Genetic polymorphisms act as the main source of
interindividual variations in P450 enzymes, affecting the therapeutic effects
of drugs as well as the metabolism of substances i.e Nicotine via CY2A6
enzymes. Polymorphisms ,by definition, are different alternative sequences at
loci within DNA that persist within a particular population over generations.
They generally appear following genetic mutations or the exposure to an
environmental factor. Such genetic mutations include deletions and insertions
that result in a frameshift or single nucleotide polymorphisms (SNPs). These
polymorphisms alter protein synthesis, protein synthesis rates, and complete loss of
proteins. Other sources of genetic polymorphisms include the exposure to
certain environmental factors such as the consumption of food whose components
may act as enzyme inducers or inhibitors. For instance, flavonoids that are
present teas that show inhibitory effects on CYP2A6; whereas drugs such as
Phenobarbitals can act as potent CYP2A6 inducers. Polymorphic variations of
CYP2A6 genes are held accountable for the interindividual variations in nicotine
metabolism, influencing nicotine dependence and smoking habit (Pouyfung et al., 2014).

CYP2A6, CYP2A7, and CYP2A13 are located on
chromosome 19q13.2 that is located within the 350-bp gene cluster Figure 2 (Reference,
2017). Both CYP2A6 and CYP2A7 have high gene homology, the only difference is
that CYP2A7 lacks a heme-binding residue which results in flawed heme
incorporation leading to enzyme inactivation. CYP2A6 constitutes 1-10% of liver
enzymatic content. CYP2A6 has been shown to be present in the olfactory mucosa
of human fetuses along with CYP2A13 which enables them to metabolize odorants
and to process olfactory signals.

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Initially, three main CYP2A6 variants were
identified: the wild type allele (wt CYP2A6*1) and two more alleles (mutant
type CYP2A6*2 and CYP2A6*3) that were defective (Yamano, Tatsuno and Gonzalez,
1990). Later, the variant strains were further explained as: (1) CYP2A6*1 the
wild type allele (2) CYP2A6*2 that is characterized by a point mutation which
leads to conversion of leucine to histidine, and encodes for proteins lacking
enzymatic activity, (3) CYP2A6*5 lacks enzymatic activity, (4) CYP2A6*3 that
was confused with the true variant, (5) CYP2A6*4 lack enzymatic activity due to
gene deletion. Individuals homozygous for CYP2A6*4 or CYP2A6*5 lack enzymatic
capacity and as a result coumarin 7-hydroxylation capacity is diminished (Tyndale and Sellers, 2001).   The different identified polymorphic forms of
CYP2A6 are illustrated in Table 2. Furthermore, individuals showing
heterozygosity for CYP2A6*2 exhibit intermediate metabolic activity. In other
words,  heterozygous individuals with CYP2A6*1/*2 and CYP2A6*1/*3
exhibit 50% of nicotine metabolizing capacity. While individuals homozygous for
CYP2A6*1/*1 show full capacity for the metabolism of nicotine (Tyndale and Sellers, 2001).  Moreover, kinetics of nicotine is greatly
influenced by the presence of variant CYP2A6 alleles. A study conducted by (Tyndale,
Pianezza and Sellers, 1999) has demonstrated that CYP2A6 variations inducing
poor metabolizers phenotype are associated with altered smoking habits in which
poor metabolizers tend to smoke less; as opposed to extensive metabolizers that
tend to smoke more and have a higher tendency to develop cancers via increasing
the rate of procarcinogen activation. The increased polymorphic variation in
CYP2A6 may predispose individuals, with smoking habits, to lung cancer. Several
pilot studies modulated by (Miyamoto et
al., 1999) and (Fernandez-Salguero
et al., 1995) provide information on the reduction of
smoking via the inhibition of CYP2A6 with or without the administration of oral
nicotine supplements (Raunio et al., 2001).

Lastly, the presence of polymorphisms is greatly
influenced by the ethnicity of a population. It has been shown that Caucasians
exhibit poor metabolizers phenotype but are prevalent in Oriental populations.
However, these data don’t reflect the disease susceptibility of a major
population, but rather reflect the effects of polymorphisms on individuals. It
is worth mentioning that there are far more undiscovered genetic mutations and
allelic encodings. Therefore, nowadays there’s an increasing aim of drawing
more attention towards the identification of these mutations which brings us
one step closer to improving  personalized medicine and smoking cessation
regimens modulation to reduce predisposition to cancer (Raunio et al., 2001).As elucidated by the science of
pharmacogenomics, individuals normally express two copies of a particular gene
in which one comes from the paternal side while the other comes from the
maternal side. Thus, enabling one to have two copies of the gene. The
possibilities of inheriting wild type of mutant type alleles is influenced by
the genotype of the parents. With regards to CYP2A6, an individual can exhibit
two wild type alleles, a heterozygous combination, a mutant type homozygous
combination (Tyndale and
Sellers, 2001).  Poor metabolizers with heterozygous allelic
distribution (CYP2A6*1/*2 or CYP2A6*1/*3) tend to smoke less and as a result
remain protected from becoming dependent tobacco smokers. It is hypothesized
that these individuals longer lasting nicotine effects as opposed to the
homozygous wild type unimpaired individuals. More specifically it was
demonstrated through a study conducted by (Rautio et al., 1998) that individuals carrying CYP2A6*3 allele
showed reduced nicotine metabolism. Also heterozygosity in one gene of CYP2A6
results in a significant decrease in tobacco consumption and the initiation of
tobacco dependence (Tyndale and Sellers, 2001). Tobacco smoking has been linked with many
cancer incidences. The relationship between tobacco smoking and cancer, lung
cancer specifically, has been well established. Following tobacco smoking,
several procarcinogens are for in the body. Such procarcinogens include
N-nitrodiethylamine, 4-(methylnitrosamine)-1-(3-pyridyl))-1-butanone (NNK) and
N’-nitrosonornicotine (NNN). NNN and NNK are formed via the a-hydroxylation by
CYP2A6 (Mittal et al., 2015). Wild type homozygous CYP2A6 allele carriers
have been shown to be able to metabolize nicotine effectively; therefore,
forming NNK and NNN, which are pronounced in cancer induction. Heterozygous
CYP2A6 allele carriers were shown to be less effective in the production of the
procarcinogens. As a result, these individuals are under lower risks of
developing cancer as they also tend to smoke less that wt homozygous
individuals (Tyndale and Sellers, 2001; Mittal et al.,
2015; Raunio et al., 2001). An enzyme inhibitor is a molecule that binds
to a certain enzyme to diminish or decrease its activity. Binding of an
inhibitor to an enzyme can lead to two possible consequences: (1) stop the
substrate from reaching to the enzyme or (2) prevent the enzyme from catalyzing
the reaction.

Basically, CYP450 enzymes can be inhibited via
various ways that are divided into reversible and irreversible enzyme
inhibition. Irreversible inhibitors usually interact with the enzyme and change
its active site chemical conformation, whereas reversible inhibitors bind
non-covalently to the enzyme and is also referred to as transient inhibition.
Competitive inhibition is a reversible inhibition in which the substrate and
the inhibitor share a very close chemical structure. They can both bind to the
active site of the enzyme. As the name implies, the substrate and the inhibitor
compete for the binding site on the enzyme. As the enzyme is not chemically
altered, it can still catalyze the chemical reaction at a much slower rate provided
the concentration of the substrate is the same. This type of inhibition is weak
and is overcome by an increase in the concentration of the substrate. As a
consequence, the maximum velocity (Vmax) of the reaction is not changed while
the apparent affinity to the substrate is extensively decreased (Figure 3).

During Non-competitive Inhibition inhibitors can
bind to the enzyme alone or to the complex formed by the enzyme and its
substrate together. Once the inhibitor binds to the enzyme and its substrate,
it  induces a conformational change in the active
site of the enzyme. The position where the inhibitor binds is called the
allosteric site on the enzyme. Once a change in the active site happens, enzyme
and substrate don’t share specificity anymore and the substrate cannot bind.
With increasing inhibition, the maximum velocity will decrease no matter how
much we increase the concentration of the substrate (Figure 3). On the other
hand, irreversible inhibition is the one in which Inhibitors usually binds
covalently to the enzyme and modifies it.Irreversible inhibitors usually contain
functional groups: nitrogen mustards, aldehydes, fluorophsphonates. They react
with amino acid side chains to form covalent bonds. It’s usually specific to
one class of enzymes and do not react and inhibit all proteins. They do not
destroy the protein structure of the enzyme, but by altering the active site of
the enzyme.

Irreversible
inhibition occurs when a molecule known as the inhibitor permanently binds to
its target enzyme. This binding is covalent most of the time. That is why this
type of inhibition is called ‘Suicide Inhibitors’ (Dewick, 2013): It kills the
enzyme as it stays with it forever. Permanency depends on the time scale:
Bacterial enzymes will be permanently inhibited for few minutes while some
eukaryotes enzyme for decades (Foye et al.,
2013). Permanent inhibitors prevent the substrate from binding to the active
site of the enzyme. As a result, the increase in the concentration of the
substrate will have no effect on the enzyme (Macdonald et al,. 2012). Table 3.1. represents a list of irreversible
inhibitors of CYP450 enzymes.

Compared
to competitive inhibition where the removal of the inhibitor from the system
relieves the inhibition, in irreversible inhibition the body needs to create
new enzymes for the chemical reaction to occur (JM et al,. 2002).

There
are 3 types of irreversible inhibitors: First, there are the group specific
reagents that react with a specific side chains to induce the inhibition. A very
common example is diisopropylphosphofluoridate (DIPF). This implies that this
side chain is present in the active site of the enzyme (Berg et al,. 2007). Second, affinity labels
have a structure that resembles to the substrate. Thus, they are more specific
to the active site of the enzyme than the group specific reagents. Finally,
Suicide inhibitors bind to the enzyme as a substrate molecule. The chemical
reaction takes place and its product bind covalently to the enzyme (Berg et al,. 2007). Figure 3.1 shows the
effect of an irreversible inhibitor on the enzyme (stahl, 2008).  Human CYP2A subfamily encompasses three main
genes (CYP2A6, 2A7, 2A13) that are mainly implicated with nicotine and cotinine
metabolism. Mice CYP2A subfamily contains four genes (CYP2A5, 2A4, 2A12, and
2A22) that are also implicated with nicotine and cotinine metabolism. CYP2A
gene family in mice is associated with the metabolism of testosterone and
xenobiotics such as nicotine and its active circulating metabolite cotinine (Zhou et al., 2010). CYP2A5 shows up to 82% homology with human
CYP2A6. This homology exists in the substrate selectivity, tissue specificity,
and amino acid composition. It was previously mentioned that both human CYP2A6
and CYP2A13 and mouse CYP2A5 are expressed in the olfactory mucosa and
respiratory tract. However, CYP2A6 is found in the human liver tissue while
CYP2A5 is expressed in the liver, kidney, esophagus and lung tissue (Aoki et al., 2006). CYP2A5 in mice metabolizes nicotine into
cotinine and activates procarcinogens such as N-nitroso-N-Diethylamine (NDEA),
aflatoxin B1 (AFB1), NNK and NNN (Visoni et al., 2008).

Remarkably, differences in the rates of
nicotine metabolism and clearance exist between the two species. In fact, mice
exhibit higher metabolic rates as compared with humans. Furthermore, this has
hindered the use of mice in studying the potential uses of nicotine in the
prevention of neurodegenerative disease such as, Parkinson’s disease. This was
shown in a study conducted by (Quik et al.,2007),
in which they were unable to extrapolate toxicological and pharmacological
findings. This was caused by differences in the two species as well as
variations in the metabolic enzymatic activity in the two species. Coumarins are natural occurring compound found
in plants such as celery, figs, and sweet clover. It’s metabolized to
7-hydroxycoumarin in humans and rodents by CYP2A6 and CYP2A5, respectively. It
has been well established that coumarin is an active ratsbane, therefore
accounting for selective toxicity in some animals and rats. In mice, the
majority of coumarin is metabolized via epoxidation to
O-hydroxyphenylacetaldehyde. While a minor portion of about 5% or less
undergoes biotransformation to coumarin 7-hydroxylation by CYP2A5. In humans,
up to 70% of coumarin undergoes coumarin 7-hydroxylation (COH). To measure the activity of coumarins, we rely
on the fluorescence of COH produced by coumarins under alkaline conditions.
Fluorometry aids the characterization and the differentiation of human and mice
liver metabolic function (Aoki et al., 2006). Spectrofluorometric procedures can be
complicated or relatively simple based on the method used. Several methods have
been devised to facilitate detection of COH, such as HPLC, capillary
chromatography, and thin layer chromatography (Rahnasto et al., 2003).

Coumarin metabolism by CYP2A6/5 enzymes is a
good indicator of the respective enzymes functionality more specifically the
CYP2A6/2A5/2A13. Despite the high structural similarity between the active site
of CYP2A6 and CYP2A5, they pose some differences. A study conducted in 2001 by
(Poso et al,. 2001) has shown that
CYP2A5 active site is larger than that of CYP2A6.  This proved that CYP2A6 enzymes poorly
tolerate the negative charges of coumarin moiety, which supports the better
inhibitory effects of coumarins on CYP2A5 in mice than CYP2A6 in humans. Fluorescent probes have shown to be more
advantageous compared with conventional screening techniques i.e.
Chromatographic techniques. The advantages of fluorescence include low cost,
easily conducted, limited number of personnel needed, efficient and rapid
screening. On the other hand, its limitations include a strict set of criteria
that the probe must conform with for it to be used during a fluorometric procedure.
Secondly, the probe must be capable of producing fluorogenic products that can
be quantified. Thirdly, they should exhibit sufficient solubility in water and
they should have high signal-to-noise ratio. Lastly and most importantly, the
fluorogenic probes shouldn’t be further metabolized during the fluorometric
procedure and must present with low background fluorescence (Donato et al,. 2004).
Fluorescent probes have set a milestone in the screening CYP450 enzymes due to
the various advantages previously mentioned which facilitates the determination
of CYP450 enzymes Xenobiotics are foreign exogenous chemical
substances that aren’t produced by body. They enter the human body via
ingestions, inhalation or topical administration. Herbal products ranging from
teas to tobacco and dietary supplements ranging from vitamins and minerals to
amino acids all fall under the umbrella of xenobiotics. Herbal products are
often perceived to be a category of dietary products.   The use of herbal products has been increasing
worldwide with total sales of approximately 130$ bn per year. This has led to a
new habit among patients that is characterized by the concomitant use of
prescription drugs with herbal products. The increasing administration of
prescription or conventional drugs with herbal products causes unwanted adverse
effects. Adverse effects are attributed to the numerous phytochemical
constituents present in the herbal product which can induce or inhibit the
action of metabolic enzymes i.e. CYP450. The changes in the activity of the
CYP450 enzymes initiated by herbal substances is correlated with the altered pharmacokinetics
(PK) or the co-administered drug or pharmacologically active compound.
Subsequently, this reduces the pharmacological effects of co-administered drug
or compounds causing toxic effects (Wanwimolruk,
Phopin and Prachayasittikul, 2014).

On the other hand, there has been an increased trend for using dietary supplements.
Dietary supplements are agents that aren’t intended for diagnosis, treatment,
curing or preventing disease. They are usually taken alongside food or medical
treatment. Examples of such compounds include tobacco, multivitamins, minerals,
amino acids, and herbs that are obtained from natural plant origins. The
dietary supplements can also be synthetically produced. The medical benefits of
dietary supplements include: (1) Providing essential nutrients to patients with endogenous
compounds deficiency, (2) Patients with eating disorders, (3) Individuals with
low meat consumption or strictly vegan individuals, (4) Allergic patients to
food constituents such as lactose, (5) During pregnancy such as folic acid, (6)U1  During decreased or diminished milk intake,
(7) After surgical procedures such as gastric sleeve. Despite some of their
seemingly essential benefits, dietary supplements are associated with
toxicities induced by either excessive use or excessive build-up in the body.
They’re also capable of hindering the absorption of pharmaceutical agents (Albert,
1987).

These products do not undergo any clinical
trials or pre-clinical testing or approval. Also, there’s a tendency for
self-prescription of dietary supplements. Therefore, their interactions should
be thoroughly discussed with medical practitioners or pharmacists to avoid any
inter-individual interactions such as the inhibition or induction of CYP450
enzymes. Drug interaction is any modification caused by
a drug on the diagnostic, therapeutic, or action of a drug in or on the body.
The risk for a drug-drug interaction increases as the number of administered
drugs increases. Statistics by a study conducted in 2002 showed that for 2 concomitantly
administered drugs the risk is 6%, while that for 8 or more concomitantly
administered drugs is 100%. The mechanisms of drug-drug interactions can be:
(1) pharmacokinetics, (2) pharmacodynamics via the combined drug actions (Kuhn,
2002). The mechanism of action herbal products hasn’t
been yet established. Herbal drugs are capable of exaggerating, inhibiting, or
diminishing the actions of a drug. Pharmacokinetic effects include absorption,
distribution, and metabolism. Psyllium is a hydrocolloidal carbohydrate
component that inhibits the absorption of drugs. While, other herbs such as
aloe vera can induce diarrhea which reduces the therapeutic index of drugs. The
distribution of warfarin is influenced by the administration of black willow
which displaces highly protein-bound drugs. Lastly, the metabolism of drugs such
as corticosteroids is reduced by the administration of herbal products such as
licorice which increases the adverse effects of corticosteroids by increasing
its accumulation in the body.

Pharmacodynamic interactions are additive such
interactions includes the enhancement of the anticoagulant action of warfarin
and ginkgo biloba (Kuhn, 2002).  Tobacco smoking and nicotine dependence is a
rising worldwide health issue, posing various adverse health effects and many
tobacco related disease. In the UK the smoking rate has reached 13.7% for
individuals aged 15 years old and above (Ec.europa.eu, 2017). Nicotine is the
main addictive constituent of tobacco. Following its inhalation nicotine
rapidly enters the systemic circulation via absorption through pulmonary blood
circulation and distributes into the brain. Tobacco-dependent smokers tend to
maintain their plasma nicotine level concentration within a narrow range to
avoid toxicity or withdrawal syndrome. Currently, the administration of nicotine via
alternative sources, for example in gums or patches, reduces the frequency of
smoking by tobacco-dependent smokers. Unfortunately, the oral administration of
nicotine is not feasible due to the intense first-pass effect metabolism of
that it encounters in vivo. Side effects such as nausea and diarrhea were
pronounced upon the administration of high doses of nicotine orally. It has
been hypothesized that the inhibition of CYP2A6 should increase the
bioavailability of nicotine with a subsequent decrease in its systemic
clearance. This can facilitate the oral administration of low dose nicotine in
order to reduce smoking incidence as well as the exposure to potential tobacco
carcinogenic metabolites. Also, CYP2A6 inhibition is solely capable of reducing
the metabolism of smoked nicotine which increases its in vitro effects from
each cigarette and proportionally decreasing the number of smoked cigarettes
per day by a dependent smokerU1 .  Examples
of herbs established to have inhibitory effects on CYP2A6 enzymes include
gingko biloba and tea flavonoid components.

The purpose of this study is to determine the
effects of drug-herb interactions on smoking cessation or reduction. This will
be investigated through the performance of in-vitro studies using
7-hydroxycoumarin, a fluorescent probe of CYP2A6 enzymes.  In this study, we will verify inhibitory
effects of quercetins in gingko biloba on CYP2A6 to validate acquired results
from previous research on the same topic in a more controlled manner.