the cigarette filter has a length of at
least, greater than, less than, equal to, or any number in between
about 15 mm to about 65 mm. In some embodiments, the cigarette
filter has a length of at least, greater than, less than, equal to,
or any number in between about 20 mm to about 40 mm. In some
embodiments, the cigarette filter has a length of at least, greater
than, less than, equal to, or any number in between about 20 mm to
about 30 mm. In some embodiments, the cigarette filter has a length
of at least, greater than, less than, equal to, or any number in
between about 25 mm to about 35 mm. In some embodiments, the
cigarette filter has a length of at least, greater than, less than,
equal to, or any number in between about 25 mm. In some embodiment,
the cigarette filter has a length of at least, greater than, less
than, equal to, or any number in between about 30 mm.
[0140] Where the filter comprises multiple sections, the length of
each section can be selected to provide optimal performance for
reducing biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells. The
total length of the combined segments results in a cigarette filter
having a length of about 15 mm to about 65 mm, as discussed above.
In some embodiments, a filter section in a multiple section filter
has a length of at least, greater than, less than, equal to, or any
number in between about 1 mm to about 65 mm. In some embodiments, a
filter section in a multiple section filter has a length of at
least, greater than, less than, equal to, or any number in between
about 1 mm to about 40 mm. In some embodiments, a filter section in
a multiple section filter has a length of at least, greater than,
less than, equal to, or any number in between about 1 mm to about
30 mm. In some embodiments, a filter section in a multiple section
filter has a length of at least, greater than, less than, equal to,
or any number in between about 1 mm to about 25 mm. In some
embodiments, a filter section in a multiple section filter has a
length of at least, greater than, less than, equal to, or any
number in between about 1 mm to about 20 mm. In some embodiments, a
filter section in a multiple section filter has a length of at
least, greater than, less than, equal to, or any number in between
about 1 mm to about 15 mm. In some embodiments, a filter section in
a multiple section filter has a length of about 1 mm to about 10
mm. In some embodiments, a filter section in a multiple section
filter has a length of at least, greater than, less than, equal to,
or any number in between about 1 mm to about 7 mm. In some
embodiments, a filter section in a multiple section filter has a
length of at least, greater than, less than, equal to, or any
number in between about 1 mm to about 5 mm. In some embodiments, a
filter section in a multiple section filter has a length of at
least, greater than, less than, equal to, or any number in between
about 4 mm to about 10 mm. In an embodiment, a filter section in a
multiple section filter has a length of at least, greater than,
less than, equal to, or any number in between about 7 mm to about
15 mm. In some embodiments, a filter section in a multiple section
filter has a length of at least, greater than, less than, equal to,
or any number in between about 12 mm to about 20 mm.
[0141] In some embodiments comprising two sections, the mouth-end
section of the filter has a length of at least, greater than, less
than, equal to, or any number in between about 1 mm to about 12 mm
and the tobacco-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about
10 mm to about 40 mm. In some embodiments comprising two sections,
the mouth-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 4
mm to about 10 mm and the tobacco-end section of the filter has a
length of at least, greater than, less than, equal to, or any
number in between about 12 mm to about 30 mm. In some embodiments
comprising two sections, the mouth-end section of the filter has a
length of at least, greater than, less than, equal to, or any
number in between about 6 mm to about 8 mm and the tobacco-end
section of the filter has a length of at least, greater than, less
than, equal to, or any number in between about 15 mm to about 20
mm. In some embodiments comprising two sections, the mouth-end
section of the filter has a length of about 7 mm and the
tobacco-end section of the filter has a length of about 18 mm. In
some embodiments comprising three sections, the mouth-end section
of the filter has a length of about 1 mm to about 12 mm, the middle
filter section has a length of at least, greater than, less than,
equal to, or any number in between about 1 mm to about 20 mm, and
the tobacco-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 1
mm to about 12 mm. In some embodiments comprising three sections,
the mouth-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 4
mm to about 10 mm, the middle filter section has a length of at
least, greater than, less than, equal to, or any number in between
about 4 mm to about 16 mm, and the tobacco-end section of the
filter has a length of at least, greater than, less than, equal to,
or any number in between about 4 mm to about 10 mm. In some
embodiments comprising three sections, the mouth-end section of the
filter has a length of at least, greater than, less than, equal to,
or any number in between about 8 mm to about 12 mm, the middle
filter section has a length of at least, greater than, less than,
equal to, or any number in between about 10 m to about 14 mm, and
the tobacco-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 8
mm to about 12 mm. In some embodiments comprising three sections,
the mouth-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 9
mm, the middle filter section has a length of at least, greater
than, less than, equal to, or any number in between about 12 mm,
and the tobacco-end section of the filter has a length of at least,
greater than, less than, equal to, or any number in between about 9
mm.
[0142] Any of a variety of multiple section cigarette filters known
in the art can be used in the compositions and methods provided
herein, for example, those multiple section cigarette filters
discussed in U.S. Pat. No. 6,779,529 to Figlar et al. and U.S.
Patent Application Publication No. 2004/0237984 to Figlar, the
contents of which are hereby incorporated by reference in their
entirety. In some embodiments, a cigarette having a multiple
section filter comprises a section containing a general adsorbent,
such as carbon, and a section containing a selective adsorbent,
such as ion-exchange resin is used.
[0143] The first and second longitudinally extending sections can
be the same or different. Additional longitudinally extending
filter sections are also contemplated. In some embodiments, the
filter comprises three or more longitudinally extending filter
sections. In some embodiments, the filter comprises four or more
longitudinally extending filter sections. In some embodiments, the
filter comprises five or more longitudinally extending filter
sections. Each filter section may be the same or different from any
other filter section. For example, a first longitudinally extending
section can be different from a second longitudinally extending
section, but similar to a third longitudinally extending section.
Various combinations of filter sections are contemplated herein. In
some embodiments, an adsorbent material, such as carbon or
ion-exchange resin, is contained within at least a portion of at
least one of the longitudinally extending sections. In some
embodiments, the carbon is present in one, two, three, four, or
five sections of a multiple section filter. In some embodiments,
the ion-exchange resin is present in one, two, three, four, or five
sections of a multiple section filter.
[0144] Any of a variety of cigarette filters comprising cavities
and/or channels known in the art can be used in the compositions
and methods provided herein, for example, those cigarette filters
comprising cavities and/or channels discussed in U.S. Pat. No.
7,240,678 to Crooks et al. and U.S. Pat. No. 7,237,558 to Clark et
al., the contents of which are hereby incorporated by reference in
their entirety.
[0145] Any section of a multiple section filter may comprise a
cavity. Filter cavities may be filled with carbon and/or
ion-exchange resin and allow larger amounts of the carbon and/or
ion-exchange resin to be present in the section that comprises the
cavity. In some embodiments, a multiple section filter comprises
one, two, three, four, or five sections that comprise a cavity. In
some embodiments, a filter section that comprises a cavity further
comprises carbon, ion-exchange resin, or a combination thereof.
Multiple section filters comprising at least one cavity are well
known in the art, and are sometimes referred to as a "compartment
filter" or a "plug/space/plug" filter.
[0146] A filter section comprising a cavity can have a variety of
lengths. In some embodiments, the length of the cavity is at least,
greater than, less than, equal to, or any number in between about 1
mm to about 20 mm. In some embodiments, the length of the cavity is
from at least, greater than, less than, equal to, or any number in
between about 3 mm to about 12 mm. In some embodiments, the length
of the cavity is at least, greater than, less than, equal to, or
any number in between about 5 mm to about 10 mm. In some
embodiments, the length of the cavity is at least, greater than,
less than, equal to, or any number in between about 5 mm. In an
embodiment, the length of the cavity is at least, greater than,
less than, equal to, or any number in between about 7 mm. In an
embodiment, the length of the cavity is at least, greater than,
less than, equal to, or any number in between about 9 mm. In some
embodiments, the length of the cavity is at least, greater than,
less than, equal to, or any number in between about 10 mm. In some
embodiments, the length of the cavity is at least, greater than,
less than, equal to, or any number in between about 12 mm.
[0147] The carbon and/or ion-exchange resin can be provided in
varying sectional amounts within the various sections of the
filter. For example any longitudinal section can comprise more or
less of an amount of carbon and/or ion-exchange resin than any
other filter section of the multiple-section filter. In some
embodiments, the filter element comprises a first section of filter
material, such as a fibrous filter material (e.g., plasticize
cellulose acetate tow) and a second section of filter material
spaced apart from the first section of filter material. The first
section of filter material is positioned at the mouth end of the
filter element and the second section of filter material is
positioned proximal to the tobacco rod. The space between the first
section of filter material and the second section of filter
material is a third section and defines a cavity. At least a
portion of the cavity contains an adsorbent material, such as
carbon, ion-exchange resin, or a combination thereof, either in
beaded granular form. Typically, substantially the entire
compartment contains an adsorbent, such as carbon, ion-exchange
resin, or a combination thereof.
[0148] In some embodiments, the cavity comprises carbon and/or
ion-exchange resin an amount from at least, greater than, less
than, equal to, or any number in between about 20 mg to about 300
mg. In some embodiments, the cavity comprises carbon and/or
ion-exchange resin an amount at least, greater than, less than,
equal to, or any number in between about 40 mg to about 250 mg. In
some embodiments, the cavity comprises carbon and/or ion-exchange
resin an amount at least, greater than, less than, equal to, or any
number in between about 60 mg to about 200 mg. In some embodiments,
the cavity comprises carbon and/or ion-exchange resin an amount at
least, greater than, less than, equal to, or any number in between
about 80 mg to about 180 mg. In some embodiments, the cavity
comprises carbon and/or ion-exchange resin an amount at least,
greater than, less than, equal to, or any number in between about
90 mg to about 160 mg. In some embodiments, the cavity comprises
carbon and/or ion-exchange resin an amount at least, greater than,
less than, equal to, or any number in between about 95 mg to about
140 mg. In some embodiments, the cavity comprises carbon and/or
ion-exchange resin an amount at least, greater than, less than,
equal to, or any number in between about 110 mg to about 130 mg. In
some embodiments, the cavity comprises carbon and/or ion-exchange
resin an amount at least, greater than, less than, equal to, or any
number in between about 95 mg. In some embodiments, the cavity
comprises carbon and/or ion-exchange resin in an amount at least,
greater than, less than, equal to, or any number in between about
124 mg. In some embodiments, the cavity comprises carbon and/or
ion-exchange resin in an amount of at least, greater than, less
than, equal to, or any number in between about 137 mg. In some
embodiments, the cavity comprises carbon and/or ion-exchange resin
in an amount of at least, greater than, less than, equal to, or any
number in between about 144 mg. In some embodiments, the cavity
comprises carbon and/or ion-exchange resin in an amount at least,
greater than, less than, equal to, or any number in between about
179 mg.
[0149] In a multiple section filter comprising a cavity, wherein
the cavity comprises carbon and/or ion-exchange resin, other filter
sections besides the cavity section my also comprise an amount of
carbon and/or ion-exchange resin. In some embodiments, the
non-cavity section comprises carbon and/or ion-exchange resin in an
amount at least, greater than, less than, equal to, or any number
in between about 20 mg to about 200 mg. In some embodiments, the
non-cavity section comprises carbon and/or ion-exchange resin in an
amount at least, greater than, less than, equal to, or any number
in between about 40 mg to about 100 mg. In some embodiments, the
non-cavity section comprises carbon and/or ion-exchange resin in an
amount at least, greater than, less than, equal to, or any number
in between about 50 mg to about 80 mg. In some embodiments, the
non-cavity section comprises carbon and/or ion-exchange resin in an
amount at least, greater than, less than, equal to, or any number
in between about 40 mg. In some embodiments, the non-cavity section
comprises carbon and/or ion-exchange resin in an amount at least,
greater than, less than, equal to, or any number in between about
50 mg. In some embodiments, the non-cavity section comprises carbon
and/or ion-exchange resin in an amount at least, greater than, less
than, equal to, or any number in between about 68 mg.
[0150] In some embodiments, the cigarette filter comprises a
mouth-end section, a cavity, and a tobacco-end section. In some
embodiments, the mouth-end section comprises cellulose acetate. In
some embodiments, the cavity section comprises carbon, ion-exchange
resin, or a combination thereof. In some embodiments, the
tobacco-end section is a Dalmatian-type section that comprises
carbon, ion-exchange resin, or a combination thereof.
[0151] In some embodiments, the cigarette filter comprises channels
within the filter. Filter channels can extend radially or
longitudinally within the cigarette filter. Channels can also be
present in filters that comprise a single section and filters that
comprise multiple sections. In multiple section filters, a channel
can extend throughout a single section of the filter, throughout
multiple sections (e.g. two, three, four, five, etc.) of the
filter, or throughout all of the sections of the filter.
[0152] In some embodiments, at least one channel extends through
the tobacco end section of filter material, the channel being
adapted for passage of mainstream smoke between the tobacco rod and
the compartment containing the adsorbent material. In some
embodiments, a single channel extends through the tobacco end
section of filter material or a plurality of channels can be
utilized. In one embodiment, a single channel proximal to the
central axis of the tobacco end section of filter material is used.
In other embodiments, a plurality of channels extend through the
filter material, either spaced along the periphery of the filter
material or grouped in the area proximal to the central axis of the
tobacco end section of filter material. The total cross-sectional
area of the one or more channels extending through the first
section of filter material may be at least, greater than, less
than, equal to, or any number in between about 0.1 to about 50
mm.sup.2, preferably about 0.5 to about 15 mm.sup.2.
[0153] The cross-sectional shape of the channels is not critical to
the invention and may be, for example, rectangular or circular. The
diameter of each channel or tube can vary. Typically, the diameter
of each channel or tube is at least, greater than, less than, equal
to, or any number in between about 0.5 to about 8 mm, frequently
about 1 to about 3 mm. The diameter of the channel or tube is
selected so as to prevent migration of the adsorbent into the
channel or tube (i.e., the diameter of the channel or tube is
smaller than the diameter of the adsorbent particles).
[0154] In some embodiments, the filter element is attached to the
tobacco rod by a tipping material, which circumscribes both the
entire length of the filter element and an adjacent region of the
tobacco rod. The inner surface of the tipping material can be
fixedly secured to the outer surface of the plug wrap and the outer
surface of the wrapping material of the tobacco rod using a
suitable adhesive. In some embodiments, a ventilated or air diluted
smoking article is provided a series of perforations for air
dilution. In some embodiments, each of the perforations extend
through the tipping material and the plug wrap. In some
embodiments, the filter element is ventilated to provide a
cigarette having an air dilution from at least, greater than, less
than, equal to, or any number in between about 0 to about 75
percent. In some embodiments, the filter element is ventilated to
provide a cigarette having an air dilution from at least, greater
than, less than, equal to, or any number in between about 15 to
about 65 percent. In some embodiments, the filter element is
ventilated to provide a cigarette having an air dilution at least,
greater than, less than, equal to, or any number in between about
25 to about 40 percent. "Air dilution" is the ratio (expressed as a
percentage) of the volume of air drawn through the perforations to
the total volume of air and smoke drawn through the cigarette and
exiting the extreme mouth end portion of the cigarette. The
perforations can be made by various techniques known to those of
ordinary skill in the art. For example, the perforations can be
made using mechanical or microlaser offline techniques or using
online laser perforation. In some embodiments, the tipping paper
circumscribes the entire filter element and at least, greater than,
less than, equal to, or any number in between about 4 mm of the
length of the tobacco rod in the region adjacent to the filter
element.
[0155] Cigarette filter elements that incorporate adsorbents, such
as carbon, ion-exchange resin, or combinations thereof, have a
propensity to remove certain gas phase components from the
mainstream smoke that passes through the filter element during draw
by the smoker. The interaction of mainstream smoke with adsorbent
substances results in a certain degree of removal of certain gas
phase compounds from the smoke. Adsorbents within a cigarette
filter are capable of removing a multitude of compounds, including
carbonyl compound, including, but not limited to, acetone,
formaldehyde, acrolein and acetaldehyde. In some embodiments, the
adsorbents are carbon, ion-exchange resin, or a combination
thereof. Examples of adsorbents useful herein include activated
charcoal, activated coconut carbon, activated coal-based carbon,
zeolite, silica gel, meerschaum, aluminum oxide, or combinations
thereof.
[0156] In embodiments in which the filter comprises carbon, the
amount and type of carbon in the filter can vary over a wide range.
The amount of carbon within the filter typically ranges from at
least, greater than, less than, equal to, or any number in between
about 5 to about 240 mg, and a person having ordinary skill in the
art would understand that any range within this broader range is
contemplated by the inventors. In some embodiments, the carbon
within the filter ranges from at least, greater than, less than,
equal to, or any number in between about 20 to about 120 mg. In
some embodiments, the carbon within the filter ranges from at
least, greater than, less than, equal to, or any number in between
about 40 to about 108 mg. In some embodiments, the carbon within
the filter comprises at least, greater than, less than, equal to,
or any number in between about 40, about 60, about 80, or about 108
mg.
[0157] Narrower ranges of smaller amounts of carbon are also
contemplated. In some embodiments, the carbon within the filter
ranges from at least, greater than, less than, equal to, or any
number in between about 35 to about 45 mg. In some embodiments, the
carbon within the filter ranges from at least, greater than, less
than, equal to, or any number in between about 55 to about 65 mg.
In some embodiments, the carbon within the filter ranges from at
least, greater than, less than, equal to, or any number in between
about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 mg. In some
embodiments, the carbon within the filter ranges from at least,
greater than, less than, equal to, or any number in between about
75 to about 85 mg. In some embodiments, the carbon within the
filter ranges from at least, greater than, less than, equal to, or
any number in between about 100 to about 115 mg. In some
embodiments, the carbon within the filter ranges from at least,
greater than, less than, equal to, or any number in between about
110 to about 130 mg. In some embodiments, the carbon within the
filter ranges from at least, greater than, less than, equal to, or
any number in between about 30 to about 70 mg. In some embodiments,
the carbon within the filter ranges from at least, greater than,
less than, equal to, or any number in between about 20 to about 50
mg. For example, the carbon within the filter can be about 20
mg.
[0158] Narrower ranges of larger amounts of carbon are also
contemplated. In some embodiments, the carbon within the filter
ranges from at least, greater than, less than, equal to, or any
number in between about 150 to about 250 mg. In some embodiments,
the carbon within the filter ranges from at least, greater than,
less than, equal to, or any number in between about 100 to about
200 mg. In some embodiments, the carbon within the filter ranges
from at least, greater than, less than, equal to, or any number in
between about 200 to about 250 mg. In some embodiments, the carbon
within the filter ranges from at least, greater than, less than,
equal to, or any number in between about 100 to about 150 mg. For
example, the carbon within the filter can be about 120 mg.
[0159] The carbon incorporated into the filter of the cigarette can
vary among a number of types and sizes. In some embodiments, the
carbon is highly activated. In some embodiments, carbon is provided
by carbonizing or pyrolyzing bituminous coal, tobacco material,
softwood pulp, hardwood pulp, coconut shells, almond shells, grape
seeds, walnut shells, macadamia shells, kapok fibers, cotton
fibers, cotton linters, or any other material that is known to be
suitable for the production of carbon particles useful in cigarette
filters. Further examples of suitable carbonaceous materials
include activated coconut hull based carbons available from Calgon
Corp. as PCB 12.times.30 and GRC-11 12.times.30. Further examples
of suitable carbonaceous materials are coal based carbons available
from Calgon Corp. as S-Sorb 12% Cu 12.times.30; BPL 12.times.30;
CRC-11F12.times.30; FCA 12.times.30, Cu, CrO.sub.3; and SGL.
Further examples of suitable carbonaceous materials are wood based
carbons available from Westvaco as WV-B, SA-20 and BSA-20. Other
carbonaceous materials are available from Calgon Corp. as HMC;
ASC/GR-1 12.times.30 Cu, Ag, CrO.sub.3; and SC II; and another
carbonaceous material includes Witco Carbon No. 637. Other
carbonaceous materials are described in U.S. patent application
Ser. No. 569,325, filed Aug. 17, 1990; U.S. Pat. No. 4,771,795 to
White, et al. and U.S. Pat. No. 5,027,837 to Clearman, et al.; and
European Patent Application Nos. 236,922; 419,733 and 419,981, the
contents of which are hereby incorporated by reference in their
entireties.
[0160] In some embodiments, the carbon is granular, particulate,
nanoparticulate, spherical, fibrous, or impregnated on paper. The
terms "granular" and "particulate" are intended to encompass both
non-spherical shaped particles and spherical particles, such as
so-called "beaded carbon" as described in WO 03/059096 A1, which is
hereby incorporated by reference in its entirety.
[0161] The size of the individual carbon particles or granules can
vary, depending upon the design of the filter element. Typically,
large size particles have a U.S. mesh size of about 6.times.16;
medium size particles have a U.S. mesh size of about 12.times.30;
and small size particles have U.S. mesh sizes of about 20.times.50
and 30.times.70. Carbonaceous materials also can have a monolithic
form, a bonded granular form, a fibrous form, or an agglomerated
form; or be combined with molecular sieves, alumina particles or
ion exchange resin particles. In some embodiments, the size of the
carbon particles is from about 4.times.6 mesh to about
100.times.300 mesh. In some embodiments, the size of the carbon
particles is from at least, greater than, less than, equal to, or
any number in between about 4.times.8 mesh to about 50.times.200
mesh. In some embodiments, the size of the carbon particles is from
at least, greater than, less than, equal to, or any number in
between about 6.times.12 mesh to about 40.times.100 mesh. In some
embodiments, the size of the carbon particles is from at least,
greater than, less than, equal to, or any number in between about
8.times.16 mesh to about 30.times.70 mesh. In some embodiments, the
size of the carbon particles is from at least, greater than, less
than, equal to, or any number in between about 4.times.6 mesh to
about 12.times.40 mesh. In some embodiments, the size of the carbon
particles is from at least, greater than, less than, equal to, or
any number in between about 12.times.40 mesh to about 40.times.100
mesh.
[0162] The amount of carbon present within the filter can also vary
depending on the filter length, and is usually measured in mg of
carbon per mm length of the filter. The amount of carbon used per
length of the filter, or sections of the filter, depends on the
desired taste characteristics, amount of desired reduction of
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells and the configuration
(e.g., Dalmatian, multiple sections, channel, cavity) of the
filter. In some embodiments, the filter or a section of a multiple
section filter comprises carbon in an amount ranging from at least,
greater than, less than, equal to, or any number in between about 1
mg/mm to about 40 mg/mm. In some embodiments, the filter or a
section of a multiple section filter comprises carbon in an amount
ranging from at least, greater than, less than, equal to, or any
number in between about 3 mg/mm to about 8 mg/mm. In some
embodiments, the filter or a section of a multiple section filter
comprises carbon in an amount ranging from at least, greater than,
less than, equal to, or any number in between about 5 mg/mm to
about 10 mg/mm. In some embodiments, the filter or a section of a
multiple section filter comprises carbon in an amount ranging from
at least, greater than, less than, equal to, or any number in
between about 15 mg/mm to about 25 mg/mm. In some embodiments, the
filter or a section of a multiple section filter comprises carbon
in an amount ranging from at least, greater than, less than, equal
to, or any number in between about 18 mg/mm to about 22 mg/mm. In
some embodiments, the filter or a section of a multiple section
filter comprises carbon in an amount ranging from at least, greater
than, less than, equal to, or any number in between about 6 mg/mm
to about 7 mg/mm. In some embodiments, the filter or a section of a
multiple section filter comprises carbon in an amount of at least,
greater than, less than, equal to, or any number in between about
20 mg/mm. In some embodiments, the filter or a section of a
multiple section filter comprises carbon in an amount ranging from
at least, greater than, less than, equal to, or any number in
between about 3 mg/mm to about 40 mg/mm.
[0163] Beaded carbon differs from granulated carbon because
granular carbon has a surface shape that is irregular from granule
to granule, whereas beaded carbon has consistent spherical form
from bead to bead. Maintenance of a uniform bead size at or about a
pre-selected diameter promotes smooth flow and consistent packing
of the beads during the manufacturing process. Any of a variety of
beaded carbon suitable for cigarette filters known in the art can
be used in the compositions and methods provided herein, for
example, those beaded carbon materials discussed in U.S. Patent
Application Publication Nos. 2006/0180164 to Paine III et al. and
2006/0201524 to Zhang et al., the contents of which are hereby
incorporated by reference in their entireties.
[0164] In some embodiments, the carbon can be surface-modified to
increase its mechanical strength. Any of a variety of
surface-modified carbon known in the art can be used in the
compositions and methods provided herein, for example, the surface
modified carbon discussed in U.S. Patent Application Publication
No. 2006/0144410 to Luan et al., the contents of which are hereby
incorporated by reference in its entirety.
[0165] The carbon can be incorporated into the filter in a
non-uniform manner. In some embodiments, granulated adsorbent, such
as carbon, can be placed in a cavity within the filter element.
However, the adsorbent could also be imbedded or dispersed within a
section of filter material, such as a fibrous filter material
(e.g., cellulose acetate tow), or incorporated into a paper, such
as the carbon-containing gathered paper described in U.S. Pat. No.
5,360,023 to Blakley et al., the contents of which are hereby
incorporated by reference in their entireties. In addition, an
adsorbent material can be placed both in a cavity and imbedded in
one or more of the sections of filter material, and the adsorbent
material in the compartment and the adsorbent imbedded or dispersed
in the filter material can be the same or different.
[0166] Certain carbonaceous materials can be impregnated with
substances, such as transition metals (e.g., silver, gold, copper,
platinum, palladium), potassium bicarbonate, tobacco extracts,
polyethyleneimine, manganese dioxide, eugenol, and 4-ketononanoic
acid. The carbon composition may also include one or more fillers,
such as semolina. Grape seed extracts may also be incorporated into
the filter element as a free radical scavenger.
[0167] In some embodiments, the carbon is coated and/or impregnated
with a coating material. In some embodiments, the coating material
is a metal. In some embodiments, the coating material is copper.
Any of a variety of coated and/or impregnated carbon known in the
art can be used in the compositions and methods provided herein,
for example, the coated and/or impregnated carbon discussed in U.S.
Pat. No. 4,636,333 to Matkin and U.S. Pat. No. 5,657,772 to Duke et
al., the contents of which are hereby incorporated by reference in
their entireties. One advantage of impregnating or coating a metal,
such as copper, onto the carbon is that a significant reduction of
negative biological materials in the cigarette smoke occurs.
[0168] Ion Exchange Resins
[0169] In addition to coating and/or impregnating the carbon
particles, it is also contemplated that the ion-exchange resin,
such as a weak base amine-containing resin, can also, or
alternatively, be coated or impregnated with a coating material. In
some embodiments, the ion-exchange resin is coated or impregnated
with a metal, such as copper. In some embodiments, both carbon and
ion-exchange resin are present in the filter, and neither, one, or
both are coated or impregnated with a coating material, such as a
metal material, including for example, copper.
[0170] Ion-exchange resin, such as resin functionalized with
primary amines, are nucleophiles capable of selectively removing
reactive electrophiles from the vapor phase of cigarette smoke. The
ion-exchange resin can comprise any polymer having active groups in
the form of electrically charged sites capable of displacement upon
interaction with ions of opposite charge. Typically, the
ion-exchange resin comprises a polymer backbone, such as styrene,
styrene-divinylbenzene copolymers, acrylates, methacrylates, phenol
formaldehyde condensates, and epichlorohydrin amine condensates,
and a plurality of electrically charged functional groups attached
to the polymer backbone. The ion-exchange resin is preferably a
weak base anion exchange resin or a strong base anion exchange
resin. Exemplary resins include DIAION.TM. ion-exchange resins
available from Mitsubishi Chemical Corp. (e.g., WA30 and DCA11) and
DUOLITE.TM. ion-exchange resins available from Rohm and Haas (e.g.,
DUOLITE.TM. A7). Other ion-exchange resins with primary amine
groups include PUROLITE.TM. A-143 and PUROLITE.TM. A-109, which is
a primary amine functionalized polystyrene crosslinked with
divinylbenzene in the form of macroporous spherical beads.
[0171] Generally, increasing the amount of the primary amines in a
resin decreases the biological insult, for example, DSBs, cell
death or perturbation of RNA transcriptome or proteome. However,
increasing the amount of primary means of the resin generally
decreases the sensory perception, that is, the taste is impaired.
Thus, although a resin such as A109 containing a high amount of
primary amines performs well in removing toxicants, the sensory
perception is not as great as resin containing mixtures of
amines.
[0172] An ion exchange resin, such as a weak base amine-containing
resin, may contain at least, greater than, less than, equal to, or
any number in between about 1.3%-1.5% wt/wt, such as 1.4% wt/wt of
nitrogen atoms (N) in the form of amine functional groups. In one
embodiment, the ion exchange at resin may contain at least, greater
than, less than, equal to, or any number in between about 0.5%,
0.75%, 1.0%, 1.5%, 2.0% or more wt/wt of nitrogen atoms (N) in the
form of amine functional groups.
[0173] A filter may contain at least, greater than, less than,
equal to, or any number in between about 0 mg-100 mg such as 10 mg,
20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or more of
an ion exchange resin.
[0174] The form of the ion-exchange resin can vary. In some
embodiments, the ion-exchange resin is in solid particulate form.
In some embodiments, the size of the ion-exchange resin particles
is from at least, greater than, less than, equal to, or any number
in between about 4.times.6 mesh to about 100.times.300 mesh. In
some embodiments, the size of the ion-exchange resin particles is
from at least, greater than, less than, equal to, or any number in
between about 4.times.8 mesh to about 50.times.200 mesh. In some
embodiments, the size of the ion-exchange resin particles is from
at least, greater than, less than, equal to, or any number in
between about 6.times.12 mesh to about 40.times.100 mesh. In some
embodiments, the size of the ion-exchange resin particles is from
at least, greater than, less than, equal to, or any number in
between about 8.times.16 mesh to about 30.times.70 mesh. In some
embodiments, the size of the ion-exchange resin particles is from
at least, greater than, less than, equal to, or any number in
between about 4.times.6 mesh to about 12.times.40 mesh. In some
embodiments, the size of the ion-exchange resin particles is from
at least, greater than, less than, equal to, or any number in
between about 12.times.40 mesh to about 40.times.100 mesh.
[0175] The ion-exchange resin can be selected taking into
consideration that the contact conditions between the tobacco smoke
and the adsorbent are dependent on a number of variables, including
how strongly the smoker pulls the smoke through the filter as the
cigarette is being smoked and how much of the tobacco rod has been
consumed prior to each puff. Thus, it is advantageous that the
ion-exchange resin have a surface area greater than or equal to
about 35 m.sup.2/g so that there is minimal diffusional resistance
and the surface area functional sites are easily accessible.
Materials with greater surface areas also demonstrate less
noticeable performance decline if part of the surface is covered
with a plasticizer.
[0176] The amount of ion-exchange resin in the filter can vary over
a wide range. The amount of ion-exchange resin within the filter
typically ranges from at least, greater than, less than, equal to,
or any number in between about 10 to about 250 mg, and a person
having ordinary skill in the art would understand that any range
within this broader range is contemplated. In some embodiments, the
ion-exchange resin within the filter ranges from at least, greater
than, less than, equal to, or any number in between about 20 to
about 200 mg. In some embodiments, the ion-exchange resin within
the filter ranges from at least, greater than, less than, equal to,
or any number in between about 40 to about 150 mg. In some
embodiments, the ion-exchange resin within the filter ranges from
at least, greater than, less than, equal to, or any number in
between about 60 to about 100 mg.
[0177] Narrower ranges of smaller amounts of ion-exchange resin are
also contemplated. In some embodiments, the ion-exchange resin
within the filter ranges from at least, greater than, less than,
equal to, or any number in between about 10 to about 50 mg. In some
embodiments, the ion-exchange resin within the filter ranges from
at least, greater than, less than, equal to, or any number in
between about 20 to about 40 mg. In some embodiments, the
ion-exchange resin within the filter ranges from at least, greater
than, less than, equal to, or any number in between about 35 to
about 45 mg. In some embodiments, the ion-exchange resin within the
filter is at least about 40 mg.
[0178] Narrower ranges of larger amounts of ion-exchange resin are
also contemplated. In some embodiments, the ion-exchange resin
within the filter ranges from at least, greater than, less than,
equal to, or any number in between about 150 to about 250 mg. In
some embodiments, the ion-exchange resin within the filter ranges
from at least, greater than, less than, equal to, or any number in
between about 100 to about 200 mg. In some embodiments, the
ion-exchange resin within the filter ranges from at least, greater
than, less than, equal to, or any number in between about 150 to
about 200 mg.
[0179] The amount of ion-exchange resin present within the filter
can also vary depending on the filter length, and is usually
measured in mg of ion-exchange resin per mm length of the filter.
The amount of ion-exchange resin used per length of the filter, or
sections of the filter, depends on the desired taste
characteristics, the amount of reduction biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in the human cells and the configuration (e.g., Dalmatian,
multiple sections, channel, cavity) of the filter. In some
embodiments, the filter or a section of a multiple section filter
comprises ion-exchange resin in an amount ranging from at least,
greater than, less than, equal to, or any number in between about 3
mg/mm to about 40 mg/mm. In some embodiments, the filter or a
section of a multiple section filter comprises ion-exchange resin
in an amount ranging from at least, greater than, less than, equal
to, or any number in between about 3 mg/mm to about 8 mg/mm. In
some embodiments, the filter or a section of a multiple section
filter comprises ion-exchange resin in an amount ranging from at
least, greater than, less than, equal to, or any number in between
about 5 mg/mm to about 10 mg/mm. In some embodiments, the filter or
a section of a multiple section filter comprises ion-exchange resin
in an amount ranging from at least, greater than, less than, equal
to, or any number in between about 15 mg/mm to about 25 mg/mm. In
some embodiments, the filter or a section of a multiple section
filter comprises ion-exchange resin in an amount ranging from at
least, greater than, less than, equal to, or any number in between
about 18 mg/mm to about 22 mg/mm. In some embodiments, the filter
or a section of a multiple section filter comprises ion-exchange
resin in an amount ranging from at least, greater than, less than,
equal to, or any number in between about 6 mg/mm to about 7 mg/mm.
In some embodiments, the filter or a section of a multiple section
filter comprises ion-exchange resin in an amount of about 20 mg/mm.
In some embodiments, the filter or a section of a multiple section
filter comprises ion-exchange resin in an amount ranging from at
least, greater than, less than, equal to, or any number in between
about 3 mg/mm to about 40 mg/mm.
[0180] The filter can also further comprise other adsorbent
materials in addition to the carbon and/or ion-exchange resin. In
some embodiments, the filter further comprises molecular sieve,
clay, alumina, silica gel, and/or modified silica gel. The particle
size of these additional adsorbents can vary over a wide range,
including the mesh ranges referenced above to the carbon particles,
and particularly from at least, greater than, less than, equal to,
or any number in between about 8.times.16 mesh to about 3.times.70
mesh.
[0181] Additional polymeric or plastic materials are also
contemplated for inclusion into the filter. In some embodiments,
the filter further comprises polymeric resin, cellulose acetate tow
plasticized using triacetin, polyethylene glycol, triethyleneglycol
diacetate, and/or other plasticizer. Where the filter material
comprises a plasticizer, such as triacetin or carbowax, the amount
of plasticizer compared to the other filter material can be up to
about a 1:1 ratio by weight. In some embodiments, the total amount
of plasticizer is at least, greater than, less than, equal to, or
any number in between about 4 to about 20 percent by weight of the
filter. In some embodiments, the total amount of plasticizer is at
least, greater than, less than, equal to, or any number in between
about 6 to about 12 percent by weight of the filter.
[0182] Further adaptations can be made to the filters described
herein. In some embodiments, the filter further comprises strands
of tobacco. In some embodiments, the strands of tobacco comprise
reconstituted tobacco. In some embodiments, the cigarette filter
comprises micro-cavity fibers. Any of a variety of cigarette
filters comprising micro-cavity fibers known in the art can be used
in the compositions and methods provided herein, for example, those
cigarette filters comprising micro-cavity fibers discussed in U.S.
Pat. No. 6,584,979 to Xue et al. and U.S. Pat. No. 6,907,885 to Xue
et al., the contents of which are hereby incorporated by reference.
Such a micro-cavity fiber may be impregnated with an adsorbent
material, such as carbon and/or ion-exchange resin, such that the
adsorbent material is inserted in the micro-cavity spaces of the
fiber. The fibers contain open or semi-open micro cavities that
include, but are not limited to, multilobal shaped fibers. One
non-limiting example of such a fiber is Honeywell's TRIAD.TM. fiber
having an internal void fractional volume from about 0.5 to about
0.6. These fibers are capable of mechanically or electrostatically
entrapping fine particles inside the fiber micro-cavity channels.
Multilobal shaped fibers containing end caps would be considered
fibers with semi-open cavities. Multilobal fibers without the end
caps could be considered fibers with open cavities. The fibers may
be constructed from any material suitable for cigarette use, such
as polyethylene, polypropylene, and polyesters. Other micro-cavity
fibers having the same performance characteristics may be used in
the practice of the present invention.
Filters and Reduced Risk Tobacco Can Be Combined to Create a
Reduced-Risk Tobacco Product
[0183] Aspects of the invention concern cigarettes that are
configured to biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome as compared to the
amount of biological insult induced by a conventional or reference
cigarette (e.g., 2R4F), in human cells, which contain the modified
tobaccos and filters discussed herein. In some embodiments, the
reduced risk tobacco product contains a cut filler composition that
comprises a cured tobacco blend having a tobacco cured to have a
reduced level of compounds that cause the induction of biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome in human cells contacted by smoke from
said tobacco, relative to conventional tobacco. For example, the
cut filler composition can comprise a cured tobacco blend having a
Burley tobacco (e.g., LA Burley 21) cured (e.g., air cured) to have
a reduced level of compounds that cause the induction of biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome in human cells contacted by smoke from
said Burley tobacco, relative to a conventional Burley tobacco, or
a Flue-cured Burley tobacco. In some embodiments, the reduced risk
tobacco product contains a cigarette wrapper with reduced ignition
propensity that circumscribes said cut filler composition. In some
embodiments, the reduced risk tobacco product contains a cigarette
filter that comprises carbon, an ion-exchange resin, or both. The
reduced-risk tobacco products designed in accordance with the
teachings provided herein possess unexpected advantages over known
tobacco products because the reduced-risk tobacco products provided
herein are configured to reduce the induction of biological insult,
for example, DSBs, cell death or perturbation of RNA transcriptome
or proteome, as compared to conventional tobacco products in human
cells contacted by cigarette smoke. That is, the cut filler
composition and the filter design selected to be incorporated into
the reduced-risk tobacco products provided herein are selected
according to their relative propensity to reduce the biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome in human cells contacted by cigarette
smoke. In some embodiments, the benefits of the cut filler
composition and the filter design selected to be incorporated into
the reduced-risk tobacco products provided herein synergistically
combine to provide even greater reduction biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells contacted by cigarette smoke.
[0184] The selection of the cut filler composition and the filter
are described elsewhere herein, and the combination thereof can be
analyzed for their relative propensity to reduce the induction of
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells contacted by cigarette
smoke according to the DNA DSB assays, clonogenic assays, and RNA
transcriptome or proteome assays provided herein and otherwise
known in the art.
[0185] The cigarettes provided herein also comprise wrappers, for
example, cigarette paper. Cigarette wrappers are generally made of
low-flash, self-extinguishing cigarette paper ranging from 10
Coresta units to 200 Coresta units, depending on the desired "tar"
delivery and filter ventilation. Coresta units are a measure of
paper permeability, which is modified either through paper porosity
or the addition of electrostatic or mechanical holes, to reduce
tobacco burnt during each puff and to alter burn rate and puff
count. In an embodiment, the cigarette wrapper comprises cigarette
paper ranging from about 10 to about 200 Coresta units. In an
embodiment, the cigarette wrapper comprises cigarette paper ranging
from about 26 to about 170 Coresta units. In an embodiment, the
cigarette wrapper comprises cigarette paper ranging from about 50
to about 150 Coresta units. In an embodiment, the cigarette wrapper
comprises cigarette paper ranging from about 80 to about 110
Coresta units. In an embodiment, the cigarette wrapper comprises
cigarette paper having about 80 Coresta units. In an embodiment,
the cigarette wrapper comprises cigarette paper having about 110
Coresta units.
[0186] The cigarette paper may be banded, non-banded, or provided
with regions that are either banded or non-banded. In an
embodiment, the cigarette paper is banded. In an embodiment, the
cigarette paper is non-banded
[0187] Any useful cigarette paper additive known in the art may be
added to the cigarette paper, including additives that modify the
burn rate of the paper. Useful additives include citrates, such as
sodium citrate, potassium citrate, magnesium citrate, citric acid,
phosphates (including alkali metal phosphates, e.g., lithium,
sodium, and potassium, magnesium phosphate, and ammonium
phosphate), and mixtures thereof. In an embodiment, the cigarette
paper comprises citrate. In an embodiment, the citrate is present
in an amount from about 0.1% by weight to about 2% by weight. In an
embodiment, the citrate is present in an amount from about 0.2% by
weight to about 1.5% by weight. In an embodiment, the citrate is
present in an amount of about 1% by weight.
[0188] One embodiment is directed to a method of making a filtered
cigarette described herein comprising: preparing a blend of cured
tobacco, wherein the blend comprises a non-transgenic Burley
tobacco and a non-transgenic Flue-cured or Bright tobacco, wherein
the non-transgenic Burley tobacco in the blend is present in an
amount of 85-92 or 45-70% by weight based on the combined weight of
the non-transgenic Burley tobacco and the non-transgenic Flue-cured
or Bright tobacco, and the non-transgenic Flue-cured or Bright
tobacco in the blend is present in an amount of 8-15 or 55-30%
respectively by weight based on the combined weight of the
non-transgenic Burley tobacco and the non-transgenic Flue-cured or
Bright tobacco; determining at least one of total pore volume or
pore volume distribution of an activated carbon; selecting an
activated carbon having a total pore volume of 0.1 mL/g to 0.9 mL/g
and/or having a certain percentage of the activated carbon having a
pore volume distribution of 0.1 mL/g to 0.9 mL/g, wherein the
percentage of carbon having the pore volume distribution is least
about 50%; optionally measuring and/or selecting an activated
carbon having an average pore diameter of 0.6 nm to 1.1 nm;
incorporating the selected activated carbon into a cigarette
filter; and generating a filtered cigarette that contains the blend
of cured tobacco and the cigarette filter. In the method, the
cigarette filter may further comprise a weak base amine-containing
resin. The ratio of the carbon, such as activated carbon, to the
weak base amine-containing resin in the filter in the method may be
from about 1:1 to 1:4. In another embodiment, the ratio of a carbon
to weak base amine-containing resin is about 1:1 to 1:3, 1:1 to
1:2, 1:2 to 1:4, 1:2 to 1:3, 1:3 to 1:4, or about 1:1, 1:2, 1:3 or
1:4, The weak base amine-containing resin used in the method may
contain at least or equal to about 50%-100% of primary amine
functional groups, such as 50-95%, 50-90%, 50-85%, 50-80%, 50-75%,
50-70%, 50-65%, 50-60%, 50-55%, 55-100%, 55-95%, 55-90%, 55-85%,
55-80%, 55-75%, 55-70%, 55-65%, 55-60%, 60-100%, 60-95%, 60-90%,
60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 6 5-100%, 6, 5-95%, 65-90%,
65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-95%, 70-90%, 70-85%,
70-80%, 70-75%, 75-100%, 7 5-95%, 75-90%, 75-85%, 75-80%, 80-100%,
80-95%, 80-90%, 80-85%, 85-100%, 85-95%, 85-90%, 90-100%, 90-95%,
95-100%. The activated carbon used in the method may have an
activity of 50-60.
[0189] In another embodiment, the method further comprises:
generating mainstream smoke from the filtered cigarette; and
measuring the presence or absence of a toxicant retained in the
filter. In addition, the method may further comprise: generating
mainstream smoke from the filtered cigarette; and measuring the
appearance of the biological insult, for example, DSBs, cell death
or perturbation of RNA transcriptome or proteome in lung cells
contacted with the mainstream smoke, a fraction of the mainstream
smoke, or a smoke condensate.
[0190] Another aspect of the invention is directed to a kit
comprising: a first cigarette comprising a first cigarette filter
that comprises a carbon or a weak base amine-containing resin, or
both; and a second cigarette comprising a second cigarette filter
that comprises a carbon, a weak base amine-containing resin, or
both, wherein the second cigarette filter is configured to retain a
greater amount of a toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells than the first cigarette filter. In the
kit, the second cigarette filter may comprise a greater amount of
the carbon or the weak base amine-containing resin or both than the
first cigarette filter.
[0191] Another aspect of the invention is directed to a method of
reducing the induction of biological insult, for example, DSBs,
cell death or perturbation of RNA transcriptome or proteome in
cells that contact cigarette smoke comprising: advising a tobacco
consumer of the need to reduce DNA DSBs in cells that contact
cigarette smoke; and replacing a cigarette habitually consumed by
the tobacco consumer with any of the cigarettes described
above.
[0192] Another aspect of the invention is directed to a method of
reducing biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of a tobacco
consumer comprising: identifying the tobacco consumer in need of a
reduction biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of the
tobacco consumer; and replacing a cigarette habitually consumed by
the identified tobacco consumer with any of the cigarettes
described above. The identifying step may comprise analyzing the
presence of a biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of the
tobacco consumer. This method may further comprise measuring the
presence of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells in the
cells of the tobacco consumer before and after providing any of the
cigarettes described above. The cells may comprise lung cells,
cheek cells, throat cells, or buccal cells.
[0193] In one embodiment, the habitually-consumed cigarette or the
first cigarette is an American Blend comprising a ratio of
approximately 40% Burley to 60% Flue-cured tobacco. In one
embodiment, the first cigarette is comprises a blend of cured
tobacco, wherein the blend comprises a non-transgenic Burley
tobacco and a non-transgenic Flue-cured or Bright tobacco, wherein
the non-transgenic Burley tobacco is present in an amount of about
45-70% by weight based on the combined weight of the non-transgenic
Burley tobacco and the non-transgenic Flue-cured or Bright tobacco;
and the non-transgenic Flue-cured or Bright tobacco is present in
an amount of about 55-30% by weight based on the combined weight of
the non-transgenic Burley tobacco and the non-transgenic Flue-cured
or Bright tobacco.
[0194] In another embodiment, a second or subsequent cigarette
comprises a blend of cured tobacco, wherein the blend comprises a
non-transgenic Burley tobacco and a non-transgenic Flue-cured or
Bright tobacco, wherein the non-transgenic Burley tobacco is
present in an amount of about 85-92% by weight based on the
combined weight of the non-transgenic Burley tobacco and the
non-transgenic Flue-cured or Bright tobacco; and the non-transgenic
Flue-cured or Bright tobacco is present in an amount of about 8-15%
by weight based on the combined weight of the non-transgenic Burley
tobacco and the non-transgenic Flue-cured or Bright tobacco.
Tobacco Products Can Be Modified Using the Filters and Reduced Risk
Tobacco Provided Herein to Gradually Acclimate a Consumer to Using
a Reduced-Risk Tobacco Product
[0195] A significant problem associated with the consumption of
reduced risk tobacco products is that consumers associate these
products with unacceptable sensory/perception properties. For
example, reduced risk cigarettes often have disagreeable tastes
and/or odors, which make it difficult for a consumer to accept
these cigarettes and, especially, to convert from consuming a
conventional cigarette to the reduced risk cigarette. Accordingly,
many smokers revert to their usual brand of high risk cigarette
after trying the reduced risk products. Thus, it is desirable to
gradually adjust a smoker's taste and/or sensory/perception over
time so as to convert or shift the smoker to the reduced risk
cigarette. The conversion from a conventional cigarette to the
reduced risk cigarette described herein can be accomplished by
gradually adjusting the tobacco blend and/or filter composition so
as to slowly change the taste and sensory/perception of the
consumer over time. By slowly evolving the taste and/or odor of a
reduced risk cigarette by changing the filter composition, tobacco
blend, or tobacco blend/filter composition, there is a reduced
ability of a tobacco consumer to notice a sensory/perception
difference between their usual high-risk cigarette and the reduced
risk cigarette, which increases the likelihood that the tobacco
consumer will continue to smoke the reduced risk product. A
step-wise program to adjust the consumers taste to a reduced risk
cigarette is contemplated.
[0196] In some embodiments, the filters and reduced risk tobaccos
described herein are combined to provide a variety of reduced-risk
cigarettes with varying blends, and amounts of carbon and low ion
exchange resin.
[0197] A step-wise program or method, may comprise gradually
reducing the exposure of a tobacco user to a toxicant that causes
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells comprising:
identifying the tobacco user to receive a gradual reduction in
exposure to a toxicant that induces biological insults such as DNA
DSBs, cell death, or perturbation of RNA transcriptome or proteome
in human cells; replacing a cigarette habitually consumed by the
identified tobacco user with a first cigarette for a predetermined
length of time, wherein the first cigarette comprises a first
cigarette filter that comprises a carbon, a weak base
amine-containing resin, or both; replacing the first cigarette with
a second cigarette after the predetermined length of time, wherein
the second cigarette comprises a second cigarette filter that
comprises the carbon, the poly-amine containing resin, or both,
wherein the second cigarette filter is configured to retain a
greater amount of the toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells in human cells than the first cigarette
filter. The predetermined length of time may be about 3-6
weeks.
[0198] In another embodiment, this method may further comprise
replacing the second cigarette after a second predetermined length
of time with a third cigarette, wherein the third cigarette
comprises a third cigarette filter that comprises the carbon, the
weak base amine-containing resin, or both, wherein the third
cigarette filter is capable of retaining a greater amount of the
toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the second cigarette filter.
[0199] Optionally, a third and/or fourth cigarette can be provided,
wherein the third and/or fourth cigarette contains a third and/or
fourth cigarette filter capable of retaining a greater amount of
the toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the second or third cigarette filter respectively.
[0200] In one embodiment, the filters, reduced risk tobaccos, and
program described above are used in methods of reducing the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells that contact
cigarette smoke (e.g., lung cells, cheek cells, throat cells or
buccal cells), wherein a tobacco consumer is advised of the need to
reduce the induction of biological insult, for example, DSBs, cell
death or perturbation of RNA transcriptome or proteome in cells
that contact cigarette smoke, by providing information or
instructions to that effect.
[0201] In one embodiment, the advising can be performed by
inclusion of a statement that the contents of the reduced-risk
cigarette reduce the induction of biological insult, for example,
DSBs, cell death or perturbation of RNA transcriptome or proteome
in cells that contact cigarette smoke in a product label, such as a
cigarette pack or carton.
[0202] In another embodiment, the filters, reduced risk tobaccos,
and program above are combined in methods of reducing the induction
of biological insult, for example, DSBs, cell death or perturbation
of RNA transcriptome or proteome in cells of a tobacco consumer
(e.g., lung cells, cheek cells, throat cells or buccal cells),
wherein a tobacco consumer is identified as one in need of such
reduction, and providing the reduced-risk cigarette comprising the
filter/reduced risk tobacco combination to the tobacco consumer,
either in the presence or absence of instructions. In one
embodiment, the identification step comprises an analysis of the
presence biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in cells of the
tobacco consumer.
[0203] By gradually decreasing the amount of an agent that induces
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells and/or increasing the
amount of ion exchange resin and/or carbon, the beneficial effects
of reduced toxicants can be obtained without a noticeable decrease
in sensory/perception properties of the cigarette.
[0204] A kit based on these methods may comprise: a first cigarette
comprising a first cigarette filter that comprises a carbon or a
weak base amine-containing resin, or both; and a second cigarette
comprising a second cigarette filter that comprises a carbon, a
weak base amine-containing resin, or both, wherein the second
cigarette filter is configured to retain a greater amount of a
toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells in human cells than the first cigarette filter. In the kit,
the second cigarette filter may comprise a greater amount of the
carbon or the weak base amine-containing resin or both than the
first cigarette filter. The kits may also include additional
cigarettes as described below in more detail.
[0205] The cigarettes and kits described herein can also be used as
part of a marketing program. Another aspect of the invention is
directed to a method of marketing a cigarette that is configured to
reduce the biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells
comprising: replacing a cigarette habitually consumed by a tobacco
consumer with a first cigarette comprising a first cigarette filter
comprising a carbon and a weak base amine-containing resin for a
predetermined length of time; replacing the first cigarette after
the predetermined period of time with a second cigarette comprising
a second cigarette filter configured to retain a greater amount of
the toxicant that induces biological insults such as DNA DSBs, cell
death, or RNA transcriptome or proteome in human cells than the
first cigarette filter; and marketing the first and second
cigarettes, wherein the first cigarette is introduced to a consumer
prior to the second cigarette and the first cigarette is marketed
for a time sufficient to adjust a tobacco consumer's taste prior to
marketing the second cigarette. The time to adjust the tobacco
consumer's taste may be less than 1 month, 2 months, 3 months, 4
months, 5 months, 6 months, 7 months, 8 months, 9 months, 10
months, 11 months, or 12 months.
[0206] In another embodiment, the method of marketing the cigarette
further comprises replacing the second cigarette with a third
cigarette that comprises a third cigarette filter capable of
retaining a greater amount of a toxicant that induces biological
insults such as DNA DSBs, cell death, or perturbation of RNA
transcriptome or proteome in human cells in human cells than the
second cigarette filter. In another embodiment, the first
cigarette, the second cigarette and the third cigarette have
substantially similar packaging. In further embodiment, the first
cigarette, the second cigarette, and the third cigarette are sold
under the same brand. In yet another embodiment, the first
cigarette, the second cigarette, and the third cigarette have the
same packaging.
[0207] The method of marketing described above they also comprise
determining a tobacco consumer's acceptance of taste (e.g., by
conducting a focus group or test analysis) of the first cigarette;
providing a second cigarette to the tobacco consumer where the
second cigarette comprises a second filter configured to retain a
greater amount of the toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells than the first cigarette filter, or wherein
the second cigarette comprises a reduced risk tobacco and/or a
second cigarette filter that comprises an ion-exchange resin and/or
carbon, wherein the second cigarette is configured to reduce the
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells in human cells or
deliver a reduced amount of an agent that induces biological
insults such as DNA DSBs, cell death, or perturbation of RNA
transcriptome or proteome in human cells relative to said first
cigarette; determining the tobacco consumer's acceptance of taste
(e.g., in a focus or test analysis) of the second cigarette; and
marketing the first and second cigarettes, wherein the first
cigarette is marketed for a time sufficient to adjust a tobacco
consumer's taste before marketing the second cigarette. A
determination of the time that is sufficient to adjust the tobacco
consumer's taste or sensory/perception can be made in the focus or
test group. Optionally, the reduced-risk tobacco and/or amount of
ion-exchange resin or carbon that will be acceptable to a
population of smokers (e.g., amount that will not substantially
alter the sensory/perception properties of the cigarette) can be
determined using standard market research surveys. Once an
acceptable amount is determined, then a second cigarette having a
second cigarette filter configured to retain a greater amount of
the toxicant that induces biological insults such as DNA DSBs, cell
death, or biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells than
the first cigarette filter, or a second cigarette comprising
reduced-risk tobacco and/or more ion-exchange resin and/or carbon
can be manufactured. Various tobaccos and/or amounts of
ion-exchange resin or carbon, as described herein, can be used in
the second cigarette, and again the optimal amounts of these
components can be determined using a market research survey, focus
groups or test analysis. The same procedure can be followed to
determine the filter that is configured to retain a greater amount
of the toxicant that induces biological insults such as DNA DSBs,
cell death, or perturbation of RNA transcriptome or proteome in
human cells than the third cigarette filter, or the type of tobacco
and/or amount of ion-exchange resin and/or carbon to use in a third
cigarette. A third cigarette is provided that comprises a third
filter configured to retain a greater amount of the toxicant that
induces biological insults such as DNA DSBs, cell death, or changes
perturbation of RNA transcriptome or proteome in human cells than
the second cigarette filter, or a third cigarette comprising
tobacco and/or a third cigarette filter that comprises an
ion-exchange resin and/or carbon, wherein the third cigarette has a
third filter configured to retain a greater amount of the toxicant
that induces biological insults such as DNA DSBs, cell death, or
perturbation of RNA transcriptome or proteome in human cells than
the second cigarette filter; and/or is configured to deliver a
reduced amount of an agent that induces biological insults such as
DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells relative to said second cigarette. By
gradually modifying the filter that is configured to retain a
greater amount of the toxicant that induces biological insults such
as DNA DSBs, cell death, or perturbation of RNA transcriptome or
proteome in human cells than the second cigarette filter, for
example, by modifying the tobacco and/or increasing the amount of
ion exchange resin and/or carbon, the beneficial effects of reduced
toxicants can be obtained without a noticeable decrease in
sensory/perception properties of the cigarette, resulting in
increased acceptance of a reduced risk cigarette by the tobacco
user.
[0208] Accordingly, some embodiments include methods of gradually
introducing the change in tobacco and filter composition to
cigarette smokers so as to adjust a consumer's sensory perceptive
behavior to the reduced risk cigarettes described herein. More such
methods are accomplished by gradually increasing the capacity of a
filter that is configured to retain a greater amount of the
toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the previous cigarette filter; for example, by
increasing the amount of reduced-risk tobacco in the blend over
time (e.g., an amount of tobacco containing a reduced number
compounds that cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells) is provided in a tobacco blend of a first
cigarette that is marketed to consumers, after one, two, three,
four, five, six, seven, eight, nine, ten, eleven, or twelve months,
a second cigarette having a second cigarette filter that is
configured to retain a progressively greater amount of the toxicant
that induces biological insults such as DNA DSBs, cell death, or
perturbation of RNA transcriptome or proteome in human cells than
the first cigarette filter, such as one having a tobacco blend with
more tobacco having a reduced number of compounds that cause the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells is
provided in a cigarette to the consumer, optionally, a third,
fourth, fifth or sixth cigarette can be provided to the consumer,
wherein said successive generations of cigarettes are also provided
after one, two, three, four, five, six, seven, eight, nine, ten,
eleven, or twelve months from the marketing of the previous
generation of cigarette and said third, fourth, fifth or sixth
cigarette has filter configured to retain a progressively greater
amount of the toxicant that induces biological insults such as DNA
DSBs, cell death, or perturbation of RNA transcriptome or proteome
in human cells than the first cigarette filter, such as one that
has progressively more tobacco having a reduced number of compounds
that cause the induction of biological insult, for example, DSBs,
cell death or perturbation of RNA transcriptome or proteome in
human cells than the preceding cigarette.
[0209] In a similar fashion, more methods are accomplished using
the cigarette designs described above by gradually increasing the
ability of a filter that is configured to retain a greater amount
of the toxicant that induces biological insults such as DNA DSBs,
cell death, or perturbation of RNA transcriptome or proteome in
human cells than the first cigarette filter, for example by
introducing more ion exchange resin and/or carbon to said tobacco
consumer in successive cigarettes. That is, a first cigarette can
have a first amount of carbon and/or resin in said filter and after
a period of one, two, three, four, five, six, seven, eight, nine,
ten, eleven, or twelve months a second cigarette can be provided to
said consumer and said second cigarette that has a second cigarette
filter is configured to retain a progressively greater amount of
the toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the first cigarette filter such as a filter that has
more carbon and/or ion exchange resin than said first cigarette.
Optionally, a third cigarette with a third cigarette filter that is
configured to retain a progressively greater amount of the toxicant
that induces biological insults such as DNA DSBs, cell death, or
perturbation of RNA transcriptome or proteome in human cells than
the second cigarette filter, such as a filter containing more
carbon or ion exchange resin is provided to the tobacco consumer
after one, two, three, four, five, six, seven, eight, nine, ten,
eleven, or twelve months of marketing the second cigarette and,
additionally, a fourth, fifth or sixth cigarette that has a fourth,
fifth, or sixth cigarette filter configured to retain a
progressively greater amount of the toxicant that induces
biological insults such as DNA DSBs, cell death, or perturbation of
RNA transcriptome or proteome in human cells than the previous
cigarette filter, such as by having progressively more carbon
and/or ion exchange resin than the preceding cigarette, is marketed
to the consumer one, two, three, four, five, six, seven, eight,
nine, ten, eleven, or twelve months after the preceding
cigarette.
[0210] Further, the reduced-risk tobacco and/or the ion exchange
resin can be gradually modified or increased in a stepwise fashion
in combination. That is, a second cigarette having a filter
configured to retain a greater amount of the toxicant that induces
biological insults such as DNA DSBs, cell death, or perturbation of
RNA transcriptome or proteome in human cells than the first
cigarette filter, such as one in which the cigarette has an amount
of tobacco having a reduced number of compounds that cause the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome in human cells and an
amount of ion exchange resin and/or carbon, can be provided in a
first cigarette and after one, two, three, four, five, six, seven,
eight, nine, ten, eleven, or twelve months of smoking the first
cigarette a second cigarette having more tobacco having a reduced
number of compounds that cause the induction of biological insult,
for example, DSBs, cell death or perturbation of RNA transcriptome
or proteome in human cells and/or ion exchange resin and/or carbon
and/or carbon pore volume can be provided to the consumer and after
one, two, three, four, five, six, seven, eight, nine, ten, eleven,
or twelve months of smoking the second cigarette, optionally, a
third cigarette having more tobacco with a reduced number of
compounds cause the induction of biological insult, for example,
DSBs, cell death or perturbation of RNA transcriptome or proteome
in human cells and/or ion exchange resin and/or carbon and/or
carbon pore volume can be provided to said tobacco consumer after
one, two, three, four, five, six, seven, eight, nine, ten, eleven,
or twelve months of smoking the second cigarette. Still further, a
fourth, fifth, or sixth cigarette can be provided wherein each
successive cigarette is provided after one, two, three, four, five,
six, seven, eight, nine, ten, eleven, or twelve months of smoking
the preceding cigarette and each successive cigarette having a
filter configured to retain a progressively greater amount of the
toxicant that induces biological insults such as DNA DSBs, cell
death, or perturbation of RNA transcriptome or proteome in human
cells than the previous cigarette filter, such as a cigarette that
has more tobacco having a reduced number of compounds that induce
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells and/or ion exchange
resin and/or carbon and/or carbon pore volume in the cigarette over
time. In this manner, an improved method of introducing a reduced
risk cigarette to the market is provided, wherein a tobacco
consumer's sensory/perception and taste behaviors are slowly
adjusted while gradually modifying filter such that it is
configured to retain a progressively greater amount of the toxicant
that induces biological insults such as DNA DSBs, cell death, or
perturbation of RNA transcriptome or proteome in human cells than
the previous cigarette filter, such as by gradually increasing the
presence of tobacco having a reduced number of compounds that cause
the induction of biological insult, for example, DSBs, cell death
or perturbation of RNA transcriptome or proteome in human cells
and/or ion exchange resin and/or carbon and/or carbon pore volume
in the cigarette after a period of time such that the consumer more
readily receives the reduced risk cigarette and replaces the
conventional cigarette used by the consumer prior to starting the
program.
[0211] Additionally, the step-wise programs described above
gradually reduce the presence of toxicants in the mainstream smoke
of the cigarette over time because as the filter is configured to
retain a progressively greater amount of the toxicant that induces
biological insults such as DNA DSBs, cell death, or perturbation of
RNA transcriptome or proteome in human cells than the previous
cigarette filter, or as the tobacco having a reduced number of
compounds that cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells and/or ion exchange resin and/or carbon
and/or carbon pore volume are increased, the amount of toxicants
present in the mainstream smoke of the cigarettes are decreased. In
some embodiments it is preferred that substantially the same
packaging is maintained during the step-wise programs above. In
some contexts, by substantially the same packaging is meant that
the cigarette brand is maintained with only slight variations in
the labeling so as to maintain customer brand recognition while
gradually changing the composition of the cigarettes, as described
above.
[0212] In the embodiments described above in this section, the
habitually-consumed cigarette or the first cigarette is an American
Blend comprising a ratio of approximately 40% Burley to 60%
Flue-cured tobacco. In one embodiment, the first cigarette
comprises a blend of cured tobacco, wherein the blend comprises a
non-transgenic Burley tobacco and a non-transgenic Flue-cured or
Bright tobacco, wherein the non-transgenic Burley tobacco is
present in an amount of about 45-70% by weight based on the
combined weight of the non-transgenic Burley tobacco and the
non-transgenic Flue-cured or Bright tobacco; and the non-transgenic
Flue-cured or Bright tobacco is present in an amount of about
55-30% by weight based on the combined weight of the non-transgenic
Burley tobacco and the non-transgenic Flue-cured or Bright
tobacco.
[0213] In another embodiment, described above in the section, a
second or subsequent cigarette comprises a blend of cured tobacco,
wherein the blend comprises a non-transgenic Burley tobacco and a
non-transgenic Flue-cured or Bright tobacco, wherein the
non-transgenic Burley tobacco is present in an amount of about
85-92% by weight based on the combined weight of the non-transgenic
Burley tobacco and the non-transgenic Flue-cured or Bright tobacco;
and the non-transgenic Flue-cured or Bright tobacco is present in
an amount of about 8-15% by weight based on the combined weight of
the non-transgenic Burley tobacco and the non-transgenic Flue-cured
or Bright tobacco.
[0214] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example I
Cigarette Design to Reduce Oxidative Stress and Subsequent
Attenuation of Genotoxicity and Cytotoxicity Effects of Cigarette
Smoke on Biological Systems
[0215] This example shows that blends having a high proportion of
Burley tobacco to Flue-cured or Bright tobacco unexpectedly induce
fewer DNA DSBs than blends having a low proportion Burley tobacco
to Flue-cured or Bright tobacco. In addition, this example shows
the unexpected result of less H2AX damage or cell death using a
carbon filter with a cigarette containing a high proportion of
Burley tobacco to Flue-cured or Bright tobacco. Further, this
example shows a synergistic effect conflict of a filter containing
a carbon and a weak base amine-containing resin.
[0216] Increased oxidative stress is a major mechanism by which
cigarette smoke causes airway damage that can lead to a host of
pathogenic conditions including asthma, pulmonary fibrosis, chronic
obstructive pulmonary disease, and lung cancer. However, a detailed
understanding of the specific molecular mechanisms that link
oxidative stress with cigarette smoke-induced pathologies is still
lacking. Increased knowledge in this area can be used developing
new approaches to mitigating or reversing the damage caused by
cigarette smoke either by chemopreventive strategies and/or
reducing the toxicity of cigarettes. Cigarette smoke is a complex
mixture of over 4000 different chemical components; some of which
are highly oxidizing in nature such as reactive oxygen species
(ROS), reactive nitrogen species (RNS) and substantial amounts of
long lived and short lived radical species, primarily carbon and
nitrogen centered. Despite efficient antioxidant defense mechanisms
in the respiratory tract, the large amounts of short lived free
radicals (such as .O2-, .NO, .OH--, etc), and more stable organic
reactive species/oxidants (such as acrolein, epoxides, etc.) that
exist in both the gas and particulate phases of cigarette smoke can
transiently, and perhaps chronically, overwhelm the cell's
steady-state antioxidant capacity. In fact, there is strong
evidence that active smoking causes a marked imbalance in an
individual's redox state and an overall increase in oxidative
stress, especially in the respiratory tract. One possible result of
this disruption to the lung's redox status is the induction of
oxidized DNA, which can manifest as missing bases, altered or
mismatched bases, deletions and insertions, strand breaks, intra
and inter-strand cross-links, structural or numerical chromosomal
aberrations, abnormal sister chromatid exchanges, and the formation
of micronuclei, as well as protein/enzyme damage through covalent
adduction, cross-linking, sulfide bond interactions and active site
modifications including DNA repair proteins. Each of these defects
can not only be genotoxic and cytotoxic, but can also play a
fundamental role in tumorigenesis.
[0217] Comprehensive design of cigarettes is based on the
individual design characteristics of traditional cigarette
technology such as blend/blend component selection, filter design
and filter additives as well as tobacco additives. Design
characteristics are selected by combining information from chemical
analysis and biological markers that support reduced oxidative
stress in biological systems. The guiding principles for the
selections are based on the predictability of specific results
based on the Chem-Bio model associated with Lipid Peroxidation
(LPO) induced oxidative stress and following the chemical markers
associated with LPO and biological markers connected to LPO induced
oxidative stress.
[0218] A549 Cell Culture and Smoke Treatment
[0219] A549 cells are purchased from American Type Culture
Collection (ATCC #CCL-185, Manassas, Va.). The cells are cultured
in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5
g/L sodium bicarbonate (ATCC, Manassas, Va.) and supplemented with
10% fetal bovine serum (ATCC, Manassas, Va.). Dual-chambered slides
(Nunc Lab-Tek II) are seeded with 1 ml of 10.sup.5 cells/ml cell
suspension per chamber 48 hours before exposure. All incubations
are at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in
air. Cells are grown to 70% conFluency, at which time they are
treated with smoke. The cell culture medium is replaced with
37.degree. C. Dulbecco's PBS (D-PBS) containing calcium and
magnesium (Sigma, St. Louis, Mo.) for the smoke exposure. Slide
chamber covers are removed and the slides were placed in a smoke
exposure chamber (20.6 cm.times.6.7 cm.times.6.3
cm-L.times.W.times.H). Smoke is generated from various cigarettes
provided below under FTC smoking conditions using a KC 5 Port
Smoker (KC Automation, Richmond, Va.). The smoke is diluted by
drawing it through a 250 mL round-bottom flask prior to its
reaching the exposure chamber. The time and distance that the smoke
travels from the end of the cigarette to the exposure chamber is
minimized by using the shortest lengths of tubing possible between
the parts of the apparatus. Cigarettes are smoked to within 3 mm of
the filter tip. Cells are exposed to smoke for up to 40 minutes.
Mock-exposed (control) cells are treated under identical conditions
as the exposed cells except for the absence of a cigarette in the
smoking port. They are mock-exposed for 10 minutes. Following
treatment or mock treatment, the D-PBS is aspirated and replaced
with 1 ml per chamber of fresh culture medium at 37.degree. C. The
slides are placed in the 37.degree. C., 5% CO.sub.2 incubator and
incubated for 15 minutes. Following incubation, the medium is
aspirated and the slides are submerged in 50 ml conical tubes
filled with 70% ethanol. The fixed slides are stored at 4.degree.
C. prior to analysis.
[0220] Immunocytochemical Detection of Phosphorylated Histone
H2AX
[0221] Cells are treated with smoke (i.e., A549) or smoke
condensate (i.e., NHBE) and fixed as described above, then rinsed
twice in PBS and immersed in 0.2% Triton X-100 (Sigma) in a
solution of 1% (w/v) bovine serum albumin (BSA; Sigma) in PBS for
30 min to suppress non-specific antibody binding. The cells are
then incubated in 100 .mu.l volume of 1% BSA containing 1:200
dilution of anti-phosphorylated histone H2AX (.gamma.-H2AX) rabbit
polyclonal Ab. After overnight incubation at 4.degree. C., the
slides are washed twice with PBS and then incubated in 100 .mu.l of
1:200 dilution of Alexa Fluor 488 goat anti-rabbit IgG (H+L)
(Molecular Probes, Eugene, Oreg.) for 45 min at room temperature in
the dark. The cells are then counterstained with 1 .mu.g/ml
4,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene,
Oreg.) in PBS for 5 min. Each experiment is performed with an IgG
control in which cells are labeled only with secondary antibody,
Alexa Fluor 488 goat anti-rabbit IgG (H+L) or FITC-conjugated
F(ab')2 fragment of goat anti-mouse immunoglobulins, without
primary antibody incubation to estimate the extent of nonspecific
binding of the secondary antibody to the cells.
[0222] Measurement of Cell Fluorescence by Laser Scanning
Cytometry
[0223] Cellular green (phosphorylated histone H2AX) fluorescence
emission is measured using a Laser Scanning Cytometer (LSC;
CompuCyte, Cambridge, Mass.), utilizing standard filter settings;
fluorescence is excited with a 488-nm argon ion laser. The
intensities of maximal pixel and integrated fluorescence are
measured and recorded for each cell. At least 3,000 cells are
measured per sample.
[0224] Phosphorylated histone H2AX may also be detected using
commercial antibodies and Western blotting.
[0225] Clonogenicity Assay
[0226] A clonogenic survival assay was used to study the ability of
tobaccos and tobacco products to impact the proliferation of cells.
The experiment involves four major steps: (1) inoculate cells into
culture dishes and incubate for 24-48 hours; (2) upon reaching the
logarithmic phase of growth, the treatment is applied; the
treatment in this case is freshly prepared and diluted CS for
increasing periods of time; (3) the cells are then allowed to
recover for a set number of hours (up to 24), then the cells are
trypsinized, replated at specific dilutions, and allowed to
continue growing for 5-7 days; the number of cells used depends
largely on the plating efficiency of the cell line and must be
determined empirically prior to the experiment; and (4) at the
conclusion of the experiment, the cells are fixed, stained, and
counted. The primary measure is to count surviving colonies of
25-50 cells which is presented as the percentage of cells which
survived the treatment. A graphical representation of survival
versus exposure time to CS is then generated. The surviving
fraction is determined by dividing the number of colonies in the
dish by the number of the colonies in the control (non-treated)
dish.
[0227] A549 cells are exposed to smoke as described above.
Following smoke exposure the medium is aspirated and the cells
rinsed refed with 37.degree. C. BEGM and placed in a 37.degree. C.,
5% CO.sub.2 humidified incubator for two to three hours. The cells
are harvested by trypsinization with trypsin-EDTA (0.25%
trypsin-0.38 mg/ml EDTA, Invitrogen). Cells are centrifuged at
260.times.g for 8 min. Cell pellets are resuspended in 1 ml of
Ham's F-12K medium, 10% FBS (complete medium) per pellet and
counted. Cells are serially diluted so that the mock treated have
.about.65 cells per well and smoke treated have .about.300 cells
per well when seeded onto 96-well flat bottom tissue culture
plates; one plate per condition. The plates are incubated for five
days in a 37.degree. C., 5% CO.sub.2 humidified incubator. The
colonies of cells are fixed with 5% formaldehyde/PBS and colored
with 0.8% crystal violet solution for visualization. The colonies
are counted with the aid of a macroscopic dissecting microscope.
The cloning efficiency results are expressed in relation to the
mock exposed cells.
[0228] Production of Cigarettes
[0229] Three test blends were produced with differing ratios of
commercially available non-transgenic Flue-cured or Bright to
non-transgenic Burley tobacco of about 90:10, 50:50 and 10:90. Each
test blend contained approximately 63-66% tobacco lamina
(Flue-cured and Burley combination), about 22% expanded stem
tobacco and about 12-15% Oriental tobacco as follows:
TABLE-US-00001 Tob. 10/90 Blend Tob. 50/50 Blend Tob. 90/10 Blend
Type: % of Blend Type: % of Blend Type: % of Blend Flue 6.56% Flue
31.53% Flue 59.07% Cured Cured Cured Burley 59.07% Burley 31.53%
Burley 6.56% Oriental 12.37% Oriental 14.94% Oriental 12.37% Cres
22.00% Cres 22.00% Cres 22.00% Stem Stem Stem Total: 100.00% Total:
100.00% Total: 100.00%
[0230] Two cigarettes were produced per sample. These samples were
designed to give similar tar and nicotine yields of about 9.0 mg
tar and 0.8 mg nicotine. All cigarette samples were machine made
with cellulose acetate filters. Samples used for H2AX and
Clonogenic assay comparisons included the machine made cellulose
acetate filter of each blend type as control and hand modified
samples of each blend type consisting of a plug space plug design
where the space was filled with 40 mg, 60 mg, 80 mg, or 100 mg TA95
carbon.
[0231] The conditions included FTC Smoke from test cigarettes at 0%
ventilation. Each of the three cigarette samples delivered
approximately 100-120% more smoke than the 2RF4 control.
[0232] H2AX Damage Decreased with Increasing Amount of Activated
Carbon
[0233] In the first experiment, cigarettes having five different
filters were used, namely one control of the specific blend type
either 90:10 flue cured/burley or 10:90 flue cured burley
containing a conventional cellulose acetate filter, and four
filters containing four different amounts of TA95 activated carbon,
namely 40 mg, 60 mg, 80 mg, or 100 milligrams.
[0234] DSBs in relative fluorescence units compared to sham control
were measured using the H2AX assay described with the following
conditions: A549 cells were cultured in Ham's F12K medium with 2 mM
L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (ATCC)
and supplemented with 10% fetal bovine serum (ATCC). All
incubations were at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 in air.
[0235] Smoke treatment for A549 cells--dual-chambered slides (Nunc
Lab-Tek II, VWR International, and West Chester, Pa.) were seeded
with 1 ml of 5.times.10.sup.4 cells/ml cell suspension per chamber
48 hours before exposure and were typically at 70% confluency at
the time of smoke treatment. The cell culture medium was replaced
with 37.degree. C. Dulbecco's PBS (D-PBS) containing calcium and
magnesium (BioSource, Rockville, Md.) for the smoke exposure. Slide
chamber covers were removed and the slides were placed in a smoke
exposure chamber (20.6 cm.times.6.7 cm.times.6.3
cm-L.times.W.times.H). Smoke was generated from 2R4F cigarettes
(Kentucky Reference Cigarette, containing 8.9 mg "tar" and 0.75 mg
nicotine per cigarette ref (37)] under Federal Trade Commission
(FTC) smoking conditions (35.+-.0.3 cc puff, one puff every 60
seconds, 2-second puff duration with none of the ventilation holes
blocked) using a KC 5 Port Smoker (KC Automation, Richmond, Va.).
Cigarettes were smoked to within 3 mm of the filter tip. All
cigarettes had been equilibrated at 23.9.degree. C..+-.1.1.degree.
C. and 60%.+-.2% relative humidity for a minimum of 24 hours and a
maximum of 14 days. The smoke exposure chamber was designed to
deliver smoke uniformly diluted with 5% CO.sub.2 in air and passed
through the cell exposure chamber at a constant flow rate of 500
cc/min. Briefly, each 35 cc puff was first drawn into a 250 cc
chamber containing 5% CO.sub.2 in air and mixed via a stir bar for
3 seconds. The diluted smoke was then passed over the cells at a
flow of 500 ml/min. The standard smoke dilution used in most of our
experiments was 35 cc delivered over 58 seconds, and the intensity
of exposure was varied by varying the length of time the cells
spent in the exposure chamber. The time and distance that the smoke
traveled from the end of the cigarette to the exposure chamber was
minimized by using the shortest lengths of tubing possible between
the parts of the apparatus.
[0236] Mock-exposed cells were treated under identical conditions
as the exposed cells except for the absence of a cigarette in the
smoking port, with cigarettes containing about 90:10
Flue-cured:Burley tobacco and filters comprising varying amounts of
activated carbon (TA95). Following treatment or mock treatment, the
D-PBS covering the cells was aspirated and replaced with 1 ml per
chamber of fresh culture medium at 37.degree. C. The cells were
placed in the 37.degree. C., 5% CO.sub.2 incubator and incubated
for various time points depending on the required experimental
conditions.
[0237] The DSBs were measured that were induced by cigarette smoke
from cigarette samples having one (1) conventional cellulose
acetate (CA) filter, and four (4) filters comprising 40 mg, 60 mg,
80 mg, or 100 mg of TA95 activated carbon. FIG. 2 illustrates the
decrease in DSBs upon increasing amount of activated carbon present
in the filter.
[0238] In a second experiment, under the same conditions results
were obtained using cigarette samples containing 10:90
Flue-cured:Burley tobacco as shown in FIG. 3. These results also
illustrate a decrease in DSBs upon increasing amount of activated
carbon present in the filter.
[0239] The H2AX damage was compared based on the blend of tobacco
in the cigarette samples. The H2AX assay described above was used
to determine the amount of DSBs as a percentage of DSBs induced by
a cigarette smoke from a cigarette sample having a cellulose
acetate control filter of a control cigarette sample. A cigarette
sample comprising the 90:10 Flue-cured:Burley tobacco blend
(left/blue) was compared to a cigarette sample comprising 10:90
Flue-cured:Burley tobacco blend (right/red). The DSB percentages
are illustrated for cigarette samples comprising four (4) filters
containing 40 mg, 60 mg, 80 mg, or 100 mg of TA95 activated carbon.
H2AX damage using the 10:90 Flue-cured:Burley tobacco blend was
significantly lower than the 90:10 Flue-cured:Burley tobacco blend
as shown in FIG. 4.
[0240] A similar comparison was made using the clonogenic assay
described above. FIG. 5 illustrates the percentage of cell death
using cigarette smoke from a control cigarette having a cellulose
acetate filter, a cigarette sample containing a 90:10
Flue-cured:Burley tobacco blend (left/blue), and a cigarette sample
containing a 10:90 Flue-cured:Burley tobacco blend (right/red). The
cell death as a percentage of total cells is illustrated in FIG. 5
for a cigarette sample comprising one (1) conventional cellulose
acetate (CA) filter as the control and two (2) filters containing
40 mg and 100 mg of TA95 activated carbon. Surprisingly, in the
cigarette sample having a filter comprising 100 mg of activated
carbon (TA95), cell death was significantly lower for the 10:90
Flue-cured:Burley tobacco blend in comparison to the 90:10
Flue-cured:Burley tobacco blend.
[0241] Synergistic Effect of Activated Carbon and Ion Exchange
Resin Filters
[0242] DSBs were measured using an H2AX assay for cigarette samples
containing 50:50 Flue-cured:Burley tobacco and having three (3)
filters containing 50 mg TA95 activated carbon, 50 mg A109 ion
exchange resin, and 50 mg each of TA95 and A109. These results were
compared with a control 2R4F cigarette sample containing a
conventional cellulose acetate (CA) filter. Both TA95 activated
carbon and A109 ion exchange resin mitigated the amount of DSBs
when added independently to a cigarette filter at equivalent 50 mg
loadings. The cigarette samples having filters containing 50 mg
TA95 activated carbon or 50 mg A109 ion exchange resin did not
result in a reduction in the DSBs in comparison to the control;
however, the cigarette sample containing a filter having a
combination of 50 mg each of TA95 and A109 illustrated a
significant reduction in DSBs. Thus, when added to a cigarette
filter in combination, activated carbon and ion exchange resin
mitigate DSBs by a larger amount than would be predicted based on
their independent loading analysis, thus illustrating an unexpected
synergistic effect. FIG. 8 shows that various ion exchange resins
similarly decrease H2AX damage, and act synergistically when
combined with activated carbon.
[0243] Similar synergistic effects were observed in the clonogenic
analysis illustrated in FIG. 7. Cloning efficiency of A549 cells
(five days post-smoke exposure) based on a percentage of cell death
was measured using a clonogenic assay described above for cigarette
samples containing 50:50 Flue-cured:Burley tobacco blends. The
results as a percentage of cell death is illustrated for cigarette
samples containing one (1) control 2R4F conventional cellulose
acetate (CA) filter, and three (3) filters containing 50 mg TA95
activated carbon, 50 mg A109 ion exchange resin, and 50 mg each of
TA95 and A109. The cigarette sample having a filter comprising 50
mg TA95 activated carbon resulted in an approximately 10% decrease
in cell death in comparison to the control. The cigarette sample
having a filter comprising 50 mg A109 ion exchange resin resulted
in a slightly higher percentage of cell death in comparison to the
control. The cigarette sample having a filter comprising a
combination of 50 mg each of TA95 and A109 resulted in a greater
than 40% reduction in cell death in comparison to the control.
Thus, A109 (50 mg) does not improve cloning efficiency by itself,
but does when mixed with TA95, illustrating a synergistic
effect.
[0244] Various Ion Exchange Resin Filters Behave
Synergistically
[0245] Three different amine containing resins (A109, Duolite and
CR20) in combination with 50 mg of TA95 carbon in a
plug-space-plug-space-plug hand modified filter design on a 50:50
flue cured burley blend were compared. The resins were added to
occupy the same volume as 50 mg of TA95 carbon (the resins are less
dense than carbon so this volume for all the resins comprises less
than 50 mg of any particular resin).
[0246] As can be seen in FIG. 8 the three resin/carbon combinations
tested reduce the number of DSBs as measured by H2AX at various
efficacies, as described above, compared to a 100 mg TA95 control
cigarette of the same blend.
[0247] Furthermore, FIG. 8 shows that the effect of adding resin to
the filter in concert with carbon is synergistic as the total
volume of "filter additives" e.g., resin plus carbon is equal to
the total volume of 100 mg of TA95 carbon, but the observed effect
to the H2AX is greater than additive.
Example II
[0248] Four cigarettes were compared using the H2AX assay described
above. All four cigarettes are comprised of the same construction
and contain the same 50:50 Flue-cured:Burley tobacco blend: 1)
control cigarette filter consisted of cellulose acetate (CA) 2) a
research 2R4F cigarette, 3) 50 mg sepiolite was added to a hand
modified CA filter in a plug-space-plug design, and 4) 40 mg of
Sepiolite +10 mg A109 was added to the filter in a plug-space-plug
design occupying the same volume as 50 mg of Sepiolite sample.
[0249] As shown in FIG. 9, no benefit was observed toward reducing
DSBs with sepiolite alone in comparison to the control cigarette,
and little benefit was observed for the research 2R4F cigarette.
Further, as shown in FIG. 7, in a separate experiment, no benefit
was observed toward reducing DSBs with 50 mg of A109 alone in
comparison to the control 2R4F cigarette. A synergistic effect was
observed upon mixing 40 mg of sepiolite with 10 mg of A109 resin
with a significant reduction in DSBs, as shown by the reduction in
FIG. 9.
[0250] Sepiolite mixed with nominal amounts of weak-base primary
amine resin has the benefits of reduced DSBs, reduced cost (of the
resin) and less objectionable sensory/taste characteristics
associated with weak-base primary amine resins.
Example III
[0251] This example describes several approaches that can be used
in addition to or in lieu of the H2AX assay and the cloning
efficiency assay to identify reduced risk prototype cigarettes, as
compared to a reference cigarette or commercially available
cigarettes (i.e., to determine whether one or more of the prototype
blends, cigarettes or filters described herein produces a cigarette
smoke that induces less of a biological insult than a reference
cigarette, such as 2R4 For a commercially available brand). The
perturbation on the transcriptome and/or proteome of multiple types
of lung cells, oral mucosal cells, and white blood cells (e.g.,
A549 cells, NHBE cells or cells obtained from a smoker) that have
been contacted with mainstream smoke obtained from a cigarette
containing a tobacco blend with or without a carbon and/or weak
base-containing resin filter, manufactured as described herein, is
compared to the impact on the transcriptome and/or proteome
obtained from mainstream smoke generated from a reference cigarette
with or without a filter (e.g., a cellulose acetate filter), such
as the 2R4F or a commercially available cigarette. Optionally, the
transcriptome and/or proteome of multiple types of lung cells that
are mock-treated (i.e., subjected to the experimental protocol
without contact with smoke (control)) can be analyzed to establish
a transcriptome and/or proteome baseline. Suitable methods for
transcriptome and proteome analysis are described in U.S. Pat. App.
Pub. No. 2008/0052789, Yang et al., BMC Cancer. 2005 Jul. 20; 5:83;
and Han et al., Am J Clin Oncol. 2008 April; 31(2):133-9, all of
which are hereby expressly incorporated by reference in their
entireties.
[0252] Accordingly, in a first set of experiments, transcriptome
and proteome measurements are obtained by generating mainstream
smoke from reference cigarettes (e.g., the 2R4F cigarette or
commercially available cigarettes) with or without a filter,
contacting lung cells with mainstream smoke generated from the
cigarettes, and conducting a microarray analysis. Cells isolated
from a smoker that has smoked a commercially available brand (e.g.,
lung cells, cells of the oral mucosa, and white blood cells) can
also be obtained by conventional techniques and analyzed, as
described above. A mock or control experiment is, optionally,
performed to determine the baseline transcriptome and proteome for
the cells that have not been contacted with cigarette smoke. If the
analysis is conducted on a human, lung cells, cells of the oral
mucosa, and/or white blood cells obtained from a non-smoker can be
analyzed. A prototype cigarette with or without a filter,
manufactured as described herein, is then analyzed by contacting
the cells with the mainstream smoke generated from the prototype
cigarette, and conducting the same microarray analysis. In some
experiments the same cells of the smoker analyzed above can be
screened after a suitable period of smoking of the prototype
cigarette (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks after transition
from commercial brand to the prototype cigarette). By comparing the
results obtained from the microarray analysis generated from the
reference cigarette to the prototype cigarette and/or the mock
experiment, differences in the impact on the transcriptome and
proteome between the reference cigarette and the prototype
cigarette are revealed and a determination that the prototype
cigarette perturbs the transcriptome and proteome less than the
reference cigarette can be made.
[0253] The microarray analysis can be conducted by harvesting the
cells after smoke treatment, extracting the total RNA, generating
fluorescently labeled cDNA and applying the nucleic acids to a
microarray chip according to the manufacturer's protocol. RNA
integrity can be assessed using capillary gel electrophoresis. A
commercially available genome-scale oligonucleotide library
containing gene-specific 70-mer oligonucleotides representing
21,329 human genes can be used for microarray production (QIAGEN
Inc., Valencia Calif.). Hybridization is performed and the
microarray is scanned using a simultaneous dual color, 48-slide
scanner (Agilent technologies). Fluorescent intensity can be
measured using commercially available software. To determine the
differentially expressed genes, the analysis is confined to the set
of genes that are expressed above background. Optionally,
quantitative reverse transcriptase PCR can be performed for
specific genes to determine differences in the level of expression.
It should also be pointed out that the above techniques can be
employed to determine not only changes in RNAs that encode proteins
but also microRNAs, which have been linked to oxidative damage
and/or carcinogenesis.
[0254] In a similar fashion, proteomic analysis can be conducted.
Accordingly, proteins obtained from multiple types of cells (e.g.,
A549, NHBE, or isolated human cells from smokers, such as lung
cells, cells of the oral mucosa, or white blood cells), which have
been contacted with mainstream smoke generated from a prototype
cigarette, manufactured as described herein, mainstream smoke
generated from a reference cigarette (e.g., 2R4F or a commercially
available brand) and, optionally, mock treated cells (control) are
applied to metal affinity or weak cation exchange (WCX2) protein
chips to generate mass spectra by surface-enhanced laser
desorption/ionization (SELDI) time-of-flight mass spectrometry, or
similar techniques that use differential hybridization to protein
chips, separately or in combination with one or more types of mass
spectrometric analyses (e.g., time-of-flight). As discussed
previously, cells isolated from a smoker that has smoked a
commercially available brand can be obtained by conventional
techniques and analyzed. A mock or control experiment is,
optionally, performed to determine the baseline proteome for the
lung cells that have not been contacted with cigarette smoke. If
the analysis is conducted on a human, lung cells, cells of the oral
mucosa, and/or white blood cells obtained from a non-smoker can be
analyzed. In some experiments, the same cells of the same smoker
analyzed above can be screened after a suitable period of smoking
of the prototype cigarette (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks
after transition from a commercial brand to the prototype
cigarette). Protein peak identification and clustering are made
using Ciphergen Biomarker Wizard and Biomarker Pattern software. In
more experiments, the isolated proteins are separated using HPLC or
2-D gel electrophoresis prior to application of SELDI (or similar
techniques that use differential hybridization to protein chips,
separately or in combination with one or more types of mass
spectrometric analyses (e.g., time-of-flight)) and immunoassays can
be performed to identify the presence, absence, and quantity of
specific proteins.
[0255] Based on previous results, it is expected that the reference
cigarette will show transcriptome and proteome changes in a
substantial proportion of the genome (e.g., up to 10%) compared to
the mock experiment. It is also expected that some of the genes in
the cells contacted with smoke from the reference cigarette will be
under-expressed, as compared to the mock experiment. Lung cells
contacted with mainstream smoke generated from one or more of the
prototype cigarettes described herein are expected to show
transcriptome and proteome changes that resemble more closely the
transcriptome and proteome profiles generated in the mock
experiment, as compared to the reference cigarette. In this
fashion, one may identify reduced risk cigarette prototypes
prepared as described herein, as compared to reference cigarettes.
As is understood by those of skill in the art, the above example
can be performed using a variety of techniques in RNA, cDNA, and
protein analysis, which provide the same information (i.e., whether
the transcriptome and proteome of lung cells contacted with
mainstream smoke generated from a prototype cigarette is perturbed
less than the transcriptome and proteome of lung cells contacted
with mainstream smoke generated from a reference cigarette).
Example IV
[0256] A filter comprising activated carbon in a range of pore
volumes or pore sizes or both are tested in cigarette samples for
cloning efficiency as measured by the clonogenic assay and the
correlation of aldehydes with DNA DSBs as measured by the H2AX
assay, or an essay that measures perturbation of RNA transcriptome
or proteome, which assays are described above.
[0257] An activated carbon having a total pore volume of 0.1 mL/g
to 0.9 mL/g and/or an activated carbon having a certain percentage
of the activated carbon having a pore volume distribution of 0.1
mL/g to 0.9 mL/g, wherein the percentage of carbon having the pore
volume distribution is least about 50%, and/or an activated carbon
having an average pore diameter of 0.6 nm to 1.1 nm produce less
fewer DNA DSBs than a conventional cellulose acetate cigarette
filter, have greater cloning efficiency by showing a lower
percentages of cell death, and/or have fewer perturbations of RNA
transcriptome or proteome in human cells.
Example V
[0258] This example provides a stepwise program to gradually reduce
the amount of toxicants in cigarette smoke, which contribute to
biological insult, for example, DSBs, cell death or perturbation of
RNA transcriptome or proteome in human cells as compared to the
amount of biological insult induced by another cigarette, such as a
habitually consumed cigarette, allowing for a process to gain wider
consumer acceptance of a reduced risk cigarette.
[0259] A product that is being sold to/used by consumers can be
modified in a gradual or stepwise fashion in order to slowly alter
the composition of the cigarette to a reduced risk cigarette
without significantly changing the taste and/or consumer perception
of the product in such a way that would lead to consumer rejection
of the product. In one embodiment, the slowly altered composition
would start from a tobacco blend and/or filter design that receives
significant market acceptance, and would change to contain a
tobacco and/or filter that is configured to reduce the biological
insult, for example, DSBs, cell death or perturbation of RNA
transcriptome or proteome in human contacted by cigarette smoke,
relative to the original cigarette product. One cigarette with
reduced ability to cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome in human cells relative to the original cigarette product
is a cigarette containing a higher amount of air-cured Burley
tobacco relative to the original product. Another cigarette with
reduced ability to cause the induction of biological insult, for
example, DSBs, cell death or perturbation of RNA transcriptome or
proteome is a cigarette product containing a filter comprising
carbon and/or resin, for example, in the ratio of 1:1 to 4:1, or a
filter containing a carbon having the total pore volume described.
The degree to which the new, reduced-risk product possesses the
property of reduced biological insult, for example, DSBs, cell
death or perturbation of RNA transcriptome or proteome in human
cells can be assessed using the H2AX assay and/or the clonogenicity
assay and/or the RNA transcriptome or proteome assay provided in
Example I.
[0260] In one example, this step program includes a replacing a
cigarette habitually slowed by a tobacco user with a cigarette
having a filter comprising 40 mg of activated carbon. The 40 mg of
carbon cigarette is then replaced after approximately 3-6 weeks,
such as 30 days, with a cigarette having a filter comprising 60 mg
of activated carbon. In another example, the 60 mg carbon cigarette
is replaced after an additional approximately 3-6 weeks, such as an
additional 30 days, with a cigarette having a filter comprising 80
mg of activated carbon. In another example, the 80 mg carbon
cigarette is replaced after an additional approximately 3-6 weeks,
such as an additional 30 days, with a cigarette having a filter
comprising 100 mg of activated carbon.
[0261] The new reduced-risk product also is tested for consumer
acceptance. A focus or test group of smokers, such as smokers of
the original product, is provided with the new reduced-risk
product. The test group is then examined by questionnaire inquiring
about the comparison of the new reduced-risk product relative to
the original product. Alternatively, the test group is monitored
for its smoking habits while using the new reduced-risk product in
comparison with the smoking habits while using the original
products. Other known focus or test group assessments of new
products also can be implemented. The results of the focus or test
group assessment will indicate whether or not the new reduced-risk
product is likely to be accepted by the general population of
consumers of the original product. Test results indicative of a low
level of consumer acceptance of the change will indicate that the
product is to be redesigned to include a smaller and/or different
change in the tobacco, and/or carbon and/or resin in the filter.
Test results indicative of a high level of consumer acceptance of
the change will indicate that the product can be released to a
larger population, including an entire national population, to
thereby provide a product with high consumer acceptance and a
reduced risk of tobacco related disease.
[0262] The gradual modification of the product can be performed on
a product having the identical packaging, or a packaging
substantially similar and not obviously denoting a modification in
the cigarette product design. In this way, a consumer accustomed to
consuming a particular brand of cigarette product can experience a
gradual decrease in the exposure to compounds that cause the
induction of biological insult, for example, DSBs, cell death or
perturbation of RNA transcriptome or proteome, and other toxicants
without the consumer noting the modification of the product in such
a way as to reduce consumer acceptance of the new reduced-risk
product.
[0263] Various types of filter designs can be modified for use with
the methods tobacco blends described in the application generally
and in the Examples above. Below are some examples of such
filters:
Example VI
[0264] A filter element having a multiple section filter is
prepared wherein a filter plug at the mouth-end is made of
cellulose acetate tow and is about 7 mm in length, a general
adsorbent section adjacent to the filter plug consists of about 40
mg of activated coconut charcoal dispersed throughout
plasticizer-treated cellulose acetate tow cut to deliver a section
about 10 mm in length, and a selective adsorbent section adjacent
to the general adsorbent section consists of about 40 mg of
Duolite.TM. A7 dispersed throughout plasticizer-treated cellulose
acetate tow cut to deliver a section about 10 mm in length. The
filter is attached to a tobacco rod having a length of about 56.5
mm and containing about 617 mg of the tobacco blend described
above.
Example VII
[0265] A filter element is prepared as in Example IV except that
about 20 mg Duolite A7 is used in the selective adsorbent section
instead of 40 mg.
Example VIII
[0266] A filter element is prepared as in Example IV except that
about 60 mg Duolite A7 is used in the selective adsorbent section
instead of 40 mg.
Example IX
[0267] A filter element having a length of about 27 mm for use with
tobacco rod length of about 57 mm is prepared. The tipping material
circumscribes the length of the filter element and extends about 4
mm down the length of the tobacco rod.
[0268] The filter element of the cigarette has a general
multi-sectional configuration. Filter elements of this general type
are available from Baumgartner Inc., Switzerland. The cigarette has
a filter element comprising a 12 mm mouth-end cellulose acetate tow
(2.5 denier per filament/35,000 total denier) segment with 7%
triacetin, a 7 mm compartment filled with granular carbon available
as G277 (85 carbon tetrachloride activity and size 20.times.50
mesh) from PICA, and an 8 mm cellulose acetate tow (8.0/32,000)
tobacco-end segment with 7% triacetin.
[0269] A ring of laser perforations is provided around the
periphery of each cigarette about 13 mm from the extreme mouth-end
thereof. The perforations penetrate through the tipping paper and
plug wrap, and can be provided using a Laboratory Laser Perforator
from Hauni-Werk Korber & Co. KG.
Example X
[0270] Cigarettes are provided as described in Example VII, except
the filter element comprises an 8 mm mouth-end end cellulose
acetate tow (8.0/32,000) segment with 7% triacetin, a 7 mm
compartment filled with granular carbon available as G277 (85
carbon tetrachloride activity and size 20.times.50 mesh) from PICA,
and a 12 mm cellulose acetate tow (2.5/35,000) tobacco-end segment
with 7% triacetin.
Example XI
[0271] A filter element having two plugs with diameter of 7.5 mm
and lengths of 14.5 mm and 8 mm, respectively, is created. The
weights of the first plug and the second plug are around 77 and 45
mg, respectively. Powders are loaded in the cylindrical space
between these two plugs which have a dimension of about 7.5 mm
OD.times.4.5 mm in length. Carbon powder is Pica coconut-based
sample #99-2-3 with a median particle diameter of about 10
micrometers and an APS silica gel is used with specific surface
treatment with median particle diameter of about 5 micrometers. The
surface area of the APS silica gel is about 300 m.sup.2/g and the
surface area of the activated carbon is about 2000 m.sup.2/g.
Example XII
[0272] A filter element according to Example IX is created, except
the adsorbent powders are loaded in an amount of 25 mg in about 100
mg of semi-open micro-cavity fibers.
Example XIII
[0273] The following Table 1 illustrates several non-limiting
embodiments of cigarettes that can be used with the methods
described herein.
TABLE-US-00002 TABLE 1 Exemplary reduced risk cigarettes Blend
Paper Filter Burley 21 LA, Flue Cured, 80 CU 1.0% citrate, banded
25 mm length with 7 mm Oriental, Conventional cellulose acetate
mouth end Burley and 18 mm cellulose acetate with 40 mg carbon
(Dalmatian) tobacco end cigarettes were made 60% dilution Burley 21
LA, Flue Cured, 80 CU 1.0% citrate, banded 25 mm length with 7 mm
Oriental, Conventional cellulose acetate mouth end Burley and 18 mm
cellulose acetate with 60 mg carbon (Dalmatian) tobacco end
cigarettes were made 60% dilution Burley 21 LA, Flue Cured, 80 CU
1.0% citrate, banded 25 mm length with 7 mm Oriental, Conventional
cellulose acetate mouth end Burley and 18 mm cellulose acetate with
80 mg carbon (Dalmatian) tobacco end cigarettes were made 60%
dilution Burley 21 LA, Flue Cured, 80 CU 1.0% citrate, banded 25 mm
length with 7 mm Oriental, Conventional cellulose acetate mouth end
Burley and 18 mm cellulose acetate with 108 mg carbon (Dalmatian)
tobacco end cigarettes were made 60% dilution Flue Cured, Oriental,
26 CU 1.0% citrate 25 mm length with 7 mm Conventional Burley
cellulose acetate mouth end and 18 mm cellulose acetate with 108 mg
carbon (Dalmatian) tobacco end Flue Cured, Oriental, 21-41 26 CU
1.0% citrate 25 mm length with 7 mm Burley, cellulose acetate mouth
end and 18 mm cellulose acetate with 108 mg carbon (Dalmatian)
tobacco end Flue Cured, Oriental, 110 CU 1.0% citrate 25 mm length
with 7 mm Conventional Burley cellulose acetate mouth end and 18 mm
cellulose acetate with 108 mg carbon (Dalmatian) tobacco end Flue
Cured, Oriental, 21-41 110 CU 1.0% citrate 25 mm length with 7 mm
Burley, cellulose acetate mouth end and 18 mm cellulose acetate
with 108 mg carbon (Dalmatian) tobacco end Flue Cured, Oriental, 26
CU 1.0% citrate 25 mm length with 8 mm Conventional Burley
cellulose acetate mouth end, 5 mm cavity with 95 mg carbon and 12
mm cellulose acetate with 68 mg carbon cigarettes were made at 26%
dilution and 60% dilution Flue Cured, Oriental, 21-41 26 CU 1.0%
citrate 25 mm length with 8 mm Burley, cellulose acetate mouth end,
5 mm cavity with 95 mg carbon and 12 mm cellulose acetate with 68
mg carbon (Dalmatian) tobacco end cigarettes were made at 26%
dilution and 60% dilution Flue Cured, Oriental, 110 CU 1.0% citrate
25 mm length with 8 mm Conventional Burley cellulose acetate mouth
end, 5 mm cavity with 95 mg carbon and 12 mm cellulose acetate with
68 mg carbon (Dalmatian) tobacco end Flue Cured, Oriental, 21-41
110 CU 1.0% citrate 25 mm length with 8 mm Burley, cellulose
acetate mouth end, 5 mm cavity with 95 mg carbon and 12 mm
cellulose acetate with 68 mg carbon (Dalmatian) tobacco end Flue
Cured, Oriental, 170 CU 1.0% citrate 25 mm length with 8 mm
Conventional Burley cellulose acetate mouth end, 5 mm cavity with
95 mg carbon and 12 mm cellulose acetate with 68 mg carbon
cigarettes were made at 0% dilution Flue Cured, Oriental, 21-41 170
CU 1.0% citrate 25 mm length with 8 mm Burley, cellulose acetate
mouth end, 5 mm cavity with 95 mg carbon and 12 mm cellulose
acetate with 68 mg carbon (Dalmatian) tobacco end cigarettes were
made at 0% dilution Flue Cured, Oriental, 170 CU 1.0% citrate, non-
25 mm length with 8 mm Conventional Burley banded cellulose acetate
mouth end, 9 mm cavity containing carbon (124 mg) and 8 mm
cellulose acetate with 40 mg carbon (Dalmatian) tobacco end Flue
Cured, Oriental, 170 CU 1.0% citrate, non- 25 mm length with 9 mm
Conventional Burley banded cellulose acetate mouth end, 7 mm cavity
containing carbon (94 mg) and 9 mm cellulose acetate with cellulose
acetate tobacco end Flue Cured, Oriental, 170 CU 1.0% citrate, non-
25 mm length with 9 mm Conventional Burley banded cellulose acetate
mouth end, 7 mm cavity containing carbon (137 mg) and 7 mm
cellulose acetate with 40 mg carbon (Dalmatian) tobacco end Flue
Cured, Oriental, 170 CU 1.0% citrate, non- 30 mm length with 9 mm
Conventional Burley banded cellulose acetate mouth end, 12 mm
cavity containing carbon (179 mg) and 9 mm cellulose acetate with
cellulose acetate tobacco end Flue Cured, Oriental, 170 CU 1.0%
citrate, non- 25 mm length with 9 mm Conventional Burley banded
cellulose acetate mouth end, 12 mm cavity containing carbon (179
mg) and 9 mm cellulose acetate with 45 mg carbon (Dalmatian)
tobacco end Flue Cured, Oriental, 170 CU 1.0% citrate, non- 25 mm
length with 10 mm Conventional Burley banded cellulose acetate
mouth end, 10 mm cavity containing carbon (144 mg) and 10 mm
cellulose acetate with cellulose acetate tobacco end Flue Cured,
Oriental, 170 CU 1.0% citrate, non- 25 mm length with 10 mm
Conventional Burley banded cellulose acetate mouth end, 10 mm
cavity containing carbon (144 mg) and 10 mm cellulose acetate with
50 mg carbon (Dalmatian) tobacco end Blends contain 5-25% Oriental
tobacco, 10-50% Burley (conventional, 21-LA or 21-41 or
combinations thereof) and 10-50% Flue cured.
[0274] Various "reduced risk" technologies that are known in the
art can be adapted for use with aspects of the invention. For
example, the tobacco blends, low alkaloid/nicotine tobacco and
blends containing this tobacco, filter technology, methods of
cigarette design, methods of gradual adjustment of blends and
filter components in successive generations of products to adjust
consumer sensory perception over time as the amount of toxicant
consumed by the consumer is adjusted, and all other aspects
described herein can be employed with or combined with known
methodologies to develop improved cigarettes and methods of use
thereof.
[0275] The following Table 2 provides a listing of references, each
of which describe a technology that can be employed with or
combined with the teachings provided herein. Each of the references
in the Table 2 is incorporated herein by reference. In some
embodiments, the following technologies known in the art can be
modified by a teaching described herein:
TABLE-US-00003 TABLE 2 Priority Technology Patent Number Date
Author Aminofunctional Silica Gel US 20050161053 18-Mar-05 Xue,
Lixin et al. Aminofunctional Silica Gel U.S. Pat. No. 6,907,885
11-Feb-03 Xue, Lixin et al. Aminofunctional Silica Gel U.S. Pat.
No. 6,911,189 28-Jan-02 Koller et al. Carbon, surface modified WO
2006/070291 A2 29-Dec-05 Luan, Z. et al. Carbon, surface modified
US 20060174899 A9 22-Dec-03 Luan, Z. et al. Carbon bead US
20060180164 A1 11-Apr-06 Paine, J. B. at al. Carbon, monolithic
pore size US 20060201524 A1 19-Dec-06 Zhuang, et al. Carbon sieve
US 20060207620 A1 15-Mar-05 Plunkett, S. B. at al. Carbon with
metal catalyst US 20060231113 A1 13-Apr-05 Newberry, p. et al.
Carbon with Zeolite membrane US 20060260626 A1 20-Dec-05 Luan, Z.
et al. Carbon with Tobacco beads for flavor US 20070000505 A1
22-Feb-06 Zhuang, S. at al. Carbon, monolithic pore size US
20070000508 A1 29-Jun-05 Xue, Lixin at al. Cellulose acetate
microcavities U.S. Pat. No. 6,779,528 16-Apr-02 Xue, Lixin et al.
Carbon with metal catalyst U.S. Pat. No. 6,789,547 30-Oct-01 Paine,
J. B. et al. Carbon bead, cigarette WO 03/059096A1 9-Jan-02 Paine,
J. B. et al. Carbon, flavored WO 03/071886A1 22-Feb-02 Yang, Z. et
al. Carbon, Activated thread WO 03/086116 A1 12-Apr-02 Xue, Lixin
et al. Resin, Polyaromatic Filter WO 2004/019709A2 30-Aug-02 Xue,
Lixin et al. Carbon, Sorbent from resin WO 2004/097309 A1 2-Apr-03
Zhuang, S. et al. Carbon, Flavored WO 2006/064371 A1 15-Dec-04
Banerjea, A. et al. Carbon, apparatus for filling filter WO
2006/072089 A1 30-Dec-05 Atwell, C. G. et al. Carbon, surface
modified WO 2006/070291 A2 30-Dec-04 Luan, Z. et al. Carbon,
Activated with molecular sieve WO 2006/072889 A1 5-Jan-05 Luan, Z.
et al. Molecular Sieve, flavored WO 2006/085142 A2 22-Dec-04 Luan,
Z. et al. Carbon and molecular sieve WO 2006/097852 A1 15-Mar-05
Plunkett, S. B. et al. Carbon, templated WO 2007/026253A2 29-Jun-05
Xue, Lixin et al. Carbon, templated WO 2007/031876 A2 29-Jun-05
Xue, Lixin et al. Carbon beads WO 2007/061094A2 13-Dec-05 Karles,
G. et al. Carbon and molecular sieve US 20050133047 22-Dec-03
Fournier et al. Molecular Sieve, ampiphilic US 20050133048
22-Dec-03 Fournier et al. Molecular sieve, zeolite US 20050133049
22-Dec-03 Fournier et al. Molecular sieve, thiol functionalized US
20050133050 23-Dec-03 Fournier et al. Alumina with carbon and
zeolite US 20050133051 A1 22-Dec-03 Luan, Z. et al. particles
Aluminosilicate molecular sieve US 20050133052 17-Nov-04 Fournier
et al. Molecular sieves, Copper impregnated US 20050133053
22-Dec-03 Fournier et al. Zeolite on cut filler US 20050133054
22-Dec-03 Fournier et al. Silica gel, surface modified US
200502050102 27-Jan-05 Yang, Z. et al. Carbon, surface modified US
20060086366 19-Oct-05 Xue, Lixin et al. Resin, Polyaromatic, Filter
U.S. Pat. No. 6,863,074 30-Aug-02 Xue, Lixin et al. Filter, Carbon
& thread WO 02/069745 A1 22-Feb-01 Jupe et al. Filter, Shaped
Microcavity WO 03/047836A1 30-Nov-01 Xue, Lixin et al. Filter
manufacturing, central flavor unit WO 03/082558 29-Mar-02 Lanier et
al. Filter, Adsorbents surface modified WO 2004/010802A1 26-Jul-02
Thomas et al. Silica powder with active protein or WO 2004/047568A1
26-Nov-02 Desisto et al. catalyst Filter, cavity filling apparatus
WO 2004/052129A2 9-Dec-02 Atwell, C. G. et al. Shaped microcavity
manufacturing WO 2004/062396A2 6-Jan-03 Xue, Lixin et al. process
Filter, nanostructured fibril WO 2005/039329A1 27-Oct-03 Saoud et
al. Process for surface modification of WO 2006/051416A1 9-Nov-04
Karles, G. et al. filter Filter, tobacco beads WO 2006/090290 A1
24-Feb-05 Zhuang, et al. Segmented rod, tobacco beads WO
2006/100605 A1 21-Mar-05 Borgogron et al. Filter, hollow fiber WO
2007/054826 A2 4-Nov-05 Rasouli et al. Filter, hollow fiber US
20070074733 A1 4-Oct-05 Rasouli et al. Flavor capsule, liquid
release WO 2007/060543 A2 15-Aug-05 Besso, C. et al. Cigarette,
coaxial tobacco rod in WO 2007/069091 A2 12-Dec-05 Zhuang, et al.
tobacco rod Filter, bicarbonate treated fibers WO 2007/069093 A2
13-Dec-05 Xue, Lixin et al. Filter, bicarbonate treated fibers US
20070181141 A1 11-Dec-05 Xue, Lixin et al. Catalyst, CO reduction
WO 2007/083195 A2 13-Dec-05 Miser, D. et al. Carbon or APS silica
gel, electrostatic US 20050126481 27-Jan-05 Xue, Lixin et al.
process Molecular sieve, functionalized SBA US 20060130855
16-Sep-05 Luan, Z. et al. 15 Carbon, surface modified US
20060144410 30-Dec-05 Luan, Z. et al. Nanofiber, polysaccharide US
20060264130 20-Dec-05 Karles, G. et al. Sorbent with flavor
additives US 20060272662 A1 3-Feb-06 Jupe et al. Filter, flavor
capsule US 20070012327 2-May-06 Karles, G. et al. Cigarette,
coaxial tobacco rod in US 20070137667 11-Dec-06 Zhuang, et al.
tobacco rod Filter, carbon, catalyst and bypass US 20070169785
22-Dec-06 Gedevanishvili et al. channel Filter, concentric with
carbon loaded U.S. Pat. No. 5,622,190 15-Nov-94 Arterbery et al.
core Filter, concentric with TOW U.S. Pat. No. 5,746,230 31-May-95
Arterbery et al. Cigarette for electrical smoking system U.S. Pat.
No. 5,915,387 31-Dec-96 Baggett et al. APS Silical gel U.S. Pat.
No. 6,209,547 29-Oct-98 Koller et al. APS Silical gel U.S. Pat. No.
6,595,218 11-May-00 Koller et al. Carbon, flavored U.S. Pat. No.
6,761,174 22-Feb-02 Jupe et al. Filter, Shaped Microcavity U.S.
Pat. No. 6,762,768 20-Apr-01 Xue, Lixin et al. Cigarette, carbon or
catalyst tip U.S. Pat. No. 6,868,855 1-Dec-03 Shafer et al.
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Shafer et al. Cigarette with thermally collapsible U.S. Pat. No.
6,883,523 14-Feb-03 Dante et al. portion Microcavity, carbon or APS
silica gel U.S. Pat. No. 6,913,784 5-Jul-05 Xue, Lixin et al.
Filter, electrostatic process carbon or U.S. Pat. No. 6,919,105
6-Jan-03 Xue, Lixin et al. APS silica gel Recess filter with cavity
U.S. Pat. No. 7,243,659 12-Jul-00 Lecoultre et al. Cavity with
central filter element WO 03/049560A1 11-Dec-01 Lauenstein, M. et
al. Filter, monolithic segments WO 2004/086888 2-Apr-03 Zhuang, et
al. Filter, Carbon & thread WO 2006/082525A1 4-Apr-05 Jupe et
al. Filter, flavor capsule WO 2006/082529 A2 4-Feb-05 Karles, G. et
al. Filter, flavor capsule WO 2006/117697 A1 3-May-05 Karles, G. et
al. Filter, flavor encapsulated in PVA WO 2007/03694A2 30-Sep-05
Becker, U. et al. Filter, peel n sniff, microcapsules WO
2007/05210A2 1-Nov-05 Wyss-Peters et al. flavor Cigarette, hollow
tube with flavorant WO 2007/099408A2 21-Dec-05 Xue, Lixin et al.
Flavored carbon US 2040226569A1 16-Jun-04 Yang, Z. et al. flavor
thread process US 20050255978 A1 6-Jul-05 Lanier et al. Flavor
Capsule US 20060112964 9-Nov-05 Jupe et al. Flavor Capsule US
20060174901 10-Aug-06 Karles, G. et al. Flavor Capsule US
2006/0174901 A1 4-Feb-05 Karles, G. et al. Nanoparticles, Fe on
filler for CO WO 03/086115 A1 12-Apr-02 Li, P. et al. reduction
Nanoparticle catalyst, CO reduction WO 03/020058 A1 31-Aug-01 Li,
P. et al. Nanoparticles, Fe on filler for CO WO 03/086112 A1
8-Apr-02 Li, P. et al. reduction Nanoparticles, on Filler WO
2004/041008 A1 4-Nov-02 Li, P. et al. Nanoparticle, Cu and Ce WO
2004/052520 A2 9-Dec-02 Deevi et al. Nanoparticle, paper
oxyhydroxide WO 2005/039326 A2 27-Oct-03 Rasouli et al.
Nanoparticle, paper, filler, WO 2005/039327 A2 27-Oct-03 Reddy, B.
et al. oxyhydroxide CO, NO reduction Nanoparticle, paper, filler,
WO 2005/039328 A2 27-Oct-03 Rabiei, S. et al. oxyhydroxide CO, NO
reduction Nanoparticle, preparation WO 2005/039331 A2 28-Oct-03
Rabiei, S. et al. Nanoparticle, paper, filler, WO 2005/039332 A2
27-Oct-03 Reddy, B. et al. oxyhydroxide CO, NO reduction
Nanoparticle, paper, filler, Silver WO 2005/122805 A2 16-Jun-04
Rangaraj, S. et al. Nanoparticle, Au--Ce CO reduction WO
2006/046145 A2 25-Oct-04 Pillai et al. nanoparticles, taste WO
2007/010405 A1 20-May-05 Rasouli et al. Metal Nanowires, filler
selective WO 2007/072231A2 20-Dec-05 Luan, Z. et al. reduction
Encapsulated catalyst, CO, NO WO 2007/083245 A2 17-Jan-06
Gedevanishvili et al. reduction Catalyst, CO reduction, filter WO
2007/096785 A2 27-Feb-06 Deevi et al. Metal oxide, filler US
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oxyhydroxide US 20050155616 25-Oct-04 Rasouli et al. Metal Oxide,
CO reduction US 20050166935 25-Oct-04 Reddy, B. et al. Metal
catalyst on tobacco powder, CO US 20060196517 30-Jan-06
Gedevanishvili et al. reduction Copper oxide, zinc oxide, cerium
oxide US 20060289024 9-Mar-06 Deevi et al. CO reduction Copper
oxide catalyst method of US 20070014711 9-Mar-06 Deevi et al.
formation Au--Ce catalyst for CO reduction US 20070056601 A1
15-Jun-06 Pillai et al. Metal Nanowires, filler selective US
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reduction, filter, paper US 20070163612 A1 11-Dec-06 Miser, D. et
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Koller et al. Preparation of intermetallics WO 2004/110591A2
13-Jun-03 Deevi et al. Nanoparticle, Palladium WO 2006/046153 A1
25-Oct-04 Deevi et al. Cu--Ce catalyst for CO reduction US
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nanoscale particles US 20050166934 Oct. 25, 2004 Deevi et al. Paper
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Example XIV
[0276] This Example provides more information on the embodiments
described herein. Differentiation among American cigarettes relies
primarily on the use of proprietary tobacco blends, menthol,
tobacco substitutes, filter ventilation, paper porosity, and paper
additives. These characteristics substantially alter per cigarette
yields of tar and nicotine in standardized protocols promulgated by
government agencies. However, studies have concluded that due to
compensatory smoking phenomena, smokers inhale similar amounts of
tar and nicotine regardless of any cigarette variable, supporting
epidemiological evidence that all brands have comparable disease
risk. Consequently, it would be advantageous to develop assays that
realistically compare cigarette smoke (CS)-induced genotoxicity
regardless of differences in cigarette construction or smoking
behavior. One significant indicator of potentially carcinogenic DNA
damage is double-strand breaks (DSBs), which can be monitored by
measuring Ser139 phosphorylation on histone H2AX. Previously we
showed that phosphorylation of H2AX (defined as .gamma.H2AX) in
exposed lung cells is proportional to CS dose. Thus, we proposed
that .gamma.H2AX may be a viable biomarker for evaluating genotoxic
risk of cigarettes in relation to actual nicotine/tar delivery.
Here we tested this hypothesis by measuring .gamma.H2AX levels in
A549 lung cells exposed to CS from a range of commercial cigarettes
using various smoking regimens. Results show that .gamma.H2AX
induction, a central element of the mammalian DNA damage response,
provides an assessment of CS genotoxicity independent of smoking
topography or cigarette type. We conclude that .gamma.H2AX
induction shows promise as a valid bioassay for the evaluation of
conventional cigarettes and prototype reduced risk tobacco
products.
[0277] Although lung cancer is among the few malignancies for which
we know the primary etiological agent (i.e., cigarette smoke, CS),
the 5-year overall survival rate of 15% underscores the fact that
the existing clinical algorithm for diagnosis, staging, and
treatment of this disease remains inadequate (Diasio 2004; Herbst
et al. 2008; Jemal et al. 2008; Spira and Ettinger 2004). Moreover,
despite high public awareness of the associated health risks,
availability of prescription and nonprescription nicotine
replacement aids, and access to community-based cessation programs,
approximately 20% of adult Americans actively smoke (Mendez and
Warner 2008). This reluctance to quit has been attributed to a
variety of causes (Gehricke et al. 2007; Hughes 2003; Irvin et al.
2003; Stevens et al. 2008; Warner and Burns 2003). Consequently,
for those individuals who do not quit there is a pressing need to
discover innovative approaches that not only increase cessation
rates, but also reduce the health risks associated with smoking.
One controversial possibility regarding the latter idea is to
significantly reduce the toxicity and carcinogenicity of cigarettes
(Hatsukami et al. 2007; Hoffmann et al. 2001; Martin et al. 2004;
Richter et al. 2008). Although there is concern that reduced risk
cigarettes may influence some individuals to initiate smoking or
refrain from quitting (Hyland et al. 2003; Stratton et al. 2001),
it is conceded that since a zero tolerance policy for 45 million
current U.S. smokers is unrealistic, such cigarettes could be one
aspect of a diverse armamentarium designed to decrease smoking
related disease (Fowles and Dybing 2003; Gray and Henningfield
2004; Hatsukami et al. 2007; Hoffmann et al. 2001; Martin et al.
2004; Stratton et al. 2001). However, before any potential benefits
of reduced risk cigarettes can be objectively promoted, an
integrated genetic, cellular, and clinical model is needed to
define the physiological processes disrupted in smokers, and the
roles these perturbations play in lung carcinogenesis. One
important element of this model will be to develop a
straightforward CS exposure system that (i) is capable of directly
comparing the genotoxic potential of various cigarettes regardless
of differences in smoking behavior or cigarette construction
(Stratton et al. 2001), (ii) assesses a valid biomarker of cancer
risk in humans, and (iii) uses whole CS similar to that which the
smoker inhales to obviate the inherent deficiency of current in
vitro tests that examine sub-fractions of CS which cannot
accurately gauge complex synergistic interactions occurring upon
exposure to nonfractionated CS (Counts et al. 2005; DeMarini et al.
2008; Stephens 2007; Witschi et al. 1997; Witschi 2000).
[0278] Current understanding of lung carcinogenesis assumes that
polycyclic aromatic hydrocarbons, in particular benzo(a)pyrene
(BaP), and tobacco specific nitrosamines such as NNK
[4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] and
NNN(N'-nitrosonomicotine) are the main causative agents in CS
(Hecht 2003; Rubin 2001; Vineis et al. 2004). However, the
multi-step process driving malignant conversion of lung cells is
arguably multifaceted, requiring not only long term exposure to
these two classes of DNA-damaging agents, but also to the mix of
tumor promoters, mutagens, organic compounds and free radicals in
CS that collectively drive the accumulation of genetic defects at
many loci (DeMarini 2004; Fowles and Dybing 2003; Hatsukami et al.
2006a; Hecht 2003; Kelloff et al. 2000; Kim et al. 2005; Laugesen
and Fowles 2005a; Pankow 2001). These defects, observed in both
malignant and premalignant asymptomatic lung tissues, clearly point
to genomic instability as an early pivotal result of CS (Franklin
et al. 1997; Fujii et al. 2002; Guo et al. 2004; Minna et al. 2002;
Sikkink et al. 2003). The tumorigenic relevance of this instability
is revealed by studies indicating that smokers with less efficient
DNA repair capacities are at higher risk for developing lung cancer
(Godtfredsen et al. 2005; Kiyohara et al. 2006; Wu et al. 2007).
Since maintaining DNA integrity is essential for the prevention of
lung cancer, defining the types of damage that result in
chromosomal instability may clarify their contributory roles in
carcinogenesis and provide viable biomarkers of cancer risk in
CS-exposed individuals (Elliott and Jasin 2002; Hatsukami et al.
2006a; Masuda and Takahashi 2002; Mills et al. 2003; Sekido et al.
2003).
[0279] Among the most hazardous DNA lesions that can result in
genetic abnormalities that are hallmarks of lung cancers and other
malignancies are DNA double strand breaks (DSBs) (Masuda and
Takahashi 2002; Mills et al. 2003; Richardson and Jasin 2000; van
Gent et al. 2001). A sensitive means to detect DSB formation is to
assess histone H2AX phosphorylation. Histone H2AX, a variant of a
family of at least eight protein species of the nucleosome core
histone H2A (Thatcher and Gorovsky 1994; West and Bonner 1980),
becomes phosphorylated upon DSB induction (Rogakou et al. 1998;
Sedelnikova et al. 2002). Phosphorylation of H2AX on Ser 139 at
sites flanking DSBs is mediated by the DNA damage checkpoint
control genes ataxia telangiectasia mutated (ATM) (Burma et al.
2001; Lee and Paull 2007; Rogakou et al. 1998; Sedelnikova et al.
2002), ataxia telangiectasia and Rad3 related (ATR) (Furuta et al.
2003), and/or DNA-dependent protein kinases (DNA-PKs) (Park et al.
2003). However, most evidence concludes that ATM mediates H2AX
phosphorylation in response to DSB formation (Abraham and Tibbetts
2005; Bakkenist and Kastan 2003; Burma et al. 2001; Ismail et al.
2005; Kitagawa and Kastan 2005; Lee and Paull 2005). The
availability of antibodies to phosphorylated H2AX (denoted
.gamma.H2AX) allows for immunocytochemical detection of DSBs
(Rogakou et al. 1999). Upon DSB induction, the appearance of
.gamma.H2AX in chromatin manifests as discrete foci (Rogakou et al.
1999; Sedelnikova et al. 2002) with each focus representing a
single DSB (Rothkamm and Lobrich 2003; Sedelnikova et al. 2002).
The intensity of .gamma.H2AX immunofluorescence (IF) measured by
cytometry was reported to strongly correlate with the number of
DSBs (MacPhail et al. 2003) and has been proposed as a surrogate
for cell killing in viability assays (Banath and Olive 2003).
Multiparameter analysis of .gamma.H2AX IF and cellular DNA content
make it possible to relate the abundance of DSBs and extent of DNA
damage to the position of the cell in the cycle (Huang et al. 2003;
Huang et al. 2004).
[0280] These observations led us to determine that exposure of A549
pulmonary adenocarcinoma cells to whole CS induces H2AX
phosphorylation in a dose-dependent manner, and to conclude that,
since ATM activation precedes or occurs coincident with H2AX
phosphorylation, the increase in .gamma.H2AX expression reflects
induction of DSBs (Albino et al. 2004; Tanaka et al. 2007). We also
showed that CS-induced DNA damage in S phase cells was more
pronounced than in other cell cycle compartments (Albino et al.
2004; Albino et al. 2006; Jorgensen et al. 2004; Tanaka et al.
2007). Since CS depresses antioxidant potential, provokes oxidative
stress, and induces lung cell proliferation in smokers (De Flora et
al. 2003; Hiroshima et al. 2002; Lee et al. 2001; Muscat et al.
2004; Sekido et al. 2003), the increased sensitivity of replicating
cells to DSB formation may play a central role in the generation of
genotoxic events that contribute to pulmonary carcinogenesis.
Support for this idea comes from recent studies showing that
induction of a DNA damage response involving .gamma.H2AX expression
occurs in precursor lung lesions, and may be a dominant barrier
preventing genetic instability and malignant conversion (Bartkova
et al. 2005; Gorgoulis et al. 2005). Thus, in the current study we
tested the hypothesis that the H2AX phosphorylation assay could
provide a novel risk assessment method for cigarettes. The data
support the important conclusions that the .gamma.H2AX assay (i)
provides a means to objectively discriminate the ability of whole
CS to cause DSBs regardless of variability in tar and nicotine
yields due to differences in smoking topography or cigarette type;
and (ii) shows promise as a valuable method to assess the genotoxic
properties of tobacco products (Albino et al. 2004) and also offers
a means to design reduced risk cigarettes with less potency to
generate DSBs and possibly pulmonary malignancies. The following
sections provide more detail on the materials and methods used in
these experiments.
Cells and Cigarette Smoke Treatment
[0281] Whole cigarette smoke (CS) treatment and culturing of A549
human lung adenocarcinoma cells was performed as described
previously (Albino et al. 2004; Albino et al. 2006). Briefly, cells
were seeded into dual-chambered slides (Nunc Lab-Tek II, VWR
International, West Chester, Pa.) such that they were typically at
70% confluency at the time of exposure to CS. The cell culture
medium was replaced with 37.degree. C. Dulbecco's PBS (D-PBS)
containing calcium and magnesium (BioSource, Rockville, Md.) and
the slides were placed in a smoke exposure chamber designed to
deliver smoke uniformly diluted with 5% CO.sub.2 in air at a
constant flow rate of 500 cc/min. Whole CS was generated under
Federal Trade Commission (FTC)/International Organization for
Standardization (ISO) (ISO Standard 3308 2000; Kozlowski and
O'Connor 2002; National Cancer Institute 1996; Thomsen 1992)
smoking conditions (35.+-.0.3 cc puff, one puff every 60 seconds,
2-second puff duration with none of the ventilation holes blocked)
or under Massachusetts Department of Public Health (MDPH) smoking
conditions (45.+-.0.5 cc puff, one puff every 30 seconds, 2-second
puff duration with 50% of the ventilation holes blocked) unless
otherwise noted using a KC 5 Port Smoker (KC Automation, Richmond,
Va.). For the specific experiments described in FIG. 4 where the
effects of changing puff volume and number of cigarettes were under
investigation, the puff interval and puff duration were maintained
under the FTC/ISO protocol, i.e. 60 s puff interval and 2 s puff
duration, with no ventilation hole blocking. CS was delivered to
the smoke exposure chamber at a constant flow rate of 500 cc/min
except under MDPH conditions, when the CS was delivered at a
constant flow rate of 1000 cc/min. Cells were exposed for 20 min,
an exposure time that generates significant levels of .gamma.H2AX
in a dose dependent manner (Albino et al. 2004; Albino et al. 2006;
Tanaka et al. 2007). Mock-exposed cells were treated under
identical conditions as the CS-exposed cells except for the absence
of a cigarette in the smoking port. Following treatment or mock
treatment, the D-PBS covering the cells was aspirated and replaced
with 1 ml per chamber of fresh culture medium at 37.degree. C. The
cells were placed in the 37.degree. C., 5% CO.sub.2 incubator and
incubated for various times (i.e., 15 min to 2 hours) after which
the medium was aspirated and the cells fixed with 1%
paraformaldehyde by gently rocking the slides at room temperature
for 15 minutes. Following aspiration of the fixative, the chamber
slides were disassembled and the slides submerged in 50 ml conical
tubes filled with 70% ethanol for storage prior to .gamma.H2AX
analysis.
[0282] Immunocytochemical detection of .gamma.H2AX was carried out
as previously described (Halicka et al. 2005). CS treated, fixed
cells were rinsed twice in PBS and incubated in 0.1% Triton X-100
(Sigma) in PBS for 15 min at room temperature, followed by
incubation in a solution of 1% (w/v) bovine serum albumin (BSA;
Sigma) in PBS for 30 min to suppress non specific antibody binding.
The cells were then incubated in 100 .mu.l volume of 1% BSA
containing 1:200 dilution of phospho-specific (Ser-139) histone
H2AX mouse monoclonal antibody (mAb) (Millipore, Temecula, Calif.).
After overnight incubation at 4.degree. C., the slides were washed
twice with PBS and then incubated in 100 .mu.l of 1:100 dilution of
Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen/Molecular
Probes, Eugene, Oreg.) for 45 min at room temperature in the dark.
The cells were then counterstained with 1 .mu.g/ml
4,6-diamidino-2-phenylindole (DAPI; Invitrogen/Molecular Probes) in
PBS for 10 min. Each experiment was performed with an IgG control
in which cells were labeled only with secondary antibody, Alexa
Fluor 488 goat anti-mouse IgG (H+L) without primary antibody
incubation to estimate the extent of nonspecific binding of the
secondary antibody to the cells.
Spectrophotometric Determination of Smoke Concentration in Aqueous
Media.
[0283] Whole CS from a reference cigarette was collected under FTC
conditions onto a 44 mm Cambridge pad. The amount of smoke
collected on the pad was determined by weight of the total
particulate matter (TPM), which is directly related to
nicotine-free dry solids, more commonly known as tar. The TPM on
the Cambridge pad was dissolved in a known amount of methanol.
Aliquots of this solution were added to PBS and the resulting
solutions were placed into a quartz cuvette and the absorbance at
300 nm was measured using an Agilent Technologies 8453
Ultraviolet-Visible (UV-vis) spectrophotometer. These measurements
were used to construct a linear calibration curve according to the
method described by Sloan et al (Sloan and Curran 1981) for the
determination of cigarette filter extracts. For all samples, the
PBS solution was removed from the dual-chambered slides containing
the cell cultures post-CS exposure and placed into a quartz cuvette
and the absorbance was measured at 300 nm. The concentration of CS
in each sample was determined by comparing the absorbance of that
sample to that of the linear calibration curve. The concentration
of all samples was reported as .mu.g is TPM/ml.
Measurement of Cell Fluorescence by Laser Scanning Cytometry
[0284] Cellular green .gamma.H2AX and blue (DAPI) fluorescence
emission was measured using a Laser Scanning Cytometer (LSC; iCys;
CompuCyte, Cambridge, Mass.), utilizing standard filter settings;
fluorescence was excited with 488-nm argon ion and violet diode
lasers, respectively (Pozarowski et al. 2006). The intensities of
maximal pixel and integrated fluorescence were measured and
recorded for each cell. At least 3,000 cells were measured per
sample.
Statistical Analysis
[0285] To compare the changes in .gamma.H2AX immunofluorescence
intensity (IF), the mean fluorescence intensity (integral values of
individual cells) was calculated for cells in each phase of the
cycle by gating G.sub.1, S and G.sub.2M cells based on differences
in DNA content (DNA index, DI). The mean of the fluorescence values
for G.sub.1, S and G.sub.2M populations of cells in the IgG control
groups were then subtracted from the respective means of the CS
treated cells. Since histone and DNA content double as cells
proceed from G.sub.1 to G.sub.2 phase, the mean values of H2AX for
the S and G.sub.2M cell populations were divided by 1.5 and 2.0,
respectively, in order to express the degree of change in
.gamma.H2AX IF, i.e., the increase in phosphorylated protein per
unit of DNA. All experiments were run under identical instrument
settings. Data are presented as mean .gamma.H2AX IF of each cell
cycle compartment or, where not indicated, of the entire population
(G.sub.1, S and G.sub.2M). The standard deviation was estimated
based on Poisson distribution of the measured cell populations.
Each experiment was run at least in triplicate, and some
experiments were additionally repeated. The inter-sample variation
in the triplicates did not exceed the value of one standard
deviation of individual samples. Other statistical details are
given in the figure legends. The following section provides more
details on the results of these experiments.
Bivariate Analysis of .gamma.H2AX IF and DNA Content in A549 Cells
Exposed to CS.
[0286] Most cells, including A549 lung adenocarcinoma cells, have a
constitutive level of expression of .gamma.H2AX IF as seen in Panel
A of FIG. 10. However, analysis of A549 cells 1 or 2 h following 20
min of exposure to whole CS (generated under the FTC/ISO protocol
as described in methods section) from 2R4F cigarettes demonstrated
increased levels of .gamma.H2AX IF in cell cycle phase-dependent
fashion. Invariably, S phase cells appear to be most sensitive to
CS exposure (FIG. 10, Panel B). Eventually, all cells increase in
.gamma.H2AX IF (FIG. 10, Panel C) relative to mock-treated control
cells. As can be seen in the plot of mean .gamma.H2AX IF versus
time since CS exposure, there is a constant increase in .gamma.H2AX
IF (FIG. 10, Panel D) that remains cell cycle phase-specific.
[0287] We next assessed the induction of .gamma.H2AX in A549 lung
cells upon exposure to whole CS generated from three main styles of
commercially available American cigarettes that primarily differ in
terms of their FTC/ISO reported delivery levels of tar and
nicotine. These are termed full-flavor, light and ultra-light
cigarettes which deliver by the FTC/ISO smoking regimen 15 mg, 10
mg, and 6 mg of tar respectively. We assessed the same brand of
cigarettes available in these three styles from the leading U.S.
manufacturer of tobacco products. FIG. 11 (Panels A and B) shows
that upon exposing A549 cells for 20 min to CS generated from these
three cigarette styles using the FTC/ISO smoking protocol, there is
a corresponding linear increase in .gamma.H2AX levels (and thus of
DSBs) as the FTC/ISO associated tar deliveries increase from
ultra-light style (6 mg tar) to the full-flavor style (15 mg tar).
However, it was of interest to determine if these data resulted
from actual differences in tar toxicities of these three cigarette
styles or simply reflected a dose-response increase as tar yields
increased from 6 to 15 mg per cigarette. Therefore, we plotted
.gamma.H2AX values measured for these cigarettes against the
estimated tar delivery for each style of cigarette during a 20
minute smoke exposure. FIG. 12 clearly shows that the total
.gamma.H2AX induced by each of the three types of cigarettes was
essentially equivalent when expressed as a function of milligram of
delivered tar. Therefore, these data support the conclusions of
various epidemiological studies that cigarette toxicity of most
commercial U.S. cigarettes, at least as measured by the
.gamma.H2AX-DNA damage assay, is essentially identical per weight
unit of tar regardless of differences in tar delivery as measured
by the FTC/ISO protocol.
DSB Formation is Proportional to Actual Tar Delivery Across Varying
Machine Smoking Parameters.
[0288] FIG. 11 shows that .gamma.H2AX levels increase
proportionally to the dose of CS. However, an important question
raised by these data is whether the FTC/ISO protocol accurately
reflects .gamma.H2AX levels induced by CS exposures that more
realistically reflect actual human smoking habits. The quantitative
comparison of the yield of chemical constituents [e.g., tar,
nicotine, carbon monoxide (CO), etc.] in mainstream CS of
commercially manufactured cigarettes sold in the U.S. and other
countries relies on the FTC/ISO protocol. However, it has been
recognized that the FTC/ISO method does not reliably predict the
variations in actual constituent uptake by individual smokers and,
thus, cannot effectively compare the toxicological potential of
different cigarette brands and types (Benowitz 1996; Hecht et al.
2005; Jarvis et al. 2001; Kozlowski et al. 1980; Roemer et al.
2004; Stephens 2007). This conviction has been confirmed by
epidemiological data showing that despite the substantial reduction
in the average FTC/ISO machine-based tar (from 38 to 13 mg) and
nicotine (from 2.7 to 0.9 mg) yields per cigarette over the past 50
years (Hoffmann et al. 1997), there has been no significant
reduction in lung cancer risk among long-term smokers because of
the phenomenon of smoking compensation (Godtfredsen et al. 2006;
Harris et al. 2004; Hatsukami et al. 2006b; Hecht et al. 2005;
National Cancer Institute 2001; Patterson et al. 2003; Thun et al.
1997; Zacny et al. 1987). Numerous studies have concluded that
regardless of the tar and nicotine levels in different types of
cigarettes, smokers will subconsciously compensate for these
differences by altering their smoking behavior (e.g., increasing
puff frequency, inhaling more deeply, smoking more cigarettes per
day, and/or blocking filter ventilation holes) to sustain a
preferred nicotine dose with smokers compensating more with low
tar/low nicotine brands than high tar/high nicotine brands
(Benowitz 2001; Fowles and Dybing 2003; Hammond et al. 2005; Harris
2001; Harris 2004; Hecht et al. 2005). The inability of the FTC/ISO
method to characterize a predictable relationship between smoking
topography, product design, CS exposure levels, and health risks
prompted the development of more intense machine smoking protocols,
notably the MDPH and Health Canada (HC) regimens which, though
still flawed, more closely approximate the highly variable smoking
behavior of humans by increasing puff volume and frequency and,
most importantly, restricting filter ventilation of machine-smoked
cigarettes (Hammond et al. 2006; Hammond et al. 2007; Health Canada
1999; Rees et al. 2008; Stephens 2007). Since FIG. 3 shows that the
levels of .gamma.H2AX are essentially equivalent across styles of
cigarettes on a per milligram tar basis, we speculated that
.gamma.H2AX levels would also be equivalent regardless of
government-defined smoking machine parameters.
[0289] To test this prediction, we used the .gamma.H2AX assay to
test two industry standard research reference cigarettes
manufactured by the University of Kentucky to be representative of
tar and nicotine deliveries of two major styles of commercially
available cigarettes: a representative light style cigarette (2R4F
which delivers an FTC/ISO determined yield of 9.70 mg tar and 0.85
mg, and has 28% filter ventilation), and a representative
ultra-light low yield style cigarette (1R5F which delivers an
FTC/ISO determined yield of 1.67 mg tar and 0.16 mg nicotine, and
has 70% filter ventilation). Since no standardized machine smoking
protocol accurately reflects the myriad habits of consumers
(Hammond et al. 2006), we chose to increase tar delivery from
machine smoked cigarettes by manipulating two compensatory
mechanisms observed to occur in smokers of low yield cigarettes,
i.e., increasing puff volume or the number of cigarettes smoked.
Additionally, in order to plot the measured .gamma.H2AX levels
versus TPM delivery for cigarettes using different smoking
parameters other than those stipulated in the FTC/ISO protocol, we
incorporated a standard methodology for determining TPM levels in
solution based on UV-vis absorbance (as described in materials and
methods). This method proved to be particularly valuable at
comparing varying tar yield cigarettes in our cell exposure system,
which does not deposit the TPM onto a pad for weight measurement
but rather deposits the TPM from the cigarette directly onto
cultured cells throughout the exposure procedure. The concentration
of all samples is reported as .mu.g/ml.
[0290] FIG. 13 (Panel A) shows a linear relationship of .gamma.H2AX
induction with actual TPM delivery from the 1R5F ultra-light
cigarette as smoking intensity rises either by increasing the
number of cigarettes or increasing the puff volume. For example, in
Panel A, when four 1R5F cigarettes are machine smoked at the lowest
puff volume of 35 cc the UV-vis absorption of the PBS solution is
0.32 absorption units corresponding to 38.4 .mu.g/ml of TPM
delivered and a .gamma.H2AX fluorescence value of 98. However,
similar TPM deliveries and .gamma.H2AX fluorescence values were
observed upon machine smoking only two 1R5F cigarettes with an
increased puff volume of 75 cc, e.g. 0.31 absorption units
corresponding to 37.3 .mu.g/ml TPM and 93 .gamma.H2AX fluorescence
units. The effect of increasing puff volumes is again clearly
demonstrated upon machine smoking three 1R5F cigarettes with 55 cc
puffs (0.39 absorption units corresponding to 46.5 .mu.g/ml TPM
delivery and 125 .gamma.H2AX fluorescence units). Finally, when
three 1R5F cigarettes were smoked at the largest puff volume
studied (i.e., 75 cc), the UV-vis absorption increased to 0.52
absorption units corresponding to 62.8 .mu.g/ml TPM delivery and
210 .gamma.H2AX fluorescence units. Comparable data were derived
from a similar assessment of the 2R4F light cigarettes (see FIG. 4,
Panel B). FIG. 13, Panel C depicts the data points collected for
both experiments graphed on the same chart. Interestingly this
graph not only shows that the amount of DNA damage induced by TPM
delivery as measured by .gamma.H2AX is linear for both styles of
cigarettes and increases with increasing smoking intensities, but
also shows that there is relatively little difference in amount of
damage between a light style of cigarette when equivalent tar
deliveries for each cigarette are measured, despite the fact that
the light cigarettes deliver nearly six times the amount of tar as
the ultra-light low tar cigarettes according to FTC/ISO reported
values. For example, FIG. 13, Panel C shows similar DSBs as
measured by .gamma.H2AX when two light style cigarettes are smoked
at 55 cc puff volume versus three ultra-light style cigarettes
smoked at more intense puffing parameters of 75 cc puff volume
(i.e., .about.200 relative .gamma.H2AX fluorescence units for the
light and .about.210 .gamma.H2AX fluorescence units for the
ultra-light cigarettes). These data suggest that a smoker may
sustain equivalent amounts of DNA damage in the form of DSBs from a
low yield cigarette as from a higher tar cigarette simply by
increasing smoking intensity (i.e., increased number of cigarettes
and/or increased puff volume).
[0291] An additional important observation to note from the data
presented in FIG. 4 is that the linear regression line intercepts
the `y` (or TPM) axis at 0.15 absorbance at 300 nm for the 1R5F
(Panel A) and 0.19 absorbance for the 2R4F (Panel B) corresponding
to 17.8 and 22.4 .mu.g/ml of TPM delivery to the dual chamber slide
respectively. This observation indicates that there is a threshold
level of tar delivery to the cells that is required for detecting
tar-induced DNA damage by the .gamma.H2AX assay. Moreover, the data
show that despite the large difference in FTC/ISO tar yields,
ultra-light cigarettes induce similar amounts of DSBs as do light
cigarettes. This tar delivery/DNA damage detection threshold
provides a reliably convenient method for comparing various styles
of cigarettes directly when utilizing the same smoking protocol.
However, it should be noted that while this DSB initiation point
can be defined using any machine smoking protocol, the same
protocol must be used when doing direct comparisons of different
styles and/or types of cigarettes.
Market Survey Shows Varying .gamma.H2AX Induction Thresholds Across
Commercially Available Brands and Styles of Cigarettes.
[0292] Upon establishing the linear relationship between
.gamma.H2AX values, tar deliveries, and machine smoking parameters,
and confirming the utility of assessing the DNA damage detection
threshold, we performed a market survey on nine commercially
available products of different cigarette styles, i.e., light and
ultra-light, from different manufacturers and compared them using
the .gamma.H2AX assay in order to test the hypothesis that the
level of DNA damage does not correspond to the amount of FTC/ISO
reported tar values. We chose to machine smoke the market survey
cigarettes by the MDPH smoking protocol since it is a widely
accepted, compensatory testing regimen for filter ventilated
products, devised to provide a more realistic estimate of the
actual yields of CS constituents reaching the smoker (Counts et al.
2005; Hammond et al. 2006; Roemer et al. 2004). Representative data
are shown in FIG. 14, which plots the amount of .gamma.H2AX IF
measured for three different commercial brands of cigarettes
representing two different styles (Marlboro Light, Marlboro
Ultra-Light, and Camel Ultra-Light) with increasing numbers of
cigarettes used for CS exposure. As observed above (see FIG. 13),
the use of more intense smoking parameters for CS generation again
resulted in no substantive difference in the level of .gamma.H2AX
between light and ultra-light styles of cigarettes. We next plotted
.gamma.H2AX IF vs. TPM from the same experiment in order to
determine the threshold of DSB detection for the different
cigarettes as shown in FIG. 6. For the three different brands of
cigarettes tested in FIGS. 5 and 6 the regression plot shows a
linear relationship between TPM and .gamma.H2AX, and when
extrapolated to the `y` intercept, reveals that the minimal tar
deposition needed to reach the threshold of DSBs detection varies
from approximately 26 to 30 .mu.g/ml TPM.
[0293] Table 3 lists the complete survey of the nine samples
tested. The majority of the cigarettes tested fall between 25-40
.mu.g/ml TPM deliveries for the .gamma.H2AX induction threshold
with one cigarette brand (Doral) requiring as little as 12 .mu.g/ml
of TPM to induce .gamma.H2AX. The results with Doral ultra-light
cigarettes could be anticipated from the data shown in FIG. 12,
which argue that even very low tar yield cigarettes can generate
significant DSBs when the CS is generated under more realistic
intense smoking parameters such as with the MDPH protocol. While a
precise mechanism explaining the result with the Doral cigarettes
is unclear at present, one possibility is that the proprietary
tobacco blend used to manufacture this brand results in increased
DNA damage upon combustion.
TABLE-US-00004 TABLE 3 Damage Initiation .mu.g/ml (TPM) Average
error Marlboro Light .RTM. 30.95 5.02 Marlboro Ult .RTM. 26.28 1.53
Camel Ult .RTM. 29.66 0.37 Doral .RTM. 12.39 0.87 Basic Ult .RTM.
25.33 0.13 USA Gold Ult .RTM. 33.56 4.96 Kent Ult .RTM. 30.68 4.00
Winston Light .RTM. 40.10 4.53 Winston Ult .RTM. 34.81 2.96
[0294] Table 3: Damage initiation measurements of market survey
cigarettes. Threshold TPM level, estimated from linear regression
analysis, required for DNA damage initiation for nine commercially
available cigarettes from the market survey. All survey samples
were smoked according to MDPH protocol. The error reflects the
range of TPM required for damage initiation between two separate
measurements.
Filter Additives can Significantly Modulate the Extent of Tar
Deposition Required to Induce .gamma.H2AX.
[0295] CS is an aerosol that contains both gaseous (i.e., a vapor
phase) and suspended particulate material (i.e., a particulate
phase). The vapor phase is primarily a mixture of gases (e.g.,
nitrogen, oxygen, carbon dioxide, carbon monoxide, etc.) and
multiple volatile and semi-volatile organic species, while the
particulate phase consists of a wide variety of condensed organic
compounds, a number of which are carcinogens (Hecht 2003;
International Agency for Research on Cancer 1986). In addition,
both the vapor and particulate phases contain large amounts of free
radicals which cause direct and indirect stress to the cell (Green
and Rodgman 1996; Smith et al. 1997). We have previously shown that
the vapor phase of CS causes DNA damage and the induction of H2AX
phosphorylation primarily via a free radical mechanism (Albino et
al. 2004; Albino et al. 2006). High activity carbon is a selective
vapor phase removal filter component that can reduce the level of
free radicals as well as other hazardous vapor phase volatile
organic compounds (VOCs) (Fowles and Dybing 2003; Laugesen and
Fowles 2005a; Polzin et al. 2008). Thus, we were interested in
determining if specific modifications to the modern standard
cellulose acetate (CA) filter, which dominates the American
cigarette market, could decrease the level of DSBs regardless of
the smoking parameter used.
[0296] We constructed prototype cigarettes using a proprietary
blend of natural tobaccos similar in composition to commercially
available American blended cigarettes that deliver tar and nicotine
amounts typical of the light and ultra-light cigarette styles used
in the market survey. The prototypes under investigation also
contain filters consisting of cellulose acetate with one or more
selective removal filtration additives (i.e., high activity carbon
and/or polystyrene divinylbenzene primary amine containing resin)
in various ratios. The MDPH smoking protocol was used during the
exposure experiments in order to extrapolate the .gamma.H2AX
initiation points of the control 1R5F cigarette and the three
prototypes chosen for filter additive analysis. In order to provide
a direct comparison with the commercial samples from the market
study described in FIGS. 14 and 15, the FTC/ISO tar and nicotine
values for the control 1R5F and prototype cigarettes are provided
in Table 4. This table also lists the type of cigarette, additive
loading ratio and TPM amount required to initiate DSBs as
determined by the .gamma.H2AX assay. We note in Table 4 that
despite the fact that the prototype cigarettes delivered
substantially more tar than the 1R5F reference cigarette as
determined by FTC/ISO smoking regimen, it still required .about.4-6
times the amount of TPM delivery to initiate DSBs by the prototype
cigarettes than by the reference cigarette or the majority of
commercial cigarette brands and styles (e.g., see Table 3). These
data suggest that the prototype cigarettes may cause considerably
less DNA damage in the form of DSBs per mg of TPM even under a more
intense compensatory smoking regimen, and that the level of DNA
damage can be directly impacted by filtration additives that
selectively remove classes of toxic CS constituents.
TABLE-US-00005 TABLE 4 FTC/ISO FTC/ISO Additive Damage Cigarette
Nicotine Tar Loading (mg) initiation Type mg mg Carbon Resin
.mu.g/ml (TPM) R{circumflex over ( )}2 1R5F 0.16 1.67 0 0 10.7 0.99
A 0.45 5.4 100 0 52.9 0.96 B 0.48 5.9 70 30 65.8 0.89 C 0.64 8.3
100 30 83.5 0.98
[0297] Table 4: Comparison of physical characteristics and damage
initiation of 1R5F and prototype cigarettes A, B, and C. Comparison
of the abundance of TPM constituents, as determined by linear
regression analysis, required for damage initiation between
industry standard 1R5F ultra-light cigarette and three prototype
cigarettes, which contain varying amounts of carbon and/or resin
additives in the filter. The 1R5F and prototypes listed in table
were smoked according to MDPH protocol. FTC/ISO nicotine and tar
deliveries are provided for comparison purposes to the market
survey described in Table 3.
Tobacco Type can also Impact .gamma.H2AX Induction.
[0298] The construction of a commercial cigarette product
regardless of manufacturer is, in general, uniform across the
industry. For an American Blended Cigarette (ABC) the components
which make up the tobacco rod include flue-cured and burley
varieties of tobacco in the highest percentage of the total rod
with both varieties contributing approximately 25-35% each of the
total content of the tobacco rod. These percentages relate to about
50-70% of the total tobacco components in the cigarette depending
on manufacturer, brand and style. The remainder of the tobacco rod
content is most often comprised of lesser amounts of oriental
tobacco type, stem from the tobacco plant and reconstituted tobacco
filler material at various percentages. Upon observing the
significant decrease of .gamma.H2AX induction by the addition of
carbon and/or resin in the cigarette filter (Table 4), we decided
to perform a preliminary .gamma.H2AX assay investigation on
laboratory cigarettes constructed from either flue-cured only or
burley only tobacco components in order to assess the potential
differences in DNA damage capacity between two of the most
prevalent tobacco components that make up a traditional ABC blend.
The TPM delivery was measured via the spectrophotometric analysis
as described previously in order to normalize any potential tar
delivery discrepancies from the laboratory prepared samples. Three
flue-cured variety cigarettes and three burley variety cigarettes
were compared on the .gamma.H2AX assay in the same fashion as was
performed for the market study, i.e. each tobacco variety was
measured for .gamma.H2AX damage from one, two and three cigarettes
followed by a linear regression plot to determine the TPM amount
required to initiate DNA damage. Analysis of the results provided
an average intercept/damage initiation point from the three
different flue-cured variety cigarettes of 43.2 .mu.g TPM/mL
.+-.11.5 and 53.1 .mu.g TPM/mL.+-.5.0 for the three different
burley variety cigarettes. As mentioned above, every cigarette
tested in the market study is an ABC style and are not composed
entirely of one type of tobacco but rather tobacco blends made up
of various percentages of tobacco and filler material. The
preliminary data from the flue-cured and burley only cigarettes
suggests the possibility that CS derived from the combustion of
blended tobaccos and filler material is a complex process that is
not simply modeled by test cigarettes containing only one of the
two most prevalent tobacco components in an ABC style product.
These complex blending phenomena could explain the results observed
in Table 3 for the range of TPM amounts required to initiate DNA
DSBs for the various cigarette brands; however since the market
study cigarette blends are proprietary, further investigation into
this question is warranted. Furthermore, it is well known that even
within specific cultivars of tobacco grown in the US, the
characteristics of the tobacco plant itself is dependent on the
various growing practices and conditions within the assorted
growing regions and that some characteristics that influence the
burning properties of the tobacco vary not only between cultivars,
growing regions or growing practices but even within the same
cultivar, in the same region on an annual basis. This natural
product diversity is compounded further when imported tobaccos from
around the world are included in the equation. However, the
preliminary H2AX data obtained from test cigarettes constructed
from only one type of tobacco suggest that trend data could be
generated and possibly related to distinct types of tobaccos. We
will evaluate additional tobacco types, e.g. oriental, stem and
reconstituted tobacco filler material, and compare the leaf
position from different tobacco types in the same assays.
[0299] We have previously described the .gamma.H2AX assay and shown
it to be a robust and reliable in vitro method to measure the
levels of DSBs induced in cultured human lung cells after exposure
to CS (Albino et al. 2004). Based on these previous data, we
proposed that this assay could provide a novel biological risk
assessment method for cigarettes that is independent of brand or
type of cigarette, and which is proportional to CS dose and
nicotine/tar delivery regardless of smoking behavior. In the
current report, we provide further evidence to support this thesis
and show that the induction of DSBs by freshly prepared whole CS is
linear with increasing tar delivery, the latter modulated by time
of exposure and puff volume. The linear responsiveness of this
assay provides a simple and straightforward method to extrapolate
and predict the cytotoxic and genotoxic consequences along the
continuum of possible human smoking patterns. Moreover, it can
yield a direct cytotoxic or genotoxic measurement of the CS
delivered to smokers on a milligram of nicotine basis, and
demonstrates the increased toxicity of whole smoke versus the vapor
or tar phase portion. The data also indicate that specific
modifications to the cigarette filter, such as inclusion of high
activity carbon, can reduce the induction of DSBs by CS.
[0300] It is well known that many complex issues arise when
attempting to compare the relative toxicities of different
cigarettes (DeMarini et al. 2008). In principle, one collects smoke
from a cigarette by various methods and performs assorted chemical,
biological or toxicological tests with the collected CS. The
initial challenge which must be addressed is to define a set of
parameters to be used for smoke collection that best reflects
actual human smoking behavior, which has been shown to be highly
variable not only between individuals, but even for an individual
smoker during a typical day of consuming cigarettes (Hammond et al.
2006; Hammond et al. 2007; Mendes et al. 2008; Stephens 2007).
Thus, it has become clear that no machine smoking procedure can
accurately mimic the wide range of human smoking characteristics.
Therefore, standardized machine generated smoking regimens (e.g.
FTC/ISO, MDPH or HC) merely reveal the relative yield of tar and
nicotine contents of different cigarettes according to a convention
of analytical standards, but are not indicative of actual human
smoking behavior. Several factors that determine per-cigarette
yields of tar and nicotine delivery to smokers include the number
and size of puffs as well as filter ventilation hole blocking that
can occur with lower-yield cigarette products (National Cancer
Institute 1996). In particular, it has been noted that the single
most effective cigarette modification that reduces FTC/ISO-measured
CS emissions is filter ventilation, a process whereby small
perforations around the filter introduce fresh air during puffs
that dilutes the amount of tar, nicotine, CO, and other hazardous
CS constituents delivered to the smoker (Counts et al. 2005;
Kozlowski et al. 2006; Kozlowski and O'Connor 2002; Stephens 2007).
However, smokers tend to cover the holes when inhaling, resulting
in significantly more exposure to CS constituents than predicted by
the FTC/ISO machine smoking regimen which does not include blocking
of filter ventilation holes as part of its protocol. There is
consensus among public health advocates that filter vent hole
obstruction by smokers is the primary factor that equalizes the
toxicity of all conventional cigarettes (Hecht et al. 2005; Jarvis
et al. 2001; Kozlowski and O'Connor 2002; Roemer et al. 2004;
Strasser et al. 2005). Consequently, when performing research in
the area of cigarette-driven toxicity, the cigarette should be
viewed solely as a reservoir capable of delivering as much tar and
nicotine as the consumer desires regardless of the style of
cigarette or FTC/ISO-determined yields of these components.
[0301] DSB formation is a valid biomarker of potential cancer risk
since these lesions are a major type of DNA damage that, if
unfaithfully repaired, can lead to translocations and chromosomal
instability, two major mechanisms involved in the etiology of
multiple types of malignancies, including lung cancers (Bonner et
al. 2008; Elliott and Jasin 2002; Kiyohara et al. 2002; Masuda and
Takahashi 2002; Mills et al. 2003; Richardson and Jasin 2000; van
Gent et al. 2001; Wu et al. 2003). Thus, the rapid induction of
high levels of DSBs by CS may present an independent risk factor to
the smoker, especially since one recent study indicated that the
probability of a DSB being inaccurately rejoined is relatively low
when DSBs are spatially separated, but increased considerably when
multiple breaks coincide (Rothkamm and Lobrich 2002), while another
study showed that heavily clustered DSBs could lead to complex
genetic changes similar to those seen in human cancers (Singleton
et al. 2002). DSBs have not yet been sufficiently assessed in
pre-malignant lung lesions or in asymptomatic bronchial epithelium
of chronic smokers (Masuda and Takahashi 2002), but recent studies
observed an activated DNA damage response in precursor lung lesions
that included the expression of phosphorylated ATM and H2AX
suggesting that DSB formation is a likely early genomic event
(Bartkova et al. 2005). The presence of an active DNA damage
response is hypothesized to be a dominant anti-cancer barrier
preventing the cell from undergoing genetic instability and
malignant conversion (Bartkova et al. 2005). Cells may breach this
barrier and undergo progression towards a more disordered state if
there are complementary mutations or other defects in key genes
within this pathway (e.g., p53, Chk2, ATM, etc.)(Bartkova et al.
2005). Evidence to support the plausibility of this model in lung
cancer comes from at least three observations: (i) p53 mutations
are among the most common genetic defects in lung cancer (Pfeifer
and Hainaut 2003; Robles et al. 2002); (ii) Chk2 kinase expression
is down-regulated in non-small cell lung cancers due to promoter
methylation (Zhang et al. 2004); and (iii) a significant reduction
in DSB repair capacity is directly associated with increased
promoter methylation of specific genes in this damage-induced
pathway which, in turn, is associated with an elevated risk of lung
cancer (Leng et al. 2008). Collectively, one of the earliest and
potentially pivotal carcinogenic DNA defects caused by CS is the
induction of large numbers of DSBs.
[0302] Consequently, reducing formation of DSBs by any toxic
stimulus could potentially mitigate long-term risk. However, it
remains to be rigorously proven whether charcoal-containing filters
can attenuate to any significant degree the deleterious effects
that CS has on the respiratory and cardiovascular systems of
long-term smokers (Coggins and Gaworski 2008; Laugesen and Fowles
2005b; Scherer et al. 2006). A major limitation to assessing any
beneficial effects of carbon-containing filters is the dearth of
reliable in vitro bioassays or in vivo dose-responsive biomarkers
that correlate with future disease risk (Coggins and Gaworski 2008;
DeMarini et al. 2008; Hatsukami et al. 2004; Hatsukami et al.
2006a; Hecht 1996; Laugesen and Fowles 2006; Rees et al. 2008). The
data presented here indicate that .gamma.H2AX assay can
discriminate a reduction in .gamma.H2AX foci (and presumably DSBs)
when using prototype cigarettes with carbon-containing filters
despite delivering substantially more tar than conventional
cigarettes, suggesting that prototype cigarettes which cause
considerably less DNA damage per mg of TPM can be quantitatively
assessed. The relevance of this assay stems from the fact that the
habitual U.S. smoker consumes an average of 16.6 cigarettes/day
(Centers for Disease Control and Prevention 2004), and probably
inhales somewhere between 100-125 puffs/day of a highly complex
mixture of reactive gases and suspended particulate matter with
carcinogenic and toxic potential that can cause direct and indirect
damage to lung cells in a repeating cycle of tissue injury and
repair that contributes to tumor induction. Yet despite a range of
studies indicating that the chemical composition (Hecht et al.
2005) and overall disease risk of CS is relatively constant across
different brands and styles of cigarettes (Godtfredsen et al. 2006;
Harris et al. 2004; Hecht et al. 2005), most long-term smokers do
not develop lung cancer. Thus, understanding the nature of DNA
damaging events induced by CS may clarify the molecular basis of
differential sensitivities among smokers. Since many CS
constituents manifest their carcinogenic potential by attacking
DNA, it is possible that the nexus of chronic CS-induced DSBs in
smokers coupled with specific haplotypes that mute prompt and
efficient DNA repair can result in a cycle of amplification and
progression of genomic defects that instigate a range of pulmonary
diseases including lung cancer (Godtfredsen et al. 2005; Kiyohara
et al. 2002; Shields 2002; Spitz et al. 1999; Wu et al. 2003; Wu et
al. 2004). The ability to directly relate DSB formation to CS
exposure using .gamma.H2AX as a surrogate biomarker may be an
important addition to the battery of available DNA damage tests
used to assess the genotoxic properties of tobacco products (Albino
et al. 2004) since it potentially reflects the formation of a type
of DNA damage responsible for chromosomal abnormalities linked to
the genesis of lung cancers and other human malignances.
[0303] All references cited herein are incorporated herein by
reference in their entirety. To the extent publications and patents
or patent applications incorporated by reference contradict the
disclosure contained in the specification, the specification is
intended to supersede and/or take precedence over any such
contradictory material.
[0304] The term "comprising" as used herein is synonymous with
"including," "containing," or "characterized by," and is inclusive
or open-ended and does not exclude additional, unrecited elements
or method steps.
[0305] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification and claims are
to be understood as being modified in all instances by the term
"about." Accordingly, unless indicated to the contrary, the
numerical parameters set forth in the specification and attached
claims are approximations that may vary depending upon the desired
properties sought to be obtained by the preferred embodiments. At
the very least, and not as an attempt to limit the application of
the doctrine of equivalents to the scope of the claims, each
numerical parameter should be construed in light of the number of
significant digits and ordinary rounding approaches.
[0306] The above description discloses several methods and
materials of the preferred embodiments. This invention is
susceptible to modifications in the methods and materials, as well
as alterations in the fabrication methods and equipment. Such
modifications will become apparent to those skilled in the art from
a consideration of this disclosure or practice of the invention
disclosed herein. Consequently, it is not intended that this
invention be limited to the specific embodiments disclosed herein,
but that it cover all modifications and alternatives coming within
the true scope and spirit of the invention as embodied in the
attached claims.
* * * * *