U.S. patent application number 12/704360 was filed with the patent office on 2010-09-16 for composition and method for the oxidative consumption of salivary biomolecules.
This patent application is currently assigned to MICROPURE, INC.. Invention is credited to Martin C. Grootveld, James L. Ratcliff, Christopher J. L. Silwood.
Application Number | 20100233101 12/704360 |
Document ID | / |
Family ID | 42730874 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100233101 |
Kind Code |
A1 |
Grootveld; Martin C. ; et
al. |
September 16, 2010 |
Composition and method for the oxidative consumption of salivary
biomolecules
Abstract
The composition and method for the use of stabilized chlorine
dioxide as an antimicrobial agent against oral microorganisms for
the treatment and prevention of halitosis and prevention of oral
diseases through its oxidative consumption and inactivation of
volatile sulfur compounds and their amino acid precursors is
disclosed. Preferred concentrations of stabilized chlorine dioxide
in this invention are in the range of 0.005% to 2.0% (w/v).
Inventors: |
Grootveld; Martin C.;
(Greater Manchester, GB) ; Silwood; Christopher J.
L.; (Middlesex, GB) ; Ratcliff; James L.;
(Pueblo West, CO) |
Correspondence
Address: |
The von HELLENS LAW FIRM, LTD.;C. Robert von Hellens
7330 N 16TH STREET, SUITE C 201
PHOENIX
AZ
85020
US
|
Assignee: |
MICROPURE, INC.
Scottsdale
AZ
|
Family ID: |
42730874 |
Appl. No.: |
12/704360 |
Filed: |
February 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61152336 |
Feb 13, 2009 |
|
|
|
Current U.S.
Class: |
424/53 |
Current CPC
Class: |
A61Q 11/00 20130101;
A61P 1/02 20180101; A61K 8/20 20130101; A61K 33/20 20130101 |
Class at
Publication: |
424/53 |
International
Class: |
A61K 8/22 20060101
A61K008/22; A61P 1/02 20060101 A61P001/02 |
Claims
1. A composition for oxidentively consuming salivary biomolecules,
said composition comprising a solution of stabilized chlorine
dioxide having a concentration in the range of about 0.05% to about
2.0% (w/v) to oxidatively consume pyruvate, methionine,
trimethylamine, tyrosine, glycine, creatine, 3-D-hydroxybutyrate,
salivary taurine, lactate, and lysine.
2. A composition for reducing halitosis and oral disease present in
the oral cavity, said composition comprising stabilized chlorine
dioxide solution having a concentration in the range of about 0.05%
to about 2.0% (w/v) for oxidatively consuming volatile sulphur
compounds and their amino acid precursors present in the oral
cavity to produce chlorine dioxide.
3. The composition as set forth in claim 2 wherein the
concentration of stabilized chlorine dioxide is approximately 0.1%
(w/v).
4. The composition as set forth in claim 3 wherein the
concentration of stabilized chlorine dioxide is approximately 0.4%
(w/v).
5. Use of a composition of stabilized chlorine dioxide solution
having a concentration in the range of about 0.005% to about 2.0%
(w/v) for oxidatively consuming salivary molecules to reduce the
presence of volatile sulphur compounds and oral diseases present in
the oral cavity.
6. The use as set forth in claim 5 wherein the salivary molecules
are selected from the group consisting of pyruvate, methionine,
trimethylamine, tyrosine, glycine, creatine, hydroxybutyrate,
salivary taurine, lactate and lysine.
7. A method for treating the oral cavity, said method comprising
the steps of: a) applying stabilized chlorine dioxide in a range of
about 0.005% to about 2.0% (w/v) to the oral cavity; b) oxidatively
consuming salivary molecules selected from the group consisting of
pyruvate, methionine, trimethylamine, tryosine, glycine, creatine,
3-D-hydroxybutyrate, salivary taurine, lactate and lysine; c)
reducing the presence of halitosis; and d) urging reduction of the
oral diseases present in the oral cavity.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application includes subject matter disclosed in
and claims priority to a provisional patent application entitled
"Oxidative Consumption of Salivary Biomolecules" filed Feb. 13,
2009 and assigned Ser. No. 61/152,336, assigned to the present
assignee.
FIELD OF THE INVENTION
[0002] The present invention relates to the oxidative consumption
of salivary biomolecules, in particular, it relates to the
generation of chlorine dioxide for antibacterial affects in the
oral cavity with a stabilized chlorine dioxide composition.
BACKGROUND OF INVENTION
[0003] Oral disease refers to a number of generally preventable
conditions of the mouth with a variety of causes. Plaque is the
most recognizable precursor to oral disease. It is the biofilm that
forms on teeth within hours after they are cleaned. The main
mineral component of teeth is hydroxyapatite (HAP) and when teeth
are cleaned, HAP becomes exposed to the oral environment. Salivary
proteins such as mucins, proline-rich proteins, statherins,
histatins, and cystatins have a strong affinity for HAP. These
proteins quickly bind or adsorb to the exposed HAP of the tooth to
form a thin coating called the acquired pellicle. Certain bacteria
in the oral cavity selectively adhere to the pellicle, begin to
divide, and form colonies. Initially, approximately 80% of the
bacteria that colonize pellicle-coated tooth surfaces are
facultative, gram-positive, non-motile cocci such as Streptococcus
sanguinis (formerly Streptococcus sanguis). The other 20% include a
variety of gram-negative bacteria such as Veillonella species. As
the colonies grow, the environment changes due to the metabolic
activities of these early colonizers and the addition of diverse
groups of other bacteria to the biofilm (plaque) mass. An important
environmental change in the plaque biofilm is the low-oxygen
environment that promotes colonization and growth of anaerobic
bacteria. Microorganisms in the biofilm synthesize a slime matrix
or glycocalyx from the abundant polysaccharides, glycoproteins, and
dietary sugars (e.g., sucrose) present in the oral environment.
Eventually, the plaque becomes a characteristic biofilm with a
highly structured, matrix-embedded, diverse microbial population in
which gene expression is severely altered. The volume and structure
of the biofilm created provides protection to the bacteria housed
within it, potentially reducing the efficacy of antimicrobials. As
a result, disruption of the biofilm of plaque is typically
accomplished by mechanical means (e.g., brushing, flossing,
professional tooth cleaning). Use of certain anti-plaque and
antiseptic agents has been suggested for prevention of biofilms,
but these treatments are typically tested in vitro using pure
strains of microbes cultured on agar. Such in vitro conditions do
not adequately simulate the biofilm environment, which may limit
the significance of the test results.
[0004] Within biofilms, continuous metabolic activity of bacteria
produces acids that can demineralize tooth enamel and dentin
leading to the development of dental caries and progressive tooth
decay. This demineralization is irreversible unless there is early
intervention by a dental professional who might recommend the
inclusion of certain fluoride-containing oral care products in the
daily dental routine. If left untouched, demineralization can
progress to the inner layers of the tooth, leading to severe pain
and increased potential for loss of the tooth.
[0005] If dental plaque is left undisturbed, deeper portions of the
plaque biofilm mineralize leading to the formation of calculus.
Calculus has two major components, organic material and inorganic
material. The organic portion of calculus consists mainly of dead
bacteria. The inorganic part of calculus is composed of several
minerals derived from calcium and phosphate present in the oral
environment. There are two types of calculus, subgingival (below
the gum line) and supragingival (above the gum line). Supragingival
calculus is highly organized, porous, and visible. Once formed,
calculus cannot be removed by conventional brushing and flossing;
the intervention of a dental professional is generally required.
Calculus retention is problematic for oral health because it
harbors biofilm-forming bacteria that can lead to the development
of periodontal (gum) infections.
[0006] Halitosis (bad breath) is caused primarily by the presence
of volatile sulfur compounds (VSCs) in expired breath.
Approximately 90% of foul odors in expired mouth air are due to the
presence of the two major VSCs: hydrogen sulfide (H.sub.2S) and
methyl mercaptan (CH.sub.3SH--also called methanethiol). The sulfur
in these VSCs comes from the breakdown by bacteria of
sulfur-containing proteins from saliva, plaque, and sloughed
epithelial cells. Increased production or build-up of any of the
protein sources will lead to higher levels of VSCs in mouth
air.
[0007] There are a number of known situations that will lead to
increased VSC production. For example, persons who do not perform
adequate oral hygiene will have abundant amounts of supragingival
and subgingival plaque biofilms on their teeth. This is especially
true in difficult-to-clean locations such as interproximal areas
between the teeth. In addition, natural teeth that support some
dental prostheses are difficult to clean. Finally, the dorsal
surface of the tongue is rough, irregular, and harbors large
quantities of microorganisms. In general, the microorganisms in
chronic intraoral biofilms will produce large quantities of VSCs.
Besides being the major contributor to halitosis, VSCs are potent
irritants and can aggravate existing inflammation of the gums. High
levels of VSCs can kill epithelial cells that may lead to increased
permeability and ulceration of the gum tissue. The existence of
open wounds coupled with increased gum tissue permeability can
promote the entry of bacteria into the bloodstream (i.e.,
bacteremia). Chronic bacteremia may increase the risk for the
development of a numbers of systemic problems such as heart
attacks, stroke, and adverse birth outcomes.
[0008] Gingivitis is defined as the presence of gingival
inflammation without loss of connective tissue attachment. The
precursor to gingivitis is undisturbed dental plaque biofilms.
Studies have shown that gingivitis will develop within 10-21 days
if all oral hygiene practices are stopped and plaque is allowed to
accumulate undisturbed. Clinical signs of gingivitis are redness,
swelling (edema), and bleeding gums.
[0009] Periodontitis refers to a group of infections in which the
supporting tissues of the teeth such as connective tissue and bone
are destroyed by plaque-induced inflammation. The most common form
is known as Chronic Periodontitis that affects approximately 20% of
the adult U.S. population. Signs of chronic periodontitis include
all of those associated with gingivitis (i.e., redness, swelling,
bleeding) plus the formation of deep periodontal pockets (increased
probing depths), gingival recession, increased tooth mobility, and
loss of bone as detected by radiographs. If left untreated, chronic
periodontitis can lead to tooth loss.
[0010] Several dozen types of oral bacteria have been implicated as
putative periodontal pathogens including gram-negative bacteria
such as: Porphyromonas gingivalis, Aggregatibacter
actinomycetemcomitans, Tannerella forsythia, Eikenella corrodens,
Prevotella intermedia, and Campylobacter rectus. Gram-positive
bacteria of importance include Streptococcus intermedius,
Micromonas micros, and Eubacterium species. Spirochetes such as
Treponema denticola are also important. Low levels of most of these
pathogens can be isolated from healthy mouths. These bacteria only
become a problem when they are left undisturbed in mature dental
plaque biofilms. Finally, chronic periodontitis is a polymicrobial
infection with multiple bacteria working together in a biofilm to
cause the disease.
[0011] Treatment of both gingivitis and chronic periodontitis is
designed to facilitate the frequent removal and disruption of
dental plaque biofilms. For gingivitis, effective oral hygiene
practices on a daily basis are usually sufficient. This involves
thorough removal of plaque from facial and lingual surfaces of the
teeth with a toothbrush and good interproximal care with dental
floss or other appropriate devices (e.g., toothpicks). Periodic
tooth cleaning by an oral health care provider is required to
remove mineralized plaque (i.e., calculus). Treatment of chronic
periodontitis is more difficult since the disease-causing plaque is
usually at subgingival sites and in deep periodontal pockets.
Standard interventions usually include oral hygiene instructions
followed by thorough subgingival debridement (i.e., scaling and
root planing). If the infection persists, surgical intervention may
be recommended to reduce the depth of the pockets and to gain
access to thoroughly remove the calculus deposits on root surfaces.
In some cases, reconstructive surgical procedures are performed in
an attempt to regain some of the lost periodontal attachment and
supporting bone. Once the infection is under control, the patient
is placed on a rigorous maintenance/recall program to reduce the
chances of recurrent infection. It is during this maintenance phase
of therapy that non-invasive over-the-counter products are
especially useful in slowing down the reformation of dental plaque
biofilms on tooth surfaces.
Chlorine Dioxide
[0012] The use of chlorine dioxide for sanitation was first
suggested in 1948 by Eric Woodward to reduce the incidence of
unpleasant taste in shrimp. Since then, chlorine dioxide use has
spread into a number of other industries. The oxidative power of
ClO.sub.2 is used in the manufacture of wood pulp as an agent for
the bleaching of cellulose fibers. In water treatment, ClO.sub.2
has become widely used for water sanitation. In this case, it has
been shown to be effective at reducing the bacterial content, algae
content, and odor associated with wastewater treatment.
Additionally, the utilization of ClO.sub.2 for treating drinking
water has been effective without adversely affecting its taste. The
benefits of ClO.sub.2 over other processes utilizing ozone or
bleach for example, are reduced cost, reduced toxicity and reduced
production of chlorinated by-products.
[0013] In 1999 the EPA published "Alternative Disinfectants and
Oxidants Guidance Manual," describing disinfectant uses for
ClO.sub.2 and containing information on the mechanism of
generation, application and standards and regulations governing use
of ClO.sub.2 and other disinfectants. Major applications listed by
table 4-5, section 4.8.2 in the manual are as follows: primary or
secondary disinfectant, taste control, odor control, TTHM/HAA
reduction (total trihalomethanes are chlorinated organics,
chloroform [CHCl.sub.3] and dichlorobromomethane [CHCl.sub.2Br] for
example; haloacetic acids are created when an atom from the halogen
group, chlorine, for example, replaces a hydrogen on the acetic
acid molecule), Fe and Mn control, color removal, sulfide
destruction, phenol destruction and Zebra mussel control [EPA 1999,
p. 4-34]. These are accomplished by oxidation of various substances
found in water. For example, unpleasant tastes and odors (sulfides,
phenols, others) can exist in water due to vegetative decay and
algae content. ClO.sub.2 reduces these tastes either by eliminating
the source (algae) or oxidizing the causative taste and odor
molecules. In the control of iron and manganese, ClO.sub.2 will
bring the dissolved ions out of solution to form precipitates,
which may be eliminated through filtration and/or sedimentation.
Zebra mussel control is important because it helps to maintain the
natural ecology of a body of water. Zebra mussels are organisms
that will infest a lake or river, strip it of nutrients and create
a pseudo-fecal mucous layer on the bottom. The use of ClO.sub.2 for
water sanitation and pulp treatment generally involves on-site
generation followed by immediate use.
[0014] The term `stabilized chlorine dioxide` on the other hand,
refers to the generation and subsequent sequestration of ClO.sub.2,
which allows for its storage and availability for later use. The
first reference to stabilized chlorine dioxide in patent was in
U.S. Pat. No. 2,482,891 in which ClO.sub.2 is stabilized in a
powder for storage. For its application, it is mixed with water to
"liberate" the chlorine dioxide. A method and composition for the
use of aqueous stabilized chlorine dioxide for antiseptic purposes
was noted in U.S. Pat. No. 3,271,242. The 1979 text Chlorine
Dioxide, Chemistry and Environmental Impact of Oxychlorine
Compounds, describes (aqueous) stabilized chlorine dioxide as
follows: [0015] "The stabilization of chlorine dioxide in aqueous
solution was proposed by using perborates and percarbonates. Thus,
a stabilized solution of ClO.sub.2 would be obtained at pH 6 to 8
by passing gaseous ClO.sub.2 into an aqueous solution containing
12% Na.sub.2CO.sub.3.3H.sub.2O.sub.2. Other variants are possible.
In reality, it seems that in these methods, the chlorine dioxide is
practically completely transformed to chlorite. Dioxide is released
upon acidification . . . " [Masschelein, 1979]
[0016] The reference to percarbonates and perborates may be
replaced by the term `peroxy compounds,` which would refer to any
buffer suitable for maintaining the pH and hence, the stability of
the ClO.sub.2 in solution. The buffer is a necessary component, as
the ClO.sub.2 is unstable at low pH. Once the solution reaches low
pH or encounters an area of low pH, the stabilized ClO.sub.2 is
released from solution and available for sanitation and
oxidation.
[0017] In oral care products, the use of stabilized ClO.sub.2 has
been suggested as an active ingredient by a number of patents: U.S.
Pat. Nos. 4,689,215; 4,696,811; 4,786,492; 4,788,053; 4,792,442;
4,793,989; 4,808,389; 4,818,519; 4,837,009; 4,851,213; 4,855,135;
4,886,657; 4,889,714; 4,925,656; 4,975,285; 5,200,171; 5,348,734;
5,489,435; 5,618,550. Additionally, the use of stabilized ClO.sub.2
has been suggested for the degradation of amino acids in U.S. Pat.
No. 6,136,348. The premise for these products is that the
stabilized chlorine dioxide will remain as such until it encounters
the localized reductions in pH. Reduced pH levels can be a result
of low pH saliva or oral mucosa, the accumulation of oral
disease-causing bacteria or the presence of plaque biofilms on
teeth and epithelial cells. Once released, the now active chlorine
dioxide is effective at killing bacteria and oxidizing VSCs. Data
have shown dramatic reduction in bacteria after exposures as short
as 10 seconds, as set forth in U.S. Pat. No. 4,689,215. Additional
data show remarkable decrease in VSCs in expired mouth air; the
mechanism is believed to be oxidation of VSCs through the cleavage
of the sulfide bonds.
[0018] The effectiveness of the chlorine dioxide is likely
dependent on the amount of ClO.sub.2 released from stabilized
chlorine dioxide when the solution is acidified. The amount of
ClO.sub.2 released depends on the initial concentration of the
solution, its pH, and the stabilizing buffer or agent used. It
could follow that that the efficacy of the chlorine dioxide as an
oral care product is dependent on the amount of ClO.sub.2 released
from the stabilized chlorine dioxide solution. As a result, it is
imperative that accurate, precise measurements are taken so the
concentration of stabilized ClO.sub.2 and of the release of
ClO.sub.2 from solution can be determined. In addition to the need
to quantify the efficacy of the solution, concentrations must be
understood to ensure the safety of the product.
[0019] A concern about the stability of stabilized ClO.sub.2 was
recited in U.S. Pat. No. 5,738,840 with reference to the inclusion
of "other oxychlorine species" which could refer to chloride
[Cl.sup.-] or chlorate [ClO.sub.3.sup.-]. The mechanism of action
was questioned and suggested that at pH between 6.2 and 7.0 "any
molecular chlorine dioxide which forms by degradation of the
chlorite is converted back to chlorite by reaction with the
residual stabilizer." This reverse reaction is unlikely due to the
lower pH in the bacteria-laden target areas of the mouth described
earlier. U.S. Pat. No. 6,231,830 calls into question the
stoichiometry and safety of the formulation presented in U.S. Pat.
No. 5,738,840. It is claimed that the formulation described is a
`chlorinator` in which " . . . a build-up of chlorate ion, an
unwanted by-product" may occur.
PRIOR ART
[0020] Previous inventions contemplate the use of stabilized
chloride dioxide as a bactericide for the treatment gingivitis as
well as a deodorizing agent for the treatment of oral malodor
(Ratcliff, U.S. Pat. No. 4,689,215; Madray, U.S. Pat. No. 6,231,830
B1; Richter, U.S. Pat. No. 5,738,840; Witt, U.S. Pat. No. 6,350,438
B1). There is a large amount of evidence that indicates chlorine
dioxide has bactericidal properties and that the chlorine dioxide
serves to attack malodorous volatile sulfur compounds in the mouth
by splitting of the sulfide bonds (Lynch et al., 1997; Silwood et
al., 2001).
[0021] Grootveld et al. (2001) demonstrated that an admixture of
oxohalogen oxidants chlorite and chlorine dioxide significantly
reduces the number of Streptococcus mutans and lactobacilli.
Candida albicans exhibited a decrease however not statistically
significant. The research collected saliva samples from 33 dental
patients prior to and following rinsing with the admixture oral
rinse and measured the levels of each organism.
[0022] Research completed by Lynch et al. (1997) evaluated the
oxidative consumption of salivary biomolecules by an oral rinse
preparation containing an admixture of stable free radical species
chlorine dioxide with chlorite anions. .sup.1HNMR spectroscopy was
used to obtain multicomponent evaluations of the actions of the
oral rinse in the treatment of periodontal diseases and dental
caries. Saliva samples were collected from 10 volunteers prior to
and following rinsing and analyzed using the .sup.1HNMR. Results
indicated that the oxidative decarboxylation of salivary pyruvate
and the oxidative consumption of urate, thiocyanate anion, and
amino acids cysteine and methionine. The reductions in biomolecules
included, but not limited to the following components: short-chain
non-volatile carboxylic acid anions. The study revealed that the
oral rinse composition of stable free radical species chlorine
dioxide with chlorite anions reduces and removes pathogenic
micro-organisms when used as an oral rinse.
[0023] Inventors, Ratcliff and Lynch, U.S. Pat. No. 6,136,348,
suggest degradation of amino acids with the use of stabilized
chlorine dioxide. The premise for the composition described in the
patent is that stabilized chlorine dioxide is chlorine dioxide
stabilized as a sodium chlorite at a neutral or alkaline pH. The
composition will remain as such until it encounters the localized
reductions in pH as in saliva. The formation of chlorine dioxide is
a slow process and the effectiveness of the chlorine dioxide is
likely dependent on the amount released from the stabilized
chlorine dioxide. The patent describes the weak bonds between some
amino acids, like cysteine, leading to susceptibility to being
destroyed by oxidative consumption.
[0024] While prior art teaches various compositions of stabilized
chlorine dioxide relative to oral health, they do not teach a
method of stabilized chlorine dioxide to oxidatively consuming
salivary biomolecules to produce antimicrobial affects for the
reduction of growth and development of oral bacteria and
microorganisms concerned with halitosis and oral disease by the
generation of chlorine dioxide.
SUMMARY OF THE INVENTION
[0025] Stabilized chlorine dioxide has a beneficial effect of
tending to prevent a number of factors of oral disease, both by
eliminating the bacteria that cause them and also by oxidizing
molecules associated with them using a solution in the form of a
wash, rinse, soak, paste, gel, aerosol spray, or other suitable
delivery system.
[0026] A buffered solution of aqueous sodium chlorite, when in
solution at neutral to alkaline pH, is considered stabilized
chlorine dioxide because it does not release the chlorine dioxide
until it is acidified. It follows that measurement of the
concentration of stabilized chlorine dioxide is not, in fact, a
measurement of chlorine dioxide (ClO.sub.2) contained in solution,
but a quantification of the concentration of (aqueous) chlorite
(ClO.sub.2--) in solution. Once acidified, the amount of ClO.sub.2
released is limited by and a direct result of the ClO.sub.2--
concentration.
[0027] For liquids such as mouthwash, the standard unit of
measurement when expressing concentration is weight-volume
percentage. That is, a certain weight of component, solid, liquid,
or dissolved in a solvent, is present in a certain volume of total
mouthwash. Preferred concentrations of stabilized chlorine dioxide
in this invention are in the range of 0.005% to 2.0% (w/v).
[0028] Halitosis is caused by the presence of volatile sulfur
compounds. By which the sulfur compounds are produced from oral
bacteria and other microorganisms, including fungi and virus forms,
in the oral cavity and when undisturbed or not removed can lead to
plaque and development of oral diseases, including gingivitis and
periodontitis. Within the diverse ecology of the oral cavity and
plaque are complex salivary biomolecules required for
microorganisms to function, grow and develop. These salivary
biomolecules act as building blocks for reproduction, increasing
numbers of microorganisms and volatile sulfur compounds in the oral
cavity leading to halitosis. By reducing or eliminating the
presence of salivary biomolecules with stabilized chlorine dioxide,
the growth and numbers of microorganisms in the oral cavity will be
reduced or eliminated and therefore treating and preventing
halitosis.
[0029] It is therefore a primary object of the present invention to
provide stabilized chlorine dioxide as an antimicrobial agent
against the oral microorganisms by generating chlorine dioxide by
the oxidative consumption of salivary biomolecules.
[0030] Another object of the present invention is to provide
stabilized chlorine dioxide as a halitosis treatment and prevention
by the oxidative consumption and inactivation of volatile sulfur
compounds and their amino acid precursors to alleviate
halitosis.
[0031] Still another object of the present invention is to
oxidatively consume and inactivate salivary biomolecules, including
pyruvate, methionine, trimethylamine, tyrosine, glycine, creatine,
3-D-hydroxybutyrate, salivary taurine, lactate, and lysine.
[0032] Yet another object of the present invention is to provide
stabilized chlorine dioxide composition in a solution or other
delivery vehicle such as in the form of a wash, rinse, soak, paste,
gel, or aerosol spray to deprive microorganisms of salivary
biomolecules as necessary compounds to grow and develop.
[0033] A further object of the present invention is to prevent
halitosis with stabilized chlorine dioxide composition by
oxidatively consuming salivary biomolecules to eliminate and
prevent microorganisms from growing and development in the oral
cavity.
[0034] A still further object of the present invention is to treat
halitosis with stabilized chlorine dioxide composition by
oxidatively consuming salivary biomolecules to eliminate and
prevent microorganisms from growth and development in the oral
cavity.
[0035] Yet a further object of the present invention is to provide
antimicrobial affects of stabilized chlorine dioxide on oral
bacterial by producing chlorine dioxide as a product of oxidatively
consuming salivary biomolecules.
[0036] These and other objects of the present invention will become
apparent to those skilled in the art as the description thereof
proceeds.
BRIEF DESCRIPTION OF FIGURES
[0037] FIG. 1, (a) and (b), illustrates the expanded 0.80-4.25 ppm
regions of the 600.13 Mhz single-pulse .sup.1H NMR spectra of a
human salivary supernatant specimen (pH value 6.78) acquired (a)
prior to and (b) subsequent to treatment with oral rinse I
according to the procedure outlined in the Materials and Methods
section. Abbreviations: A. Acetate-CH.sub.3; Ala I and II,
alanine-CH.sub.3 and --CH group proton respectively; Bu I,
.beta.-hydroxybutyrate proton .gamma.-CH.sub.3 group protons; Bu
II, III and IV, .beta.-hydroxybutyrate .beta., .beta.', and .alpha.
protons respectively (ABX coupling system); iso-But I and II,
iso-butyrate-CH.sub.3 and --CH group protons respectively; n-But I,
II and III, n-butyrate .gamma., .beta., and .alpha. protons
respectively; Chol, choline-N.sup.+(CH.sub.3).sub.3; Cit,
Citrate-AB-CH.sub.2--CO.sub.2.sup.-; DMeN, dimethylamine-CH.sub.3;
Eth I and II, ethanol-CH.sub.3 and --CH.sub.2 group protons
respectively; Form, formate-H; Gly, glycine-CH; H is I and II,
histidine ABX system .beta. protons; Lac I and II, lactate-CH.sub.3
and --CH protons respectively; Leu I, II, III and IV, leucine
.delta., .gamma., .beta., and .alpha. protons respectively; MeGu,
methylguanidine-CH.sub.3; MeN, methylamine-CH.sub.3; Meth,
methanol-CH.sub.3; N--Ac, spectral region for acetamido methyl
groups of N-acetyl sugars; Phe I and II, phenylalanine ABX .beta.
protons; Prop I and II, propionate-CH.sub.3 and --CH.sub.2 group
protons respectively; Pyr, pyruvate-CH.sub.3; Sar I and II,
sarcosine-CH.sub.3 and --CH.sub.2 group protons respectively; Suc,
succinate-CH.sub.2; Tau I and II, Taurine-CH.sub.2NH.sub.3.sup.+
and --CH.sub.2SO.sub.3.sup.- protons respectively; TMeN,
trimethylamine-CH.sub.3, Tyr I and II, tyrosine ABX .beta. protons;
Tyr III, tyrosine ABX .alpha. proton; n-Val I and II, n-valerate
.delta. and .gamma. protons respectively.
[0038] FIG. 2 illustrates a plot of absorbance at 262 nm
(A.sub.262) versus chlorite concentration for a series of
calibration standards in the 1.60-8.00 mM concentration range
[0039] FIG. 3(a) illustrates a reversed-phase (RP) ion-pair (IP)
chromatograms of a 1.00 mM chlorite standard solution. The
retention time of ClO.sub.2.sup.- was 6.90 min.
[0040] FIG. 3(b) illustrates a reversed-phase (RP) ion-pair (IP)
chromatograms of oral rinse I formulation (diluted 1/4 with
doubly-distilled water prior to analysis). The retention time of
ClO.sub.2.sup.- was 6.90 min.
[0041] FIG. 3(c) illustrates a reversed-phase (RP) ion-pair (IP)
chromatograms of a typical salivary supernatant sample (0.10 ml)
pre-treated with 0.50 ml of the above oral rinse I. The retention
time of ClO.sub.2.sup.- was 6.90 min.
[0042] FIG. 4 illustrates a plot of chlorite peak area
(.mu.V.s.sup.-1) obtained from the HPLC analysis versus chlorite
concentration for a series of chlorite calibration standards
DESCRIPTION OF THE INVENTION
[0043] This invention relates to the discovery through research of
the composition for and methodology of generating of chlorine
dioxide by a stabilized chlorine dioxide composition through the
oxidatively consuming salivary biomolecules in the oral cavity and
producing antimicrobial affects on oral bacteria and microorganisms
concerned with halitosis and oral disease with the reduction of
growth and development. Chlorine dioxide is known to be a strong
oxidizer and is capable of oxidizing amino acids. The work of Lynch
et al. proves so with the degradation of cysteine and methionine
into pyruvate in the presence of an admixture of stable free
radical species chlorine dioxide with chlorite anions (1997). This
was confirmed with the following evidence of research suggesting
oxidative consumptions of salivary biomolecules and interactions of
stabilized chlorine dioxide as chlorite with human salivary
biomolecules. The oxidative decarboxylation of salivary pyruvate by
stabilized chlorine dioxide composition indicates a mechanism of
action of the interaction of this invention with salivary
biomolecules as an antimicrobial agent.
[0044] The specific mechanism of action of `stabilized` chlorine
dioxide (specifically, chlorite anion) on oral organisms and
biomolecules has not been fully investigated. The present invention
research evidence suggests that stabilized chlorine dioxide
oxidatively consumes salivary biomolecules and creates products
that may exert bactericidal and bacteriostatic effects on the oral
bacterial cells which ultimately gives rise to cell death. These
effects can lead to control over the formation of bacterial plaque
and the adverse generation of malodorous volatile sulfur compounds,
major contributors to oral diseases.
[0045] The purpose of researching the oxidentive consumption of
salivary biomolecules this investigation was to determine: (1) the
metabolic profile of human saliva and the capacity of salivary
biomolecules to react with stabilized chlorine dioxide oral rinse,
(2) the amount of chlorine dioxide generated from chlorite when the
oral rinse is mixed with saliva, and how much chlorine dioxide is
consumed or chlorite remains, and (3) an assay technique for
monitoring chlorine dioxide activity in saliva, as well as
determining the level of volatile sulfur compounds after being
treated with a stabilized chlorine dioxide rinse. The oral rinse
compositions included a concentration of 0.1% (w/v) and 0.4% (w/v)
stabilized chlorine dioxide. These formulations are designated as
oral rinse I and II, respectively.
[0046] This research suggested that the stabilized chlorine dioxide
composition has the capacity to clinically alleviate oral malodor
by the direct oxidative inactivation of volatile sulfur compounds
and their amino acid precursors. These results also reveal a new
mechanism of action of stabilized chlorine dioxide (chlorite),
specifically its reaction with human salivary biomolecules to
produce chlorine dioxide.
Materials and Methods
Spectrophotometric Determination of Chlorite Concentrations in Oral
Rinse Formulations
[0047] For oral rinse I, 1.00 ml aliquots were diluted to a total
volume of 3.00 ml with doubly-distilled water and electronic
absorption spectra of these solutions were recorded on a Unicam
UV-2 spectrophotometer in the 190-400 nm wavelength range.
Similarly, 0.20 ml volumes of oral rinse II were diluted to a final
volume of 3.00 ml with doubly-distilled water and electronic
absorption spectra were also acquired in this manner. Chlorite
concentrations were determined via measurement of its absorbance at
262 nm [.epsilon.=160 M.sup.-1cm.sup.-1, as determined in this
study]. A further series of these oral rinse solutions were
pre-treated with the amino acid L-glycine (final concentration 2.00
mM) to remove hypochlorous acid/hypochlorite (HOCl/OCl.sup.-) and
chlorine dioxide (ClO.sub.2.), the former generating glycine
monochloroamine via equation A.
H.sub.3N.sup.+--CH.sub.2--CO.sub.2.sup.-+OCl.sup.-.fwdarw.Cl--NH--CH.sub-
.2--CO.sub.2.sup.-+H.sub.2O (A)
[0048] Results acquired revealed that there were no differences
between spectra obtained before and after glycine treatment,
indicating that these potentially interfering, further oxohalogen
oxidants were absent from the oral rinse formulations examined.
Volunteer Recruitment and Collection of Samples
[0049] A series of non-medically-compromised volunteers (n=20)
without any form of active periodontal disease or active dental
caries were recruited to the study. To avoid interferences arising
from the introduction of exogenous agents into the oral
environment, volunteers were requested to collect all saliva
available, i.e., (`whole` saliva expectorated from the mouth) into
a plastic universal tube immediately after waking in the morning on
a pre-selected day.
[0050] Each volunteer was also requested to refrain completely from
oral activities (i.e., eating, drinking, tooth-brushing, oral
rinsing, smoking, etc.) during the short period between awakening
and sample collection (ca. 5 min.). Each collection tube contained
sufficient sodium fluoride (15 .mu.mmol.) to ensure that
metabolites are not generated or consumed via the actions of
micro-organisms or their enzymes present in whole saliva during
periods of sample preparation and/or storage.
[0051] Saliva specimens were transported to the laboratory on ice
and then centrifuged immediately (3,000 r.p.m for 15 min.) on their
arrival to remove cells and debris, and the resulting supernatants
were stored at -70.degree. C. for a maximum duration of 18 hr.
prior to analysis. The pH values of each supernatant were
determined prior to .sup.1H NMR analysis.
Spectrophotometric Analysis of Residual (Unreacted) Chlorite Anion
(ClO.sub.2.sup.-) in Oral Rinse/Salivary Supernatant Mixtures
[0052] An ATI Unicam UV-VIS UV-2 spectrophotometer was employed for
the determination of residual chlorite in each of the salivary
supernatants collected in order to determine its level of
consumption by biomolecules therein on equilibration.
[0053] 0.09 ml aliquots of each salivary supernatant specimen were
treated with 0.450 ml of oral rinse I. This mixture was thoroughly
rotamixed and diluted to a final volume of 1.20 ml to yield an
absorbance value of approximately 1 at 262 nm. The reference cell
contained an equivalent volume of corresponding salivary
supernatant diluted to a final volume of 1.20 ml with
doubly-distilled H.sub.2O. Initially, scans were made over the
wavelength range of 190-300 nm.
[0054] Since oral rinse II contained exactly four times the
concentration of ClO.sub.2.sup.- [0.4% (w/v)], 0.10 ml aliquots of
each salivary supernatant specimen were treated with 0.500 ml of
this product, and once thoroughly rotamixed, a 0.135 ml aliquot of
this mixture was diluted to a final volume of 1.20 ml with
H.sub.2O. The reference cell contained 22.5 .mu.l of salivary
supernatant diluted to 1.20 ml with H.sub.2O.
[0055] ClO.sub.2.sup.- has a wavelength of maximum absorbance
(.lamda..sub.max) at 262 nm (.epsilon.=160 M.sup.-1 cm.sup.-1) and
therefore was readily detectable at the volumes (and hence
concentrations of ClO.sub.2.sup.-) of each oral rinse added.
[0056] Where required, the pH value of samples were adjusted to a
value of 1.00 and samples were then equilibrated at ambient
temperature for a 24 hr. period (to ensure conversion of each mole
of ClO.sub.2.sup.- remaining to 0.50 of an equivalent of
ClO.sub.2.) in order to improve the sensitivity of this assay
system [ClO.sub.2. has a .lamda..sub.max value in the visible
region (360 nm) with .epsilon.=1,150 M.sup.-1 cm.sup.-1].
HPLC Monitoring of the Interaction of the Oral Rinse Oxohalogen
Oxidants with Intact Human Saliva
[0057] The chlorite level remaining in each salivary supernatant
sample was also determined using a novel high-performance liquid
chromatographic (HPLC) technique employing a reversed-phase C18
column with the ion-pair reagent hexadecyl-trimethylammonium
bromide (HTB) present in the mobile phase. The operating system
utilised was a Waters Millennium HPLC system, consisting of a
Waters 626 Pump, Waters 996 Photodiode Array Detector and a Waters
in-line degasser remotely operated using Waters unique Millennium
software.
[0058] Samples were prepared via the treatment of 0.10 ml volumes
of saliva supernatants with 0.50 ml aliquots of 1/4 diluted oral
rinses I and II. Once thoroughly rotamixed, 10 .mu.l aliquots of
the resulting solutions were injected using a remotely-operated
automated auto-sampler with injector onto a reversed-phase C18 ODS
Column (4.6.times.75 mm). A Spherisorb S5-ODS 1 guard column was
employed to remove any potential analytical column
contaminants.
[0059] The mobile phase was de-gassed using an in-line degasser.
The mobile phase consisted of 2% (w/v) borate/gluconate buffer with
2% (v/v) butan-1-ol and 12% (v/v) acetonitrile (final pH 7.2) and
operated at a flow rate of 1.10 ml/min. The ion-pair reagent
(Hexadecyl-trimethylammonium Bromide) was added at a final
concentration of 50.00 mM in order to ensure that ClO.sub.2.sup.-
is readily separated from interfering salivary components. This
analyte was identified by comparisons of its peak's absorption
spectrum generated by the photo-diode array detector
(.lamda..sub.max. 262 nm) with that of an authentic chlorite
standard.
Preparation of Human Salivary Supernatant Samples for .sup.1H NMR
Analysis
[0060] Each individual salivary supernatant sample was divided into
three equivalent portions (0.60 ml). In total, there were three
separate specimen reaction mixtures: 3.0 ml of oral rinses I and II
were added to the first and second salivary supernatant samples
respectively, whilst the third served as an untreated control in
which 3.0 ml of H.sub.2O was added to the original 0.6 ml volume of
salivary supernatant. The samples were then thoroughly rotamixed to
ensure a homogenous mixture and then equilibrated at 37.degree. C.
for a period of 30 s.
[0061] Samples were prepared by adding 0.05 ml of deuterium oxide
(2H.sub.2O, providing a field frequency lock) and 0.05 ml of a 5.0
mM solution of sodium 3-trimethylsilyl [2,2,3,3-.sup.2H.sub.4]
propionate [TSP, chemical shift reference (.delta.=0.00 ppm) and
internal quantitative standard] in .sup.2H.sub.2O to a 0.60 ml
volume of each sample examined.
[0062] Each sample was then subjected to multicomponent high
resolution .sup.1H NMR analysis in order to identify the nature of
salivary biomolecules which react with ClO.sub.2.sup.- and/or
ClO.sub.2. i.e, via oxidative consumption or otherwise, together
with the products generated from such reaction systems.
.sup.1H NMR Measurements
[0063] One-dimensional (1-D) .sup.1H NMR spectra were acquired on a
Bruker AMX-600 spectrometer (ULIRS, Queen Mary, University of
London facility, U.K) operating at a frequency of 600.13 MHz and a
probe temperature of 298 K. The intense water signal (.delta.=4.80
ppm) was suppressed by presaturation via gated decoupling during
the delay between pulses.
[0064] Pulsing conditions for 1-D spectra acquired on salivary
supernatant and oral rinse samples were: 128 free induction decays
(FIDS); 16,384 data points; 3-7 .mu.s pulses; 1.0 s pulse
repetition rate. Line-broadening functions of 0.30 Hz were
routinely utilised for the processing of experimental NMR data.
Where present, the methyl group resonances of lactate
(.delta.=1.330 ppm) and alanine (.delta.=1.481 ppm) served as
secondary internal references for the control and oral
rinse-treated salivary supernatant samples examined.
Results
.sup.1H NMR Analysis of Oral Rinse Formulations I and II
[0065] .sup.1H NMR spectra acquired on the oral rinse I formulation
contained clear, prominent resonances ascribable to citrate
[--CH.sub.2CO.sub.2.sup.- protons, .delta.=2.65 ppm (dd, AB
coupling system)] which serves as a buffering agent, with lower
intensity signals arising from acetate [--CH.sub.3 group, singlet
(s) located at 1.92 ppm] and formate [.sup.-O.sub.2C--H singlet
(s), .delta.=8.46 ppm]. Ethanol [--CH.sub.3 and --CH.sub.2OH group
protons, .delta.=1.21 (t) and 3.66 (q) respectively] was also
detectable at trace levels.
[0066] Spectra acquired on the oral rinse II product also contained
resonances ascribable to citrate [--CH.sub.2CO.sub.2.sup.- protons,
.delta.=2.65 ppm (dd, AB coupling system)] and lower intensity
signals arising from trace levels of acetate [--CH.sub.3 group,
singlet (s) located at 1.92 ppm] and formate [.sup.-O.sub.2C--H
singlet (s), .delta.=8.46 ppm].
.sup.1H NMR Analysis of the Interaction of
ClO.sub.2.sup.--containing Oral Rinse Formulations with Human
Salivary Supernatant Specimens
[0067] 600 MHz .sup.1H NMR spectra were acquired for every salivary
supernatant sample examined (i.e., a total of 60, 3 daily specimens
collected from each of 20 human volunteers). A typical .sup.1H NMR
spectrum of a human salivary supernatant sample is shown in FIG.
2(a); that of the same saliva specimen pre-treated with Oral Rinse
I is displayed in FIG. 2(b). These .sup.1H NMR investigations [of
the oxidative consumption of salivary biomolecules by oxohalogon
oxidants present in Oral Rinses I and II tested (predominantly
ClO.sub.2.sup.-)] revealed that: [0068] 1. Pyruvate was oxidatively
decarboxylated to acetate and CO.sub.2 [0069] 2. The volatile
sulphur compound (VSC) precursor methionine was oxidised to its
corresponding sulphoxide [0070] 3. A resonance ascribable to
malodorous trimethylamine (s, .delta.=2.91 ppm) was reduced in
intensity (a process presumably resulting in its transformation to
trimethylamine oxide) [0071] 4. Tyrosine was oxidised (presumably
to a quinone species) [0072] 5. The Glycine .alpha.-CH.sub.2 group
resonance was reduced in intensity, an observation possibly
attributable to its reaction with trace levels of
hypochlorite/hypochlorous acid present in the oral rinses
(generating mono- and/or dichloroamine species) [0073] 6. The
concentrations of creatinine and 3-D-hydroxybutyrate were
diminished following treatment with each oral rinse, an observation
consistent with their oxidative consumption by oxohalogen species
present therein. [0074] 7. Salivary taurine decreased in
concentration post treatment. [0075] 8. Lactate-CH.sub.3 and --CH
signals were diminished in intensity following treatment. [0076] 9.
Resonances ascribable to lysine were reduced in intensity
post-treatment.
[0077] With regard to these .sup.1H NMR analysis results acquired,
the consumption of salivary methionine by chlorite is of much
importance to oral hygiene and clinical periodontology since both
CH.sub.3SH and H.sub.2S are generated from this amino acid via
metabolic pathways operational in gram-negative micro-organisms.
Hence, data acquired here indicates that the oral rinses examined
have the capacity to clinically alleviate oral malodour via the
direct oxidative inactivation of VSCs and their amino acid
precursors.
[0078] As demonstrated here, the techniques employed are of much
value concerning multicomponent assessments of the interactions of
chlorite with human salivary biomolecules, and the oxidative
decarboxylation of salivary pyruvate by this oxohalogen oxidant
serves as an important example of this which may be of some
relevance to its mechanisms of action.
Spectrophotometric Analysis of Chlorite Calibration Standards
[0079] Prior to spectrophotometric analysis of Oral Rinses I and
II, the extinction coefficient of chlorite (ClO.sub.2.sup.-) was
determined at its .lamda..sub.max value of 262 nm. This was
conducted by analysing authentic ClO.sub.2.sup.- calibration
standards (1.60-8.00 mM, Table 1 and FIG. 2). Each measurement was
made in triplicate in order to ensure the reproducibility of data
acquired. Plots of absorbance at 262 nm (A.sub.262) versus chlorite
concentration were clearly linear: the extinction coefficient was
determined as .epsilon.=160 M.sup.-1 cm.sup.-1, and the correlation
coefficient (r) for the plot shown in Table 1 was 0.9955.
TABLE-US-00001 TABLE 1 Absorbance values at 262 nm for replicate (n
= 3) determinations obtained for a series of chlorite calibration
standards (1.60-8.00 mM) Concentration (mM) 1st 2nd 3rd 1.60 0.274
0.274 0.273 2.40 0.405 0.404 0.403 3.20 0.509 0.509 0.508 4.00
0.632 0.63 0.631 4.80 0.784 0.783 0.783 5.60 1.032 1.034 1.033 6.40
1.055 1.056 1.055 7.20 1.161 1.163 1.161 8.00 1.253 1.252 1.252
[0080] Treatment of the water diluent with up to 20% (v/v) ethanol
exerted no influence on the final absorbance values obtained, an
observation which confirmed that this potential contaminant exerted
no influence on the spectrophotometric assay of chlorite performed
in this manner (i.e., no reaction between these agents was noted
under our experimental conditions).
Spectrophotometric Determination of the Consumption of Oral Rinse
Chlorite by Human Salivary Supernatant Specimens
[0081] Following the establishment of ClO.sub.2.sup.-'s extinction
coefficient (via the acquisition of electronic absorption spectra
on a series of its calibration standards), difference
spectrophotometric analysis of chlorite in each of the salivary
supernatant/oral rinse mixtures was performed in order to determine
its level of consumption by biomolecules therein on equilibration.
In this manner, the decrease in absorbance at 262 nm observed
following equilibration of the oral rinse formulations with human
salivary supernatants according to the procedure outlined in
methods was employed to estimate the level of oral rinse chlorite
(ClO.sub.2.sup.-) consumption by this biofluid. Table 2(a) gives
the concentrations of chlorite consumed (per ml of saliva) for
reaction mixtures containing a 5:1 volume ratio of oral
rinse:salivary supernatant.
TABLE-US-00002 TABLE 2(a) Spectrophotometric determination of the
consumption of oral rinse ClO.sub.2.sup.- by human salivary
supernatant samples (.mu.mol. ClO.sub.2.sup.- consumed per ml of
saliva). Patient Code Oral Rinse I Oral Rinse II J1 0.1640 0.1504
0.1776 0.0944 0.1168 0.1192 J2 0.0200 0.0096 0.0176 0.0360 0.0280
0.0472 J3 0.3040 0.3152 0.3040 0.3552 0.3024 0.3024 BR1 0.0400
0.0760 0.0600 0.0696 0.0584 0.1136 BR2 0.1136 0.0752 0.1008 0.1392
0.1136 0.1808 BR3 0.2968 0.2800 0.3096 0.0976 0.0504 0.0776 G1
0.0008 0.0104 0.0168 0.0584 0.1448 0.1168 G2 0.0168 0.0080 0.0112
0.1528 0.1664 0.1640 G3 0.0392 0.0624 0.0584 0.0864 0.0720 0.0920
U1 0.0200 0.0112 0.0200 0.0528 0.0360 0.0248 U2 0.0168 0.0040
0.0072 0.0168 0.0304 0.0392 U3 0.0168 0.0216 0.0144 0.4504 0.3888
0.4056 M1 0.0696 0.0728 0.0704 0.1000 0.1528 0.1280 M2 0.0072
0.0016 0.0080 0.2024 0.1504 0.1608 M3 0.0000 0.0048 0.0016 0.0192
0.0224 0.0224 L1 0.0064 0.0040 0.0128 0.0248 0.0024 0.0112 L2
0.0104 0.0064 0.0056 0.0416 0.0832 0.0696 L3 0.0296 0.0320 0.0352
0.0664 0.064 0.0608 SB1 0.1408 0.1576 0.1272 0.0720 0.0416 0.0664
SB2 0.1400 0.1496 0.1336 0.2888 0.3584 0.2776 SB3 0.0240 0.0240
0.0264 0.0224 0.0248 0.0336 I1 0.0336 0.0424 0.0296 0.1112 0.1336
0.1528 I2 0.0856 0.0752 0.0544 0.0448 0.0336 0.0248 I3 0.0264
0.0216 0.0240 0.072 0.0976 0.0808 R1 0.0376 0.0536 0.0368 0.0080
0.0224 0.0056 R2 0.0056 0.0016 0.0000 0.1000 0.0752 0.0888 R3
0.0056 0.0104 0.0104 0.0224 0.0112 0.0192 ZK1 0.0088 0.0096 0.0096
0.0664 0.0552 0.0608 ZK2 0.1032 0.1376 0.1248 0.0976 0.0752 0.1136
ZK3 0.0232 0.0192 0.0264 0.1168 0.0808 0.0832 V1 0.2000 0.2128
0.2264 1.4808 1.4888 1.4696 V2 0.0328 0.0408 0.0472 0.2168 0.1552
0.1976 V3 0.0704 0.0680 0.0672 0.7000 0.6528 0.6304 Z1 0.0120
0.0096 0.0128 0.0664 0.0528 0.0552 Z2 0.0240 0.0224 0.0184 0.0056
0.0024 0.0000 Z3 0.0232 0.0184 0.0104 0.0504 0.0472 0.0552 GG1
0.0344 0.0216 0.0328 0.2000 0.1752 0.1944 GG2 0.1400 0.1296 0.1296
0.2392 0.2584 0.2472 GG3 0.0088 0.0136 0.0104 0.0024 0.0136 0.008
N1 0.0056 0.0064 0.0040 0.1976 0.2080 0.2000 N2 0.0112 0.008 0.0048
0.0696 0.0752 0.0528 N3 0.0184 0.0200 0.0120 0.0112 0.0168 0.0056
ED1 0.0792 0.0576 0.0920 0.2752 0.2720 0.2448 ED2 0.3184 0.3352
0.3288 0.3168 0.4640 0.4808 ED3 0.0288 0.0224 0.0160 0.1080 0.1248
0.0608 AB1 0.0112 0.0232 0.0256 0.2112 0.1608 0.1080 AB2 0.0024
0.0032 0.0048 0.0584 0.1000 0.0944 AB3 0.008 0.0104 0.0072 0.0552
0.0696 0.0696 S1 0.2512 0.2736 0.2624 0.964 0.9832 1.0304 S2 0.1952
0.1728 0.1584 0.1472 0.1696 0.1056 S3 0.1176 0.1744 0.1440 0.5448
0.5776 0.5696 DG1 0.1104 0.1144 0.0880 0.0504 0.0392 0.0664 DG2
0.0192 0.0328 0.0208 0.0552 0.1360 0.0080 DG3 0.0088 0.0120 0.0096
0.0808 0.0832 0.0888 SG1 0.0336 0.0456 0.0544 0 0 0.0024 SG2 0.0744
0.0944 0.0656 0.1136 0.1080 0.1080 SG3 0.0144 0.0120 0.0120 0.1504
0.1168 0.1392 P1 0.0128 0.0112 0.0152 0.1112 0.1336 0.1304 P2
0.0176 0.0200 0.0248 0.0472 0.0392 0.0504 P3 0.0600 0.0504 0.0408
0.0640 0.1024 0.1472 Abbreviations: patient codes in the rows refer
to volunteers, whilst columns represent oral rinse treatments, with
three independent sampling days `nested` within each treatment.
Multifactorial Analysis-of-Variance of Difference
Spectrophotometric Data Involving the Determination of
ClO.sub.2.sup.- Consumption by Salivary Biomolecules
[0082] Statistical analysis of data acquired regarding the
difference spectrophotometric determination of ClO.sub.2.sup.-
consumption by salivary biomolecules [i.e., multifactorial
analysis-of-variance (ANOVA)] revealed highly significant
differences between (1) the ClO.sub.2.sup.- content of each oral
rinse investigated (p<<0.001), (2) volunteers (p<0.01) and
(3) `days nested within volunteers` (p<0.001). Indeed, estimates
of the overall mean consumption of ClO.sub.2.sup.- determined for a
reaction mixture containing a 5:1 (v/v) ratio of oral rinse:human
salivary supernatant were 6.334.times.10.sup.-2 and
1.626.times.10.sup.-1 .mu.mol. ClO.sub.2.sup.- per ml of salivary
supernatant for Oral Rinses I and II respectively. The `between
replicates` mean square value was only 1.266.times.10.sup.-4,
indicating a high level of reproducibility on repeat (triplicate)
determinations conducted on each sample tested. The full ANOVA
table is shown in Table 2(b).
TABLE-US-00003 TABLE 2(b) Multifactorial analysis-of-variance
(ANOVA) table for data acquired from the study involving the
difference spectrophotometric determination of ClO.sub.2.sup.-
consumption by salivary biomolecules. Source of Variation d.f SS MS
F p EMS (1) Between 1 1.3839 1.3839 64.37 <<0.001
ClO.sub.2.sup.- concen- trations (Fixed Effect) (2) Between 19
6.5421 0.3443 2.54 <0.01 .sigma..sup.2 + Volunteers
6.sigma..sub.o.sup.2 + (Random Effect) 18.sigma..sup.2.sub.v (3)
Between 40 5.4146 0.1354 6.30 <0.001 .sigma..sup.2 +
6.sigma..sub.o.sup.2 Sampling Days within Volunteers (Random
Effect) (4) Error 295 6.3504 0.0215 .sigma..sup.2 (Residual) (5)
Between 4 5.065 .times. 1.266 .times. Replicates 10.sup.-4
10.sup.-4 Total 359 19.6915 Abbreviations: d.f., degrees-of
freedom; SS, sum of squares values; MS, mean square values; F, F
variance ratio statistic; EMS, expected mean square.
Development of a Novel HPLC Method for Monitoring Oral Rinse
Chlorite Consumption and its Oxidative Interaction with Salivary
Biomolecules
[0083] In this section, the development of an HPLC method for the
determination of ClO.sub.2.sup.- in human saliva specimens (i.e.,
prior and subsequent to its treatment with the oral rinse
formulations) is described.
[0084] The chlorite level remaining in each salivary supernatant
sample was determined using a high-performance liquid
chromatographic (HPLC) technique employing a reversed-phase C18
column with the ion-pair reagent hexadecyl-trimethylammonium
bromide (HTB) present in the mobile phase.
[0085] Experiments involving alteration of the ion pair reagent
concentration from 5.00 to 50.00 mM showed that a concentration of
50.00 mM gave rise to a good resolution of ClO.sub.2.sup.- from
salivary components in all samples investigated. Identification of
the ClO.sub.2.sup.- peak was based on its retention time (6.9 min)
and the diode-array spectrum of its HPLC peak (.lamda..sub.max 262
nm). Injection of authentic sodium chlorite calibration standards
(1.00-10.00 mM) demonstrated a clear linear relationship between
peak intensity and concentration. Typical chromatograms of a 1.00
mM chlorite standard solution, the oral rinse I formulation
(diluted 1/4 with doubly-distilled water prior to analysis) and a
typical salivary supernatant sample (0.10 ml) pre-treated with 0.50
ml of the above oral rinse (I) are shown in FIGS. 3(a), (b) and (c)
respectively. The retention time of ClO.sub.2.sup.- was 6.90
min.
[0086] Plots of chlorite peak area (Table 3) versus its
concentration were clearly linear (FIG. 4).
TABLE-US-00004 TABLE 3 Area under chlorite peak (.mu.V/sec.) values
obtained via HPLC analysis of known chlorite calibration standards
Concentration (mM) Mean value uV/sec 0.80 70833 69102 69879 1.60
151673 151878 153334 2.40 208530 209419 210975 3.20 259823 258413
259662 4.00 326322 326592 326771 4.80 394229 394023 394386 5.60
514239 510086 513058 6.40 535511 535418 530565 7.20 586871 592830
585209 8.00 628810 628254 622356
CONCLUSIONS
[0087] Results acquired on the consumption of (relatively) simple
amino acids such as glycine, alanine and taurine by the oral rinse
tested here (predominantly containing ClO.sub.2.sup.- as an
oxidant) are explicable by previous investigations conducted on the
kinetics and mechanisms of the reactions of such biomolecules with
oxyhalogen oxidants (including ClO.sub.2.sup.-) as outlined
below.
[0088] Of much relevance to the substantial extent of salivary
taurine consumption by the oral rinses investigated in the studies
are experiments reported by Chinake and Simoyi (1997) on the
oxidation of this .beta.-amino acid by ClO.sub.2.sup.- (at neutral
to acidic pH values, i.e., those which are relevant to the oral
environment). Indeed, the stoichiometry of this reaction system was
found to involve the consumption of 3 molar equivalents of
ClO.sub.2.sup.- per mole of taurine to generate 1 of taurine's
N-monochloroamine [Cl(H)NCH.sub.2CH.sub.2SO.sub.3H] and 2 of
ClO.sub.2. (the production of N-monochlorotaurine is rapid when
expressed relative to that of ClO.sub.2. accumulation); at the
lower pH values investigated, N-monochlorotaurine disassociated to
taurine and N-dichlorotaurine. An important characteristic of this
reaction system involves a significant induction period in which
both HOCl and the reactive intermediate
H(OH)NCH.sub.2CH.sub.2SO.sub.3H are produced, a process leading to
the formation of N-chlorotaurine and ClO.sub.2. autocatalytically.
As expected for redox reactions involving ClO.sub.2.sup.-, this
autocatalysis is mediated by a Cl.sub.2O.sub.2 intermediate
species, and interestingly, taurine's C--S bond is not cleaved,
despite the availability of the powerful oxidant HOCl.
[0089] Hence, these previously reported studies clearly explain the
substantial .sup.1H NMR-detectable reductions in salivary taurine
observed on treatment of human salivary supernatant specimens with
the tested oral rinse ClO.sub.2.sup.-. They also indicate that the
oral rinse-induced oxidative consumption of a range of
.alpha.-amino acids present in this biofluid also detected in this
investigation, specifically free (non-protein-incorporated)
alanine, arginine, aspartate, cysteine, glutamate, glutamine,
histidine, hydroxyproline, isoleucine, leucine, lysine, methionine,
ornithine, phenylalanine, proline, tyrosine and valine, also
proceed via this mechanism.
[0090] However, since many N.sup..alpha.-monochloroamines generated
in this manner are unstable at physiological temperature
(37.degree. C.) (Hazen et. al. (1998)), and decompose to
corresponding aldehydes (equation 1), and hence further
investigations focused on the detection and quantification of such
species corresponding to the side-chains of .alpha.-amino acids
(e.g., formaldehyde from salivary glycine, acetaldehyde from
alanine, etc.) are required in order to demonstrate this.
Cl(H)N--CHR--CO.sub.2.sup.-+H.sub.2O.fwdarw.RCHO+NH.sub.3+CO.sub.2+Cl.su-
p.- (1)
[0091] Interestingly, it is well known that aldehydes act as potent
microbicidal agents, and hence those derived from the above
processes may also exert this activity in the oral environment.
Indeed, a 2.0% (w/v) solution of this agent is frequently employed
as a disinfectant (Follente et. al.).
[0092] Similarly, the oxidative consumption of
.gamma.-aminobutyrate (GABA) noted here is likely to proceed via a
similar mechanism. However, the amino acids cysteine, methionine
and tyrosine, each with redox-active side-chains can, of course,
also be oxidatively modified by ClO.sub.2.sup.- (and also
ClO.sub.2. and HOCl/OCl.sup.- produced via its reaction with these
and/or further .alpha.-amino acids, together with GABA and
particularly taurine) to cysteine sulphonate (and cystine),
methionine sulphoxide (equation 2) and a tyrosine-derived quinone
species respectively.
H.sub.3N.sup.+CH(CH.sub.2CH.sub.2SCH.sub.3)CO.sub.2.sup.-+ClO.sub.2.sup.-
-.fwdarw.H.sub.3N.sup.+CH(CH.sub.2CH.sub.2SOCH.sub.3)CO.sub.2.sup.-+OCl.su-
p.- (2)
[0093] With regard to the oxidative consumption of salivary
.alpha.-keto acid anions, particularly pyruvate and
.alpha.-ketoglutarte, by ClO.sub.2.sup.- present in the tested oral
rinses, which was also observed in our investigations, it has been
previously noted that an intense green Cl.sub.2/OCl.sup.-
colouration is generated on reaction of ClO.sub.2.sup.- with
pyruvate (equation 3) [Lynch et. al. 1997]. Hence, such reaction
systems clearly generate HOCl/OCl.sup.- which can, of course,
subsequently produce N-monochloro- and -dichloroamines from free
or, in selected cases, protein-incorporated amino acids, the former
decomposing to corresponding aldehydes under physiological
conditions.
CH.sub.3COCO.sub.2.sup.-+ClO.sub.2.sup.-.fwdarw.CH.sub.3CO.sub.2.sup.-+C-
O.sub.2+OCl.sup.- (3)
[0094] Therefore, it should be noted that the production of
reactive HOCl/OCl.sup.- during an induction period observed during
the reaction of ClO.sub.2.sup.- with the .beta.-amino acid taurine
(Chinake and Simoyi (1997)) (and also presumably the salivary
.alpha.-amino acids and .gamma.-aminobutyrate consumed on reaction
with tested oral rinse ClO.sub.2.sup.-) will also serve to further
reduce the amino acid concentrations of human saliva. Indeed, even
if this mechanistic process only proceeds in the reactions of
selected free amino acids with ClO.sub.2.sup.- (or those located at
the N-termini of salivary proteins), the HOCl/OCl.sup.- generated
will, of course, be available to react with a much wider range of
such HOCl/OCl.sup.- `scavenger` species in a (relatively)
unselective manner to form N.sup..alpha.-monochloro- and
dichloroamines, together with N.sup..epsilon.-monochloro- and
-dichloroamines in lysine residues (either free or
protein-incorporated). As noted above, specific aldehydes arising
from the decomposition of their parent amino acid
N.sup..alpha.-monochloroamine precursors will serve as valuable
indicators of the activity of HOCl/OCl.sup.- arising from these
reaction systems (RCHO, where R represents an amino acid side-chain
moiety).
[0095] Aldehydes produced from the interaction of HOCl/OCl.sup.-
with salivary .alpha.-amino acids and the decomposition of the
primary N.sup..alpha.-monochloroamine products can also react with
ClO.sub.2.sup.-, and the oxidation of formaldehyde (HCHO) by this
oxyhalogen oxidant was critically examined by Chinake et. al.
(1998) in both mildly acidic and alkaline media. This reaction gave
rise to CO.sub.2 and ClO.sub.2. as products, the latter in
virtually quantitative yield, and was autocatalytic with respect to
hypochlorous acid/hypochlorite (HOCl/OCl.sup.-). Indeed, the
primary phase of the process generated HOCl which facilitated
(catalysed) the production of ClO.sub.2. and the additional
oxidation of formic acid/formate (HCO.sub.2H/HCO.sub.2.sup.-);
ClO.sub.2. rapidly accumulated in view of its (relative) lack of
reactivity towards both HCHO and HCO.sub.2H/HCO.sub.2.sup.-.
Although with excess HCHO the stoichiometry of this process was
determined to be
3ClO.sub.2.sup.-+HCHO.fwdarw.HCO.sub.2H+2ClO.sub.2..sub.(aq.)+Cl.sup.-+2H-
.sub.2O, when large excesses of ClO.sub.2.sup.- were present [as,
of course, expected in the case of in the case of 5:1 (v/v)
mixtures of tested oral rinses: human salivary supernatant], the
stoichiometric profile involved in the consumption of 6 molar
equivalents of ClO.sub.2.sup.- per mole of HCHO to generate 4 of
ClO.sub.2., 2 of Cl.sup.- and 1 of CO.sub.2.
[0096] With regard to the oral rinse-mediated decrease in the
intensities of salivary cysteine resonances observed here (and also
in previously-conducted chemical model studies (Lynch et. al.,
1997), Darkwa et. al. (2003) investigated the oxidative consumption
of N-acetylcysteine by ClO.sub.2.sup.-, and found that the final
product generated from this reaction system was N-acetylsulphonate
and that the process had a stoichiometry of
3ClO.sub.2.sup.-+2RSH.fwdarw.3Cl.sup.-+2RSO.sub.3H; as expected,
there was no evidence for the production of N-chloroamine
derivatives. This oxidation proceeds via a mechanism involving a
stepwise S-oxygenation process involving the consecutive generation
of sulphenic and sulphonic acid adducts. Intriguingly, a notable
characteristic of the reaction is the rapid, immediate formation of
chlorine dioxide (ClO.sub.2.) without a monitorable induction
period since oxidation of the thiol by this oxyhalogen free radical
species is sufficiently slow for it to accumulate without such a
time lag which, in general, represents a characteristic of the
oxidation of organosulphur compounds by ClO.sub.2.sup.-. A full
description of the `global` dynamics of this system involves 8
reactions in a truncated mechanism.
[0097] In conclusion, evidence provided in our investigations
clearly demonstrate that the generation of ClO.sub.2. from
ClO.sub.2.sup.- in the oral environment is not entirely dependent
on entry of the latter into acidotic environments therein
(equations 4 and 5, the pK.sub.a value of the
ClO.sub.2.sup.-/HClO.sub.2 system being 2.31 (Lynch et. al. 1997)).
Although the mean pH value of this biofluid is ca. 7 when
unchallenged with oral stimuli (i.e., `resting`), the consumption
of relatively large volumes of beverages of lower pH value (ca. pH
4) can clearly exert a significant influence on this parameter.
However, it should also be noted that the pH value of primary root
caries lesions can approach a limit of 4.5, and therefore this
represents an environment in which there are expected to be marked
elevations in the level of HClO.sub.2 generated (i.e., from 0.0020%
at pH 7.00 to 0.64% of total available oxyhalogen oxidant at pH
4.50), although it should be noted that, in view of the pK.sub.a
value of the ClO.sub.2.sup.-/HClO.sub.2 couple, this value still
remains very low when expressed relative the total amount of
oxyhalogen oxidant available (the remainder being ClO.sub.2.sup.-
in the absence of alternative means of producing ClO.sub.2., or
HOCl/OCl.sup.-, from the interaction of ClO.sub.2.sup.- with
.alpha.-, .beta.- and .gamma.-amino acids available). Of course,
from the stoichiometry of equation 5, 2 molar equivalents of
ClO.sub.2. are generated per 4 of HClO.sub.2, and hence the above
figures for HClO.sub.2 generation represent double that of the
total ClO.sub.2. producable (i.e., maximum percentages of 0.0010
and 0.32% of total oxyhalogen oxidant at pH values of 7.00 and 4.50
respectively). Clearly, the rate of ClO.sub.2. generation from
HClO.sub.2 should also be considered in view of the short oral
rinse-salivary supernatant equilibration time involved in our
studies.
ClO.sub.2.sup.-+H.sup.+.fwdarw.HClO.sub.2 (pK.sub.a=2.31) (4)
4HClO.sub.2.fwdarw.2ClO.sub.2.+ClO.sub.3.+Cl.sup.-+H.sub.2O (5)
* * * * *