U.S. patent application number 10/484321 was filed with the patent office on 2005-02-17 for treatment of nail infections with no.
Invention is credited to Benjamin, Nigel.
Application Number | 20050037093 10/484321 |
Document ID | / |
Family ID | 9919789 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050037093 |
Kind Code |
A1 |
Benjamin, Nigel |
February 17, 2005 |
Treatment of nail infections with no
Abstract
Nitrogen oxide generating compositions are useful in the
treatment of subungual infections, as NO has surprisingly been
found to be able to penetrate the nail to exert an anti-fungal
effect.
Inventors: |
Benjamin, Nigel; (London,
GB) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
9919789 |
Appl. No.: |
10/484321 |
Filed: |
August 2, 2004 |
PCT Filed: |
August 2, 2002 |
PCT NO: |
PCT/GB02/03575 |
Current U.S.
Class: |
424/718 |
Current CPC
Class: |
A61K 47/38 20130101;
A61P 31/10 20180101; A61P 43/00 20180101; A61P 17/00 20180101; A61K
33/00 20130101; A61K 47/12 20130101; A61K 47/32 20130101; A61K 8/19
20130101; A61K 9/0012 20130101; A61K 31/04 20130101; A61Q 3/00
20130101 |
Class at
Publication: |
424/718 |
International
Class: |
A61K 033/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2001 |
GB |
0119011.5 |
Claims
1. A method for the treatment or prophylaxis of a subungual
infection, the method comprising: generating nitrogen oxide by
combining a nitrite and an organic acid, wherein the nitrite and
the organic acid are separately disposed from each other prior to
being combined, and combining the nitrite and the organic acid
forms a composition.
2. The method according to claim 1, wherein the organic acid is
present in sufficient quantity that the composition formed by
combining the nitrite and the organic acid is at a pH of 5.5, or
below.
3. The method according to claim 1, wherein the organic acid is
selected from: formic acid, malic acid, maleic acid, acetic acid,
lactic acid, citric acid, benzoic acid, tartaric acid and salicylic
acid, ascorbic acid, ascorbyl palmitate, and mixtures thereof.
4. The method according to any preceding claim 1, wherein the
nitrite is selected from the alkali metal nitrites and the alkaline
earth metal nitrites.
5. The method according to claim 4, wherein the nitrite is selected
from: sodium, potassium, magnesium and barium nitrites.
6. The method according to claim 1, wherein the organic acid
comprises citric acid and the nitrite comprises sodium nitrite, at
least one being present in an aqueous vehicle.
7. The method according to claim 1, wherein the subungual infection
is onychomycosis.
8. The method according to claim 1, wherein the nitrite is
formulated with an excipient selected from: Eudragits, carbopol,
carboxymethylcellulose, hydroxymethylcellulose, and mixtures
thereof.
9. The method according to claim 1, wherein the organic acid is
formulated with an excipient selected from: carbopol,
carboxymethylcellulose, hydroxymethylcellulose, methylcellulose,
and mixtures thereof.
10. The method according to claim 1, wherein the organic acid and
the nitrite are separately disposed in aqueous based formulations
prior to being combined.
11. The method according to claim 10, wherein each preparation is
in a form separately selected from gels, creams, lotions, ointments
and paints suitable for mixing each with the other.
12. The method according to claim 1, wherein the organic acid and
the nitrite are each separately formulated as a gel, paint or
lacquer prior to being combined.
13. The method according to claim 1, wherein, prior to being
combined, the organic acid and the nitrite are each separately
formulated as a liquid or gel which, when mixed, solidify or form a
gel or paint.
14. The method according to claim 1, wherein the nitrite is
approximately 0.5 to 30%, by weight, of the composition formed by
combining the nitrite and the organic acid.
15. The method according to claim 14, wherein the nitrite is 5 to
15%, by weight, of the composition formed by combining the nitrite
and the organic acid.
16. The method according to claim 1, wherein the organic acid is
approximately 5 to 30%, by weight, of the composition formed by
combining the nitrite and the organic acid.
17. The method according to claim 16, wherein the organic acid is
approximately 10 to 15%, by weight, of the composition formed by
combining the nitrite and the organic acid.
18. (Cancelled).
19. The method according to claim 1, wherein the composition formed
by combining the nitrite and the organic acid is applied to an
infected nail in an effective amount.
20. A kit comprising: a nitrite; and an organic acid kept separate
from the nitrite, wherein the kit is configured so that the nitrite
and the organic acid can be combined to generate nitrogen oxide,
and the nitrogen oxide can be used for the treatment or prophylaxis
of a subungual infection.
21. A kit according to claim 20, comprising an aqueous preparation
of the nitrite and an aqueous preparation of the organic acid,
separately disposed one from each other, the two preparations each
being suitable to apply to a nail to be treated such that the
nitrite and acid can react to release nitrogen oxides for
penetration into the nail.
22. A kit according to claim 21, wherein each preparation is in a
form selected from lotions, gels, creams and lacquers.
23. A kit according to claim 21, wherein the preparations are
provided in resealable containers.
Description
[0001] The present invention relates to methods of treatment of
infections of finger and toe nails, medications for use in such
treatment, and methods for the preparation of such medication.
[0002] Nail infections are common and, when serious, can be painful
and disfiguring, affecting the quality of life of patients. The
fungi involved in nail infections are mainly those that cause
athlete's foot (or tinea pedis) spreading from the toe cleft to the
nail. Fungal infection of the nail is known as onychomycosis, which
is also known as tinea unguium, dermatophytic onychomycosis or nail
"ringworm".
[0003] The most frequently isolated pathogens in onychomycosis are
dermatophytes, especially Trichophyton rubrum (toe nails 56%,
finger nails 36%) and Trichophyton mentagrophytes (toe nails 19%,
finger nails 11%). Yeast infections are less common but are usually
associated with Candida albicans (toe nails 10%, finger nails
30/o).
[0004] It is estimated that at least 15 to 20% of the population
aged 40 to 60 has onychomycosis, with 25 to 40% of those over 60
years suffering this condition, but only 3% or less of under 18's.
However, it is difficult to put a precise figure on the actual
occurrence of onychomycosis, as at least 50% of sufferers fail to
seek medical advice.
[0005] Mild onychomycosis may simply be restricted to white patches
or pits in the nail's surface but, in more established disease, the
symptoms include nail bed hyperkeratosis, nail plate thickening,
discolouration and onycholysis (separation of the nail plate from
the nail bed).
[0006] Many factors predispose patients to onychomycosis, including
diabetes mellitus, increasing age, hyperhydrosis, onychogryphosis,
trauma, poor peripheral circulation and immunosuppression. It is
more common in men, and is rare before puberty and in
pre-menopausal women.
[0007] Onychomycosis is a fungal condition, and conditions which
suit fungal growth tend to encourage the development of
onychomycosis. Accordingly, 80% of cases involve the foot,
especially the hallux, or big toe, and are commonly associated
with, for example, tight fitting footwear and excessive sweating,
such as may commonly be encountered in sporting activity. However,
trauma is also a significant aetiologic factor, especially in the
toenail, the longest toe being particularly susceptible.
[0008] It is not completely certain how the condition is acquired,
but onychomycosis is contagious. Infections may come from the
spread of the fungi from the skin to the nails or directly from
other people with skin or nail infections. In the case of toenail
infection, athlete's foot fungus can spread to the nail, nail
trauma often being present, thereby allowing entry to the
fungus.
[0009] Treatments for onychomycosis, despite the prevalence of the
disease, are somewhat limited, the condition being highly resistant
to topical medication. Topical treatments include Loceryl
(amorolfine) and Penlac (ciclopirox), but cure rates are low
(<10%), and treatment times long (up to 0.12 months), due to
poor penetration through the nail, as well as poor activity against
the causative organisms.
[0010] One particular problem with treating onychomycosis and other
nail infections is that the infection is generally located in, or
proximal to, the nail bed, as well as in the nail itself. Thus, the
infection is protected from external attack by the very nail which
it is disfiguring. Treatment may include removal of nail material
to expose the infectious organisms, although it is undesirable to
remove too much of the nail. In addition, duration of treatment is
generally up to a year, or longer.
[0011] More recently, oral treatments have been developed
(terbinafine and itraconazole) that have achieved higher cure rates
(.about.70%) and shorter treatment periods (12-16 weeks). However,
there are safety concerns with these newer oral therapies,
including liver toxicity, severe skin reactions and drug
interactions.
[0012] Thus, there is a need for a topical, or transungual, therapy
that provides cure rates similar to or better than such oral
therapies, but with reduced safety concerns.
[0013] Surprisingly, we have now found that nitrogen oxides are
capable of penetrating the nail and are effective in the treatment
of the causative organisms of subungual infections.
[0014] Thus, in a first aspect, the present invention provides a
nitrogen oxide generating composition for use in the treatment of
subungual infections.
[0015] Nitric oxide (NO) is a major product of the compositions of
the invention, and is well known to have antimicrobial and wound
healing effects [c.f. WO 95/22335; and Hardwick et al., (2001),
Clin. Sci., 100, 395-400].
[0016] Nitric oxide is synthesised in the body in the vascular
endothelium and neurons, as well as in activated macrophages.
Relatively high levels of NO are observed in sweat Although it is
not known precisely how NO kills micro-organisms, it is speculated
that NO serves to disrupt bacterial DNA, or interfere with the
function of bacterial enzymes which contain transition metals.
[0017] Nitric oxide, for therapeutic use, is most conveniently
produced by the reaction of nitrite with an acid, particularly an
inorganic nitrite with an organic acid. This results in the
production of the molecular form of nitrous acid, which readily
dissociates into a molecule of water and a molecule of dinitrogen
trioxide, the latter, in turn, dissociating to form NO and nitrogen
dioxide. The reactions are shown below.
NO.sub.2.sup.-+H.sup.+HNO.sub.2
2HNO.sub.2N.sub.2O.sub.3+H.sub.2O
N.sub.2O.sub.3NO+NO.sub.2
[0018] Although N.sub.2O.sub.3 is an intermediate in this reaction,
there is evidence that it is capable of independent existence, and
that it may be at least partially responsible for the fungicidal
effects associated with the compositions of the present
invention.
[0019] In the presence of a reducing agent, such as ascorbic acid,
the reaction of dinitrogen trioxide to form NO is more efficient,
and can be represented, for example, as follows:
N.sub.2O.sub.3+C.sub.6H.sub.8O.sub.6.fwdarw.2NO+H.sub.2O+C.sub.6H.sub.6O.s-
ub.6
[0020] Nitrous acid may suitably be generated, for example, by the
action of an acid on a nitrite, particularly where the resulting
salt is insoluble.
[0021] It will be appreciated that, whilst any suitable source of
nitrogen oxides, preferably providing at least a fraction of NO,
may be employed in the present invention, it is generally preferred
that any nitrogen oxides be generated in accordance with one or
more of the above reactions.
[0022] The compositions of the present invention may be any that
are suitable to provide nitrogen oxides. In one embodiment, the
proportion of NO generated by the compositions of the present
invention is preferred to be at least 50% and, more preferably, at
least 80%. Where the only acid used is a reducing acid, then this
proportion may rise to anything up to 100% NO content of the
nitrogen oxides generated.
[0023] By "nitrogen oxide generating" is meant that compositions of
the present invention serve to release nitrogen oxide in situ, i.e.
at the location where they are applied, which will generally be on
an infected nail. At its simplest, and in one embodiment, this may
comprise an ointment or gel or, indeed, any other suitable, topical
vehicle, in which gaseous NO has been dissolved, for example, and
which, once applied to the nail, releases NO.
[0024] Given that only quite small amounts of nitrogen oxides are
required in order to be effective, then it does not matter if gas
escapes other than at the nail interface, or if only small
quantities actually permeate across the nail, or if the amount of
nitrogen oxides are attenuated in their passage across the nail,
provided that sufficient nitrogen oxides reach the site of action
to have a cidal or inhibitory effect.
[0025] Although NO and its precursors are generally short lived
moieties, with half lives as short as just a few seconds, we have
established that they can pass through human nails in sufficient
quantities to treat subungual infections. This is all the more
surprising, as not only may it may take several hours for there to
be any evidence of NO or other nitrogen oxides passing across the
nail but, once NO/other nitrogen oxides start to be released, the
release can continue for up to 10 hours, or even longer. Without
being bound by theory, it appears that the nail is acting as a
reservoir, or sink, and adsorbs or absorbs the NO or a precursor
therefor. The NO or precursor travels across the nail, and NO and
other nitrogen oxides released at the other side. Given the short
half lives of the gases, it is possible that they are complexing
protein in the nail, and diffusing slowly across.
[0026] Although it is known that NO has an antifungal effect, it is
not clear that it is necessarily NO that is permeating across the
nail, and it may be that NO is only regenerated once the precursor
has passed across the nail. Indeed, compositions producing large
amounts of NO do not necessarily have the greatest effect in the
present invention. Without being bound by theory, it is possible
that it is not advantageous to efficiently and quickly generate NO
at the surface of the nail, as NO, owing to its short half life,
may not diffuse in quantity across the nail. Instead, those
compositions taking longer to generate NO appear more efficient at
delivering the cidal element across the nail, whether that element
be NO or another nitrogen oxide.
[0027] Compositions of the invention comprising essentially only
ascorbic acid or other similar reducing acid and a nitrite tend to
produce large amounts of NO rapidly. Although the nail is somewhat
porous, if the NO is produced too quickly there may be insufficient
time for the nail to adsorb much of the NO produced, and
experiments show that these compositions are associated with a
lower overall flux of NO across the nail.
[0028] Silver nitrite is capable of producing NO in the presence of
acids, but has relatively low efficacy, so is not generally
preferred. However, both sodium nitrite and potassium nitrite were
found to react with acetic, citric, maleic and malic acid, for
example, to produce zones of inhibition in fungicidal tests.
[0029] The levels of kill of the fungal mycelium for sodium and
potassium nitrite, when combined with acetic, citric, maleic or
malic acid, were found to be similar, all giving large zones of
inhibition. Likewise, experiments conducted with spores were found
to show similar anti-fungal effects for the same acid-nitrite
mixtures. General findings were that the solutions produced greater
anti-fungal activity compared to creams, whilst mycelia were found
to be more susceptible to the anti-fungal effects than spores.
However, the creams generally produce nitrogen oxides for longer,
which can be an advantage where it is desired to prolong NO or
other nitrogen oxide generation at the nail surface to enhance
permeation across the nail.
[0030] The amount of NO produced by the acid-nitrite mixtures does
not necessarily correlate to the size of the zone of inhibition,
although there is a general correlation with the overall amount of
nitrogen oxides produced. For example, ascorbic acid-nitrite
solutions producing large amounts of NO only had little anti-fungal
activity in some tests, while potassium nitrite and malic acid
combinations only produce approximately half the amount of NO as
citric acid, yet the sizes of zones of inhibition were not
necessarily any smaller.
[0031] The delayed release of nitrogen oxides from the far side of
the nail is particularly useful, as it generally takes at least 5
minutes exposure to the active substance to produce kill. Peak
killing is observed at around 30 minutes, although anti-fungal
activity generally continues to increase up to 2 hours after
exposure to the active gas.
[0032] The nitrogen oxide generating composition may take any
suitable form. However, it will be appreciated that, where the
generation of nitrogen oxides is active, then the reactants should
be kept separate one from the other until nitrogen oxide is
actually required. Although this is generally a preference, it need
not necessarily always apply. For example, an occlusive patch may
be constructed with a gel, or matrix, into which nitrogen oxide
generating ingredients are loaded, the patch then being protected
by a suitable webbing to prevent gaseous release.
[0033] In such a patch, it is preferred that the matrix or gel is
adhesive, and that the strength of the adhesion is sufficient to
overcome any tendency of the nitrogen oxide to escape and push away
the webbing, although it will be appreciated that the strength of
the adhesive should not be such that the webbing cannot be
satisfactorily removed to allow application of the patch. Further,
it is preferred to provide suitable stabilisers, such as chelating
agents, in the gel or matrix, in order to prolong the life of the
NO or to reduce the rate at which it is produced. Additionally, as
NO is more soluble in non-aqueous and lipid substances, the
addition of such substances to the treatment may prolong the
activity and delivery of NO to the affected nail and nail bed.
[0034] Nevertheless, compositions already comprising free NO will
not generally be stable for any great length of time, and should
preferably be used by the patient as soon as possible after
preparation.
[0035] More preferred is to provide the compositions of the present
invention in multiple parts. These parts may each, separately,
comprise actives or reactants, which, when mixed, serve to generate
nitrogen oxides. Thus, a first composition may comprise a suitable
nitrite provided in a suitable vehicle. A second composition may
comprise a suitable acid. The two compositions can then be mixed,
preferably intimately, and then applied to the infected nail, or
may be mixed in situ. Although it is generally desired to minimise
the number of components that it is necessary to mix in order to
achieve the final nitrogen oxide generating composition, it will be
appreciated that any number may be provided. In particular, it may
be preferred to provide a third composition comprising a reducing
acid, for example. However, where a reducing acid, such as
ascorbate, is used, then it is generally preferred to either use it
as the acid, in its own right, or to provide it together with the
primary acid in a separate composition from the nitrite.
[0036] The nitrite component may be incorporated in a range of
excipients, including, for example, Eudragit L100, carbopol,
carboxymethylcellulose, or hydroxymethylcellulose, and the acid
component may be incorporated in another suitable excipient, such
as carbopol, carboxymnethylcellulose, hydroxymethylcelluilose,
methylcellulose or in an aqueous base. Other excipients, such as
polyvinyl alcohol, propylene glycol, polyvinylpyrrolidone
(povidone), gelatin, guar gum, and shellac have use in assisting
film formation, useful to maintain the composition in situ.
[0037] In particular, compositions suitable for introducing
substances across the dermis and the stratum corneum may not be
generally suitable for the nail, as the nail is effectively a
hydrophilic substance, while the stratum corneum is generally
hydrophobic. Thus, ionic and ionisable substances readily soluble
in water may preferably achieve uptake in the nail, and excipients
encouraging this are preferred.
[0038] It is generally preferred to use aqueous based formulations
to assist in permeation. Such formulations may be mobile solutions
but, as it appears that a certain minimum length of exposure of NO
and/or other nitrogen oxides to the nail is optimal, then it is
generally preferred that any generation of nitrogen oxides does not
finish too quickly, in order to allow sufficient time for uptake at
the nail interface, and it is generally preferred the formulations
be in the form of gels, creams, lotions, ointments, or other
thickened form, such as lacquers. Suitable thickening and other
characteristics may be achieved by the use of suitable excipients,
as described above. For example, it has been established that
Eudragits have the ability to alter the release profile of nitrogen
oxides from a formulation.
[0039] In a preferred embodiment, the composition comprises
separate aqueous preparations of an organic acid and a nitrite.
More preferably, each preparation is in the form of a gel, cream,
lotion, ointment or paint suitable for mixing with the other, which
is also selected from a similar group. It is particularly preferred
that one or both preparations comprises an excipient suitable to
retard the release of nitrogen oxides on mixing. In the case of
nitrite, a preferred excipient is a Eudragit, such as Eudragit
L100. Other preferred excipients for both are as exemplified
above.
[0040] The excipients chosen may simply be in order to delay oxide
release, but will generally also possess thickening qualities,
amounts of the excipient being generally determined by the amount
needed to provide a suitable gel. This may vary within well known
limits, as readily determined and recognised by those skilled in
the art. However, as a guide, suitable amounts may vary between
about 1% and about 40%, although there is a large discrepancy
between excipients. In general, the preferred excipients need only
be used in amounts of between 2% and 10%, such as 3% to 5%, while
those used primarily for gelling will be used in amounts suitable
to achieve that purpose, whether in combination, or separately. For
example, polyethylene glycol is suitably used as a viscosifying
agent in amounts ranging from about 10% to 50%, but more preferably
about 20% to 35% w/v. Suitable thickening agents include carbopols
and the cellulose derivatives, and these are typically employed in
amounts of between about 2% and 10%, according to the nature of the
formulation required. A preferred formulation is a mobile gel.
[0041] It will be understood that the compositions of the invention
may comprise other components as desired, such as antioxidants,
preservatives, colourants and perfumes, as well as surface active
agents and/or penetrating agents, as desired, although aqueous
combinations of nitrite and acid are readily able to provide
nitrogen oxides across the nail without any need for such
additional components.
[0042] Although the present invention is generally illustrated
herein in respect of two compositions being mixed to provide the
final, nitrogen oxide generating composition, it will be
appreciated that such references include references to more than
two initial compositions, unless otherwise apparent, or
indicated.
[0043] Compositions of the present invention may comprise any
suitable vehicles for mixing. What is important is that the acid
and the nitrite, or nitrite precursor, be able to react in such a
manner as to generate the desired nitrogen oxides. Thus, at least
one of the initial compositions providing the final composition
should preferably comprise an aqueous component, in order to allow
the nitrogen oxide generating reaction to take place. More
preferably, both of the initial compositions should comprise
aqueous components to facilitate the mixing of the ingredients
although, where it is desired that the ingredients should only
react slowly, the amount of water may be minimised in one or both
of the initial compositions.
[0044] There is no restriction on the types of initial compositions
that may be mixed in order to achieve the final composition,
provided that the final composition serves to generate nitrogen
oxides. In this respect, and throughout, it will be appreciated
that reference to "nitrogen oxides" includes reference to 100% NO,
although this is not necessarily desirable or preferred.
[0045] For example, the initial compositions may be in any suitable
form, such as liquid, gel or solid although, where one is solid,
then the other is preferably liquid or gel. In the category of
liquid, are included solutions, suspensions and colloids, and such
considerations also apply to gels, which generally comprise any
state between liquids and solids.
[0046] More particularly, gels include such states as creams,
ointments, tinctures, waxes and lotions, although the latter may
fall under liquids, depending on the properties thereof. It will be
appreciated that there is no specific exclusion, provided that the
liquid gel solid serves as a vehicle for the active.
[0047] Solid vehicles may include matrices in patches, for example,
or longer chain waxes.
[0048] The initial compositions may suitably be mixed, either
before application or in situ, in order to provide the final
composition to generate nitrogen oxides. Such mixtures may be
straightforward gel/gel mixtures, for example, which can then be
applied to the nail, and left in place. They may also comprise two
liquids which, between them form a gel, lacquer, solid or paint
and, likewise, two gels, or a liquid and a gel, may serve to
solidify, form a lacquer, or otherwise form a protective
environment to generate, hold and dispense nitrogen oxides.
[0049] In one preferred embodiment, a gel may be applied to the
nail and then a patch, such as a plaster, carrying a matrix
containing the other active is applied over the gel and, once in
contact, the actives slowly interact to generate nitrogen
oxide.
[0050] In another preferred embodiment, the actives may be
dispensed as paints or lacquers. Suitably, one component, the
nitrite for example, may be applied and allowed to dry, and then
the second painted on top. The water in the second allows the
reaction to proceed. If desired, a quick drying solvent, such as an
alcohol or acetate, may be employed, although it is an advantage of
the present invention that such solvents and permeation enhancers
are not necessary. It may be desirable, however, to provide
ingredients of a film in the separate preparations, so that a
polymerisation reaction occurs, for example, on mixing. A catalyst
may be provided in one preparation, while a selection of
polymerisable monomers may be provided in the other. Alternatively,
evaporation of the solvent may allow a polymer in the preparation
to gel further, or harden.
[0051] The nitrogen oxide generating phase of the compositions of
the invention is generally on the wane by two or three hours, and
frequently less, although the sinking effect of the nail provides
nitrogen oxides at the other side of the nail for considerably
longer than this, and often not until oxide production has
effectively ceased. During oxide production, it is preferred to
keep the blended composition in situ, which may be achieved by the
composition setting. Alternatively, it may desirable to protect the
nail with an occlusive dressing, for example.
[0052] In another preferred embodiment, the matrix of the patch, or
plaster, is non-aqueous but hydrophilic, and contains a mixture of
the actives in substantially dry form. In this case, the term "dry
form" may include crystals incorporating water of crystallisation,
for example. Thus, although both of the actives are present in the
matrix of the patch, or plaster, they cannot react in the absence
of suitable quantities of water to act as solvent to provide a
reactive environment. When it is desired to apply the patch, or
plaster, any protective webbing can be removed and a suitable
quantity of water, such as a few drops, can be applied to the
matrix to activate the active ingredients. The activated patch may
then be applied to the nail to allow the nitrogen oxides generated
to have their effect.
[0053] This principle of providing a substantially dry composition
to which water is added may also apply to other compositions. In
such cases, it will be appreciated that the term "dry" applies to
the free water content, so that, whilst a composition may be a gel,
for example, the water content will be extremely low, such as 1%,
or even lower. It is preferred that such compositions are
substantially anhydrous.
[0054] The active ingredients of the compositions of the present
invention may be present in any suitable quantities, as will be
apparent to those skilled in the art. In general, it is preferred
that the quantity of nitrite is approximately 0.5 to 30%, by
weight, of the final composition. More preferably, the amount of
nitrite, or its precursor, is 1 to 20%/o and, particularly, 1 to
15%, preferably 5 to 15%. A preferred range is 1 to 10%, or 2 to
10%. Higher concentrations are generally preferred, and a minimum
concentration of 8-10% is preferred. In creams, lotions and gels,
it is envisaged that an upper limit is about 13.5%, although
suitable formulation may permit higher levels.
[0055] It is generally preferred that the composition of the
present invention be provided as two aqueous gels, lotions or
creams. More preferably, one contains citric acid at a
concentration of between 0.5% and 20%, such as 0.75%, 2.25%, 4.5%,
9% and 13.5% w/w, and the other contains sodium nitrite as
described above, for example, 0.5%, 1.5%, 3.0%, 6% and 9% w/w.
Preferred concentrations are in the range of 10% for each active
component, this providing a suitable excess of the acid. It is also
preferred that both actives be present in at least 2%, preferably
5% Y w/w or greater, to provide an effective dose of nitrogen
oxides across the nail. In a preferred embodiment, the acid is
present in an amount of 13.5% and the nitrite at 9% while, in
another, each is present in amounts of 10%. The preferred acid is
citric acid, while the preferred nitrite is sodium nitrite.
[0056] The gels, lotions or creams may be mixed in any suitable
quantity, by the patient, for example, to cover the affected part
of the toe or finger nails(s). Suitable quantities of each gel,
lotion or cream may be in the range of 0.05 to 1 g, more preferably
0.1-0.5 g, the components reacting to produce nitrogen oxides.
[0057] It is preferred that the acid be present in at least
stoichiometric amounts by comparison to the nitrite, or its
precursor. More preferably, the acid is present in a stoichiometric
excess, sufficient to ensure an acidic environment for a sufficient
quantity of the nitrite to generate nitrogen oxides. Although it is
not necessary for the whole of the nitrite to generate nitrogen
oxides, it is generally inefficient to allow too much of the
nitrite to go unreacted, and it is preferred that the majority of
the nitrite be converted to nitrogen oxides.
[0058] In general, it is preferred that the acid be present in
sufficient quantity that the final composition be at a pH of 5, or
below, especially pH 4, or below. However, the nitrogen oxide
generating reaction may take place a higher pH's, and a pH of 5.5
or even 6 may be acceptable, especially in the presence of excess
reducing acids, so that it will be appreciated that the pH of the
final composition does not form an essential part of the present
invention.
[0059] There is no restriction on the nitrite other than that it be
generally pharmaceutically acceptable. Even this requirement is not
a major consideration, as the final compositions will generally be
applied to the nail of the patient, so that dermal contact is
minimised, thereby concomitantly minimising potential systemic
exposure. Nevertheless, for safety considerations, it is preferred
that the nitrites, or their precursors, be generally safe for
topical administration.
[0060] The nature of the nitrite, for simplicity's sake, will
generally be inorganic and at least partially soluble in water.
Preferred are the alkali metal nitrites and the alkaline earth
metal nitrites, although other suitable nitrites, such as the
transition metal compounds, may also be used, subject to
suitability, especially solubility. In particular, the sodium,
potassium, magnesium and barium compounds may be used, the sodium
and potassium compounds generally being preferred from the point of
view of expense and availability.
[0061] Suitable acidifying agents include inorganic acids but,
owing to their general pharmaceutical unacceptability, are not
generally preferred. Thus, more preferred are the organic acids,
especially those capable of forming a solution with water and
yielding a pH of 4 or below. Such acids include formic acid, malic
acid, maleic acid, acetic acid, lactic acid, citric acid, benzoic
acid, tartaric acid and salicylic acid, and it will be appreciated
that this list is inclusive, rather than exclusive. Other suitable
acids include ascorbic acid and ascorbyl palmitate which do not
necessarily form such acidic solutions, but which are reducing
acids and have the advantage of increasing the amount of NO
generated, and which may also serve to stabilise the NO, once
generated. It will be appreciated that reference to acids herein
includes reference to any form of the acid suitable provide an
aqueous solution of the acid, either with water alone, or with a,
preferably physiologically acceptable deprotecting agent, which may
be present initially in the nitrite solution or preparation, prior
to mixing. Examples of suitable forms include the hydrated and
anhydrous forms of the acid, such as citric acid monohydrate and
its anhydrous form.
[0062] Owing to the advantageous qualities of the reducing acids,
in one embodiment it is preferred to provide a reducing acid in
addition to the primary acid when forming the final composition.
Suitable proportions are between about 5% and about 200% of the
primary acid, with 5% to about 150% more preferred; and
particularly between 5 and 40% of the primary acid and, more
particularly, between 10 and 20%.
[0063] It will be appreciated that the present invention extends to
any composition capable of producing a zone of inhibition, in
accordance with the accompanying Examples, especially where the
organism is T. rubrum.
[0064] The present invention also extends to methods of treatment
of subungual infections wherein the compositions described herein
are applied to the infected nail in effective amounts.
[0065] The present invention also extends to use of nitrogen oxide
generating components in the manufacture of a medicament for the
treatment or prophylaxis of a subungual infection.
[0066] It will be appreciated that the present invention includes
kits of parts comprising compositions as defined herein. In
particular, in a preferred embodiment, the present invention
provides a kit comprising an aqueous preparation of a nitrite and
an aqueous preparation of an organic acid, separately disposed one
from the other, the two preparations each being suitable to apply
to a nail to be treated such that the nitrite and acid can react to
release nitrogen oxides for penetration into the nail. The
preparations, are preferably in concentrations and/or forms as
described herein, especially lotions, gels, creams or lacquers, and
are suitably provided in resealable containers such that each kit
may provide multiple doses or applications.
[0067] Any form of subungual infection may be treated using
compositions of the present invention. In general, however, it is
preferred to treat onychomycosis.
[0068] Suitable durations of treatment will generally be readily
determined by the skilled physician. In general, however, it is
preferred to continue treatment until either an actual cure or a
fall, clinical cure is achieved. In the former case, the causative
organisms are killed, but the nail may still be disfigured, as
finger nails can take 6 months to grow out, while toe nails can
take up to a year. A clinical cure is achieved when the affected
nail shows no further signs of infection and, as this depends on
the nail growing out, can take significantly longer than an actual
cure.
[0069] It is preferred, in general, to continue treatment for at
least two months, more preferably three months, and especially
between 3 and 6 months. In fact, our tests indicate that the
causative organisms are likely to be killed within a few days of
commencing treatment, so that treatment for one week may well be
sufficient, especially if compositions are applied two or three
times a day, for example. It is envisaged that a three month
treatment regimen will be adequate to effect a cure, this also
permitting the patient to be able to observe that healthy nail is
growing through. However, it will be appreciated that treatment may
be continued for as long as desired, for example, until a clinical
cure is achieved, which may be up to 14 months or longer, allowing
for cure and nail growth.
[0070] Doses and amounts of composition to be applied may be
dependent on parameters such as the age and weight of the patient
but, more particularly, may be dependent on nail dimensions, such
as thickness and area, of the recipient, and will be readily
determined by the skilled physician. It is an advantage of the
present invention that only small amounts of nitrogen oxides are
necessary to be effective, so that it is not necessarily a
requirement that there be differing prescriptions for different
patients, and one type of formulation may be used for all patients.
However, different strengths may be employed, such as higher oxide
producing formulations for persistent conditions in toes, and lower
strength formulations for finger nails, for example. The strength
does not necessarily relate to the amount of oxides produced, but
may equally relate to the length of time oxides are generated by a
given composition.
[0071] Administering the compositions of the invention two or more
times a day, preferably two or three times a day, forms a preferred
embodiment of the invention. This may effectively provide a boost
of levels of nitrogen oxides at the infected side of the nail at
around the time the effect of the previous dose wears off. Doses
may be selected to maintain continuous transfer of nitrogen oxides
across the nail, or discontinuous, as desired.
[0072] Compositions of the present invention may be made by any
suitable means. Where the compositions comprise aqueous components,
then it is generally preferred to dissolve the active ingredients
in water, or an aqueous preparation, which may then be kept
separate from the other actives until required. Any excipients may
generally be added after solution of the primary active ingredient.
Dry formulations may be made up substantially complete, save for
the addition of water, which is added when it is desired to
activate the composition.
[0073] Where the final composition comprises liquids or gels, these
may be applied by any suitable means, including manual mixing.
Other means may comprise a double barrelled syringe or a dual
actuated dispenser, for example, with final mixing by a finger or
spatula, or any other means appropriate.
[0074] It will be understood that the following Examples are
non-limiting on the present invention. The Examples are illustrated
with reference to the accompanying drawings, in which:
[0075] FIG. 1 illustrates the test used for measuring zones of
inhibition;
[0076] FIG. 2 shows a Franz cell set up to measure evolution of
nitrogen oxides from compositions of the invention;
[0077] FIG. 3 shows production of NO with respect to time, from a
selection of geling agents containing nitrites and acids;
[0078] FIG. 4 shows the zones of inhibition for certain solutions
and gel based formulations;
[0079] FIG. 5 shows the zones of inhibition for certain
formulations investigated in the presence of reducing agents;
[0080] FIG. 6 shows the zones of inhibition for certain
formulations investigated in the presence of .alpha.-tocopherol and
other reducing agents;
[0081] FIG. 7 shows the average peak NO and NO.sub.2/NO.sub.3
concentrations produced by various formulations;
[0082] FIG. 8 shows the average time to reach the maximum NO
production from the various formulations investigated;
[0083] FIG. 9 shows the amount of NO which passed through a nail
from 10% acid and 10% nitrite solutions over an 18 hour period;
[0084] FIG. 10 shows the amount of NO which passed through a nail
from 5% acid and 5% nitrite solutions over an 18 hour period;
[0085] FIG. 11 shows the set up used to measure the amounts of NO
passing through the nail in FIGS. 9 and 10;
[0086] FIG. 12 shows the amount of NO which passed through a nail
from a 10% ascorbic acid and 10% nitrite solution over an 18 hour
period; and
[0087] FIG. 13 shows the Franz cell set up used to measure the
zones of inhibition achieved by compositions of the invention
applied to a human nail.
METHOD EXAMPLE 1
[0088] Analytical in vitro Model for Screening Gas Producing
Formulations
[0089] The method for assaying the zone of killing of various
creams and solutions was as shown in FIG. 1. The key shows the
components of the test. A lid from a 2 ml Eppendorf tube was
embedded in a layer of Sabouraud dextrose agar, and the surface of
the agar coated with A. niger spores. Aspergillus niger spores were
used, as these are known to be far more resistant to antimicrobial
activity compared to the mycelium. The dishes were either then
incubated to obtain a mycelium, or used straight away to test the
ability of the mixtures of the invention to kill spores. The
mixtures of the invention were placed in the Eppendorf cap and
stirred 10 times with the end of a pipette. These test plates are
also referred to herein as walled well zone of inhibition plates.
Each formulation was tested three times.
[0090] This technique provides excellent results, as the NO
producing mix does not come into contact with the agar, and
controls showed that there was no inhibition of growth by the empty
well. This assay is also sensitive.
[0091] The controls used were as follows:
[0092] i) an empty Eppendorf lid only in Sabouraud dextrose
agar;
[0093] : ii) 0.1 ml sodium nitrite (10%) in an Eppendorf lid;
[0094] iii) 0.11 ml citric acid (13.5%) in an Eppendorf lid;
[0095] iv) 0.1 ml sodium nitrite (9%) in an Eppendorf lid; and
[0096] v) 0.1 ml citric acid (10%) in an Eppendorf lid.
[0097] No inhibition of growth was seen with any of the
controls.
METHOD EXAMPLE 2
[0098] Indicator Organism
[0099] The dermatophytes associated with onychomycosis are slow
growing organisms taking a minimum of 5 days to produce a full
carpet of growth on a Sabouraud dextrose agar plate incubated at
25.degree. C. This is a limiting factor in screening active
formulations, as it takes at least a week to produce one set of
results. Therefore, it was generally decided to use a faster
growing indicator organism to assay the effectiveness of the
formulations, although effectiveness of some compositions was
confirmed on T rubrum. The organism chosen was the fungus,
Aspergillus niger, an organism often used to monitor preservative
and anti-microbial efficiency of cosmetic and topical
formulations.
[0100] A. niger was used as the indicator organism for fungi
associated with onychomycosis, and tests were carried out on both
the fungal spores and mycelium. Where the mycelium is used, the
killing zone is indicated by the lack of development of fungal
spores (black/brown), a white zone showing inhibition of growth. On
fungal spore plates, inhibition of growth is indicated by no
development of mycelium (white/cream), so that only agar is
seen.
METHOD EXAMPLE 3
[0101] NO Detection
[0102] NO produced was measured using a WPI (World Precision
Instruments, Inc.) NO detector. Measurements of NO need to be
carried out in an aqueous environment when using this sensor, and
the experiments were adapted to suit this requirement These devices
also have the ability to monitor NO.sub.2/NO.sub.3 production by
addition of chemicals and minor modifications of the methods.
[0103] A Franz cell was assembled and positioned onto a magnetic
stirrer at room temperature with the de-ionised water in the lower
receptor containing a magnetic follower to ensure dispersion of the
gas penetrating the membrane. A 1/2 inch (13 mm) filter paper disk
was impregnated with the sodium nitrite component of the NO
producing formulation. The impregnated disk was placed in the upper
compartment of the Franz cell on top of the membrane before
pipetting an equal volume of citric acid component onto it. The
amount of NO produced was monitored using the WPI NO probe. The
experiment was set up as shown in FIG. 2.
[0104] When measured NO levels had reached a plateau, the disk
containing the mixture of sodium nitrite and citric acid was
removed and 0.1 ml of a 0.1 M H.sub.2SO.sub.4+0.1 M KI solution was
added to the receptor compartment and the amount of
NO.sub.2/NO.sub.3 was measured (by conversion) using the WPI
detector.
EXAMPLE 1
[0105] Effect Of Acid Preparations Mixed With Nitrite Solutions On
A. niger Spores
[0106] The acids selected were: citric, acetic, ascorbic, maleic
and malic acid. The nitrites selected were sodium, potassium and
silver.
[0107] The assay set up described in Method Example 1 was used. All
Examples herein involving zones of killing used the set up of
Method Example 1, unless otherwise indicated.
[0108] The following acid solutions (w/v) were prepared in
distilled water:
[0109] Citric acid 2.5%
[0110] 2) Citric acid 5%
[0111] 3) Citric acid 7.5%
[0112] 4) Citric acid 10%
[0113] 5) Ascorbic acid 2.5%
[0114] 6) Ascorbic acid 5%
[0115] 7) Ascorbic acid 7.5%
[0116] 8) Ascorbic acid 10%
[0117] 9) Maleic acid 2.5%
[0118] 10) Maleic acid 5%
[0119] 11) Maleic acid 7.5%
[0120] 12) Maleic acid 10%
[0121] 13) Malic acid 2.5%
[0122] 0.14) Malic acid 5%
[0123] 15) Malic acid 7.5%
[0124] 16) Malic acid 10%
[0125] 17) Acetic acid 2.5%
[0126] 18) Acetic acid 5%
[0127] 19) Acetic acid 7.5%
[0128] 20) Acetic acid 10%
[0129] The lids of 2 ml Eppendorf micro tubes were removed,
sterilised and incorporated into a Sabouraud dextrose agar plate,
by placing the lids in a petri dish and pouring 25 ml of the agar
around them. The agar was allowed to set and was then seeded with
A. niger. Each of the solutions indicated above were mixed with
2.5, 5, 7.5 and 10% sodium/potassium/silver nitrite solutions by
adding exact 0.1 ml quantities of each solution into the lid of the
micro tube and mixing by gently rotating the plate. Separate plates
were set up for each of the acid solutions listed above and were
incubated at 32.degree. C. Zones of inhibition of growth on each
plate were measured after 24 hours. The results are shown in Tables
1 and 2, below.
1TABLE 1 Sodium Nitrite Zone Of Inhibition Results - Aspergillus
niger Spores Sodium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 2.2
3.0 2.9 2.8 5% 2.8 3.6 3.9 3.9 7.5% 2.9 3.7 4.5 4.6 10% 3.0 4.1 4.4
5.4 Ascorbic 2.5% 0 0 0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0
Maleic acid 2.5% 2.5 2.6 3.9 3.0 5% 3.0 3.2 3.7 3.1 7.5% 3.4 3.9
4.6 4.9 10% 3.1 4.4 5.1 5.8 Malic acid 2.5% 2.4 2.9 3.8 3.6 5% 2.9
3.4 3.8 4.2 7.5% 2.9 3.8 4.6 4.3 10% 3.2 4.2 5.8 5.1 Acetic acid
2.5% 2.5 2.7 3.4 3.6 5% 2.8 3.3 4.0 4.0 7.5% 2.6 3.5 4.1 4.1 10%
3.4 3.6 4.4 5.2
[0130]
2TABLE 2 Potassium Nitrite Zone Of Inhibition Results - Aspergillus
niger Spores Potassium nitrite 2.5% 5% 7.5% 10% Citric 2.5% 0 0 0
1.5 acid 5% 0 1.5 1.8 2.8 7.5% 0 1.7 2.1 3.4 10% 0 2.2 3.2 3.8
Ascorbic 2.5% 0 0 0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0
Maleic acid 2.5% 0 1.3 1.5 1.4 5% 0 2.5 3.0 2.5 7.5% 0 2.5 3.2 3.2
10% 0 3.0 3.2 5.0 Malic acid 2.5% 0 1.3 1.5 1.8 5% 0 2.1 2.5 2.2
7.5% 0 2.4 2.5 2.7 10% 0 2.8 2.7 3.5 Acetic acid 2.5% 1.7 2.3 2.5
2.5 5% 2.1 3.5 3.2 4.0 7.5% 3.0 3.7 4.0 4.5 10% 3.5 4.0 4.5 4.6
[0131] No Table is shown for silver nitrite, as no zones of
inhibition were seen for any of the acids tested with silver
nitrite.
[0132] Some ascorbic acid mixtures swelled on mixing to such an
extent that they domed above the tops of the wells, occasionally
resulting in small amounts of over-spill on to the agar during
transfer of the plates to the incubator, which produced zones of
inhibition. No zones of inhibition were seen with ascorbic acid
solutions in the absence of spillage. No other acid evinced any
sign of over-spill or of swelling. From these tests, it would
appear that all mixtures tested, apart from those involving either
silver nitrite or ascorbic acid, are effective.
EXAMPLE2
[0133] Effect Of Acid Preparations Mixed With Nitrite Solutions On
A. niger Mycelium
[0134] Acid solutions were prepared as described in Example 1.
Prior to addition of gas producing formulations, the plates were
incubated at 32.degree. C. overnight to establish a full carpet of
growth. Solutions were then added to the wells as described in
Example 1. The plates were then incubated at 32.degree. C. for a
further 24 hours. Zones of inhibition of growth were measured, as
the point where the area of spore formation (black), and no spore
formation (white/cream) met. Results are shown in Tables 3, 4 and
5.
3TABLE 3 Sodium Nitrite Zone Of Inhibition Results - Aspergillus
niger Mycelium Sodium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 3.0
0 0 0 5% 2.8 1.2 1.8 2.4 7.5% 3.2 1.4 3.1 3.4 10% 3.4 3.1 3.7 4.6
Ascorbic 2.5% 0 0 0 0 acid 5% 0 (2) 0 0 (2.1) 0 (2.5) 7.5% 0 (2.5)
0 (3.4) 0 (2.5) 0 10% 0 (2.0) 0 (1.9) 0 (3.0) 0 (3.9) Maleic acid
2.5% 3.4 3.5 3.4 3.0 5% 3.9 4.4 4.6 4.4 7.5% 4.0 5.0 4.8 6.0 10%
4.2 5.4 6.2 8.0 Malic acid 2.5% 3.5 3.7 3.3 3.1 5% 3.7 4.2 4.1 4.1
7.5% 3.5 5.0 4.9 6.0 10% 3.7 5.6 5.0 6.4 Acetic acid 2.5% 2.6 3.8
3.7 3.3 5% 3.0 3.6 3.6 4.0 7.5% 3.0 4.3 4.1 4.4 10% 3.6 4.5 4.7
6.2
[0135]
4TABLE 4 Potassium Nitrite Zone Of Inhibition Results - Aspergillus
niger Mycelium Potassium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5%
2.7 3.1 3.2 3.5 5% 3.1 3.9 4.2 5.0 7.5% 3.6 3.9 4.2 5.4 10% 3.7 3.9
5.3 6.2 Ascorbic 2.5% 0 0 0 0 acid 5% 0 (1.5) 0 0 (2.0) 0 (2.4)
7.5% 0 (3.1) 0 (2.5) 0 0 (3.1) 10% 0 (2.0) 0 (2.4) 0 (3.0) 0 (3.2)
Maleic acid 2.5% 3.5 3.5 3.4 3.5 5% 3.8 3.8 5.0 5.0 7.5% 4.1 3.9
6.5 7.8 10% 4.5 4.3 8.0 9.0 Malic acid 2.5% 3.4 3.9 3.8 3.4 5% 3.7
4.0 5.2 5.0 7.5% 3.8 4.4 5.4 5.8 10% 4.2 5.0 6.3 6.4 Acetic acid
2.5% 2.7 2.8 3.4 3.6 5% 3.4 3.0 4.2 4.4 7.5% 3.6 3.7 4.3 4.7 10%
4.4 4.9 4.6 5.1
[0136]
5TABLE 5 Silver Nitrite Zone Of Inhibition Results - Aspergillus
niger Mycelium Silver nitrite 0.025% 0.05% 0.075% 0.10% Citric acid
2.5% 0 0 0 0 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Ascorbic 2.5% 0 0
0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid 2.5% 0 0 0
0 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Malic acid 2.5% 0 0 0 0 5% 0
0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Acetic acid 2.5% 0 0 0 1.1 5% 0 0 0
1.2 7.5% 0 0 0 1.2 10% 0 0 0 1.3
[0137] From these tests, it would appear that all mixtures tested,
apart from most of those involving either silver nitrite or
ascorbic acid, are effective.
EXAMPLE 3
[0138] Effect Of Acid Aqueous Cream Preparations Mixed With Nitrite
Solutions On A. niger Spores
[0139] The same procedure as described in Example 1 was followed,
except that aqueous creams were used in place of solutions, using
the same concentrations of both acids and nitrites. The results are
shown in Tables 6 and 7.
6TABLE 6 Effect of Sodium Nitrite Creams On Aspergillus niger
Spores Sodium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 0 0 0 0 5%
0 0 0 1.5 7.5% 0 0 1.6 2.3 10% 0 0 1.7 2.9 Ascorbic 2.5% 0 0 0 0
acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid 2.5% 0 0 0 0
5% 0 0 0 1.6 7.5% 0 0 1.7 2.2 10% 0 0 1.9 2.5 Malic acid 2.5% 0 0 0
0 5% 0 0 1.2 2.0 7.5% 0 0 1.6 2.4 10% 0 0 1.7 2.7 Acetic acid 2.5%
0 0 0 0 5% 0 0 0 1.7 7.5% 0 0 1.7 2.3 10% 0 0 2.2 2.8
EXAMPLE 4
[0140] Effect Of Acid Aqueous Cream Preparations Mixed With Nitrite
Solutions On A. niger Mycelium
[0141] The same procedure as described in Example 2 was followed,
except that aqueous creams were used in place of solutions, using
the same concentrations of both acids and nitrites. The results are
shown in Tables 8, 9 and 10.
7TABLE 8 Effect of Sodium Nitrite Creams on Aspergillus niger
Mycelium Sodium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 0 0 1.2
1.4 5% 0 1.2 1.4 3.1 7.5% 0 1.8 3.1 3.7 10% 0 2.4 3.4 4.6 Ascorbic
2.5% 0 0 0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid
2.5% 0 0 1.2 1.6 5% 0 1.3 2.7 3.2 7.5% 0 1.9 4.1 4.4 10% 0 2.1 4.4
4.5 Malic acid 2.5% 0 0 1.6 2.6 5% 0 1.2 3.1 3.5 7.5% 0 1.3 3.6 4.4
10% 0 1.65 4.3 5.2 Acetic acid 2.5% 1.2 1.3 2.0 2.4 5% 2.0 2.1 2.7
3.2 7.5% 2.6 2.2 3.0 4.0 10% 2.4 3.0 3.7 4.7
[0142]
8TABLE 7 Effect of Potassium Nitrite Creams On Aspergillus niger
Spores Potassium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 0 0 0 0
5% 0 0 1.6 1.7 7.5% 0 1.7 1.65 1.8 10% 0 1.8 1.8 1.9 Ascorbic 2.5%
0 0 0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid 2.5% 0
0 0 1.5 5% 0 0 1.5 1.7 7.5% 0 0 1.6 2.0 10% 0 0 2.3 2.9 Malic acid
2.5% 0 1.4 1.4 1.6 5% 0 1.5 2.1 1.8 7.5% 0 1.65 2.3 2.3 10% 0 1.7
2.35 2.7 Acetic acid 2.5% 0 0 1.3 1.5 5% 0 1.3 2.0 1.7 7.5% 0 1.8
2.4 2.5 10% 1.7 2.4 2.6 2.7
[0143] No Table is shown for silver nitrite, as no zones of
inhibition were seen for any of the acid creams mixed with silver
nitrite. From these tests, it would appear that all mixtures
tested, apart from those involving either silver nitrite or
ascorbic acid, are effective.
9TABLE 9 Effect of Potassium Nitrite Creams on Aspergillus niger
Mycelium Potassium nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 0 0 0
0 5% 0 1.5 1.7 2.2 7.5% 0 1.8 2.1 3.2 10% 1.5 2.8 3.4 3.8 Ascorbic
2.5% 0 0 0 0 acid 5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid
2.5% 0 0 0 0 5% 1.3 2.5 2.5 3.0 7.5% 1.5 3.0 3.2 3.2 10% 1.4 2.5
3.2 5.0 Malic acid 2.5% 0 0 0 0 5% 1.3 2.1 2.4 2.8 7.5% 1.5 2.5 2.5
2.7 10% 1.8 2.2 2.7 3.5 Acetic acid 2.5% 1.7 2.1 3.0 3.5 5% 2.3 3.5
3.7 4.0 7.5% 2.5 3.2 4.0 4.5 10% 2.5 4.0 4.5 4.6
[0144]
10TABLE 10 Effect of Silver Nitrite Creams on Aspergillus niger
Mycelium Silver nitrite 2.5% 5% 7.5% 10% Citric acid 2.5% 0 0 0 0
5% 0 0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Ascorbic 2.5% 0 0 0 0 acid 5% 0
0 0 0 7.5% 0 0 0 0 10% 0 0 0 0 Maleic acid 2.5% 0 0 0 0 5% 0 0 0 0
7.5% 0 0 0 0 10% 0 0 0 0 Malic acid 2.5% 0 0 0 0 5% 0 0 0 0 7.5% 0
0 0 0 10% 0 0 0 0 Acetic acid 2.5% 0 0 1.1 0 5% 0 0 1.8 1.2 7.5% 0
1.6 2.0 1.6 10% 1.2 2.0 2.5 2.5
[0145] From these tests, it would appear that all mixtures tested,
apart from most of those involving either silver nitrite or
ascorbic acid, are effective.
EXAMPLE 5
[0146] Amount of NO Produced by Mixtures of Acids and Nitrites
[0147] A rough estimation of the amounts of NO produced by each
acid and nitrite solution was calculated using the WPI NO probe, as
described in Method Example 3. The nitrite component (50 .mu.l) was
added to 10 ml of the acid solution in each experiment.
[0148] Initially the NO probe was immersed in the acid component (a
10% solution for each experiment). The probe was left to
equilibrate in the acid before nitrite was added. Once a baseline
had been established, the instrument was zeroed, and the nitrite
was added. The lowest concentration was added first, and subsequent
additions were made once the curve began to flatten. The graphs
produced only provided a rough indication of the amount of NO
produced, the values not being measured, on this occasion. A
calibration curve was produced prior to each set of readings using
the standard protocol detailed by WPL to allow for any slight
changes in NO detection at different temperatures, as suggested by
WPI.
[0149] For sodium nitrite and potassium nitrite, 2.5, 5, 7.5 and
10% solutions were added at various points to each acid. Silver
nitrite is very insoluble and only very low concentrations could be
used, so only 0.025, 0.05, 0.075 and 0.1% solutions were added to
the acids.
[0150] Calibration curves and NO release profiles were generated
for all the acids and nitrites. For both sodium nitrite and
potassium nitrite mixed with ascorbic acid, bubbles formed around
both the magnetic follower and around the NO probe.
[0151] A brief study was performed to estimate the relative amounts
of NO.sub.2/NO.sub.3 in some of the formulations. This was done, as
described in Method Example 3 above, by adding 1 ml of 0.1 M
H.sub.2SO.sub.4 and 0.1 M KI, once the NO producing formulation had
reached a maximum. The 0.1 M H.sub.2SO.sub.4 and 0.1 M KI converts
NO.sub.2/NO.sub.3 to NO which is recorded using the WPI NO probe.
The results obtained were:
[0152] 10 ml citric acid (10%)+50 .mu.l NaNO.sub.2 (10%) produced
4000 pA of NO. On addition of 0.1 M H.sub.2SO.sub.4 and 0.1 M KI
the peak value reached 20000 pA. It was estimated that there is
approximately 20% production of NO with 80% to
NO.sub.2/NO.sub.3.
[0153] 10 ml citric acid (10%)+50 .mu.l KNO.sub.2 (10%) produced
2200 pA of NO. On addition of 0.1 M H.sub.2SO.sub.4 and 0.1 M KI,
the peak reached 10700 pA. It was estimated that there is
approximately 20% production of NO, with 80% to
NO.sub.2/NO.sub.3.
[0154] 10 ml ascorbic acid (10%)+50 .mu.l AgNO.sub.2 (0.1%)
produced 5200 pA of NO. On addition of 0.1 M H.sub.2SO.sub.4 and
0.1 M KI no change was observed in the peak. It was estimated that
there was approximately 100% production of NO, with no
NO.sub.2/NO.sub.3.
[0155] 10 ml acetic acid (10%)+50 .mu.l AgNO2 (0.1%) produced 70 pA
of NO. On addition of 0.1 M H.sub.2SO.sub.4 and 0.1 M KI, the peak
reached 2700 pA. It was estimated that there is approximately 3%
production of NO, and 97% to NO.sub.2/NO.sub.3.
[0156] It was surprising to note that, despite the results of the
previous Examples, where no zones of inhibition were identified for
mixtures employing ascorbic acid, combinations using ascorbic acid
appeared to produce considerable amounts of NO. However, ascorbic
acid produced little or no NO.sub.2/NO.sub.3 with silver nitrite.
All of the other acids tested produced larger quantities of
NO.sub.2/NO.sub.3 than NO.
[0157] It is also noteworthy that the malic acid-potassium nitrite
NO profile and the citric acid-potassium nitrite NO profile show
little correlation between the amounts of NO produced and the zone
sizes seen in the earlier Examples. These two acids produced very
similar zone sizes, but malic acid produced only approximately a
third of the amount of NO with sodium nitrite.
EXAMPLE 6
[0158] Formulation Evaluation
[0159] Eudragits
[0160] Eudragit based lacquers were investigated for their ability
to alter the NO release profile of a formulation. Generally,
polymethacrylates (Eudragits) are used for oral tablet/capsule
formulations as film coating agents. Selection of different films
can produce different drug release rates. Different Eudragits
available include; Eudragit E, Eudragit L and Eudragit S. Eudragit
E is used as a plain insulating filn former and is soluble in
gastric fluid below pH 5. Eudragits L and S are used as enteric
coating agents, and are also resistant to gastric fluid. Different
forms of Eudragits L and S are soluble at different pH levels, for
example Eudragit L 100 is soluble >pH 6, and Eudragit S 100 is
soluble >pH 7. Eudragits can be combined to obtain different
drug release characteristics. Investigations were performed on
Eudragit formulations containing both acid and nitrite for the
production of the active gas.
[0161] Eudragit L100 was tested in combination with sodium nitrite
and prolonged the release of NO from 5 to 25-30 minutes (data not
shown). Formulating the acid component in a reverse Eudragit (E100)
appeared to have little or no effect on prolonging the production
of NO. Thus, a suitable formulation is sodium nitrite in L100, with
the acid component present in abundance in a gel.
EXAMPLE 7
[0162] Alternative Gel Formulations
[0163] Combinations of nitrite and acid in formulations of various
gelling agents were visually assayed. The gelling agents used were
as follows:
[0164] 3% Carboxymnethylcellulose (CMC);
[0165] 3% Methylcellulose (MC);
[0166] 3% Carbopol 934;
[0167] 3% Gelatin A;
[0168] 3% Gelatin B;
[0169] 20% Polyethylene glycol (PEG) 400;
[0170] 20% PEG 600;
[0171] 20% PEG 1000;
[0172] 3% Hydroxymethylcellulose (HMC); and
[0173] 3% Polyvinyl alcohol (PA).
[0174] All gels were prepared with:
[0175] (i) a 10% solution of citric acid, and
[0176] (ii) a 10% solution of sodium nitrite.
[0177] A visual assessment and a pH test (using litmus paper) were
also carried out. Results are shown in Tables 11 and 12.
11TABLE 11 10% Sodium nitrite gels Gelling agent pH Characteristics
CMC 8 Formed thick faint yellow gel MC 8 Watery and yellow in
colour, also precipitate seen Carbopol 934 9.5 Thick faint yellow
opaque gel Gelatin A 8.5 Orange clear liquid Gelatin B 8 Yellow
clear liquid PEG 400 8.5 Faint yellow clear liquid PEG 600 8.5
Faint yellow clear liquid PEG 1000 9 Faint yellow clear liquid HMC
8.5 Thick faint yellow gel PA 8 Faint yellow liquid, with
precipitate From Table 11, it can be seen that CMC, HMC and
Carbopol all formed stable gels in the presence of 10% sodium
nitrite.
[0178]
12TABLE 12 Characteristics of 10% Citric acid gels Gelling agent pH
Characteristics CMC 2 Mobile gel, opaque (colourless) MC 1 Mobile
gel, clear Carbopol 934 1 Mobile gel opaque Gelatin A 2 Faint
yellow clear liquid Gelatin B 2 Faint yellow clear liquid PEG 400 1
Clear liquid PEG 600 1 Clear liquid PEG 1000 2 Clear liquid HMC 1
Clear thick gel PA 2 Clear liquid From Table 12, it can be seen
that HMC, CMC, MC and Carbopol all formed stable gels in the
presence of 10% citric acid.
[0179] These tests provide some preliminary indications. In
particular, gelatin is a preferred gelling agent, and it is
preferred to use this at conventionally higher levels, such as 20%
to 40%, more generally around 30%.
EXAMPLE 8
[0180] Effects Of NO-Producing Formulations On T. rubrum
[0181] The method described above, in Method Example 2, using a
walled well zone of inhibition plate was adapted to test the
dermatophyte cidal activity of various NO-producing formulations.
The following formulations were made and added to a walled well
agar plate, pre-seeded with T. rubrum:
[0182] Carbopol containing 10% citric acid (5011) mixed with the
Eudragit L100 containing 10% sodium nitrite (50 .mu.l).
[0183] CMC containing 10% citric acid (50 .mu.l) mixed with CMC
containing 10% sodium nitrite (501).
[0184] Aqueous cream containing 10% citric acid (50 .mu.l) mixed
with aqueous cream containing 10% sodium nitrite (50 .mu.l).
[0185] HMC containing 10% citric acid (50 .mu.l) mixed with HMC
containing 10% sodium nitrite (50 .mu.l).
[0186] Eudragit E100 containing 10% citric acid (50 .mu.l mixed
with the Eudragit L100 containing 10% sodium nitrite (50
.mu.l).
[0187] MC containing 10% citric, acid (50 .mu.l) mixed with the
Eudragit L100 containing 10% sodium nitrite (50 .mu.l).
[0188] 50 .mu.l of 10% citric acid mixed with a solution of 10%
sodium nitrite (50 .mu.l).
[0189] 50 .mu.l of 10% citric acid mixed with the Eudragit L100
containing 10% sodium nitrite (50 .mu.l).
[0190] Positive control (no formulation added).
13TABLE 13 Results - Zone Sizes After Five Days NO Producing
formulation Zone of 10% Citric acid 10% sodium nitrite inhibition
(cm) Carbopol L100 7.7 CMC CMC 5.6 Aqueous cream Aqueous cream 3.4
HMC HMC 8.5 E100 L100 0 MC L100 7.7 Solution Solution 8.5 Solution
L100 8.5 None None 0
[0191] These results show that T. rubrum is highly susceptible to
killing by the formulations of the invention, large zones of kill
being seen.
EXAMPLE 9
[0192] WPI Measurement of NO Production from Lacquers/Gelling
Agents
[0193] An experiment was set up to analyse the length of time of
release of NO from NO-producing formulations. Placing the
formulation directly onto the surface of a 0.2 .mu.m pore filter in
a Franz cell resulted in the formulations leaching through the
filter, with the reactions taking place in the lower reservoir of
the Franz cell. Accordingly, antibiotic disks were impregnated with
the sodium nitrite component of the NO producing formulation, the
disk being placed in the upper compartment of the Franz cell, on
top of the membrane, and then pipetting an equal volume of the
citric acid component onto the impregnated disk. The amount of NO
produced was monitored using the WPI NO probe. The experiment was
set up as shown in FIG. 2.
[0194] Three formulations were assessed:
[0195] 10% Citric acid in Carbopol 934 and 10% sodium nitrite in
Eudragit L100
[0196] 10% Citric acid in HMC and 10% sodium nitrite in Eudragit
L100
[0197] 10% Citric acid in solution and 10% sodium nitrite in
solution
[0198] The results are shown in, FIG. 3, which shows production of
NO with respect to time, from a selection of gelling agents
containing nitrites and acids. The carbopol and L100 NO producing
formulation reached a peak at 50000 pA, which is above the
detection limit of the WPI meter. However, it can be seen from the
graph that this formulation released NO at a fairly steady and at a
consistent rate for 23 minutes, at which time the experiment was
terminated. By comparison, the NO produced by the solutions began
to slow down far earlier, and it is probable that most of the NO
produced bubbled out of the solutions and was released into the
atmosphere. Similar patterns were seen for the sample with HMC.
This shows that a formulation approach can be used to obtain
different release profiles of NO.
EXAMPLE 10
[0199] Formulation for Testing
[0200] The formulations listed in Table 14 were investigated for
zone of inhibition, NO release profiles, and NO.sub.2/NO.sub.3
release. The concentration of the components listed in Table 14 are
those (% w/w in de-ionised water) before mixing.
14TABLE 14 Sample Formulations containing 10% Formulations
containing 10% No. sodium nitrite (% w/w) citric acid (% w/w) 1
Carboxymethylcellulose + Carbopol (3%) (CMC) (3%) 2 CMC (3%) +
Hydroxymethylcellulose (HMC) (3%) 3 CMC (3%) + Methylcellulose (MC)
(3%) 4 CMC (3%) + CMC (3%) 5 CMC (3%) + Sol'n (DW) 6 L100
(Eudragit) (5%) + Carbopol (3%) 7 L100 (5%) + HMC (3%) 8 L100 (5%)
+ MC (3%) 9 L100 (5%) + CMC (3%) 10 L100 (5%) + Sol'n (DW) 11
Hydroxymethylcellulose + Carbopol (3%) (HMC) (3%) 12 HMC (3%) + HMC
(3%) 13 HMC (3%) + MC (3%) 14 HMC (3%) + CMC (3%) 15 HMC (3%) +
Sol'n (DW) 16 Sol'n (Distilled water (DW)) + Carbopol (3%) 17 Sol'n
(DW) + HMC (3%) 18 Sol'n (DW) + MC (3%) 19 Sol'n (DW) + CMC (3%) 20
Sol'n (DW) + Sol'n (DW) 21 Cream (50% Aqueous cream + Cream (50%
aqueous cream dissolved in DW) dissolved in DW) 22 Sol'n (DW) + 1 M
Sodium ascorbate 23 Sol'n (DW) + 1 M Sodium hydrogensulphite
(SHS)
[0201] The combinations of Table 15 were assessed only for the zone
of inhibition.
15TABLE 15 Sample no. Formulations (% w/w) 24 citric acid (20%) +
ascorbic acid (20%) + sodium nitrite (20%) 25 citric acid (20%) +
ascorbic acid (16%) + sodium nitrite (20%) 26 citric acid (20%) +
ascorbic acid (12%) + sodium nitrite (20%) 27 citric acid (20%) +
ascorbic acid (8%) + sodium nitrite (20%) 28 citric acid (20%) +
ascorbic acid(4%) + sodium nitrite (20%) 29 citric acid (16%) +
ascorbic acid (20%) + sodium nitrite (20%) 30 citric acid (12%) +
ascorbic acid (20%) + sodium nitrite (20%) 31 citric acid (8%) +
ascorbic acid (20%) + sodium nitrite (20%) 32 citric acid (4%) +
ascorbic acid (20%) + sodium nitrite (20%) 33 citric acid (20%) + 1
M Na.sub.2S.sub.2O.sub.4 + sodium nitrite (20%) 34 citric acid
(20%) + 1 M sodium ascorbate + sodium nitrite (20%) 35* citric acid
(20%) + 1 M Na.sub.2S.sub.2O.sub.4 36* citric acid (20%) + 1 M
sodium ascorbate 37** DW The individual components listed represent
the initial concentration (% w/w in deionised water (DW)), before
mixing. * and ** represent positive and negative controls,
respectively.
[0202] Zones of inhibition were also assessed for an alternative
reducing agent, .alpha.-tocopherol. Combinations for assessment
using this agent are listed in Table 16. Formulations containing
combinations of sodium nitrite and citric acid with either ascorbic
acid (Sample no. 24), Na.sub.2S.sub.2O.sub.4 (Sample no. 33) or
sodium ascorbate (Sample no. 34) were also included as
comparators.
16TABLE 16 Sample no. Formulation (% w/w) 38 9 g DW + 1 g Citric
acid + 50 .mu.l .alpha.- tocopherol + sodium nitrite (20%) 39 4 g
DW + 1 g Citric acid + 5 g Ethanol + 50 .mu.l .alpha.-tocopherol +
sodium nitrite (20%) 40* DW + DW The individual components listed
represent the initial concentrations (% w/w), before mixing.
*represents the negative control.
EXAMPLE 11
[0203] A) Zones of Inhibition
[0204] FIGS. 4, 5 and 6 show the effect of the formulations of
Example 10 on the zone of inhibition. Clearly, it can be concluded
that there is no apparent difference in the zone of inhibition
observed for most of the formulations investigated, although Sample
no. 32 (a low concentration of citric acid with a high
concentration of ascorbic acid) and Sample no. 39
(.alpha.-tocopherol formulated in ethanol) exhibited lower activity
than other formulations. However, other formulations containing
combinations of a high concentration (20%) of ascorbic acid
(Samples 24-31) showed no apparent reduction in antifungal
activity, as shown in the zone of inhibition assay.
[0205] FIG. 4 shows the zones of inhibition for solutions and gel
based formulations listed in Table 14.
[0206] FIG. 5 shows the zones of inhibition for formulations
investigated in the presence of reducing agents listed in Table
15.
[0207] FIG. 6 shows the zones of inhibition for formulations
investigated in the presence of .alpha.-tocopherol and other
reducing agents listed in Table 16.
[0208] B) NO and NO.sub.2/NO.sub.3 release
[0209] The data generated from the WPI instrument for the amount of
NO produced was quantified in units of pA. A calibration plot was
constructed to calculate the concentration of NO released (data not
shown). In general, the profiles obtained show that solution based
formulations released NO more quickly than gel based formulations.
Amongst the solution based formulations, Sample no. 20 (a solution
of sodium nitrite and citric acid) showed a high release of NO in
the shortest time. However, combinations of solution formulations
containing sodium nitrite solution and citric acid in carbopol
(Sample no. 16) and CMC (Sample no. 19) appeared to produce the
highest amount of NO over an extended period of time. The lowest
amount of NO released was generally found to occur with the CMC
based formulations.
[0210] FIG. 7 shows the average peak NO and NO.sub.2/NO.sub.3
concentrations produced by the various formulations investigated.
Of the gel based formulations investigated, CMC formulations were
found to produce lower concentrations of NO compared to the
remaining gel based formulations (L100, HMC and Carbopol). The
average peak NO concentrations produced as a function of time from
the various formulations investigated are shown in FIG. 8. With the
exception of Sample no. 19 (which contained CMC) the data
demonstrated that the solutions (Sample no. 5, 10, 1620) were
significantly faster in achieving peak NO concentration when
compared to the gel formulations.
[0211] FIG. 7 shows the average peak NO and NO.sub.2/NO.sub.3
concentration produced from the various formulations
investigated.
[0212] FIG. 8 shows the average time to reach the maximum NO
production from the various formulations investigated.
EXAMPLE 12
[0213] Passage of NO Across Human Nail
[0214] 18 h, 10% solutions,
[0215] The set up shown in FIG. 11 was employed. At To, one
application of 100 .mu.l of 10% sodium nitrite and. 100 .mu.l of
10% citric acid were pipetted onto the surface of a piece of human
nail, mounted in the nail Franz cell (exposure surface 0.1963
cm.sup.2). The amount of NO which passed through the nail was
monitored using the WPI NO detector, over an 18 hour period. The
results are shown in FIG. 9.
[0216] 18 h, 5% Solutions
[0217] The set up shown in FIG. 11 was employed. At To, one
application of 100 .mu.l of 5% sodium nitrite and 100 .mu.l of 5%
citric acid were pipetted onto the surface of a piece of human
nail, mounted in the nail Franz cell (exposure surface 0.1963
cm.sup.2). The amount of NO which passed through the nail was
monitored using the WPI NO detector, over an 18 hour period. The
results are shown in FIG. 10.
[0218] Negative Control
[0219] The negative control comprised 200 .mu.l of de-ionised water
pipetted onto the surface of a piece of human nail, mounted in the
nail Franz cell, otherwise employing the same conditions as for the
10% and 5% procedures.
[0220] As can be seen from the Figures, the 10% solutions provided
an apparent initial drop in NO. However, after about 4 hours
(different experiments yield different lag times, data not shown),
NO abruptly increases and is then maintained at high levels for a
period of about 10 hours. The 5% solutions produced positive levels
of NO, albeit somewhat lower than the 10% formulations. The
negative control never deviated from the baseline, after an initial
dip at the beginning of the experiment (data not shown).
EXAMPLE 13
[0221] Measurement Of NO Passage Across Human Nail Using 10%
Ascorbic Acid And 10% Sodium Nitrite
[0222] In the first part of the experiment, the dual arm nail Franz
cell of FIG. 11 was set up, but using a piece of gas permeable
membrane in place of the human nail. The system was left for
approximately 40 min before the formulations were added. 100 .mu.l
of 10% ascorbic acid and 100 .mu.l of a 10% sodium nitrite solution
were added to the top of the Franz cell as in Example 12, and the
top of the Franz cell was covered with parafilm.
[0223] The amount of NO produced during this experiment exceeded
the detection limit of the NO detector, and the plot went off scale
(10 .mu.M) after just over one hour. No initial drop in the peak
was observed. The amount of NO produced was approximately 20 times
higher than that seen with citric acid (10%) and sodium nitrite
(10%).
[0224] The experiment was then repeated, this time using apiece of
human nail. 100 .mu.l of 10% ascorbic acid and 100 .mu.l of 10%
sodium nitrite were then applied directly to the top surface of the
nail and mixed. The Franz cell was covered with parafilm, and the
amount of NO passing through the nail monitored using the WPI
meter. The results are shown in FIG. 12.
[0225] The drop in the peak appears to be a standard artefact in
experiments involving human nail and NO producing formulations. The
increase in the peak following the decrease was very sudden, with
no gradual change, from one to the other.
[0226] From FIG. 12, it can be seen that, while mixtures involving
ascorbic acid can provide sustained release of NO across the nail,
the levels are about 10.times. lower than when using citric
acid.
EXAMPLE 14
[0227] Franz cell Nail Bioassay for Anti-Fungal Gas Penetration
across a Human Nail
[0228] Franz cells were set up as shown in FIG. 13, the lower
section being filled with molten Sabouraud dextrose agar up to 8 mm
below the outlet hole. After the agar had cooled and set, 25 .mu.l
of an A. niger spore suspension was pipetted onto the surface of
the agar and a section of human nail fitted in an air-tight manner
above the outlet hole, and clamped in place. The cells were then
incubated at 32.degree. C. for 24 h to obtain a carpet of
mycelium.
[0229] Once the mycelium had grown, the cells were removed from the
incubator, and 0.1 ml of citric acid and 0.1 ml of sodium nitrite
were pipetted into the funnel of the Franz cell and mixed using the
pipette tip. Negative controls had either a 0.2 ml solution of 10%
sodium nitrite or 0.2 ml of 10% citric acid added. The top of the
funnel for all of the Franz cells was then covered with Parafilm.
The cells were left at room temperature. After a period of 2 h 10
min, the solutions in each Franz cell were removed and replaced
with an equivalent solution and the process repeated every 2 h and
10 min for a further two applications, giving a total of 4
applications. After the final application of solution, the cells
were left covered at room temperature, to allow slow growth of the
organism for 24 h.
[0230] After 24 hours, all six cells with mixtures of citric acid
and sodium nitrite had a central region of the colony of A. niger,
closest to the nail/formulation, which had been killed by the
active gas. This was evidenced by a halo effect, where an area of
dead, white mycelium was surrounded by a ring of black, sporulating
mycelium. After a further 24 hours, the A. niger recovered by
growing from the outmost region, which was not killed, towards the
centre.
[0231] All of the cells with either sodium nitrite or citric acid
showed a full carpet of growth of mycelium, which had formed
spores, as evidenced by a solid black circle.
[0232] This result clearly demonstrates the ability of the
formulations of the invention to generate an effective gaseous
component across a nail. It definitively shows that the active gas
is penetrating a complete human toe nail, and directly affecting
the growth of A. niger. In addition, the formulation is penetrating
the nail and affecting the growth of an organism over an additional
8 mm distance, the separation in vivo being virtually negligible.
The controls confirm that it is the active gas that is causing the
halo effect.
EXAMPLE 15
[0233] Kits
[0234] A kit comprising two aqueous gel preparations was made up.
The preparations had the following formulations:
17 A. Sodium Nitrite Gel (2 ml) Sodium Nitrite 6.6%
Hydroxymethylcellulose 10.0% Polyvinyl Pyrrolidone 5.0%
Polyethylene glycol 20.0% Benzyl Alcohol 1.0% Colour 0.005% Water
qs to 100% B. Citric Acid Gel (2 ml) Citric acid 10.0% Carbopol
5.0% Polyvinyl pyrrolidone 5.0% Polyethylene glycol 30.0% Methyl
paraben 0.2% Propyl paraben 0.02% Water qs to 100% Colour
0.005%
[0235] Formulations A and B were separately provided in resealable,
squeezable tubes for mixing either immediately before application-
to the nail, or on the nail itself.
* * * * *