U.S. patent application number 10/815995 was filed with the patent office on 2005-01-27 for therapeutic and diagnostic methods dependent on cyp2a enzymes.
This patent application is currently assigned to Nicogen, Inc.. Invention is credited to Sellers, Edward Moncrieff, Tyndale, Rachel F..
Application Number | 20050020641 10/815995 |
Document ID | / |
Family ID | 34084801 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020641 |
Kind Code |
A1 |
Sellers, Edward Moncrieff ;
et al. |
January 27, 2005 |
Therapeutic and diagnostic methods dependent on CYP2A enzymes
Abstract
A method of regulating the activity of human cytochrome P450
isozyme CYP2A6 to control nicotine metabolism or decrease the
production of carcinogens from procarcinogens, such as those
present in tobacco smoke, in an individual by selectively
inhibiting CYP2A6. Various prophylactic (i.e., prevention and
treatment) compositions and methods are also described, including
an improved oral nicotine composition and method comprising the use
of nicotine together with an inhibitor of the CYP2A6 enzyme.
Furthermore, it has been discovered that the presence in an
individual of a mutant allele of human cytochrome P450 enzyme
CYP2A6 (referred to throughout this specification as "CYP2A6" for
brevity) is predictive of an individual who: (1) has a decreased
risk of becoming a smoker, (ii) will smoke less if he/she becomes
dependent, and/or (iii) may be at relatively lower risk for cancer
due to both decreased smoke exposure and decreased CYP2A6-mediated
activation of tobacco smoke and other procarcinogenic substrates.
This invention provides diagnostic methods for predicting tobacco
dependence risk and risk for cancers related to CYP2A6 substrates
in an individual by analyzing for the presence of a mutant genotype
for human cytochrome P450 enzyme CYP2A6 in an individual, ranging
from gene duplication (multiple copies of CYP2A6) to single or even
no copies due to null alleles or gene deletion.
Inventors: |
Sellers, Edward Moncrieff;
(Toronto, CA) ; Tyndale, Rachel F.; (Toronto,
CA) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP
INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Nicogen, Inc.
St. Lurent
CA
|
Family ID: |
34084801 |
Appl. No.: |
10/815995 |
Filed: |
April 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10815995 |
Apr 2, 2004 |
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09584669 |
Jun 1, 2000 |
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09584669 |
Jun 1, 2000 |
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PCT/CA98/10193 |
Dec 1, 1998 |
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60067020 |
Dec 1, 1997 |
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60067021 |
Dec 1, 1997 |
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60084847 |
May 8, 1998 |
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60107392 |
Nov 6, 1998 |
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Current U.S.
Class: |
514/343 |
Current CPC
Class: |
A61K 31/4525 20130101;
A61K 31/131 20130101; A61K 31/4439 20130101; A61K 31/27 20130101;
A61P 25/24 20180101; A61K 31/37 20130101; A61K 31/131 20130101;
A61P 25/32 20180101; A61K 31/4178 20130101; A61K 31/37 20130101;
A61K 31/351 20130101; A61P 25/18 20180101; A61K 31/122 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61P 25/16 20180101; A61K
31/351 20130101; A61K 31/27 20130101; A61K 31/4439 20130101; A61K
31/7032 20130101; A61P 25/26 20180101; A61K 31/122 20130101; A61K
31/4178 20130101 |
Class at
Publication: |
514/343 |
International
Class: |
A61K 031/4439 |
Claims
1. A method for the treatment of a condition requiring a reduction
in the activity of a CYP2A enzyme comprising administering to an
individual in need thereof an effective amount of one or more
substances selected from the group consisting of (i) a substances
which inhibits CYP2A activity; (ii) a substances which inhibit
transcription of a gene encoding CYP2A, (iii) a substance which
inhibits translation of a gene encoding CYP2A, and (iv) a
substances which deletes all or a portion of the gene encoding
CYP2A.
2. The method according to claim 1 wherein said CYP2A enzyme is
CYP2A6.
3. The method according to claim 33 wherein said condition is
smoking and inhibition of the CYP2A enzyme inhibits the conversion
of nicotine to cotinine.
4. (Cancelled)
5. The method according to claim 3, wherein said nicotine is
formulated for oral administration.
6. The method according to claim 5, wherein said one or more
substances and said nicotine are formulated in a single
composition.
7. The method according to claim 1, wherein said substance is
psoralen, tranylcypromine, pilocarpine, coumarin, chromone,
esculetin, phenelzine, paroxetine, selegiline, pargyline, or
combinations thereof.
8. The method according to claim 1, wherein said substance
is-tranylcypromine.
9. The method according to claim 8, wherein tranylcypromine is
administered in an amount from 0.1 mg to 80 mg.
10-11. (Cancelled)
12. The method according to claim 1, further comprising
contemporaneously administering an inhibitor of CYP2B6.
13. The method according to claim 1, wherein said condition is
cancer and inhibition of the CYP2A enzyme inhibits the metabolism
of a procarcinogen to a carcinogen.
14. The method according to claim 13, wherein said procarcinogen is
a N-nitrosodialkylamine selected from the group consisting of
N-nitrosodiethylamine, N- nitrosodimethylamine, and
4-(methylnitrosamino)-1-) (3-pyridyl)-1-butanone.
15. The method according to claim 1, wherein said individual is
suffering from a condition selected from (i) addiction to tobacco,
(ii) risk of developing an addiction to tobacco, (iii) risk of
developing a smoking associated cancer, and (iv) exposure to one or
more compounds which are converted to carcinogens by CYP2A6.
16. A composition comprising an effective amount of one or more
substances in admixture with a suitable diluent or carrier wherein
said substance is selected from the group consisting of (i) a
substance which inhibits CYP2A activity; (ii) a substance which
inhibits transcription of a gene encoding CYP2A, (iii) a substance
which inhibits translation of a gene encoding CYP2A, and (iv) a
substance which deletes all or a portion of the gene encoding
CYP2A.
17. The composition of claim 16 wherein said CYP2A enzyme is
CYP2A6.
18. The composition of claim 16, wherein said condition is smoking
and inhibition of the CYP2A enzyme inhibits the conversion of
nicotine to cotinine.
19. The composition of claim 18, wherein said composition further
comprises nicotine.
20. (Cancelled)
21. The composition of claim 16, wherein said substance which
inhibits CYP2A is selected from psoralen, tranylcyprornine,
pilocarpine, coumarin, chromone, esculetin, phenelzine, paroxetine,
selegiline and pargyline.
22. A method for enhancing the effectiveness of a nicotine
replacement therapy comprising contemporaneously administering to
an individual in need thereof (a) nicotine and (b) one or more
substances selected from the group consisting of (i) a substance
which inhibits CYP2A activity; (ii) a substance which inhibits
transcription of a gene encoding CYP2A, (iii) a substance which
inhibits translation of a gene encoding CYP2A, and (iv) a substance
which deletes all or a portion of the gene encoding CYP2A.
23. The method according to claim 22, wherein said substance which
inhibits CYP2A6 and is selected from psoralen, tranylcypromine,
pilocarpine, coumarin, chromone, esculetin, phenelzine, paroxetine,
selegiline and pargyline.
24. A kit for use in the method of claim 22 comprising (a) nicotine
and (b) one or more substances selected from the group consisting
of (i) a substance which inhibits CYP2A activity, (ii) a substance
which inhibits transcription of a gene encoding CYP2A, (iii) a
substance which inhibits translation of a gene encoding CYP2A, and
(iv) a substance which deletes all or a portion of the gene
encoding CYP2A.
25. A method for determining the risk of an individual becoming a
smoker or for developing cancer comprising determining the genotype
or phenotype of a CYP2A allele in the individual wherein the
presence of a mutant allele is predictive of a decreased risk of
smoking.
26. (Cancelled)
27. A method according to claim 25, wherein said CYP2A enzyme is
CYP2A6.
28. A method according to claim 25, comprising analyzing a
DNA-containing bodily sample from the individual for the presence
of a mutant allele of human cytochrome P450 isozyme CYP2A6.
29. A method according to claim 28, wherein said DNA-containing
bodily sample is selected from a bodily fluid, a blood sample,
blood plasma, and peripheral leukocytes.
30. A kit for use in the method of claim 25, comprising means to
identify a mutant allele of CYP2A6.
31. The method of claim 25, the method comprising the steps of:
administering a dose of a CYP2A6 substrate to the individual and
determining in a bodily sample from the individual the level of
said CYP2A6 substrate or a metabolite of said CYP2A6 substrate.
32. A kit for use in the method of claim 31, comprising a CYP2A6
substrate and means for quantifying said CYP2A6 substrate or a
metabolite of said CYP2A6 substrate.
33. A method according to claim 2, further comprising
contemporaneously administering nicotine.
34. The method according to claim 1, wherein said substance is
coumarin.
35. The method according to claim 34, wherein coumarin is
administered in an amount from 1 mg to 1000 mg.
36. A composition comprising nicotine and an effective amount of
one or more substances in admixture with a suitable diluent or
carrier wherein said one or more substances are selected from the
group consisting of (i) a substance which inhibits CYP2A activity;
(ii) a substance which inhibits transcription of a gene encoding
CYP2A, (iii) a substance which inhibits translation of a gene
encoding CYP2A, and (iv) a substance which deletes all or a portion
of the gene encoding CYP2A.
37. The method according to claim 1, wherein said individual is
suffering from a condition selected from malignant disease,
psychosis, schizophrenia, Parkison's disease, anxiety, depression,
alcoholism, opiate dependence, memory deficits, ulcerative colitis,
and cholinergic deficits.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods and compositions for
regulating nicotine metabolism in an individual; methods and
compositions for enhancing nicotine replacement therapies; methods
and compositions for diagnosing tobacco risk dependence and risk
for cancers and methods for treating or preventing cancer.
REVIEW OF THE ART
[0002] Nicotine is one of the most widely used psychoactive drugs
in the world. The World Health Organization reports that there are
currently in excess of 1 billion smokers worldwide, or roughly
one-third of the global population aged 15 years and older. It is
well established that smoking is associated with a higher incidence
of many diseases, including various types of cancers, respiratory
diseases, cardiovascular diseases, gastrointestinal disorders, as
well as many other medical complications (Lee et al., Arch. Intern.
Med., "Cigarette smoking, nicotine addiction, and its pharmacologic
treatment," 153(1): 34-48 (1993)).
[0003] Nicotine is the primary compound present in tobacco that is
responsible for establishing and maintaining tobacco dependence
(Henningfield et al., J. Pharmacol. Exp. Ther., "Abuse liability
and pharmacodynamic characteristics of intravenous and inhaled
nicotine," 234(1): 1-12 (1985)). Specifically, it has been
established in the art that dependent smokers adjust their smoking
behaviour to maintain central nicotine levels (McMorrow M J, et
al., "Nicotine's role in smoking: an analysis of nicotine
regulation," Psychological Bulletin. 93(2):302-27 (1983); Russell M
S H, "Nicotine intake and its regulation by smokers. Tobacco
smoking and nicotine," Advances in behavioural biology, Martin W R,
et al., New York, Plenum Press, 31:25-50 (1987)). It has been
further established that: (i) smoking increases if nicotine content
in cigarettes is decreased (Benowitz N L, "Drug Therapy.
Pharmacologic Aspects of Cigarette Smoking and Nicotine Addiction,"
New Engl. J. Med., 319(20): 1318-30 (1988)), (ii) smoking increases
if nicotine excretion is increased by urine acidification (Benowitz
N L, "The Use of Biologic Fluid Samples in Assessing Tobacco Smoke
Consumption," NIDA Res. Monogr., 48:6-26 (1983)), and (iii) smoking
decreases with administration of nicotine via concurrent I.V. or
patch nicotine (Benowitz, N L et al., "Nicotine Metabolic Profile
in Man: Comparison of Cigarette Smoking and Transdermal Nicotine",
J. Pharmacol. Exp. Ther., 268(1):296-303 (1994); and Benowitz, N L
et al., "Intravenous Nicotine Replacement Suppresses Nicotine
Intake From Cigarette Smoking", J. Pharmacol. Exp. Ther.,
254(3):1000-5 (1990)).
[0004] In light of the key role of nicotine in producing tobacco
dependence and regulating smoking behaviour, it is important to
understand the pattern of nicotine metabolism and the sources of
variation of this metabolism in humans.
[0005] In humans, 60-85% of nicotine is metabolized to the inactive
metabolite cotinine (Benowitz, et al. 1994). The cytochrome P450
(CYP) system has been implicated in the metabolism of nicotine.
Evidence for CYP involvement in nicotine metabolism has come from
rat liver studies in which reconstituted purified CYPs, and
specific antibodies were shown to inhibit nicotine metabolism. In
particular, rat studies have shown that phenobarbital inducible
CYPs (i.e., the CYPs; -2B1, -2B2, -2C6, and -3A2) are involved in
nicotine metabolism. Of 12 human CYPs forms tested, CYP2B6 showed
the highest nicotine oxidase activity while CYP2E1 and CYP2C9
showed intermediate levels (Flammang et al., "Nicotine metabolism
by cDNA-expressed human cytochrome P450s," Biochem. Arch., 8:1-8
(1992)). cDNA studies have implicated CYP2B6, CYP2C9, CYP2D6 and
CYP2E1 and have provided a possible role for CYP2A6 in nicotine
metabolism in isolated expression systems (Flammang et al., 1992;
McCracken et al., "Cotinine formation by cDNA-expressed human
cytochromes P450," Med. Sci. Res., 20:877-878 (1992)).
[0006] In copending International patent application S.N.
PCT/CA97/00506 (filed Jul. 17, 1997), the contents of which are
hereby incorporated by reference, the present inventors teach that
the genetically polymorphic CYP2A6 enzyme is the major enzyme
responsible for this metabolic conversion. In human populations
there is considerable interindividual variability in hepatic CYP2A6
function measured in vivo and in vitro (Yamano S, et al., "The
CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human
liver microsomes," Biochemistry, 29:1322-1329 (1990); Cholerton S,
et al., "Comparison of a novel thin-layer
chromatographic-fluorescence detection method with a
spectrofluorometric method for the determination of
7-hydroxycoumarin in human urine," Journal of Chromatography,
575(2):325-30 (1992); Rautio A, et al., "Interindividual
variability of coumarin 7-hydroxylation in healthy volunteers,"
Pharmacogenetics 2(5):227-33 (1992); and Iscan et al.,
"Interindividual variability of coumarin 7-hydroxylation in a
Turkish population," Eur. J. Clin. Pharmacol. 47(4):315-318
(1994)).
[0007] Tobacco products are vehicles for the delivery of nicotine
to the bloodstream which quickly carries nicotine to the brain and
other organs. Nicotine produces many physiological and behavioural
effects, including alteration of brain chemistry and function,
which leads to an individual's dependence on nicotine. Dependent
smokers adjust their smoking behaviour to regulate nicotine in the
brain and body. Evidence includes increased smoking if nicotine
content in cigarettes is decreased (Benowitz 1988), increased
smoking if nicotine excretion is increased by urine acidification
(Benowitz 1983), and decreased smoking with concurrent I.V. or
patch nicotine (Benowitz, et al. 1994; Benowitz N L, et al.
1990).
[0008] While the art has made strides in gaining an understanding
of the pattern of nicotine metabolism and the sources of variation
of this metabolism in humans, there is still room for improvement.
One area which has received little or no attention is in the
diagnosis of risks for smoking and tobacco-related cancers, for
example in non-smokers of relatively young age. In particular, it
would be desirable to have a means by which it would be possible to
readily identify individuals who: (i) have a decreased risk of
becoming smokers, (ii) smoke less if they become dependent, and/or
(iii) may be at relatively lower risk for cancer due to both
decreased smoke exposure and decreased enzyme-mediated activation
of tobacco smoke procarcinogens.
[0009] Other than nicotine dependence as a result of tobacco use,
nicotine itself is not considered hazardous, namely it is not
considered to be a causative agent in cancer and heart and lung
disease. It is the other products which are found in tobacco
products which are considered to be harmful, including combustion
products such as carbon monoxide, gases and tar.
[0010] Nicotine replacement therapies (also referred to throughout
this disclosure as "NRT's") are used to deliver nicotine to
individuals in an attempt to assist an individual in abstaining
from tobacco products. Recently, in a United Nations Conference on
Trade and Development, entitled "Roundtable on Social and Economic
Aspects of Reduction of Tobacco Smoking by Use of Alternative
Nicotine Delivery Systems", Sep. 22-24, 1997, in an attempt to
reduce tobacco-related morbidity and mortality, it was recommended
that nicotine replacement therapies be made more easily available
than tobacco.
[0011] Smoking tobacco products amount to a rapid delivery
mechanism of nicotine to the bloodstream since almost all of the
nicotine absorbed from tobacco smoke reaches systemic circulation
without the need to initially pass through liver. For this reason,
conventional nicotine replacement therapies have been based on the
use of a delivery system (e.g., transdermal, etc.) which will
systemically deliver nicotine.
[0012] Unfortunately, current commercially available NRT's are
relatively inconvenient to use and administer, and are not liked by
many patients. For example, transdermal (e.g., transdermal, chewing
gum, etc.) NRT's are associated with occasional skin irritation and
chewing gum (and other buccal delivery systems) NRT's are perceived
as having a bad taste. Further, transdermal and chewing gum NRT's
are plagued by the delivery of inconsistent nicotine levels to the
patient. Still further, alternative delivery NRT systems such as
inhalers and nasal sprays have failed to achieve patient
acceptability.
[0013] Of note is that, to the knowledge of the inventors, an oral
nicotine replacement therapy is not currently commercially
available. While not wishing to be bound by any particular theory
or mode of action, the reason for this is believed to be as
follows. Oral nicotine must first pass through the liver before
entering the systemic circulation. As a result, extensive
metabolism of nicotine occurs. In particular, oral nicotine is
about 60-85% metabolized from nicotine to continue by the liver so
only 15-40% of oral nicotine reaches the systemic circulation
(Benowitz, et al., "Stable isotope studies of nicotine kinetics and
bioavailability," Clin. Pharmacol. Ther., 49(3):270-7 (1991);
Svensson, "Clinical pharmacokinetics of nicotine," Clin.
Pharmacokinet., 12(1):30-40 (1987); and Zins, et al.,
"Pharmacokinetics of nicotine tartrate after single-dose liquid
enema, oral, and intravenous administration," J. Clin. Pharmacol.,
37(5):426-36 (1997)). Because the first-pass metabolism of nicotine
is so effective and high concentrations of nicotine can not be used
without irritating the digestive system, oral administration (e.g.,
a pill) has, heretofore, been an ineffective delivery system for
nicotine. In light of this, there is no known effective oral
nicotine replacement therapy. It would be desirable to have such a
therapy since it would be much more convenient for the patient and
would be more precisely controlled by the physician (e.g.,
prescribing dosage based on body weight and related factors which
are difficult to take into account when prescribing nicotine patch
or chewing gum).
SUMMARY OF THE INVENTION
[0014] The present inventors have found that variation in nicotine
metabolism among individuals is due to variable expression of CYP2A
isozymes; CYP2A6 has been shown to be the major nicotine
metabolizing enzyme in human livers. Coumarin, a specific CYP2A6
substrate, was found to specifically and selectively inhibit
nicotine metabolism to cotinine by 84%.+-.11% in test livers, and
addition of orphenadrine (a CYP2B6 inhibitor) enhanced the
inhibition. Methoxsalen and tranylcypromine have also been found to
be potent inhibitors of CYP2A6 and thus of nicotine to cotinine
metabolism. The data indicate that variability in CYP2A6 expression
results in inter-individual variation in nicotine metabolism, which
in turn, can have behavioural consequences such as smoking more or
less cigarettes. Therefore, inhibitors of CYP2A6 can be used to
regulate nicotine metabolism, and in particular substantially
decrease nicotine metabolism, thereby affecting tobacco use.
[0015] Broadly stated, the present invention relates to the
diagnosis, prophylaxis and treatment of conditions requiring a
reduction in the activity of a human cytochrome P450 enzyme CYP2A
(referred to as "CYP2A" for brevity). The term "CYP2A" as used
herein means all isoforms of CYP2A including but not limited to
CYP2A(CYP1), CYP2A6, CYP2A7, CYP2A12, CYP2A13 and CYP2A16.
Preferably the enzyme is CYP2A6.
[0016] The inventors have determined that the presence in an
individual of a mutant allele of human cytochrome P450 enzyme
CYP2A6 (referred to throughout this specification as "CYP2A6" for
brevity) is predictive of an individual who: (i) has a decreased
risk of becoming a smoker, (ii) will smoke less if he/she becomes
dependent, and/or (iii) may be at relatively lower risk for cancer
due to both decreased smoke exposure and decreased CYP2A6-mediated
activation of tobacco smoke and other procarcinogenic
substrates.
[0017] In one embodiment, this invention provides a diagnostic
method for tobacco dependence risk and for cancers related to
CYP2A6 substrates in an individual by analysing a DNA-containing
bodily sample from the individual for the presence of a mutant
allele of human cytochrome P450 enzyme CYP2A6. Preferably this
method comprises genotype assaying the bodily sample, which may be
genomic DNA isolated from peripheral leukocytes in the bodily
sample. Alternatively the method comprises phenotype assaying the
bodily sample, which may be a fluid, such as a blood sample or
blood plasma. This invention also provides diagnostic kits for use
in the analysis. The invention also provides a diagnostic method
for tobacco dependence risk and for cancers related to human
cytochrome P450 enzyme CYP2A6 substrates in an individual by
administering a dose of a CYP2A6 substrate to the individual and
determining in a bodily sample from the individual the level of
said CYP2A6 substrate or a metabolite of said CYP2A6 substrate.
[0018] The invention specifically demonstrates that individuals who
are carry CYP2A6 deficient alleles are less likely to become
smokers and will smoke less cigarettes if tobacco-dependent. In
addition, because CYP2A6 is known to activate procarcinogens, such
as those found in tobacco-smoke, the diagnostic aspect of the
invention will be useful for identifying the contribution of this
polymorphic locus to the genetic risk of an individual for
cancer.
[0019] If the result of the diagnostic assay is that the individual
possesses wild-type CYP2A6 (i.e., the individual contains no mutant
alleles of CYP2A6), the present diagnostic method and kit is
predictive of an individual who: (i) has an increased risk of
becoming a smoker, (ii) will smoke more if he/she becomes
dependent, and/or (iii) may be at relatively higher risk for cancer
due to both decreased smoke exposure and decreased enzyme mediated
activation of procarcinogens. Once this individual is identified,
he/she may be treated prophylactively with effective quantities of
CYP2A6 inhibitors described in detail in copending International
patent application Ser. No. PCT/CA97/00506 (filed Jul. 17, 1997)
and U.S. provisional patent application Ser. No. 60/067,021 (filed
on Dec. 1, 1997), which lead to other aspects of the present
invention.
[0020] Thus, the invention also provides a smoking prevention
composition or a smoking regulation composition comprising a CYP2A6
inhibitor, together with a carrier therefor, along with methods for
preventing or regulating smoking by administering a CYP2A6
inhibitor to an individual. Likewise, this invention provides
methods for cancer prevention or treatment or the regulation of the
formation of carcinogens by administering a CYP2A6 inhibitor to an
individual. Compositions containing a CYP2A6 inhibitor are also
provided for use in these methods.
[0021] This invention provides methods for enhancing oral nicotine
therapy, such as oral administration of nicotine bitartrate, by
inhibiting nicotine metabolism through selective inhibition of
CYP2A6, optionally with further selective inhibition of CYP2B6.
Preferred inhibitors of CYP2A6 include coumarin, methoxsalen and
tranylcypromine.
[0022] This method may be used to treat a condition requiring
nicotine administration, preferably by administering a CYP2A6
inhibitor taken together with an oral formulation of nicotine,
optionally also administering a CYP2B6 inhibitor. Preferred
inhibitors of CYP2A6 include coumarin, methoxsalen and
tranylcypromine.
[0023] The present inventors have surprisingly found that several
natural products, are inlubitors of the enzyme CYP2A. Accordingly,
the present invention provides a method of inhibiting CYP2A
comprising administering an effective amount of a natural product
or an extract of a natural product to an individual in need
thereof, this method being useful in treating conditions requiring
regulation of CYP2A activity. In one embodiment, the natural
product is Hypericum or a Hypericum extract. In another embodiment,
the natural product is Cichorium intybus or Bougainvllra
spectabillis or an extract thereof.
[0024] This invention also provides a composition comprising an
oral formulation of nicotine and a CYP2A6 inhibitor, optionally
also containing a CYP2B6 inhibitor. Preferred inhibitors of CYP2A6
include coumarin, methoxsalen and tranylcypromine.
[0025] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. Aspects of this invention may be more fully described
in one or more of U.S. Provisional Patent Applications Nos.
60/067,20; 60/067,021; 60/084,847; and 60/107,392, which are each
incorporated herein by reference in their entirety. It should be
understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the invention
are given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention will be better understood with reference to
the drawings in which:
[0027] FIG. 1 illustrates the results of a study showing CYP2A6
activity in heterozygous CYP2A6 individuals and wild-type CYP2A6
individuals as a function of time after administration of a CYP2A6
substrate;
[0028] FIGS. 2A-2D show chemical structures of some representative
CYP2A6 inhibitors;
[0029] FIG. 3 is a graph illustrating a correlation between fasted
morning and non-fasted afternoon coumarin (C) testing sessions;
[0030] FIG. 4 is a graph showing a time course of total
7-hydroxycoumarin concentration detected in the plasma of subjects
given 100 mg of coumarin;
[0031] FIGS. 5 and 6 illustrate results of a study described in
Example 1;
[0032] FIG. 7 illustrates mean plasma nicotine concentrations in
the study reported in Example 8;
[0033] FIG. 8 illustrates current desire to smoke in the study
reported in Example 8;
[0034] FIG. 9 illustrates mean breath carbon monoxide in the study
reported in Example 9;
[0035] FIG. 10 illustrates the ratio of increased plasma nicotine
to increased breath carbon monoxide in Example 9;
[0036] FIG. 11 illustrates mean number of cigarettes consumed
during the smoking period in Example 9;
[0037] FIG. 12 illustrates the mean number of cigarette puffs taken
in each 10 min. period during the smoking period in Example 9;
[0038] FIG. 13 illustrates the mean latency period between the
first two cigarettes in Example 9;
[0039] FIG. 14 illustrates the mean grams of tobacco burned in
Example 9.
[0040] FIG. 15 illustrates the effect of CYP2A6 inhibitors
methoxsalen and tranylcypromine on increasing the bioavailability
of nicotine supplied orally, with concommitant reduction in the
desire to smoke.
[0041] FIG. 16 is a graph illustrating the effect of Hypericum
extracts on nicotine metabolism by expressed human cDNA CYP2A6.
[0042] FIG. 17 is a graph showing the mean plasma concentration of
nicotine versus time, in the presence of St. John's Wort or a
placebo.
[0043] FIG. 18 is a bar graph showing the mean plasma concentration
of nicotine in the presence of St. john's Wort or a placebo.
[0044] FIG. 19 are graphs illustrating effect of esculetin on
nicotine metabolism by human liver micorsomes.
[0045] FIG. 20 shows the chemical structure of various compounds
found in natural products.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] Broadly stated, the present invention relates to the
diagnosis, prophylaxis and treatment of conditions requiring a
reduction in the activity of a CYP2A enzyme. The term "CYP2a" as
used herein means all isoforms of CYP2A including but not limited
to CYP2A(CYP1), CYP2A6, CYP2A7, CYP2A12, CYP2-13 and CYP2A16.
Preferably enzyme is CYP2A6.
[0047] As described in copending International patent application
S.N. PCT/CA97/00506, the contents of which are hereby incorporated
by reference, inhibition of CYP2A6 (and optionally CYP2B6) inhibits
the metabolism of nicotine. In particular, it was found that CYP2A6
is a major nicotine metabolizing enzyme in human livers and that by
inhibiting CYP2A6 the metabolism of nicotine to continue in the
liver is inhibited.
[0048] Diagnostic Methods
[0049] The present inventors have shown that individuals who carry
CYP2A6 mutant alleles (i) have a decreased risk of becoming a
smoker, (ii) will smoke less if he/she becomes dependent and/or
(iii) may be at relatively lower risk for cancer due to both
decreased smoke exposure and decreased CYP2A6-mediated activation
of tobacco smoke and other procarcinogenic substrates.
[0050] Accordingly, the present invention provides a method for
determining the risk of an individual becoming a smoker comprising
determining the genotype or phenotype of a CYP2A allele in the
individual wherein the presence of a mutant allele is predictive of
a decreased risk of smoking. Preferably, the CYP2A enzyme is
CYP2A6.
[0051] Tobacco smoke contains a number of tobacco-specific
procarcinogen nitrosamines, for example the N-nitrosodiethylamine
and 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK). These
compounds are termed pro- or precarciniogens, as they are activated
by the body. Specifically, these tobacco smoke procarcinogens can
be activated by CYP2A6 (Crespi, et al., "Human cytochrome P450IIA3:
cDNA sequence, role of the enzyme in the metabolic activation of
promutagens, comparison to nitrosamine activation by human
cytochrome P450IIE1," Carcinogenesis 11(8):1293-1300 (1990);
Yamazaki, et al., "Cytochrome P450 2E1 and 2A6 enzymes as major
catalysts for metabolic activation of N-nitrosodialkylamines and
tobacco-related nitrosamines in human liver microsomes,"
Carcinogenesis 13(10):1789-94 (1992)). Therefore individuals who
have CYP2A6 null alleles may also be less efficient at
bioactivating tobacco smoke procarcinogens to carcinogens. This is
of particular interest as ethnic variation in frequencies for
CYP2A6 variant alleles exist (Nowak et al., 1998;
Fernandez-Salguero P, et al., "A genetic polymorphism in coumarin
7-hydroxylation: sequence of the human CYP2A genes and
identification of variant CYP2A6 alleles," Am. J. Hum. Genet.,
57(3):651-60 (1995); Yoloi and Kamataki, 1998) and may be related
to the ethnic differences in lung cancer incidence and histology
(Groeger et al., 1997). Thus, individuals carrying CYP2A6 defective
alleles may have a decreased risk of developing tobacco-related
cancers and other medical complications for three reasons. 1) They
have a decreased risk of becoming a smoker. 2) If they do become
tobacco-dependent, they smoke less than those without impaired
nicotine metabolism resulting in lower exposures to tobacco-related
procarcinogens (Law, et al., "The dose-response relationship
between cigarette consumption, biochemical markers and risk of lung
cancer," Br. J. Cancer 75(11):1690-1693 (1997)). 3) They may
activate fewer tobacco-related procarcincogens. These three factors
suggest a significant reduction in tobacco-related cancers for
carriers of a CYP2A6 defective allele(s).
[0052] Accordingly, the present invention provides a method for
determining the risk of an individual for developing cancer
comprising determining the genotype or phenotype of a CYP2A allele
in the individual wherein the presence of a mutant allele is
predictive of a decreased risk of developing cancer. Preferably,
the CYP2A enzyme is CYP2A6.
[0053] The diagnostic aspect of this invention includes both
phenotypic and genotypic methods for determining whether an
individual has wild-type or mutant alleles for CYP2A6. The
phenotypic assay may be performed by a metabolic study which is in
effect an in vivo enzyme assay for CYP2A6 activity. This assay may
be performed by administering a dose of a CYP2A6 substrate, for
example nicotine or coumarin, and monitoring the physiological
levels of the substrate and/or the product of enzymatic metabolism
of the substrate in the individual at one or more time points
during and subsequent to administration of the test dose.
Typically, the levels will be measured in a biological fluid, such
as blood, plasma, or urine, using well known assays for the
particular components, examples of which are disclosed herein. An
example of an in vivo phenotype and enzyme activity assay is
provided in Example 3 below. This phenotypic assay can be used to
classify individuals based on their normally expressed level of
CYP2A6, which will correspond generally with the genotype of the
individual as homozygous for fully active CYP2A6, heterozygous or
homozygous for a lower activity allele, in decreasing order of
nicotine metabolic rate.
[0054] The diagnostic aspect is also based on analysing a
DNA-containing bodily sample from the individual for the presence
of a mutant allele of human cytochrome P450 enzyme CYP2A6. As used
throughout this specification, the term "mutant allele" is meant to
encompass any allele having decreased or absent CYP2A6 activity,
i.e., including null alleles. The presence of the mutant allele of
CYP2A6 can be determined by conventional genotyping or phenotyping
assays.
[0055] Many CYP2A6 alleles have been identified including, but not
limited to, the wild-type allele (referred to throughout this
specification as "CYP2A6*1"), and two defective or null mutant
alleles ("CYP2A6*2" and "CYP2A6*3", respectively (see,
Fernandez-Salguero, et al. 1995), the contents of which are hereby
incorporated by reference). The CYP2A6*2 allele differs from the
wild-type allele by a single point mutation which leads to a
leucine to histidine amino acid change at codon 1609. In vitro and
in vivo studies have demonstrated that this allele is a null
allele. Mutations in the CYP2A6*3 allele occur in exons 3, 6, and
8. Very recently an additional CYP2A6 allele was identified which
consists of an entire CYP2A6 gene deletion (Nunoya K et al., 1998
"A new deleted allele in the human cytochrome P450 2A6 (CYP2A6)
gene found in individuals showing poor metabolic capacity to
coumarin and (+)-cis-3,5-dimethyl-2-(3- -pyridyl)thiazolidin-4-one
hydrocholoride (SM-12502). Pharmacogenetics 1998, 8: 239-249. Of
course, additional mutant alleles which encode CYP2A6 enzymes
with-reduced activity may be found in individuals identified by the
phenotypic and/or genotypic methods of this invention, and these
individuals will also be expected to have lower risk of developing
cancer and decreased risk of smoking.
[0056] The individual contemplated for the diagnostic methods of
this invention (as well as the prophylactic and therapeutic methods
described below) may be any type of mammal, but is preferably a
primate, and more preferably a human.
[0057] Preferably, the bodily sample is a fluid such as blood or
blood plasma. Alternatively, the bodily sample can be tissue. See,
for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989),
the contents of which are hereby incorporated by reference, for
discussion of general assay techniques useful with the diagnostic
methods described herein.
[0058] With reference to FIG. 1, there is illustrated the result of
CYP2A6 activity in a group (Group I) of individuals having
heterozygous CYP2A6 activity (i.e., each individual in this group
had a single mutant allele of CYP2A6 and a single active allele of
CYP2A6) and a group of individuals (Group II) having wild type
CYP2A6 activity (i.e. each individual in this group had two active
alleles of CYP2A6). Blood plasma samples from each of the
individuals in both groups were take post-administration of
coumarin (100 mg) at 35 minutes, 45 minutes and 75 minutes.
Coumarin 7-hydroxylation was used to assess the compliment activity
of CYP2A6. The results of the phenotyping assay clearly show that
the Group I individuals have a significantly lower CYP2A6 activity
(less than half at 35 and 45 minutes) than the Group II
individuals.
[0059] Alternatively, the subject is an individual having a CYP2A6
genotype associated with an active form of the enzyme. The CYP2A6
genotype of an individual and the existence of an active CYP2A6
enzyme in an individual may be determined using procedures using
techniques described herein. For example, coumarin 7-hydroxylation
has been used to measure CYP2A6 activity (see, Cholerton, et al.
(1992) and Rautio, et al. (1992)).
[0060] The recognition by the present inventors that CYP2A6 is the
major nicotine metabolizing enzyme in human livers suggests that
the enzyme can be assayed in an individual to determine the
individual's risk of developing tobacco dependence. Determination
of CYP2A6 levels may also be used to select and monitor in an
individual appropriate conventional nicotine replacement therapies
such as the nicotine patch and nicotine gum. It is unlikely that
conventional nicotine replacement therapies (e.g. nicotine gum,
nicotine patch, spray, pulmonary inhalation or other forms) will
have a high success outcome if an individual has high levels of
CYP2A6, although such individuals may be good candidates for
enhanced NRT according to the methods described herein. Conversely,
if an individual has very low levels of CYP2A6, administering
nicotine at high dosages will likely result in increased toxicity,
and side effects. Furthermore, the co-administration of a CYP2A6
inhibitor with an existing NRT would be expected to decrease the
kinetics of nicotine from that source and to enhance the efficacy
of the NRT (discussed below under Therapeutic Methods).
[0061] Prophylactic and Therapeutic Methods
[0062] As mentioned previously, the present invention relates to
methods for the prophylaxis and treatment of conditions requiring a
reduction in the activity of a CYP2A enzyme. In particular, the
prophylactic/therapeutic aspect of the present invention relates to
treatment and prevention of smoking, in vivo carcinogen formation
and cancer in an individual. Each of this involves administration
to an individual of a CYP2A inhibitor, preferably a CYP2A6
inhibitor.
[0063] In one aspect, the present invention provides a method of
preventing, treating or regulating smoking in an individual
comprising administering an effective amount of one or more
substances selected from the group consisting of (i) substances
which inhibit CYP2A activity; (ii) substances which inhibit
transcription, translation of the gene encoding CYP2A, or both;
(iii) substances which delete all or a portion of the gene encoding
CYP2A. Preferably, the CYP2A is CYP2A6.
[0064] As used throughout this specification, the terms 'smoking
prevention" and "preventing smoking", as used throughout this
specification, are intended to mean that the likelihood of the
onset of smoking (i.e., the progression from a cigarette to regular
smoking) in a current non-smoking individual (i.e., a person who
has never smoked or is a ex-smoker) and the return to smoking of a
previous smoker (i.e. relapse prevention) is substantially
mitigated.
[0065] The terms "smoking regulation" and "regulating smoking", as
used throughout this specification, are intended to mean that the
amount smoked by a current smoking individual is reduced or, at
least, fails to increase.
[0066] The terms "smoking treatment" or "treatment of smoking"
means the stopping of all smoking or the reduction in amount of
smoking as reflected in less use of tobacco products, a decrease in
pattern of use or a decrease in tobacco smoke exposure. The measure
of tobacco smoke exposure can be measured by analyzing breath
carbon monoxide.
[0067] In another aspect, the present invention provides a method
of regulating the formulation of a carcinogen in an individual
comprising administering an effective amount of one or more
substances selected from the group consisting of (i) substances
which inhibit CYP2A activity; (ii) substances which inhibit
transcription, translation of the gene encoding CYP2A, or both;
(iii) substances which delete all or a portion of the gene encoding
CYP2A. Preferably, the CYP2A is CYP2A6.
[0068] The terms "carcinogen formation regulation" and "regulating
formation of a carcinogen", as used throughout this specification,
are intended to mean that the occurrence of carcinogen formation in
an individual is reduced. This may be achieved, for example, by
using CYP2A6 inhibition to inhibit activation of procarcinogens
present in the individual. As used throughout this specification,
the term "procarcinogen" is meant to encompass any substance which
is at least one of procytotoxic, promutagenic and progenotoxic
("pro" means the metabolite is more active that the parent
compound).
[0069] In a further aspect, the present invention provides a method
of preventing cancer in an individual comprising administering an
effective amount of one or more substances selected from the group
consisting of (i) substances which inhibit CYP2A activity; (ii)
substances which inhibit transcription, translation of the gene
encoding CYP2A, or both; (iii) substances which delete all or a
portion of the gene encoding CYP2-A. Preferably, the CYP2A is
CYP2A6.
[0070] The terms "cancer prevention" and "preventing cancer", as
used throughout this specification, are intended to mean that the
likelihood of the onset of cancer in a current cancer-free
individual (i.e., a person who has never had cancer or whose cancer
is in remission) is substantially mitigated.
[0071] The terms "inhibitor" and "inhibition", in the context of
the present invention, are intended to have a broad meaning and
encompass substances which directly or indirectly (e.g., via
reactive intermediates, metabolites and the like) act on CYP2A to
inhibit or otherwise regulate the ability of CYP2A to catalyze
metabolism of a substrate. Other substances which act indirectly on
CYP2A include those substances which inhibit transcription and/or
translation of the gene encoding CYP2A. In particular, the terms
"CYP2A6 inhibition" and "CYP2A6 inhibitor" are intended to have a
broad meaning and encompass any substance which: (i) inhibits
CYP2A6 activity; (ii) inhibits transcription and/or translation of
the gene encoding CYP2A6; or (iii) deletes or removes the gene
encoding CYP2A6. Particularly preferred substances are those which
alter the kinetics for metabolism of nicotine to cotinine, alter
smoking behavior, alter the likelihood of addiction to smoking in a
population of non-smokers, or alter the kinetics of formation for
carcinogens whose formation from procarcinogens is catalyzed by
CYP2A, and more preferably exhibit the biological altering effect
without producing other biological effects at significant
levels.
[0072] A substance will "selectively" inhibit CYP2A activity when
the substance can alter the kinetics for metabolism of nicotine to
cotinine, alter smoking behavior, alter the likelihood of addiction
to smoking in a population of non-smokers, or alter the kinetics of
formation for carcinogens whose formation from procarcinogens is
catalyzed by CYP2A generally at a dosage level which is lower than
the dosage of the substance which is effective for production of
another biological effect. For example, it is shown below that
administration of methoxsalen acted to increase plasma levels of
nicotine and to reduce desire to smoke in dependent smokers at
levels that were one-fourth the therapeutic dose for treatment of
psoriasis by methoxsalen.
[0073] The term "effective amount" as used herein means an amount
effective and at doses and for periods of time necessary to achieve
the desired results; this may mean limiting doses where the desired
result is selective inhibition and selectivity is achieved through
differential inhibition of CYP2A. Preferably, the substances
inhibit CYP2A6.
[0074] CYP2A6 Inhibitors
[0075] As hereinbefore mentioned, in one of its aspects, the
present invention relates to a method of regulating nicotine
metabolism to cotinine in an individual comprising selectively
inhibiting CYP2A6. Inhibition of CYP2A6 may be achieved using one
or more of the following (i) substances which inhibit CYP2A6
activity; or (ii) substances which inhibit transcription and/or
translation of the gene encoding CYP2A6.
[0076] Substances which inhibit CYP2A6 activity include substances
which specifically bind to CYP2A6 and thereby inhibit its activity.
Examples of such substances include antibodies which are specific
for CYP2A6 including for example, the monoclonal antibody described
by Pearce R, et al. ("Species differences and interindividual
variation in liver microsomal cytochrome P450 2A enzymes: effects
on coumarin, dicumarol, and testosterene oxidation," Arch. Biochem.
Biophys., 298(1): 211-225 (1992)), and commercially available
antibodies such as MAB2A6 and monoclonal CYP2A6, sold by Gentest
Corporation, Woburn, Mass., U.S.A; XenoTech 2A6 sold by XenoTech
LLC, Kansas City, Kans., U.S.A and polyclonal CYP2A6 sold by
Research Diagnostics, Inc, Flanders, N.J., U.S.A.
[0077] Preferred inhibitors of CYP2A6 include methoxsalen,
psoralen, tranylcypromine, pilocarpine, coumarin, chromone,
esculetin, phenelzine, paroxetine, selegiline and pargyline.
[0078] Substances which inhibit CYP2A6 activity also include
substances having a lactone structure with a carbonyl oxygen.
Non-limiting examples of such substances include coumarin (The
Merck Index, Eleventh Edition Budavari, S., ed. Merck & Co.
Inc., 1989, No. 2563), furanocoumarin, methoxsalen (The Merck
Index, No. 5911), imperatorin (The Merck Index, No. 4839), psoralen
(The Merck Index, No. 7944), .alpha.-naphthoflavone,
isopimpinellin, .beta.-naphthoflavone, bergapten (The Merck Index,
No. 1173), sphondin, coumatetralyl (racumin), and
(+)-cis-3,5-dimethyl-2-(3-p- yridyl)-thiazolidim-4-one (SM-12502)
(Nunoya, et al., J. Pharmscol. Esp. Ther., 277(2):768-74 (1996)).
Other substances which inhibit CYP2A6 and can be used in the
methods and compositions of the invention include naringenin and
related flavones, diethyldithiocarbamate, nicotine (useful
primarily in the screening methods of the invention),
N-nitrosodialkylamine (e.g. N-nitrosodiethylamine (The Merck Index,
No. 6557), N-nitrosodimethylamine (The Merck Index, No. 6558)),
nitropyrene, menadione (The Merck Index, No. 5714), imidazole
antimycotics, miconazole (The Merck Index, No. 6101), clotrimazole
(The Merck Index, No. 2412), pilocarpine (The Merck Index, No.
7395), hexamethylphosphoramide,
4-methylnitrosamine-3-pyridyl-1-butanol, aflatoxin B (The Merck
Index, No. 168), tranylcypronune (the Merck Index, No. 9491),
including cis, trans, (+) and (-) isomers, trioxsalen, alaproclate,
phenelzine, pargyline, paroxitine, selegiline, amphetamine,
bupropion, buspirone, citalopram, desmethylcitalopram, doxeprine,
fluoxetin, naltrexone, norfluoxetine, nortriptyline, sertraline,
trazodone, viaqualine, zimelidine, chromone, bergapten and
narigenin. All of the substances thati nhibit CYP2A6 activity
include racemic mixtures of the compounds as well as the cis,
trans, (+) and (-) isomers. See FIGS. 2A to 2D for the chemical
structures of these and other non-limiting representative
inhibitors. Selective and non-selective monoamine oxidase
inhibitors (e.g., alaproclate, phenelizine, deprenyl, pargyline,
selegiline and the like) are particularly preferred. Various
isomers of the above compounds which can be shown to inhibit CYP2A6
as described below are within the contemplation of this
invention.
[0079] Derivatives and analogs of these substances may also be used
in the methods and compositions of the invention. Derivatives and
analogs include compounds that are structurally similar to the
compounds described herein and can bind to the CYP2A6 active site.
For example, derivatives of tranylcypromine, coumarin and
methoxsalen include pharmaceutically acceptable salts, esters and
complexes of tranylcypromine, coumarin and methoxsalen including
potassium and sodium salts, and anino acid, carbohydrate and fatty
acid complexes. By way of example, suitable analogs of coumarin may
be selected based upon their functional similarity to coumarin,
including the ability to inhibit the metabolism of nicotine to
continue by CYP2A6. Examples of functional analogs of coumarin
include 7-methoxycoumarin, 7-methylcoumarin, and 7-ethoxycoumarin
and all structures shown in FIGS. 2A, 2B, 2C. Analogs of coumarin
may also be selected based upon their three dimensional structural
similarity to coumarin--i.e., the lactone/carbonyl structure.
[0080] The present inventors have surprisingly found that natural
products and extracts of natural products inhibit CYP2A6 activity
in both human liver microsomes and pure full-length human cDNA
expressed cytochromes. Accordingly, CYP2A6 inhibitors of the
present invention include natural products or extracts of a natural
product capable of inhibiting CYP2A6, such as Hypericum or a
Hypericum extract or Cichorium intybus or Bougainvllra spetabillis
or an extract thereof.
[0081] The term "Hypericum" as used herein as synonymous with
Hypericum perforatum, St. John's Wort, Goatweed and Klamath Wee.
The phrase "Hypericum or an extract of Hypericum" as used herein
includes the whole plant Hypericum perforatum or a derivative,
extract, isolate or purified component thereof that can inhibit
CYP2A activity. This includes natural components of the plant and
synthetic analogues. A preferred extract of Hypericum is a methanol
extract.
[0082] Derivatives of Hypericum which may be used in the methods
and compositions of the invention include hypericin,
pseudohypericin, quercetin, hyperoside, quercitrin, isoquercitrin,
rutin, campherol, luteolin, 13-II8-biopigenin,
1,3,6,7-tetrahydroxyxanthone, procyanidines, hyperforin, ethereal
oil, phenol carbonic acids (e.g. chlorogenic acid), xanthone,
phenylpropanes, flavonol derivatives, biflavones,
proanthocyanidins, xanthones, phloroglucinols, naphthodianthrones
and essential oil constitutes. Also included are the
pharmaceutically acceptable salts, esters and complexes of the
derivatives including potassium and sodium salts, and amino acid,
carbohydrate and fatty acid complexes. Suitable derivatives of
Hypericum may be selected based upon their ability to inhibit CYP2A
with greater than 50% inhibition, and/or a Ki less than 300
.mu.M.
[0083] The phrase "Cichorium intybus or Bougainvllra spectabillis
or an extract thereof" as used herein includes the whole plants
Cichorium intybus or Bougainvllra specablluis or a derivative,
extract, isolate or a purified component thereof that can inhibit
CYP2A activity. This includes natural components of the plants as
well as synthetic analogues. A preferred extract from Cichorium
intybus or Bougainvllra spectabillis is esculetin, esculin or
esculin monohydrate.
[0084] Other extracts of natural products that may be useful in the
present invention are shown in FIG. 20, and in U.S. provisional
application Ser. No. 60/084,847, which is incorporated herein by
reference.
[0085] The above lists of substances which inhibit CYP2A6 are
provided by way of example only and should not be seen as limiting
the scope of this invention. Additional substances which inhibit
CYP2A6 activity may be identified using the screening methods
described herein.
[0086] Substances which inhibit transcription and/or translation of
the gene encoding CYP2A6 include a nucleic acid sequence encoding
the CYP2A6 gene (GenBank Accession No. HSU22027) or parts thereof
(e.g., the region which is about 20 nucleotides on either side of
nucleotide 790 (ATG), and the splice sites 1237, 2115, 2499, 3207,
4257, 4873, 5577 and 6308), inverted relative to their normal
orientation for transcription--i.e., antisense CYP2A6 nucleic acid
molecules. Such antisense nucleic acid molecules may be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed with CYP2A6 mRNA or the CYP2A6 gene. The antisense
sequences may be produced biologically using an expression vector
introduced into cells in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense sequences are
produced under the control of a high efficiency regulatory region,
the activity of which may be determined by the cell type into which
the vector is introduced.
[0087] A nucleic acid molecule containing the antisense sequences
may be introduced into cells in a subject using conventional
techniques, such as transformation, transfection, infection, and
physical techniques such as electroporation or microinjection.
Chemical methods such as coprecipitation and incorporation of DNA
into liposomes may also be used to deliver antisense sequences. The
molecules may also be delivered in the form of an aerosol or by
lavage. Suitable vectors or cloning vehicles for transferring the
nucleic acid molecules are known in the art. Examples of suitable
vectors include retroviral vectors, adenoviral vectors, and DNA
virus vectors.
[0088] The ability of a substance to selectively inhibit CYP2A6 and
thus regulate nicotine metabolism to cotinine may be confirmed
using the methods described herein for screening for an
inhibitor.
[0089] In one embodiment of the invention, the CYP2A6 inhibitor is
at least one member selected from the group comprising coumarin,
methoxsalen, tranylcypromine, derivatives thereof and analogs
thereof (see FIG. 2A). Initial in vitro screening and clinical
studies have identified that methoxsalen is a potent inhibitor of
CYP2A6.
[0090] CYP2A6 may also be selectively inhibited in the method of
the invention by interfering with the transcription of the gene
encoding CYP2A6 using gene transfer methods such as targeted gene
mutagenesis using allelic replacement, insertional inactivation, or
deletion formation. For example, allelic gene exchange using
non-replicating or conditionally-replicating plassiids has been
used widely for the mutagenesis of eukaryotes. Allelic exchange can
be used to create a deletion of the CYP2A6 gene. Exemplary methods
of making the alterations set forth above are disclosed by
Sambrook, et al. (1989).
[0091] CYP2B6 Inhibitors
[0092] CYP2B6 inhibitors may also be used in combination with
inhibitors of CYP2A6 to provide an enhanced inhibitory effect.
Inhibitors of CYP2B6 include one or more of the following (i)
substances which inhibit CYP2B6 activity; or (ii) substances which
inhibit transcription and/or translation of the gene encoding
CYP2B6. CYP2B6 inhibitors may also be used alone to inhibit
nicotine metabolism in an individual.
[0093] Substances which inhibit CYP2B6 activity include substances
which specifically bind to CYP2B6 and thereby inhibit its activity.
Examples of such substances include antibodies which are specific
for CYP2B6 including for example, commercially available antibodies
such as anti-CYP2B6 sold by Gentest Corporation, Woburn, Mass.,
U.S.A.
[0094] Substances which inhibit CYP2B6 activity also include
substances selected from phenylethyl amines, diphenylbarbiturates,
diethyl substituted barbiturates and hydantoins. In particular,
diphenhydramine and its derivatives, including orphenadrine (The
Merck Index, No. 6831), and derivatives or analogs of orphenadrine,
and other antihistamines, anticholinergic substances such as
cholines and analogs and derivatives thereof may be used as CYP2B6
inhibitors in various embodiments of the methods and compositions
of the invention. Antibodies, such as polyclonal CYP2B1/2,
polyclonal CYP2B1 and polyclonal CYP2B6 sold by Gentest
Corporation, Woburn, Mass., U.S.A., also bind specifically to
CYP2B6 such that they also inhibit the activity of CYP2B6.
[0095] Derivatives of orphenadrine which may be used in the methods
and compositions of the invention include pharmaceutically
acceptable salts, esters and complexes of orphenadrine including
potassium and sodium salts, and amino acid, carbohydrate and fatty
acid complexes. In one embodiment, suitable analogs of orphenadrine
may be selected based upon their functional similarity to
orphenadrine, including the ability to inhibit CYP2B6. Analogs of
orphenadrine may also be selected based upon their three
dimensional structural similarity to orphenadrine.
[0096] Substances which inhibit transcription and/or translation of
the gene encoding CYP2B6 include a nucleic acid sequence encoding
the CYP2B6 gene (see FIG. 2B, GenBank Accession No. HSP452B6 for
the mRNA sequence of CYP2B6), or parts thereof (e.g., the region
which is on either side of nucleotide 9 (ATG), and the sites 111,
274, 424, 585, 762, 904, 1092, and 1234 nt), inverted relative to
their normal orientation for transcription--i.e., antisense CYP2B6
nucleic acid molecules. Such antisense nudeic acid molecules may be
produced and introduced into cells using conventional procedures as
described herein.
[0097] CYP2B6 may also be selectively inhibited in a method of the
invention by interfering with the transcription of the gene
encoding CYP2B6 using conventional gene transfer methods as
discussed herein.
[0098] In preferred embodiments of the invention the CYP2B6
inhibitor employed is orphenadrine and derivatives or analogs of
orphenadrine.
[0099] An inhibitor of CYP2A6 or CYP2B6 may be targeted to the
enzyme using antibodies specific to an epitope of the enzyme. For
example, bispecific antibodies may be used to target an inhibitor.
The bispecific antibodies contain a variable region of an antibody
specific for at least one epitope of CYP2A6 or CYP2B6, and a
variable region of a second antibody which is capable of binding to
an inhibitor. The bispecific antibodies may be prepared by forming
hybrid hybridomas, using procedures known in the art such as those
disclosed in Staerz, et al. ("Hybrid hybridoma producing a
bispecific monoclonal antibody that can focus effector T-cell
activity," Proc. Natl. Acad. Sci. USA, 83(5):1453-7 (1986)) and
Staerz, et al. (Immunology Today, 7:241 (1986)). Bispecific
antibodies may also be constructed by chemical means using
conventional procedures such as those described by Staerz, et al.
("Hybrid antibodies can target sites for a attack by T cells,"
Nature, 314(6012):628-31 (1985)) and Perez, et al. ("Specific
targeting of cytotoxic T cells by anti-T3 linked to anti-target
cell antibody," Nature, 316(6026):354-6 (1985)), or by expression
of recombinant immunoglobulin gene constructs.
[0100] Nicotine Replacement Therapy
[0101] An oral nicotine replacement therapy containing nicotine
alone would be ineffective due to the extensive metabolism of
nicotine in the liver which significantly decreases the systemic
availability of the nicotine. However, administering the nicotine
with a CYP2A inhibitor would increase the bioavailability and the
effectiveness of the oral nicotine therapy.
[0102] The present invention also includes a nicotine replacement
therapy comprising contemporaneously administering to an individual
in need thereof (a) oral nicotine and (b) one or more substances
selected from the group consisting of (i) substances which inhibit
CYP2A activity; (ii) substances which inhibit transcription,
translation of the gene encoding CYP2A, or both; (iii) substances
which delete all or a portion of the gene encoding CYP2A.
[0103] Preferably, the inhibitor is an inhibitor of CYP2A6 such as
methoxsalen or tranylcypromine.
[0104] As used herein, "contemporaneous administration" of two
substances to an individual means providing each of the two
substances so that they are both biologically active in the
individual at the same time. The exact details of the
administration will depend on the pharmacokinetics of the two
substances in the presence of each other, and can include
administering the two substances within a few hours of each other,
or even administering one substance within 24 hours of
administration of the other, if the pharmacokinetics are suitable.
Design of suitable dosing regimens are routine for one skilled in
the art, in view of the details provided herein on the biological
activities of CYP2A6 substrates and inhibitors. In particular
embodiments, two substances will be administered substantially
simultaneously, i.e., within minutes of each other, or in a single
composition that contains both substances. On the other hand, a
CYP2A6 inhibitor which acts by deleting or removing the gene
encoding CYP2A6 could be administered months or even years before
administration of nicotine or a procarcinogen that would otherwise
be converted to a carcinogen by CYP2A6, and the effects due to the
two administrations may still be contemporaneous.
[0105] Screening for Inhibitors
[0106] In addition to the CYP2A inhibitors listed above, substances
which may be used in the methods of this invention include other
substances that alter the kinetics for metabolism of nicotine to
cotinine, alter smoking behavior, alter the likelihood of addiction
to smoking in a population of non-smokers, alter the kinetics of
formation for carcinogens whose formation from procarcinogens is
catalyzed by CYP2A. All of these substances have in common an
ability to reduce the activity of CYP2A enzymes in an individual.
The present disclosure therefore provides a method of screening for
a substance that inhibits a CYP2A enzyme in an individual
comprising assaying for a substance which selectively (i) inhibits
CYP2A6 activity, (ii) inhibits transcription and/or translation of
the gene encoding CYP2A6, or (iii) deletes or removes the gene
encoding CYP2A6.
[0107] The inhibitory activity of a particular substance identified
herein or an analog or derivative thereof may be confirmed by
testing in experimental model systems and in clinical studies, for
example as outlined below and exemplified in the Examples herein.
Furthermore, specificity or selectivity of a substance listed above
or a substance newly identified by screening as described herein
may be determined or confirmed as described hereinbelow. While no
particular test is mandated by this invention, the usefulness of a
particular substance (e.g., a substance not specifically listed
hereinabove or referred to in FIGS. 2A-2D) as a CYP2A6 inhibitor
may be readily determining by testing the substance as follows.
[0108] In Vitro Inhibition
[0109] An initial screen to select candidate inhibitors for use in
the methods according to this invention comprises:
[0110] (a) reacting, in the presence of a test substance, a
substrate of CYP2A6 with a source of CYP2A6 under conditions such
that CYP2A6 is capable of converting the substrate into a reaction
product;
[0111] (b) assaying for reaction product, unreacted substrate or
unreacted CYP2A6;
[0112] (c) comparing the results of such assay to controls in the
absence of the substance to determine if the test substance
inhibits CYP2A6 and thereby is capable of inhibiting CYP2A
enzymes.
[0113] Substrates of CYP2A6 which may be used in the in vitro test
for identification of substances for use in methods of the
invention, as well as in the in vivo tests below, include nicotine,
coumarin, analogs thereof and derivatives thereof. The
corresponding reaction products for nicotine and coumarin are
cotinine and 7-hydroxycoumarin, respectively.
[0114] CYP2A6 used in the method of the invention may be obtained
from natural, recombinant, or commercial sources. For example
CYP2A6 may be obtained by recombinant methods such as those
described by Nesnow S, et al. ("N-nitrosodiethylamine and
4-(methylnitrosamino)-1-(3-pyridyl)-1-but- anone induced
morphological transformation of C3H/10T1/2CL8 cells expressing
human cytochrome P450 2A6," Mutation Research, 324:93-102 (1994)).
Cells or liver microsomes expressing CYP2-A6 may also be used in
the method.
[0115] Conditions which permit the formation of a reaction product
may be selected having regard to factors such as the nature and
amounts of the test substance and the substrate. The results using
the substrates in the presence and absence of the test substance
may be compared to results using methoxsalen or tranylcypromine as
controls which show positive inhibition tests.
[0116] The reaction product, unreacted substrate, or unreacted
CYP2A6; may be isolated by conventional isolation techniques, for
example, salting out, chromatography, electrophoresis, gel
filtration, fractionation, absorption, polyacrylamide gel
electrophoresis, agglutination, or combinations thereof.
[0117] To facilitate the assay of the reaction product, unreacted
substrate, or unreacted CYP2A6; antibody against the reaction
product or the substance, or a labeled CYP2A6 or substrate, or a
labeled substance may be utilized. Antibodies, CYP2A6, substrate,
or the substance may be labeled with a detectable marker such as a
radioactive label, antigens that are recognized by a specific
labeled antibody, fluorescent compounds, enzymes, antibodies
specific for a labeled antigen, and chemiluminescent compounds.
[0118] The substrate used in the method of the invention may be
insolubilized. For example, it may be bound to a suitable carrier.
Examples of suitable carriers are agarose, cellulose, dextran,
Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter
paper, ion-exchange resin, plastic film, plastic tube, glass beads,
polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
carrier may be in the shape of, for example, a tube, test plate,
beads, disc, sphere. etc. The insolubilized CYP2A6, substrate, or
substance may be prepared by reacting the material with a suitable
insoluble carrier using known chemical or physical methods, for
example, cyanogen bromide coupling.
[0119] In Vivo Inhibition
[0120] Substances which pass the above-mentioned in vitro screening
test are then preferably subjected to an in vivo test to confirm
their suitability for use in the methods of this invention. A
suitable in vivo test method comprises the steps of:
[0121] (a) administering a subtherapeutic dose of nicotine (e.g.,
1.0, 2.0 or 4.0 mg expressed as the base) in an oral formulation to
an individual, together with the test substance;
[0122] (b) collecting pre-nicotine and post-nicotine plasma samples
from the individual (e.g., 30, 60 and 90 minutes after (a));
[0123] (c) determining the plasma nicotine concentration using a
conventional analytical technique (e.g., HPLC, gas chromatography
and the like), and
[0124] (d) comparing the plasma nicotine concentration to a control
(i.e., nicotine given without test substance) to assess whether the
test substance results in a statistically significant increase in
the plasma nicotine concentration at one or more time points, more
preferably the later time points.
[0125] Genetic Level Effectors
[0126] Analogous methods may be used for screening for a substance
that regulates nicotine metabolism to cotinine in an individual by
inhibiting transcription and/or translation of the gene encoding
CYP2A6. A screening method for such substances comprises the steps
of:
[0127] (a) culturing a host cell comprising a nucleic acid molecule
containing a nucleic acid sequence encoding CYP2A6 and the
necessary elements for the transcription or translation of the
nucleic acid sequence, and optionally a reporter gene, in the
presence of a test substance; and
[0128] (b) comparing the level of expression of CYP2A6, or the
expression of the protein encoded by the reporter gene with a
control cell transfected with a nucleic acid molecule in the
absence of the test substance.
[0129] A host cell for use in the method of the invention may be
prepared by transfecting a suitable host with a nucleic acid
molecule comprising a nucleic acid sequence encoding CYP2A6. A
nucleic acid sequence encoding CYP2A6 may be constructed having
regard to the sequence of the CYP2A6 gene (see the sequence under
Genbank Accession number HUS22027, incorporated herein by
reference) following procedures known in the art. Suitable
transcription and translation elements may be derived from a
variety of sources, including bacterial, fungal, viral, mammalian,
or insect genes. Selection of appropriate transcription and
translation elements is dependent on the host cell chosen, and may
be readily accomplished by one of ordinary skill in the art
Examples of such elements include: a transcriptional promoter and
enhancer or RNA polymerase binding sequence, a ribosomal binding
sequence, including a translation initiation signal. Additionally,
depending on the host cell chosen and the vector employed, other
genetic elements, such as an origin of replication, additional DNA
restriction sites, enhancers, and sequences conferring inducibility
of transcription may be incorporated into the expression vector. It
will also be appreciated that the necessary transcription and
translation elements may be supplied by the native CYP2A6 gene
and/or its flanking sequences.
[0130] Examples of reporter genes are genes encoding a protein such
as B-galactosidase, chloramphenicol acetyltransferase, firefly
luciferase, or an immunoglobulin or portion thereof such as the Fc
portion of an immunoglobulin, preferably IgG. Transcription of the
reporter gene is monitored by changes in the concentration of the
reporter protein such as .beta.-galactosidase, chloramphenicol
acetyltransferase, or firefly luciferase. This makes it possible to
visualize and assay for expression of CYP2A6 and in particular to
determine the effect of a substance on expression of CYP2A6.
[0131] Suitable host cells include a wide variety of prokaryotic
and eukaryotic host cells, including bacterial, mammalian, yeast or
other fungi, viral, plant, or insect cells.
[0132] Protocols for the transfection of host cells are well known
in the art (see, Sambrook, et al. (1989)). By way of example, Nanji
M, et al. ("Expression in a baculovirus system of a cDNA encoding
human CYP2A6," Biochem. Soc. Trans., 22 (1994)) describe the
expression of a cDNA encoding human CYP2A6 in a baculovirus system;
Nesnow, S., et al. (1994) and Tiano H F, et al. ("Retroviral
mediated expression of human cytochrome P450 2A6 in C3H/10T1/2
cells confers transformability by
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),"
Carcinogensis, 14:1421-7 (1993)) describe the expression of CYP2A6
from a retroviral vector in transformable C3H/10T/2 mouse embryo
fibroblasts; and Salonpaa P, et al. ("Retrovirus-mediated stable
expression of human CYP2A6 in mammalian cells," Eur. J Pharmacol.,
248:95-102 (1993)) describe the preparation of arnphotropic
recombinant retroviruses containing CYP2A6 using LXSN vector and
PA317 packaging cells.
[0133] Host cells which are commercially available may also be used
in the method of the invention. For example, the h2A3 (now known as
h2A6) and h2B6 cell lines available from Gentest Corporation are
suitable for the screening methods of the invention.
[0134] Substances which pass the in vitro screening test for
alteration of expression of CYP2A6 preferably are then subjected to
an in vivo test to confirm their suitability for use in the methods
of this invention, by analogy to the in vivo test for inhibitors of
CYP2A enzyme activity.
[0135] The above mentioned methods may be used to identify negative
regulators of nicotine metabolism to cotinine in brain and liver
thereby affecting conditions requiring regulation of nicotine
metabolism. Further confirmation of the suitability of the
substances, and/or demonstration of the selectivity of the effects,
may be achieved by population studies of the effects of the
substances on the kinetics for metabolism of nicotine to cotinine,
on smoking behavior, on the likelihood of addiction to smoking in a
population of non-smokers, and/or on the kinetics of formation for
carcinogens whose formation from procarcinogens is catalyzed by
CYP2A. Such studies are a routine matter for the skilled clinician
in view of the guidance provided herein and the exemplary studies
described in the Examples below.
[0136] Compositions
[0137] Substances which inhibit CYP activity described in detail
herein, or substances identified using the methods of the invention
may be incorporated into pharmaceutical compositions. Therefore the
invention provides a pharmaceutical composition for use in treating
a condition requiring a reduction in the activity of a CYP2A enzyme
comprising an effective amount of one or more substances which
selectively inhibit CYP2A6, and a pharmaceutically acceptable
carrier, diluent, or excipient. In one of its aspects, the
invention provides a pharmaceutical composition for use in smoking
prevention, smoking treatment, smoking regulation, regulating
carcinogen formation, cancer prevention and/or cancer treatment. A
method of treatment using such a composition is also provided.
Further, the treatment methods and compositions of the invention
may also be used together with other active compounds, including
such other active compounds which are susceptible to
CYP2A6-mediated metabolism leading to an inhibition or reduction in
effectiveness of the other active compound.
[0138] Conditions requiring regulation of nicotine metabolism to
cotmine include nicotine use disorders--i.e., dependent and
non-dependent tobacco use, and nicotine-induced disorders--i.e.,
withdrawal. The conditions may develop with the use of all forms of
tobacco (e.g., cigarettes, chewing tobacco, snuff, pipes, and
cigars) and with prescription medications (e.g. nicotine gum,
nicotine patch, spray, pulmonary inhalation or other forms). In
particular, the pharmaceutical compositions and treatment methods
of the invention may be used to diminish a subjects desire to smoke
and thereby alter smoking behaviour. The pharmaceutical
compositions and treatment methods of the invention may also be
used together with other centrally active pharmaceutical
compositions that modify smoking behaviour (e.g. bupropion (a.k.a.
Wellbutrin.RTM.) in its various formulations), to decrease the dose
of the centrally active composition or to increase its
effectiveness in the treatment of tobacco dependence.
[0139] The compositions and treatment methods of the present
invention by regulating nicotine metabolism in an individual are
highly effective. The methods and compositions maintain the
behavioural components of smoking and modify them by reducing
nicotine metabolism to cotinine. An individual with reduced
nicotine metabolism following administration of a composition of
the present invention, will alter smoking behaviour and smoke
exposure because of modification of nicotine requirements. The
methods and compositions of the invention show patterns of
reduction, more sustained abstinence, and lower tobacco smoke
exposure than obtained with prior art methods in particular those
using nicotine deprivation.
[0140] The behavioural component of smoking is particularly
important in some groups of individuals, and thus the methods and
compositions of the invention in modifying and maintaining
behavioural components may be particularly useful in reducing
smoking in those individuals. For example, it has been found that
behavioural components are significant in tobacco use by women. The
present invention permits the development of behavioural learning
on an individual/or group basis.
[0141] The compositions and treatment methods of the invention are
also particularly suited to regulate nicotine metabolism in
individuals or populations having high levels of CYP2A6. For
example, Caucasians in North America have high levels of CYP2A6. An
individual or population having a high level of CYP2A6 can be
identified using our methods for measuring CYP2A6.
[0142] The compositions and methods of the invention also have the
advantage of individualization and flexibility in treatment
duration. The compositions and treatment methods are particularly
suitable for severely dependent individuals, previous treatment
failures, individuals unable to accept the current approach of
complete cessation, treatment/prevention of relapse, or concurrent
treatment with other methods such as the nicotine patch. It is
expected that the compositions and treatments of the invention will
decrease the doses of nicotine patch and all other forms of
nicotine replacement therapies that are needed and will prolong the
duration of action of the therapy and/or enforce their
effectiveness in the treatment of tobacco dependence.
[0143] The methods and compositions of the invention in treating
individuals with nicotine use disorders and nicotine-induced
disorders are also useful in the treatment and prophylaxis of
diseases or conditions, including nicotine-related disorders such
as opioid related disorders; proliferative diseases; cognitive,
neurological or mental disorders; and other drug dependencies in
the individuals. Examples of such underlying diseases or conditions
include malignant disease, psychosis, schizophrenia, Parkinson's
disease, anxiety, depression, alcoholism, opiate dependence, memory
deficits, ulcerative colitis, cholinergic deficits, and the
like.
[0144] The methods and compositions of the invention may also be
used in the prophylaxis and treatment of individuals having a
condition which requires a reduction in CYP2A6 or CYP2B6. For
example, CYP2A6 is known to metabolize several procarcinogens such
as NNK (Crespi C L, et al., "A tobacco smoke-derived nitrosamine,
4-(methylnitrosamino)-1-(3-pyridyl)-1-- butanone, is activated by
multiple human cytochrome P450s including the polymorphic human
cytochrome P4502D6," Carcinogenesis, 12(7):1197-201 (1991)),
aflaxtoxin B1 (Yun C H, et al., "Purification and characterization
of human liver microsomal cytochrome P450 2A6," Molec. Pharmacol.,
40(5):679-85 (1991)); hexamethylphosphoramide (Ding X, et al.,
"Mossbauer studies on the metal-thiolate cluster formation in
Fe(II)-metallothionein," Eur. J. Biochem., 171(3):711-4 (1988)),
and nitrosodimethylamine (Davies R L, et al., "Development of a
human cell line by selection and drug-metabolizing gene
transfection with increased capacity to activate promutagens,"
Carcinogenesis, 10:885891 (1989); Fernandez-Salguero, et al.
(1995)). Therefore, inhibitors of CYP2A6 may be useful in the
prophylaxis (e.g., inhibition of CYP2A6 substrates thereby
decreasing genotoxicity, cytotoxicity and/or mutagenicity) and
treatment of malignant diseases, and, without limitation, the
above-mentioned conditions and diseases.
[0145] Formulation and Dosing
[0146] The pharmaceutical compositions of the invention contain
substances which inhibit CYP2A described in detail herein or
substances identified using the methods of the invention. The
active substances can be administered alone, but are generally
administered with a pharmaceutical carrier etc. (see below),
selected on the basis of the chosen route of administration and
standard pharmaceutical practice.
[0147] The dosage administered will vary depending on the use and
known factors such as the pharmacodynamic characteristics of the
particular substance, and its mode and route of administration;
age, health, and weight of the individual recipient; nature and
extent of symptoms, kind of concurrent treatment, frequency of
treatment, and the effect desired.
[0148] In some instances, instead of increasing the dosage of a
compound, the kinetics of inhibition created by certain chemical
compounds can be altered or enhanced by adding to the treatment
protocol a second inhibitor to a substance (e.g., enzyme) that is
capable of inhibiting the metabolism of the CYP2A6 inhibitor. By
adding such a second inhibitor, the quantity of the CYP2A6
inhibitor will be maintained thus prolonging the beneficial effect
of maintaining an elevated plasma concentration of nicotine. The
use of such a second inhibitor is very beneficial since it
facilitates treatment of individuals by maintaining substantially
constant nicotine levels and acting locally on the kinetics of the
CYP2A6 inhibitor. By using this approach, large dosages of
centrally active compounds can be avoided.
[0149] Similarly, preexposure of an individual to an inhibitory
substance sometimes can result in an inhibitory effect that will
outlast the presence of the drug in the plasma or that will have a
persistent effect in the individual despite the inhibitor's half
life in the plasma. This phenomenon caused by preincubation or
preexposure of an inhibitory substance can help increase the dose
interval at which a dosage of the substance must be administered,
decrease the chronic dose or enhance CYP2A6 inhibition.
Furthermore, preexposure of an individual to one inhibitory
substance can subsequently decrease the needed dose of a second
inhibitor.
[0150] The appropriate dosage of a substance which selectively
inhibits CYP2A6 is dependent upon the amount of CYP2A6 that is
present in an individual's body. This amount is in turn dependent
upon whether the individual contains two mutant alleles, one mutant
allele or no mutant alleles at the CYP2A6 gene locus. In Example 1,
we confirmed that such variations can exist in the genetic material
of a population. It is, therefore, an aspect of this invention to
provide a method for determining the CYP2A6 activity in an
individual containing two mutant alleles, one mutant allele or no
mutant alleles at a gene locus for the CYP2A6 gene, the method
comprising the steps of:
[0151] (a) assaying a bodily sample containing deoxyribonucleic
acid (i.e. a "DNA-containing bodily sample") from the individual to
determine whether the individual contains two mutant alleles, one
mutant allele or no mutant alleles at the CYP2A6 gene locus;
[0152] (b) determining the amount of CYP2A6 present in the
individual; and
[0153] (c) correlating the results of assaying in step (a) and the
amount of CYP2A6 in step (b) to determine an appropriate dosage for
that individual of a substance which (i) selectively inhibits
CYP2A6 activity, or (ii) selectively inhibits transcription and/or
translation of the gene encoding CYP2A6.
[0154] The individual recipient may be any type of mammal, but is
preferably a human. Generally, the recipient is an individual
having a CYP2A6 genotype associated with an active form of the
enzyme. The CYP2A6 genotype of an individual and the existence of
an active CYP2A6 enzyme in an individual may be determined using
procedures described herein. For example, coumarin 7-hydroxylation
has been used to measure CYP2A6 activity (Cholerton, et al. (1992);
and Rautio, et al., (1992)). As discussed above, the methods and
compositions of the invention may be preferably used in individuals
or populations having high levels of CYP2A6, or in individuals
where the behavioural components of smoking are significant.
[0155] For use in the treatment of conditions requiring regulation
of nicotine metabolism to cotinine, by way of general guidance, a
daily oral dosage of an active ingredient such as coumarin or
methoxsalen can be about 0.01 to 80 mg/kg of body weight,
preferably 0.01 to 20, more preferably 0.05 to 3 mg/kg of body
weight. Ordinarily a dose of 0.03 to 50 mg/kg of coumarin,
methoxsalen or tranylcypromine per day in divided doses one to
multiple times a day, preferably up to four times per day, or in
sustained release form is effective to obtain the desired results.
In accordance with a particular regimen, coumarin or methoxsalen or
tranylcypromine is administered once to four times daily for as
long as necessary. While standard interval dose administration may
be used the compositions of the invention may be administered
intermittently prior to high risk smoking times, e.g., early in the
day and before the end of a working day.
[0156] More than one substance described in detail herein or
identified using the methods of the invention may be used to
regulate metabolism of nicotine to cotinine. In such cases the
substances can be administered by any conventional means available
for the use in conjunction with pharmaceuticals, either as
individual separate dosage units administered simultaneously or
concurrently, or in a physical combination of each component
therapeutic agent in a single or combined dosage unit. The active
agents can be administered alone, but are generally administered
with a pharmaceutical carrier selected on the basis of the chosen
route of administration and standard pharmaceutical practice as
described herein.
[0157] The substances for the present invention can be administered
for oral, topical, rectal, parenteral, local, inhalant or
intracerebral use. In an embodiment of the invention, the
substances are administered in intranasal form via topical use of
suitable intranasal vehicles, or via transdermal routes, using
forms of transdermal skin patches known to those of ordinary skibl
in that art. To be administered in the form of a transdermal
delivery system, the dosage administration will be continuous
rather than intermittent throughout the dosage regimen. The
substances can also be administered by way of controlled or slow
release capsule system and other drug delivery technologies.
[0158] For example, for oral administration in the form of a tablet
or capsule, the active substances can be combined with an oral,
non-toxic, pharmaceutically acceptable, inert carrier such as
lactose, starch, sucrose, glucose, methyl cellulose, magnesium
stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol
and the like; for oral administration in liquid form, the oral
active substances can be combined with any oral, non-toxic,
pharmaceutically acceptable inert carrier such as ethanol,
glycerol, water, and the like. Suitable binders, lubricants,
disintegrating agents, and colouring agents can also be
incorporated into the dosage form if desired or necessary. Suitable
binders include starch, gelatin, natural sugars such as glucose or
beta-lactose, corn sweeteners, natural and synthetic gums such as
acacia, tragacanth, or sodium alginate, carboxymethylcellulose,
polyethylene glycol, waxes, and the like. Suitable lubricants used
in these dosage forms include sodium oleate, sodium stearate,
magnesium stearate, sodium benzoate, sodium acetate, sodium
chloride, and the like. Examples of disintegrators include starch,
methyl cellulose, agar, bentonite, xanthan gum, and the like.
[0159] Gelatin capsules may contain the active substance and
powdered carriers, such as lactose, starch, cellulose derivatives,
magnesium stearate, stearic acid, and the like. Similar carriers
and diluents may be used to make compressed tablets. Tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of active ingredients over a period
of time. Compressed tablets can be sugar coated or film coated to
mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
may contain colouring and flavouring agents to increase patient
acceptance.
[0160] Water, a suitable oil, saline, aqueous dextrose, and related
sugar solutions and glycols such as propylene glycol or
polyethylene glycols, may be used as carriers for parenteral
solutions. Such solutions also preferably contain a water soluble
salt of the active ingredient, suitable stabilizing agents, and if
necessary, buffer substances. Suitable stabilizing agents include
antioxidizing agents such as sodium bisulfate, sodium sulfite, or
ascorbic acid, either alone or combined, citric acid and its salts
and sodium EDTA. Parenteral solutions may also contain
preservatives, such as benzalkonium chloride, methyl- or
propyl-paraben, and chlorobutanol.
[0161] The substances described in detail herein and identified
using the methods of the invention can also be administered in the
form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles, and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine, or phosphatidylcholines.
[0162] Substances described in detail herein and identified using
the methods of the invention may also be coupled with soluble
polymers which are targetable drug carriers. Examples of such
polymers include polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmeth-acrylamideph- enol,
polyhydroxyethylaspartamidephenol, or polyethyl-eneoxide-polylysine
substituted with palmitoyl residues. The substances may also be
coupled to biodegradable polymers useful in achieving controlled
release of a drug. Suitable polymers include polylactic acid,
polyglycolic acid, copolymers of polylactic and polyglycolic acid,
polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates,
and crosslinked or amphipathic block copolymers of hydrogels. The
substances can also be affixed to rigid polymers and other
structures such as fullerenes or Buckeyballs.
[0163] Pharmaceutical compositions suitable for administration
contain about 1 milligram to 1500 milligrams of active substance
per unit. In these pharmaceutical compositions, the active
ingredient will ordinarily be present in an amount of about 0.5-95%
by weight based on the total weight of the composition.
[0164] Suitable pharmaceutical carriers and methods of preparing
pharmaceutical dosage forms are described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, a standard
reference text in this field.
[0165] Co-administration with Oral Nicotine
[0166] In a particular embodiment, it has been found that specific
inhibitors of CYP2A6, preferably methoxsalen and/or
tranylcypromine, are particularly effective inhibitors of CYP2A6
and of the metabolism of an oral formulation of nicotine and as
such, enhance the effect of oral nicotine replacement therapies. In
other words, it has been found that these inhibitors are effective
in inhibiting nicotine metabolism and thereby increasing plasma
concentrations of nicotine, particularly when the nicotine is
orally ingested thereby enhancing oral nicotine replacement
therapies.
[0167] Thus, this invention provides a composition for enhancing
the effect of oral nicotine replacement therapy, comprising an
inhibitor of CYP2A6 and nicotine formulated for oral ingestion. In
this method, the substances described in detail herein and/or
identified using the screening method described above, together
with nicotine, form the active ingredient, and are typically
administered in admixture with suitable pharmaceutical diluents,
excipients, or carriers suitably selected with respect to the
intended form of administration, that is, oral tablets, capsules,
elixirs, syrups and the like, consistent with conventional
pharmaceutical practices.
[0168] Those of skill in the art will recognize that oral
formulation within the invention can be in the form of: (i) a
single composition comprising both the CYP2A6 inhibitor and
nicotine, or (ii) a kit comprising independently administered
compositions comprising the CYP2A6 inhibitor and nicotine,
respectively. For independently administered compositions, the
administration is preferably substantially contemporaneous. When
the preferred CYP2A6 inhibitors methoxsalen and/or tranylcypromine
are administered with oral formulations of nicotine the plasma
concentrations of nicotine have increased over the plasma
concentrations when nicotine is orally digested without
administering the CYP2A6 inhibitor(s).
[0169] Combination of Inhibitors
[0170] The combination of an CYP2A6 inhibitor (e.g., coumarin,
methoxsalen), and a CYP2B6 inhibitor (e.g., orphenadrine) enhances
inhibition of nicotine metabolism to cotinine. Thus, a preferred
embodiment of the invention provides a method for treating
conditions requiring regulating nicotine metabolism to cotinine
comprising administering an effective amount of a CYP2A6 inhibitor
and an effective amount of a CYP2B6 inhibitor to selectively
inhibit nicotine metabolism to cotinine. In a preferred embodiment
of the invention, the CYP2A6 inhibitor is methoxsalen or an analog
or derivative thereof, and the CYP2B6 inhibitor is orphenadrine, or
an analog or derivative thereof. The inhibitors may be administered
concurrently, separately or sequentially. Preferably, the
administration of the inhibitors is substantially contempraneous.
The doses of the CYP2A6 inhibitor and the CYP2B6 inhibitor are each
selected so that each inhibitor alone would not show a full effect.
The effective doses are those which are approximately the minimum
doses adequate for enhanced inhibition of nicotine metabolism to
cotinine. In one mode, the combination of inhibitors may be
administered substantially contemporaneously with a source of
nicotine, preferably nicotine formulated for oral administration.
Pharmaceutical compositions containing combinations of CYP2A6 and
CYP2B6 inhibitors may be prepared, and administered as described
herein for the compositions containing CYP2A6 inhibitors. The
pharmaceutical compositions preferably contain methoxsalen or an
analog or derivative thereof, and orphenadrine, or an analog or
derivative thereof, in concentrations of 1 to 1500 mg, and 25 to
400 mg, respectively.
[0171] Embodiments of the present invention will be illustrated
with reference to the following examples which should not be
construed as limiting the scope of the invention.
EXAMPLES
Example 1
[0172] Epidemiology Study
[0173] We examined the prevalence of CYP2A6 gene mutations in 126
tobacco dependent Caucasian smokers and 143 Caucasian individuals
who had tried smoking, but who had never became tobacco dependent
smokers (e.g., exposure controls). The objectives were two fold.
The first was to determine the incidence of individuals who were
deficient in CYP2A6 activity (e.g., homozygous for null CYP2A6
alleles). The second was to determine if slower CYP2A6 mediated
nicotine metabolism, due to having null CYP2A6 alleles, decreased
the chances of becoming a tobacco dependent smoker.
[0174] In this Example a study was conducted to assess the CYP2A6
genotype in a group of individuals and the effect of the CYP2A6 on
the smoking behaviour of the individuals.
[0175] Subjects were unrelated healthy individuals each with 4
Caucasian grandparents and were divided into three groups. The
first group comprised tobacco Dependent only (TD, DSM-IV
("DSM"=Diagnostic Statistician Manual of the American Psychiatric
Association)) subjects including 76 males aged 19 to 52 years old
(mean (SD): 31.1 years old (85 years)), and 57 females aged 20 to
70 years old (mean (SD): 31.4 years old (10.2)). The second group
comprised Alcohol and Tobacco Dependent (AT, DSM-W) subjects
including 60 males aged 17 to 61 years old (mean (SD): 37.2 years
old (9.94 years)), and 10 females aged 19 to 66 years old (mean
(SD): 41.4 years old (11.89 years)). The third group was an
exposure control group consisting of Never-Tobacco Dependent (NTD(
subjects, who had previously tried smoking, but had never become
dependent. This group included 86 males 19 to 59 years old (mean
(SD): 29.2 years old (8.6 years)), and 77 females 19 to 58 years
old (mean (SD): 27.4 years old (8.4 years)). All subjects completed
a drug questionnaire and tobacco module (Heatherton, et al., "The
Fagerstrom Test for nicotine Dependence: a revision of the
Fagerstrom Tolerance Questionnaire," Br. J. Addict.,
86(9):1119-1127 (1991)). All subjects had no other psychoactive
drug dependencies, including alcohol (except of course for the AT
group).
[0176] CYP2A6 genotyping of each subject was performed on genomic
DNA isolated from peripheral leukocytes as described by
Fernandez-Salguero, et al. (1995). Briefly, the assay consisted of
a CYP2A6 gene-specific nested PCR amplification followed by a RFLP
analysis.
[0177] Materials and Methods:
[0178] Primers Used for PCR Genotyping Assays:
1TABLE 2 Assay Name Sequence (5'-3') CYP2A6*2 (v.sub.1) and F4
CCTCCCTTGCTGGCTGTGTCCCAAGCT CYP2A6*3 (v.sub.2) TAGGC R4
CGCCCCTTCCTTTCCGCCATCCTGCCC CCAG E3F GCGTGGTATTCAGCAACGGG E3R
TCGTGGGTGTTTTCCTTC
[0179] CYP2A6 Genotype
[0180] DNA is extracted from blood samples and quantified using
routine extraction procedures. CYP2A6 genotype was determined using
nested PCR and RFLP as described by Fernandez-Salguero, et al.
(1995). The first amplification, which is CYP2A6 gene-specific, was
used to increase the specificity for the CYP2A6 gene (versus other
CYP2A genes). Exon 3 was utilized in the second amplification
because both the CYP2A6*2 and CYP2A6*3 mutant alleles contain
nucleotide changes leading to amino acid changes in this region of
the CYP2A6 gene.
[0181] The first amplification was performed using the XL-PCR kit
(Parkin-Elmer Co., Norwalk, Conn.). A 100 .mu.l reaction mixture of
0.2 .mu.M of primer F4 and R4, 200 .mu.M dNTPs, 0.8 mM magnesium
acetate, and 2 U of rTth1 DNA polymerase and 400 to 600 ng of
genomic DNA used. The amplification was performed in a MJ DNA
Engine (MJ Research, Inc., Watertown, Mass.) at 93.degree. C. for 1
minute, 66.degree. C. for 6 minutes and 30 seconds for 31
cycles.
[0182] The second amplification was performed in a reaction mixture
containing 0.5 .mu.M of primers E3F and E3R, 200 .mu.M dNTPs, 1.5
mM MgCl.sub.2, 2.5 U of Taq DNA polymerase (Gibco BRL, Life
Technologies, Burlington, Ontario), and 2.5 .mu.l of first
amplification product, which was the template for the reaction. The
reaction conditions were as follows: 94.degree. C. for 3 minutes,
followed by 31 cycles of 94.degree. C. for 1 minute, 60.degree. C.
for 1 minute and 72.degree. C. for 1 minute.
[0183] The second amplification yielded a PCR product 201 bp in
length which was digested with Xcm I (New England Biolabs) and Dde
I (New England Biolabs and Pharmacia Biotech) to detect the
CYP2A6*2 and CYP2A6*3 mutations, respectively (cutting indicates
the presence of the mutation). Concentrations of enzymes and PCR
product, total volume and digestion time were determined
empirically to optimize cutting efficiency with a minimal amount of
time and enzyme. Xcm I digestion reactions were carried out at
37.degree. C. for 2 hours in a 30 .mu.l reaction mixture containing
1.times.NEBuffer 3 (100 mM NaC, 50 mM Tris-HCl, 10 mM MgCl.sub.2, 1
mM DIT pH 7.9@25.degree. C.), dH.sub.2O, and 2 U of Dde I
digestions were carried out at 37.degree. C. for 2 hours in a 30
.mu.l reaction mixture containing One-Phor-All (OPA) buffer
(Pharmacia Biotech) and 2 U of Dde I. Digestion products were
analysed on ethidium-stained 3% agarose gels.
[0184] Blood samples were obtained under consent from all subjects.
Positive controls were donated by Drs. P. Fernandez-Salguero and H.
Raunio. Negative controls used water in place of genomic DNA. Every
genotyping reaction carried four randomly selected samples from a
previous reaction to check for reproducibility.
[0185] Chi-Square tests were performed comparing distribution of
CYP2A6 genotypes and alleles between groups (SAS). F-tests (check
for unequal variance [SAS]), followed by a two-sample t-test (SAS)
were used in comparing smoking patterns within smoke. Significance
was at 5%.
[0186] It was postulated that individuals with impaired nicotine
metabolism (i.e., carriers of at least one CYP2A6 defective or
mutant allele) would experience greater aversive effects due to
higher nicotine levels and not become smokers or would smoke at a
decreased level when compared to individuals with active nicotine
metabolism (i.e., individuals having CYP2A6*1/CYP2A6*1 genotype).
Specifically, it was hypothesized that there would be an
under-representation of individuals carrying defective CYP2A6
alleles in a tobacco dependent population.
[0187] With reference to the results of the study, among the total
dependent smokers (TD+AT), the frequency of individuals carrying 1
or 2 of the CYP2A6 defective alleles was lower than in the exposure
control group (NTD): 13.3% vs. 20.2%, p=0.076, .chi.-square; Odds
Ratio of 1.66, C.I. 0.95-2.89--see FIG. 5. Further, both the
CYP2A6*2 and CYP2A6*3 allele frequencies were lower in the
dependent smokers (TD and AT) than in the exposure control group
(NTD): CYP2A6*2: 3.0% versus 3.1% and CYP2A6*3: 4.4% versus
7.7%--see FIG. 5.
[0188] We further postulated that, within the group of those who
smoke, those with deficient nicotine metabolism (i.e., carriers of
at least one CYP2A6 defective or mutant allele) would smoke fewer
cigarettes.
[0189] With further reference to the results of the study, within
the TD group, those subjects which were homozygous for CYP2A6
active alleles smoked significantly more cigarettes per day and per
week when compared with smokers who were heterozygous carrying a
single CYP2A6 defective allele (i.e., one or both of CYP2A6*2 and
CYP2A6*3): 23 versus 19 cigarettes per day, t test P=0.02, 125
versus 161 cigarettes per week, t test P=0.009--see FIG. 6.
[0190] As discussed above, nicotine is important in establishing
and maintaining tobacco dependence; variability in nicotine
pharmacokinetics could have a profound influence on whether
individuals become smokers. The data produced in this study
demonstrates an under-representation of individuals carrying 1 or 2
of the CYP2A6 defective alleles in a tobacco dependent population
(TD+AT) when compared to a never tobacco dependent (NTD) control
population. While not wishing to be bound by any particular theory
or mode of action, this may be caused when individuals who carry
CYP2A6 defective alleles, upon smoking, experience higher nicotine
levels and greater aversive effects to the nicotine. As a result,
the individual may discontinue smoking and be less likely to become
tobacco dependent. Therefore, the data produced in this study
indicates that individuals who carry 1 or 2 of the CYP2A6 defective
or mutant alleles, and who try smoking, are at lesser risk for
becoming tobacco dependent than individuals who have two active
CYP2A6 alleles.
[0191] Further, as discussed above, it is well known that dependent
smokers adjust their smoking behaviour in order to maintain blood
and brain nicotine concentrations, and thus, variable nicotine
metabolism could play a role in altering smoking patterns. The
observation that dependent smokers who carry a single defective
CYP2A6 allele smoke significantly fewer cigarettes when compared to
homozygous wild-type smokers indicates that nicotine metabolism, as
mediated by CYP2A6, is a significant determinant in the amount that
dependent smokers smoke. In other words, heterozygosity in a single
gene, namely the CYP2A6 gene, is affecting this complex drug taking
behaviour.
[0192] The clinical implications of CYP2A6 genotype on tobacco
dependence and smoking behaviour disclosed herein are widespread.
Individuals who carry CYP2A6 defective alleles may have a decreased
risk for cancer development because they have a decreased risk of
becoming tobacco-dependent smokers. If they do become dependent
smokers, the data produced in this study demonstrates that they
would smoke less than those homozygous for active CYP2A6 alleles.
There is clear evidence that the amount of tobacco smoked is
related to increased risk for lung cancer (Law, et al. 1997))--see
FIG. 6.
[0193] In addition, tobacco smoke contains a number of tobacco
specific procarcinogen nitrosamines, such as
N-nitrosodialkylamines--e.g., N-nitrosodiethylamine (the Merck
Index, No. 6557), N-nitrosodimethylamine (The Merck Index, No.
6558) and 4-methylnitrosamino)-1-)3-pyridyl)-1-buta- none (Crespi,
et al. 1990; Yamazaki, et al. 1992). As these procarcinogens can be
activated by CYP2A6, individuals who carry CYP2A6 defective alleles
will be advantageously inefficient at bioactivating tobacco smoke
procarcinogens to carcinogens.
[0194] Thus, in summary the data produced in the study of this
Example demonstrates that a single genetically polymorphic gene,
the CYP2A6 gene, is related, and in some cases predictive, of
whether an individual becomes a smoker. In addition, if an
individual becomes dependent on tobacco alone, the CYP2A6 gene
variants alter the number of cigarettes that he/she smokes.
Accordingly, the CYP2A6 genotype directly influences the risk for
tobacco dependence, alters the amount of tobacco consumed, and
plays a role in tobacco-related cancer susceptibility.
[0195] One envisaged application of the present invention is the
genetic identification of an individual's risk for smoking and
related cancers. Identification of high and low risk individuals
will allow targeted prevention, treatment and education.
Specifically this will involve the identification of an individual
with a high and low risk for: (i) becoming a smoker (if the
individual is a non-smoker), (ii) higher tobacco-consumption (if
the individual is a smoker), and (iii) CYP2A-related cancers.
EXAMPLE 2
[0196] Coumarin Phenotyping Test and CYP2A6 Genotyping Test
[0197] A. Coumarin Test
[0198] Coumarin is a selective and specific substrate for human
CYP2A6 and can be used to: (1) identify individuals who are
potential therapeutic exclusions for use of CYP2A6 inhibitors; (2)
for dosage refinement based on the initial level of activity of
CYP2A6; and (3) for risk factor assessment in identifying
individuals who will not benefit from the treatment or who may be
at risk to toxicity from agents which are inhibitors and substrates
themselves of CYP2A6. The Coumarin Test exists in two forms:
[0199] (1) Coumarin Test When Only Urine is Available
[0200] Coumarin 5 mg formulated in a capsule or other dose form is
administered orally to fasted individuals after voiding of residual
bladder urine. Urine is collected for the first 2 hours and for the
subsequent 6 hours. The amount of urinary excretion of the coumarin
metabolite 7 hydroxy-coumarin (free and conjugated) is determined
by determining the concentration of these metabolites on the urine
using an HPLC assay as described in an earlier example. The
relative activity of CYP2A6 is reflected in the total amounts of 7
hydroxy-coumarin excreted in the sampling periods separately and
combined and the activity can be expressed as the ratio of the
percent coumarin excretion (amount excreted in the first 2
hours/amount excreted in 8 hours).times.100. This percent excretion
ranges from values less then 20% in individuals without CYP2A6
activity to >80% in individuals with high activity. This test
can be equally effectively and reliably be applied to smokers and
non-smokers and may be used at any time of day with out apparent
effect of the smoking condition or time of day on the results. The
test demonstrates high within subject reproducibility with a linear
r of >0.9. See FIG. 3 for results of a study in which smokers
and nonsmokers were given coumarin in the morning and afternoon on
each of 2 separate days. High within subject reproducibility and
reliability is demonstrated.
[0201] (2) Coumarin Test When Plasma Samples Can Be Taken
[0202] In some clinical situations blood samples can be easily
taken or are necessary as part of other clinical tests. In this
situation, a plasma-based test of CYP2A6 activity has been
developed and applied to individuals of known genotype. Individuals
ingest coumarin 5.0 mg orally and 45 minutes later a blood sample
is drawn in a heparinized (or other anticoagulant containing tube).
The sample is spun and the plasma separated. The plasma is analysed
by HPLC to quantitate 7 hydroxycoumarin (total after deconjugation
with beta glucuronidase incubation). High analytical sensitivity is
required in order to use 5.0 mg of coumarin. When such sensitivity
is not available, the dose of coumarin may be increased up to 50
mg.
[0203] HPLC Analysis of 7-hydroxycoumarin in Urine and Plasma:
[0204] (1) Sample Preparation:
[0205] Urine or plasma samples (0.5 ml) are hydrolyzed with 0.2 ml
of 9-glucuronidase acetate buffer solution (15 mg/ml acetate
buffer, 0.2 M, pH 5.0) at 37.degree. C. for 30 min. Extraction is
followed with 2 ml ether by vortex for 5 min and centrifuged at
3000 rpm for 10 min. Ether extract (1.2 ml) is transferred to
another clean tube and dried down under nitrogen gas. The residue
is reconstituted in the HPLC mobile phase (see below), and injected
onto HPLC.
[0206] (2) HPLC Analysis:
[0207] The HPLC system consists of Hewlett Packard 1050 HPLC system
(pump, autosampler and UV detector) and HP339611 integrator. The
chromatographic separation was performed with an HP Spherisorb-ODS2
column (125.times.4 mm I.D., 5 .mu.m). Samples were eluted with a
mobile phase of acetonitrile:water:acetic acid of 150:850:2 (v/v/v)
at a flow rate of 1.0 ml/min, and monitored by a UV detector at a
wavelength of 324 nm for 7-hydroxycoumarin and 280 nm for coumarin.
Samples are quantitatively determined by an external standard
method.
[0208] The CYP2A6 activity is expressed as the concentration of 7
hydroxycoumarin in the plasma at various points in time (e.g. 20,
30, 45 and 75 minutes) or as the ratio of coumarin/7
hydroxy-coumarin in the plasma at that time.
[0209] The preferred mode of use is a simple plasma sample at 20 or
30 minutes after the oral administration of coumarin in which both
coumarin and 7-hydroxycoumarin are quantified and in which the
coumarin to 7-hydroxycoumarin ration is used as the index of CYP2A6
activity.
[0210] Results:
[0211] Blank urine or plasma samples showed no interfering peak for
7-hydroxycoumarin or coumarin. Sensitivity of this method is 1
ng/ml urine or plasma. Intraday and inter-day variations are less
than 10%. This analysis is linear from 1 ng to 4000 ng/ml.
[0212] FIG. 4 is a graph showing a time course of total
7-hydroxycoumarin concentration detected in the plasma of subjects
given coumarin. FIG. 4 illustrates various time courses based on
corresponding genotypes for CYP2A6.
[0213] B. CYP2A6 Genotyping Test
[0214] As for the CYP2A6 genotyping test, mutant alleles which
decrease CYP2A6 activity in an individual can be screened in a DNA
sample using the materials and screening method described in
Example 1.
Example 3
[0215] In Vivo Phenotype Assay
[0216] The plasma kinetics of nicotine and coumarin were compared
after oral administration in 10 smokers and 9 non-smokers (12
males, 7 females) of known CYP2A6 genotype. The dose of nicotine
was 4.0 mg (expressed as base) and the dose of coumarin was 50 mg.
The plasma concentration of nicotine, cotinine, coumarin and
7-OH-coumarin were measured as described above.
[0217] Optimal separation of *1/*1 (wild type homozygotes, n=13)
and heterozygotes (*1/*2; *1/*3, n=4) and homozygotes (*2/*2, n=2)
was found at 45 min with coumarin by measuring its metabolite
7-OH-coumarin (7-OH-coumarin [.mu.M]*1/*1=5.6.+-.2.9; *1/*2 or
*1/*3-3.8.+-.1.1, p=0.04). Optimal separation was found at 90 min
with nicotine (nicotine [nM]*1/*1=24.+-.15; *1/*2 or
*1/*3=29.+-.12; *2/*2=52.+-.3; *2/*2 vs. *1/*2 or *1/*3, p=0.01;
*2/*2 vs. *1/*1, p=0.0001). The use of the coumarin/7-OH-coumarin
or nicotine/cotinine ratio did not improve separation.
[0218] Cotinine (nicotine metabolite) was significantly more slowly
produced in *1/*2 or *1/*3 initially, but late in sampling cotinine
was actually higher in *1*/2 or *1/*3, suggesting a role for CYP2A6
in cotinine metabolism. The slope of the curve for appearance vs.
time was significantly less for 7-OH-coumarin and greater for
nicotine in *1/*2 or *1/*3 compared to *1/*1, as were the areas
under the curves, indicating differences in bioavailability, rates
of absorption and metabolism. The area of the curves for
7-OH-coumarin and for nicotine were inversely correlated (p=0.08
(Spearman rank), n=18). One *1/*1 individual with 4fold greater
7-OH-coumarin production and one *2/*2 individual with high
coumarin and low nicotine metabolism were identified, suggesting
that CYP2A6 gene variants not detected with current PCR procedures
exist.
[0219] These data indicate CYP2A6 genotype is an important
determinant of time course for nicotine disposition in vivo. These
data also indicate that an in vivo assay of CYP2A6 reflects the
phenotype of the individual, and that the phenotypic determination
provides information on the genotype of that individual.
Example 4
[0220] Tissue Localization of CYP2A6 Expression
[0221] It was determined if the CYP2A6 enzyme was expressed in
tissues in which tobacco-related cancers occur (e.g., lung and
bladder). In order to determine whether CYP2A6 was expressed in
tissues which were subject to tobacco-related cancers, the tissue
distribution of the CYP2A6 mRNA was examined in various human
tissues using Northern blot analysis. Briefly mRNA from numerous
tissues was loaded onto gels and separated by electrophoresis. The
mRNA was then transferred to membranes and probed with radioactive
CYP2A6 cDNA probe. Evidence was found for the expression of CYP2A6
in uterus, ovaries, colon, small intestine, testis, bladder, heart,
stomach, prostate, skeletal muscle, pancreas and lung. These data
suggest that in addition to the possibility that CYP2A6-activated
carcinogens from the liver might cause cancer in various tissues
there could also be in situ activation of the procarcinogens in a
number of human tissues.
Example 5
[0222] Epidemologic Study for Cancer Risk
[0223] In addition to the role that null CYP2A6 alleles have in
reducing the rate of nicotine-dependence and the amount smoked if
one becomes dependent, procarcinogens found in tobacco-smoke can be
activated by CYP2A6. In order to determine whether there was a
significant contribution to cancer rates due to activation of
procarcinogens by CYP2A6, independent of its role in smoking, a
study was made of individuals with a tobacco-related cancer who
were non-smokers. These individuals were passively exposed to
tobacco smoke procarcinogens, therefore providing a group in which
the role of the CYP2A6 null alleles in the activation of
procarcinogens could be tested independently of the role of this
enzyme on smoking behaviour. It was found that 14.3% of non-smokers
who had bladder cancer (a tobacco-related cancer) carried a null
allele for CYP2A6. In contrast, in the control population who were
non-smokers and did not have bladder cancer, the frequency of null
allele carriers was 24.1%. This demonstrates that individuals who
carry null alleles for CYP2A6 are less likely to get a
tobacco-related cancer due to their decreased activation of
procarcinogens to cancer-causing carcinogens.
Example 6
[0224] Effect of Methoxsalen, a CYP2A6 Inhibitor, on Activation of
NNK, a Procarcinogen, to its Metabolites NNAL and NNAL
Glucuronide
[0225] The effect of methoxsalen, CYP2A6 inhibitor, on nicotine
metabolism and NNAL production was studied in eleven (n=6 females,
5 males) tobacco dependent smokers. Subjects were recruited only if
they smoked at least 15 cigarette per day, and were required to
smoke the same number of cigarettes each day, on all four study
days. During assessment, blood was taken to be analyzed for plasma
nicotine and cotinine levels, as well as for CYP2A6 genotyping.
Breath carbon monoxide was also measured.
[0226] The four test days were broken down into one placebo day (no
drug given) (day 1) and three methoxsalen (10 mg t.i.d. p.o.)
treatment days 2, 3, 4), such that medication was taken at 8:00
a.m., 3:00 p.m. and 10:00 p.m. each day. During each study day a
smoking log was completed. This log asked subjects to document the
number of cigarette smoked from 8:00 a.m. to 3:00 p.m. to 10.00
p.m., and 10:00 p.m. to 8:00 a.m. Study days 1 and 2 could be
separated, but days 2,3 and 4 were required to be consecutive.
[0227] On both study days 1 (placebo) and 4 (treatment), a 24 h
urine collection was initiated on waking. At 2:00 p.m. blood was
drawn for nicotine and cotinine analysis and breath carbon monoxide
was measured. On day 4, a second blood sample was taken for
methoxsalen analysis. Urine samples can be analyzed for 24 h NNAL,
NNAL glucuronide, creatinine and cotinine.
[0228] Methoxsalen increased plasma nicotine by 17% (23.0 to
27ng/ml); decreased breath CO by 11% (22.5 to 20.5 ppm) and
increased the ratio of plasma nicotine to breath CO (an index of
tobacco smoke exposure) by 30% (1.1 to 1.43, p=0.033). The larger
decrease in the index of smoke exposure indicates: 1) the smokers
decreased the intensity of their smoking due to inhibition of
CYP2A6 and slowed nicotine elimination despite being told to not
change their smoking; 2) methoxsalen in these doses is an effective
inhibitor of CYP2A6; and 3) methoxsalen and other CYP2A6 inhibitors
will decrease production of NNAL or related substances and the
activation of other carcinogens in vivo.
Example 7
[0229] Influence of the CYP2A6 Null Alleles on Tobacco-smoke
Exposure Leading to Lung Cancer
[0230] The effect of the CYP2A6 null alleles on activation of
procarcinogens was examined in an epidemiological study of lung
cancer. The allele frequencies in DNA samples from 227 individuals
with lung cancer was determined. Following diagnosis of lung cancer
and resection of the tumor and surrounding lung tissue, the DNA was
extracted and genotyped for CYP2A6. The population was principally
smokers or ex-smokers. Among those from whom detailed smoking
histories were available, we were able to assess the number of
pack-years of smoking (20 cigarettes/day for one year=one
pack-year) as a measure of procarcinogen exposure, prior to
detection of the lung cancer. Those individuals who had wt/wt
CYP2A6 activity required an average of only 45 pack-years prior to
lung cancer detection. In contrast, those individuals with a CYP2A6
null allele, who activate less of the procarcinogens, required
considerably more procarcinogen exposure prior to detection of lung
cancer (e.g., 54 pack-years). Thus those individuals with decreased
CYP2A6 activity activate less of the tobacco-smoke procarcinogens
and require greater exposure before lung cancer is detected.
[0231] It was also observed that the number of individuals with the
CYP2A6V1 allele in the lung cancer population was decreased
relative to. a non-lung cancer control population, consistent with
the relative protection against cancer offered by decreased
procarcinogen activation. This was observed in both non-smokers and
smokers with lung cancer relative to their respective controls.
[0232] In addition, all of the individuals who had ras oncogene
mutations in the tumor tissues were full activity CYP2A6 wildtype
indicating a decreased risk for oncogene mutations in those
individuals with decreased CYP2A6 activity (e.g., carriers of the
CYP2A6 null alleles). Ras oncogene mutations are predictive of a
poorer treatment outcome and faster growing tumors relative to
those tumors without ras oncogene mutations. This suggests that
individuals with CYP2A6 null alleles, were less likely to have
mutated ras oncogenes, and were more likely to have a better
treatment prognosis.
[0233] These data indicate the utility in assessing CYP2A6 alleles
for estimation of cancer risk (null alleles being associated with
decreased risk for lung cancer) and that inhibition of CYP2A6 will
decrease procarcinogen activation decreasing the risk for
cancer.
Example 8
[0234] Effect of CYP2A6 Inhibition on the Bioavailability of Oral
Nicotine
[0235] An in vivo study was undertaken to determine the effect of
CYP2A6 inhibition on the bioavailability of oral nicotine. In
particular, the study compared the kinetic effects of nicotine
cotreatment with methoxsalen 30 mg, tranylcypromine 10 mg and of
placebo (i.e., nicotine only) p.o. on the bioavailability of
nicotine 4 mg p.o (expressed as base; nicotine bitartrate salt was
actually administered), and the acute safety and acceptability of
the three cotreatments. Additionally, preliminary information was
obtained about effects on nicotine craving following these
cotreatments.
[0236] The subjects for the study were smokers following a
specified abstinence regimen as set out below. There were 12
subjects.
[0237] The smokers underwent placebo p.o. and two separate
cotreatments 30 (methoxsalen 30 mg and tranylcyprornine 10 mg)
accompanying nicotine 4 mg p.o. During a 4-hour test session, blood
and urine samples were collected for kinetic measures, and
physiologic and subjective measures were collected. The treatment
order was randomized and counterbalanced and the drug presentations
were single-blind, namely, the subjects were unaware of what drug
they were taking.
[0238] All of the subjects had the following characteristics:
[0239] (a) age at least 21;
[0240] (b) current consumption of at least 25 cigarettes per
day;
[0241] (c) DSM IV current tobacco dependence;
[0242] (d) nicotine dependence as indicated by a score of at least
3 on the Fagerstrom Test of Nicotine Dependence (FIND);
[0243] (e) no regular use of tobacco in any form other than
cigarettes;
[0244] (f) ability to abstain from cigarette smoking and from
caffeine for up to 12 hours;
[0245] (g) agreement and ability to maintain the use of any
therapeutic drugs on a consistent schedule across the study
days.
[0246] All of the subjects did not have any of the following
characteristics:
[0247] (a) known sensitivity to methoxsalen or chemically similar
compounds;
[0248] (b) known excessive photosensitivity;
[0249] (c) current use of any antidepressants, sympathomimetics,
CNS depressants, hypotensive agents, or antiparkinsonian drugs
(because of possible adverse tranylcypromine interactions);
[0250] (d) body weight <51 kg (30 mg methoxsalen is not
recommended for use below this body weight);
[0251] (e) pregnancy or lactation;
[0252] (f) risk of pregnancy (females who are sexually active with
male partners and not using highly effective contraceptive
precautions, defined as surgical sterilization of either partner,
oral contraceptives, barrier and spermicide, or condom and
spermicide);
[0253] (g) liver damage, blood dyscrisias (counterindications for
tranylcypromine);
[0254] (h) symptoms suggestive of cardiac disease or
hypertension;
[0255] (i) any other medical or psychiatric condition that requires
further investigation or treatment or that is a contraindication
for any of the proposed study drugs;
[0256] (j) current desire or attempts to quit smoking within the
expected duration of the study series; and
[0257] (k) any other condition likely to interfere with compliance
with the study schedule or successful collection of study
measures.
[0258] There were two separate study schedules for subjects tested
in the morning and in the afternoon. On both schedules, each
subject abstained from tobacco, food, beverages (i.e., other than
water), and any inconsistently used drugs from midnight before each
study day but continued to take any regularly scheduled drugs
allowed by the protocol (e.g., oral contraceptives, daily
vitamins).
[0259] Subjects on the morning schedule continued such abstinence
until arrival at the test site at approximately 8 am. Subjects on
the afternoon schedule ate a normal-sized breakfast, including at
most one cup of a caffeinated beverage and one cigarette before 9
am. From 9 am then to approximately 1 pm subjects in the afternoon
session resumed the abstinence regimen until arrival at the test
site.
[0260] Before baseline measures were taken, a breath CO sample
(Ecolyzer) was taken to assess compliance with the smoking
abstinence (<10 ppm expected). The subsequent daily schedule is
set out in Table 3. All measurements were with respect to a time
zero at 9:00 am or 2:00 pm, at which time a nicotine capsule and a
cotreatment are taken p.o. Each SMS cycle consisted of heart rate,
blood pressure, and subjecive measures. A standard breakfast or
lunch (but without caffeine) was served after the blood sample at
+1:00 h.
2TABLE 3 Time Elapsed Event -00:45 Prepare the subject -00:30 Blood
sample (8 mL) taken - #1 -00:15 SMS cycle #1 00:00 All capsules
p.o. 00:30 Blood sample (8 mL) taken - #2 00:50 SMS cycle #2 01:00
Blood sample (8 mL) taken - #3 01:30 Blood sample (8 mL) taken - #4
01:50 SMS cycle #3 02:00 Blood sample (8 mL) taken - #5 2:50 SMS
cycle #4 03:00 Blood sample (8 mL) taken - #6
[0261] Subjects were medically assessed for discharge no earlier
than +2:00 h and were discharged after all measures were complete
at +3:00 h. Subjects were not allowed to smoke until after their
discharge. The schedule was in certain circumstances delayed by up
to 20 minutes, consistent across the four days, in order to allow
three subjects to be tested on the same day.
[0262] Each subject was tested on two non-consecutive days in a
week for three separate weeks and maintained a morning or afternoon
schedule consistently.
[0263] Sterile nicotine bitartrates was obtained by the Pharma
Centre from Sigma Chemical. The reported purity was >99.5% which
was confirmed by HPLC. The nicotine bitartrate powder was measured
on a precision balance accurate to within 1 .mu.g, measured into
portions containing 4 mg of the nicotine base, and then
encapsulated. Capsules were filled to a tolerance of +2% of their
nominal mass of nicotine powder.
[0264] Methoxsalen is marketed in Canada in two forms, one of
relatively low bioavailability (trade name Oxsoralen.RTM.) and two
of approximately twice as high a bioavailability
(Oxsoralen-Ultra.RTM. and Ultra MOP.RTM.). The package insert for
Ultra MOP.RTM.) (Canderm Pharmacal Ltd.) recommends a daily dose of
30 mg to 50 mg for any patient weighing 51 kg or more. Subjects
were restricted to a minimum body weight of 51 kg, in order to
allow the use of a fixed 30 mg dose for all subjects; the dose was
not adjusted for larger subjects.
[0265] Capsules of methoxsalen 10 mg and tablets of tranylcypromine
10 mg (Parnate.RTM.) were used in their marketed forms. Because it
was a randomized but single-blind study, subjects were able to
recognize that the capsule forms and number varied from day to day,
but they did not know which form represented which drug.
[0266] Lactose tablets were used for the placebo.
[0267] For the first four days, each day's drug supply consisted of
one or three capsules, as set out in Table 4, in addition to
nicotine 4 mg. The investigators were unaware of the distribution
to preserve random allocation.
[0268] The nicotine and other capsules were taken
simultaneously.
[0269] A separate, printed, reference copy of each subject's
treatment randomization code was provided to the investigators
after the drugs were dispensed.
3 TABLE 4 Active Drug, Capsule Form Administered Dosage placebo 1
.times. coumarin-size placebo methoxsalen, 30 mg 3 .times.
methoxsalen, 10 mg tranylcypromine, 10 mg 1 .times.
tranylcypromine, 10 mg
[0270] Using an indwelling venous catheter, 8 mL blood samples were
collected at the elapsed times set out in Table 3. These samples
were analyzed for nicotine, cotinine and inhibitor
concentration.
[0271] Two separate urine samples were collected, a baseline just
prior to the capsules and a three-hour pooled sample. Each urine
sample was analyzed for nicotine and cotinine content.
[0272] Plasma and urinary nicotine and cotinine (and also the
conjugates in urine) were determined using an HPLC method with a UV
detector. Specifically, 1 mL of sample, 50 .mu.L (2 .mu.g/mL) of
the internal standard (N-ethylnomicotine) and 1 mL of
trichloroacetic acid (10%) were pipetted into each tube (12 mL).
The tube was capped, vortex-mixed for a few seconds, and then
centrifuged at 30,000 g for 5 minutes. The clear supernatant was
decanted in a second tube. To this protein-free plasma extract was
added 0.5 mL of a 5 M potassium hydroxide solution and 6 mL of
methylene chloride. The second tube was then capped, agitated for
30 minutes in a horizontal shaker and then centrifuged to separate
the phases. The aqueous phase (the top layer) was aspirated, and
3.0 mL of 0.5 N hydrochloric acid solution was added to the organic
phase and vortex-mixed for 30 seconds. The phases were separated by
centrifugation, and the aqueous phase was transferred to a clean
tube with 0.5 mL of 5 M potassium hydroxide solution, followed by
addition of 5 m L of methylene chloride and vortex mixing for 30
seconds. The phases were separated by centrifugation, the aqueous
(top) layer was aspirated, and 200 .mu.l methanolic hydrochloric
acid (10 mmol HCl in methanol) was added to the remaining solution
and mixed gently. The organic solvent was then evaporated under
nitrogen in a water bath at 40.degree. C. The sides of the tube
were washed with 200 .mu.L of methanolic hydrochloric acid and the
solution was evaporated. The residue was reconstituted in 100 .mu.L
of 30% methanol and 90 .mu.L thereof was injected in the HPLC
column.
[0273] The chromatographic separation was performed with a
Supelco.TM. 5-8347 LC-8-DB (150.times.4.6 mm, 5 .mu.m). The sample
was eluted with a mobile phase of 0.34 M citric acid
buffer:acetonitrile, 800 :45 (v/v) containing 0.34 M
KH.sub.2PO.sub.4, 1-heptane sulphonate (671 mg) and triethylamine
(5 mL) with a flow rate of 1.3 mL/min, and monitored by a UV
detector at .lambda.=260 nm.
[0274] The sensitivity of the nicotine assay is <1 ng/mL and
that of the cotinine is <5 ng/mL. Conjugates in urine were
determined after hydrolysis with .beta.-glucuronidase, when
appropriate.
[0275] Plasma nicotine concentration was determined using the
above-mentioned assay, and the results are illustrated in FIG. 7 as
the mean for all subjects.
[0276] Cardiovascular measures, transduced by a Hewlett/Packard
78352C Adult Patient Monitor and recorded directly into a computer
included heart rate and blood pressure (while seated).
[0277] Using a visual analog scale, each subject was asked to
evaluate, on a scale of 0 to 100, his/her current desire to smoke a
various points in time, both pre- and post drug administration
(Appendix A).
[0278] For the purposes of establishing clinical kinetic
differences and describing kinetic parameters, the primary
dependent variable was the 3-hour trapezoidal-rule nicotine
AUC.
[0279] FIG. 7 illustrates the mean plasma nicotine concentrations
measured just prior to oral drug administration and for three hours
thereafter (during which no smoking was allowed). As illustrated,
the combined methoxsalen/nicotine and tranylcypromine/nicotine
treatments both induce an increase in mean plasma nicotine
concentration that is at least four times as large as that induced
by the placebo/nicotine combination.
[0280] FIG. 8 illustrates the self-rated "Current desire to smoke"
evaluation, using a visual analog scale scored from 0 to 100. As
illustrated, both the methoxsalen/nicotine and
tranylcypromine/nicotine combination reduced the desire to smoke
significantly more than does the placebo/nicotine combination.
Example 9
[0281] Effects of Metabolically Enhanced Oral Nicotine Replacement
Therapy on Short-term Smoking Behaviour
[0282] A study was undertaken to determine the effects of
metabolically enhanced oral nicotine replacement therapy on
short-term smoking behaviour.
[0283] Because nicotine is the addictive agent in tobacco
dependence, and smokers regulate their brain nicotine within a
fairly narrow individual concentration band, any effective
non-smoking method of nicotine delivery should result in a decrease
in smoking by allowing smokers to maintain plasma nicotine without
resorting to smoking and, in some cases, reduce the secondary
reinforcement of smoking behaviour as a component of eventual
smoking cessation. This effectiveness can only be enhanced if the
mechanism for ensuring effective delivery also delays the clearance
of plasma nicotine after the absorption stage. A model for testing
the acute behavioural effects of this treatment strategy is
provided in a previous study of the effectiveness of nicotine gum
(Nemeth-Coslett R, et al., "Nicotine gum: dose-related effects on
cigarette smoking and subjective ratings," Psychopharmacology,
92(4):424-30 (1987)), where smoking in a 90-minute test period was
significantly affected by the nicotine content of the gum.
[0284] In this Example, all four combinations of
placebo/methoxsalen and placebo/nicotine were tested (i.e., (i)
placebo methoxsalen and nicotine; (ii) methoxsalen and nicotine,
(iii) methoxsalen and placebo nicotine; and (iv) placebo
methoxsalen and placebo nicotine).
[0285] This Example demonstrates: the kinetic effectiveness of
methoxsalen-enhanced oral nicotine replacement therapy in briefly
abstinent smokers; and the behavioural effectiveness of
methoxsalen-enhanced oral nicotine replacement therapy in briefly
abstinent smokers.
[0286] There were 11 subjects. The subject inclusion and exclusion
criteria used in Example 8 were used in this Example.
[0287] The subjects each underwent four separate sessions of 90
minutes smoking abstinence followed by 90 minutes ad lib smoking,
where the following four treatments were each presented
double-blind once during the first four study days, in randomized,
counterbalanced order, during the abstinence period: (i) placebo
methoxsalen with placebo nicotine, (ii) placebo methoxsalen with 4
mg nicotine, (iii) methoxsalen 30 mg with placebo nicotine, and
(iv) methoxsalen 30 mg with 4 mg nicotine. Methoxsalen 10 mg
capsules were used, as in Example 1, and capsules of royal jelly
were used as the placebo. Capsules were dispensed in an opaque
vial, and neither subjects nor investigators viewed the capsules
prior to the subjects placing them directly into their mouths.
[0288] Nicotine 4 mg capsules were prepared as in Example 8, and
corresponding royal jelly capsule placebos containing only lactose
were also prepared. Subjects took the three methoxsalen/placebo
capsules and one nicotine/placebo capsule either all at once or
consecutively.
[0289] The treatment order was counterbalanced across the two sexes
and the two times of day to the extent possible. The order of the
treatments was determined by a computerized randomization program.
The treatment was double-blind.
[0290] As in Example 8, there was a morning and afternoon
schedule.
[0291] Study sessions were scheduled twice each day, with three
subjects running simultaneously, beginning at 8:30/8:40/8:50 am and
at 1:00/1:10/1:20 .mu.m. Sessions lasted for about 4 hours. Prior
to each session, subjects were allowed to smoke, eat, and drink
caffeine as they desired up to 90 minutes before each session, at
which time they stopped eating and drinking (other than water).
Each session began 60 minutes before a drug/placebo/nicotine were
taken. The precise schedule is set out in Table 5.
4TABLE 5 Time Elapsed Event -00:40 Pre-abstinence cigarette -00:30
Abstinence begins -00:10 1. Blood sample (8 mL) taken - #1 2. CO
measured 3. Questions 1-9 in Appendix C 00:00 1. CO measured 2. All
capsules p.o. 00:50 1. Blood sample (8 mL) taken - #2 2. CO
measured 3. Questions 1-12 in Appendix C 00:59 1. CO measured 2.
Video camera on 01:00 1. Abstinence ends 2. Free smoking begins
02:30 1. Video camera off 2. Free smoking ends 3. Blood sample (8
mL) taken - #3 4. CO measured 5. Questions 1-17 in Appendix C 6.
Abstinence resumes 02:40 CO measured 02:50 CO measured 03:00 CO
measured
[0292] The first post-cigarette abstinence period lasted 90
minutes, of which the last 60 minutes were post administration of
placebo/drug/nicotine. The second abstinence period was included to
facilitate repeated measures of the post-smoking breath carbon
monoxide (CO). On the three occasions when blood samples were
collected, the subject also answered a brief questionnaire about
possible study drug symptoms and effects and about desire to smoke.
Additionally, on the third occasion, there were also questions
about perception of the cigarettes smoked during the free smoking
period. This questionnaire (see Appendix A) is based on the one
used in the nicotine gum study (Nemeth-Coslett, et al. (1987)).
[0293] During the free smoking period, subjects were allowed to
smoke as they wished, providing only that they stay within the area
of the room visible to the video camera, and to eat light snacks
and drink non-caffeinated beverages.
[0294] The primary dependent variable measured was the change in
breath CO during the free-smoking period, measured as the mean of
the three samples 10, 20, and 30 minutes post-smoking minus the
mean of the two samples 10 and 0 minutes pre-smoking. Other
dependent variables measured were the change in plasma nicotine
between 0 and 150 minutes post-drug, the responses to the symptom
and rating scales, the consumption of tobacco in the free smoking
period (measured as the weight of butts remaining subtracted from
the weight of the same number of unsmoked cigarettes), and the puff
timing and count from analysis of the videotapes.
[0295] Dependent variables were evaluated in an analysis of
variance, with the treatment drug combinations as the primary
independent variable of interest, with sex, morning/afternoon
schedule, and treatment order as additional explanatory variables
removed from the error term.
[0296] The results of the study of Example 10 will now be discussed
with reference to FIGS. 9-14.
[0297] FIG. 9 illustrates the mean breath carbon monoxide
concentration measured just prior to oral drug administration, 60
minutes later (no smoking allowed) and after the 90 minute free
smoking period. As illustrated, the combined methoxsalen/nicotine
treatment results in a significant and large reduction in the
increased in breath carbon monoxide during the smoking phase. The
carbon monoxide levels increased in the combined
methoxsalen/nicotine treatment group only 30% of that seen in the
other conditions, reflecting a large reduction in smoking and smoke
exposure. This reduction may be attributable due to any combination
of fewer puffs, shallower puffs and/or puffs held for a shorter
duration before exhalation.
[0298] FIG. 10 illustrates the ratio of the increased plasma
nicotine concentration to increased breath carbon monoxide
concentration over the 90 minute free smoking period. In other
words, this Figure illustrates the measure of potential reduction
in smoke exposure that might occur while dependent smokers
replenish their systemic plasma nicotine content. As illustrated,
the methoxsalen/nicotine treatment stands apart from all three of
the other treatments and from their mean, more than doubling the
gain in nicotine per unit of increase in breath carbon monoxide
concentration.
[0299] FIG. 11 illustrates the commonly used measure of smoking:
the mean number of cigarettes smoked during the 90 minute period.
As illustrated, the combined methoxsalen/nicotine treatment is
associated with the least smoking, however number of cigarettes
consumed is an insensitive measure of smoke exposure and smoking
behaviour compared to breath carbon monoxide concentration.
[0300] FIG. 12 illustrates the mean cumulative number of puffs
taken by the end of each 10 minute period during the 90 minute free
smoking period, and the data is summarized by the area under this
curve. This area would increase if either the total number of puffs
increased, or if the same number were consumed earlier thereby
shifting the curve to the left. As illustrated, the
methoxsalen/nicotine treatment is associated with the fewest
cumulative number of cigarette puffs.
[0301] FIG. 13 illustrates that the number of grams of tobacco
burned is significantly less in the methoxsalen/nicotine treatment
group. Again, this measure is less sensitive than direct measures
of smoking behaviour and smoke exposure (e.g., breath carbon
monoxide concentration and nicotine/breath carbon monoxide
concentration).
[0302] FIG. 14 illustrates that the inhibition of CYP2A6 metabolism
of nicotine achieves the reduction in breath carbon monoxide by
changing nicotine-regulated smoking behaviour. The latency between
cigarettes is significantly prolonged by the combined
methoxsalen/nicotine treatment.
[0303] In summary, the results illustrated in FIGS. 9-14 show a
modification in smoking behaviour by subjects who were treated with
a combination of methoxsalen and nicotine. Specifically, key
objective indicators such as plasma nicotine concentration and
breath carbon monoxide were significantly reduced compared to the
other treatment regimens.
[0304] CYP2A6 metabolizes approximately 75% of nicotine in vivo.
Tobacco-dependent smokers regulate their smoking to maintain
nicotine levels; CYP2A6 inhibition should decrease nicotine
metabolism, decrease smoking and smoke exposure (e.g. breath CO).
Overnight nicotine-abstinent dependent smokers (6 males, 5 females)
smoked one cigarette followed by one of four oral drug combinations
in crossover counter-balanced order. methoxsalen 30 mg (CYP2A6
inhibitor K; =0.2 .mu.M) or placebo with either nicotine 4.0 mg or
placebo. Sixty minutes later, subjects started 90 mins of ad
libitum smoking. Subjects receiving methoxsalen with oral nicotine
smoked less than in the placebo/placebo (e.g. breath CO 50% less
increase; latency to the second cigarette 83% increase, number of
cigarettes smoked 24% decrease; tobacco burned (grams) 24%
decrease, total number of puffs taken decrease 25% [all
p<0.05]). In addition, on several measures (e.g. latency to
second cigarette) the rank order of response was
methoxsalen/nicotine>methoxsalen/placebo>placebo/nicot-
ine>placebo/placebo suggesting a methoxsalen effect on systemic
clearance of nicotine. CYP2A6 inhibition alone or combined with
oral nicotine decreases smoking and could have a role in tobacco
smoking cessation, exposure reduction or relapse prevention
strategies.
Example 10
[0305] Inhibitors Of Nicotine's Metabolism: Potential New
Treatments For Tobacco Dependence
[0306] Nicotine is inactivated by CYP2A6 to cotinine. As nicotine
bioavailability is low (20-35%), an oral nicotine replacement is
feasible. Ini vitro inhibition of nicotine metabolism was studied
in expressed CYP2A6 using tranylcypromine, methoxsalen, and as
inhibitors (K.sub.i=0.05-6 .mu.M). In dependent smokers
methoxsalen, 30-50 mg orally 30 min prior to nicotine 31 .mu.g/kg
subcutaneously (3 doses, hourly), increased the 8-hour mean plasma
nicotine by 49% (p<0.01) while coumarin (225 mg hourly.times.6)
increased it by 15% (p<0.05) compared to placebo. Using
methodology described above, studies conducted in regular smokers
(n 7-12) given nicotine (4.0 mg p.o.) alone and concurrently with
placebo, methoxsalen (3 mg, 10 mg or 30 mg) or tranylcypromine (2.5
mg or 10 mg) significantly increased the plasma nicotine compared
to placebo (see FIG. 15). Specifically, methoxsalen 10 and 30 mg
(M10 and M30, FIG. 15) and tranylcypromine 25 and 10 mg (T2.5 and
T10, FIG. 15) produced approximate 100% increases in plasma
nicotine (p<0.01). In addition, these increases in oral nicotine
bioavailability were associated with a significant (p<0.05)
decrease in desire to smoke for M10 and M30 and T25 (p=0.17) (see
FIG. 15, solid circles).
[0307] From these studies it is evident that methoxsalen and
tranylcypromine in doses 1/4 and 1/8, respectively, of their
current therapeutic doses used for treating psoriasis and
depression, respectively, can be used to inhibit nicotine
first-pass metabolism and systemic metabolism. Other CYP2A6
inhibitors and their isomeric forms can be expected to do the
same.
Example 11
[0308] Extraction of Hypericum
[0309] The following procedures were used to prepare an extract of
Hypericum perforatum:
[0310] An extraction mixture of 10 Hypericum capsules (0.3%
hypericin) and 50 ml of 80% methanol (water 20%, v/v) was placed in
a beaker (125 ml). In one protocol the extraction was carried out
using cold methanol. In a second protocol, the extraction mixture
was placed in a water-bath and heated until boiling (about
80.degree. C.). The mixture was kept boiling for 30 min, keeping
the volume to 50 ml by adding methanol, then cooled down at room
temperature, and centrifuged at 3,000 g for 5 min. The resulting
extract was then blown to dryness and resuspended in trisbuffer pH
7.4.
Example 12
[0311] Effect of Hypericum Extracts on CYP2A6 Activity
[0312] Nicotine metabolism was monitored in vitro. Inhibitor
assessment was carried out by comparing the assay including 50
.mu.l tris buffer with the extracts prepared in Example 11 (50
.mu.l, diluted in tris buffer). Human liver microsomes or CYP2A6
microsomes were used. The concentration of inhibitor in Hypericum
was calculated on the basis of an apparent molecular weight of the
inhibitor of 504 and a concentration of 0.3% active material in the
plant material when diluting to yield concentrations of 20, 10, 5,
1, 0.1 and 0.01 and 0.01 .mu.M.
[0313] Incubation Mixture
[0314] Substrate 80 .mu.M (50 .mu.l tris buffer)
[0315] NADPH1 mM (50 .mu.l tris buffer)
[0316] Microsomes 80 .mu.g (30 .mu.l tris buffer)
[0317] Cytosol (rat) 20 .mu.l (tris buffer)
[0318] Tris buffer 50 .mu.l (pH7.4)
[0319] Final volume 200 .mu.l
[0320] Incubation Procedure:
[0321] The incubation was carried out at 37.degree. C. for 30 min.
An internal standard (50 .mu.l, caffeine, 0.05 mg/ml) was added to
the incubation mixture, followed by DCM (1 ml). The mixture was
shaken for 10 min and centrifuged for 5 min at 3,000 g. The top
layer was aspirated. HCl (0.01 N, 100 .mu.l) was added, followed by
shaking for 1 min, then centrifuge for 5 min at 3,000 g. 30 .mu.l
of the HCl layer was injected for HPLC.
5 HPLC Conditions Column: Supelco; 5-8347; LC-8-DB; 150 .times. 4.6
mm; 5 .mu.m .lambda.: 260 nm Flow rate: 1.3 ml/min Mobile phase:
1000/120 (citric buffer/acetonitrile, v/v) Citric buffer: Citric
Acid 7.14 g (0.34 M) KH.sub.2PO.sub.4 4.627 g (0.34 M) 1-Heptane
sulphonate 670 mg Triethylamine 5 ml Final volume 1000 ml Adjust
the pH to 4.40 with 5 N KOH Retention times (min): 2.2 (nicotine
iminium ion) 2.7 (nicotine) 3.4 (continine) 3.9 (caffeine)
(internal standard)
[0322] FIG. 16 demonstrates the inhibition of nicotine metabolism
with (1) cold methanol extract of Hypericum; (2) a hot methanol
extract of Hpericum; (3) purified Hypericin; and (4) a negative
control. The results indicate that both methanol extracts of
Hypericum can inhibit CYP2A6 activity.
Example 13
[0323] Effect of St. John' Wort on Nicotine Metabolism
[0324] Twelve subjects received placebo or 3 St. John's Wort
capsules 300 mg plus oral nicotine 4.0 mg as base orally. Blood
samples were taken at designated times over 3 hours.
[0325] The results shown in FIGS. 18 and 19 demonstrate that St.
John's Wort increases the bioavailability of nicotine int vivo. The
mean plasma nicotine concentrations were 64% higher after St.
John's Wort.
Example 14
[0326] Effect of Esculetin on CYP2A6 Activity
[0327] Esculetin, a compound found in Cichorium intybus and
Bougainvllra spectabillis, was tested for its ability to inhibit
nicotine metabolism by CYP2A6 using the methods set forth in
Example 12.
[0328] The results, shown in FIG. 19, demonstrate that esculetin is
a potent inhibitor of CYP2A6.
[0329] Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated to
those skilled in the art that the invention can be modified in
arrangement and detail without departure from such principles. We
claim all modifications coming within the scope of the following
claims.
[0330] All publications, patents and patent applications referred
to herein are incorporated by reference in their entirety to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference in its entirety.
6 TABLE 1 Average Number of Cigarettes Genotype Smoked Per Day
Frequency TD AT NTD TD AT Males Females Total Males Females Total
Age 28.3 31.2 37.8 31.1 31.4 31.2 37.2 41.4 37.8 wt/wt 130 114 62
26.1 19.5 23.2 28.9 27.8 28.7 wt/mut 31 17 7 19.3 18.7 19.1 29.7
50.0 32.6 mut/mut 2 2 1 22.5 -- 22.5 25 -- 25 n 163 133 70 76 57
133 60 10 70 wt/wt: CYP2A6 * 1/CYP2A6 * 1 wt/mut: CYP2A6 * 1/CYP2A6
* 2 + CYP2A6 * 1/CYP2A6 * 3 mut/mut: CYP2A6 * 2/CYP2A6 * 2
[0331]
Sequence CWU 1
1
4 1 32 DNA artificial sequence Primer 1 cctcccttgc tggctgtgtc
ccaagcttag gc 32 2 31 DNA artificial sequence Primer 2 cgccccttcc
tttccgccat cctgccccca g 31 3 20 DNA artificial sequence Primer 3
gcgtggtatt cagcaacggg 20 4 18 DNA artificial sequence Primer 4
tcgtgggtgt tttccttc 18
* * * * *