Note: Descriptions are shown in the official language in which they were submitted.
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Use of Copper Silicate for the Control of Herpes Infections
Field of the Invention
The present invention relates to the use of copper silicate for treating. and
preventing Herpes virus (HV) infections and diseases and in particular
diseases
caused by HSV-1 or HSV-2 such as herpes and Varicella zoster (VZV) such as
shingles. The present invention also relates to copper silicate compositions
specifically adapted to treat HV infections.
Background Art
Herpes simplex virus (HSV), Herpesviridae Simplexvirus, enters the host by
direct
contact, is spread to a target tissue only, spreads within the host via
neuronal
axonal flow, targets the dorsal root ganglia and after recovery of the host
from an
acute infection, remains latent in the targeted tissue.
HSV disrupts host cell molecular functions and host cellular structure and is
manifested clinically as host cellular death, resulting in shallow, painful
vesicular
ectodermal lesions or by hemorrhagic encephalitic necrosis of the brain.
Target
tissues for HSV are the skin or mucous membranes usually derived from
embryonic ectoderm: mouth, skin, vagina, conjunctiva, cornea, etc. The virus
enters the host cell by direct mucosal contact or by direct contact of abraded
skin.
In the skin the virus- replicates in epithelial cells and then enters local
sensory
neurons. The virus travels to the dorsal root ganglia via retrograde axonal
flow
where it establishes permanent residency. There it establishes latency a state
in
which the viral lytic genes are silenced and only the latency locus is
transcriptionally active. Although latent most of the time, it reactivates
intermittently, travels down the sensory nerve and causes vesicular eruptions
at
or near the site of initial invasion. Alternatively the virus may invade the
CNS and
cause encephalitis.
The rate of seropositivity to HSV varies widely from country to country: from
relatively low in Japan where Herpes simplex Type 1(HSV-1) seroprevalence for
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men and women has decreased from 75.3 and 80.6% in 1973 to 54.4 and 59.6%,
respectively in 1993 and where Herpes simplex Type 2 (HSV-2) seroprevalence
has decreased from 10.2 and 9.9% in 1973 to 1.8 and 1.2%, respectively in
1993,
to quite high in Africa where all adult study groups have a high HSV-1
seroprevalence of >80%. HSV infects more than 50% of the adult population, but
some infections may be unrecognized. About half of these develop clinical
manifestations of the disease. It's most significant manifestations are
keratitis,
genital lesions and labial vesicular lesions ("cold sores").
A common manifestation of HSV-1 infection is cold sores. However, HSV-1 also
causes herpes keratitis (cornea). This disease is identified by a typically
bizarre
dendritic-patterned corneal ulcer that tends to be recurrent and very often -
leads to
scarring with a reduction of vision, sometimes to the level of legal
blindness.
HSV-1 also causes herpes labialis, peri-orbital, peri-oral, peri-nasal skin
eruptions
and, in older patients, the 'virus has been associated with herpes zoster
("shingles") infection of the upper trunk.
HSV-2 causes the most prevalent sexually transmitted disease in the United
States and visits to physicians for genital herpes simplex virus infection
continue
to increase. As many as 30 million Americans are infected with HSV-2. About
half of these carriers are symptomatic. The clinical manifestations range from
mild genital inflammation to severe, very painful, vesicular lesions and
ulceration.
Systemic involvement in the most severe cases may include hepatitis. Brain
damage and death often are the result of HSV-2 acquired by a newborn infant as
it passes through an infected birth canal. HSV-2 can also cause cold sores but
this is relatively uncommon.
Aciclovir is used to treat HSV-1 and HSV-2 as well as other virus infections.
The
mode of action appears to be interference with viral DNA polymerase resulting
in
premature termination of the DNA chain and a reduction of viral replication.
However, this agent is relatively expensive and there have been concerns of
general cytotoxicity and the rapid, irreversible development of resistant
viral
strains. Furthermore, the topical version of Aciclovir is poorly absorbed
dermally.
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Varicella-zoster virus (VZV) is spread by the respiratory route and
disseminates
to lymph nodes and then via lymphocytes back to the skin, resulting in the
rash of
chickenpox. Like HSV, VZV infects the neurones of the dorsal root ganglia,
where it causes lifelong latency. However, VZV reactivates much less often
than
HSV - in association with waning T-cell immunity - usually causing only one
lifetime episode of herpes zoster (shingies). Nevertheless, VZV causes more
severe damage to the nerve and dorsal root ganglia than HSV, leading to pain
and often neural dysfunction. Prolonged pain may result from scarring of
neural
tissue.
Most people in developed countries are infected with VZV in childhood, with
90%
seropositive by adulthood. Herpes zoster can develop at any age, but the
highest
incidence is after 60 years. Overall, it occurs in 20% of the population, with
more
than one recurrence in 4%.
The present invention seeks to overcome the above problems by providing a
safe, relatively inexpensive and effective treatment for viral skin diseases
such as
those caused by HSV and VZV.
Suonrnary of the Invention
The present invention provides for the use of copper silicate to control
Herpes
virus (HV).
The anti-HV activity of copper silicate renders it useful for treating HV
infections.
Thus, the present invention also provides a method of treating a HV infection
in a
subject, the method comprising the step of administering to said subject an
effective amount of copper silicate.
The present invention further provides for the use of copper silicate for
preparing
a medicament for treating the clinical manifestations of HV infections such as
cold
sores (predominantly caused by HSV-1) or the sores associated with genital
herpes (predominantly caused by HSV-2).
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The present invention still further provides a method of treating or
preventing HV
infection in a subject, the method comprising the step of repeatedly topically
administering an effective amount of copper silicate.
The copper silicate may be used as part of combination therapy. Thus, the
present invention also provides a method of treating or preventing HV
infection in
a subject, the method comprising the step(s) of topically administering an
effective amount of copper silicate and (ii) administering to said subject an
effective amount of at least one other anti-viral agent or an effective amount
of at
least one anaesthetic.
The present invention also provides for the use of copper silicate for
preparing a
medicament for treating or preventing HV infection or a disease or disorder
caused by HV.
The present invention further provides a composition adapted for topical
administration to a site of HV infection comprising an anti-viral . effective
amount of
copper silicate.
Brief Description of the Figures
Figure 1 is a graph comparing the anti-viral activity of the first copper
silicate
formulation and a copper sulfate solution;
Figure 2 is a graph depicting the anti-viral activity of different
concentrations of the
first copper silicate formulation over time;
Figure 3 is a graph comparing the anti-viral activity of the first copper
silicate
formulation and a copper sulfate formulation;
Figure 4 is a graph depicting the anti-viral activity of a second copper
silicate
formulation;
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Figure 5 is a graph depicting the anti-viral activity of a copper silicate
formulation
according to one embodiment of the present invention against HSV1 in
comparison to copper sulfate (Kill rate of 100% = no plaques observed);
Figure 6 is a graph depicting the anti-viral activity of a copper silicate
formulation
according to one embodiment of the present invention against HSV2 in
comparison to copper sulfate (Kill rate of 100% = no plaques observed);
Figure 7 is a graph depicting the anti-viral activity of a copper silicate
formulation
according to one embodiment of the present invention against HSV2 ACR in
comparison to copper sulfate (Kill rate of 100% = no plaques observed); and
Figure 8 is a graph depicting the cell toxicity of a copper silicate
formulation
according to one embodiment of the present invention in comparison to copper
sulfate. Viability in % was assessed after 30 minute exposure to Cu (750-
0.00125
ppm).
Detailed Description of the Invention
Use of copper silicate as an anti-viral
The present invention provides for the use of copper silicate to control
herpes
virus (HV).
For the purposes of the present invention, the phrase "controlling herpes
virus"
and similar phrases such as "control herpes virus" means one or more. of the
following: at least reducing the number of viable viruses; at least reducing
viral
replication; and at least reducing the clinical manifestations of HV infection
such
as reducing the number or size of lesions, reducing the pain associated with a
clinical outbreak and/or reducing the healing time associated with the lesion.
For the purposes of the present invention HV includes HSV such as Herpes
simplex virus 1(HSV-9 ), Herpes simplex virus 2 (HSV-2), Varicella zoster
virus
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(VZV) and closely related viruses that cause similar infections/diseases in
humans such as other members of the Alphaherpesvirinae subfamily.
The present invention also provides a method of treating a HV infection in a
subject, the method comprising the step of administering to said subject an
effective amount of copper silicate.
For the purposes of the present invention "HV infection" includes (i) clinical
manifestations of HV infection in epithelial - derived tissues (mucous
membrane
or dermal origin) ; and (ii) sites where it is apparent that a clinical
manifestation is
likely to appear. Target tissues for HV are the skin or mucous membranes
usually derived from embryonic ectoderm: mouth, skin, vagina, conjunctiva and
cornea. Clinical manifestations include lesions e.g. coid sores and the
lesions
associated with genital herpes.
Particular disorders associated with HSV-1 infection that can- be treated
according
to the present invention include: gingivostomatitis, genital and other
cutaneous
lesions, herpes keratitis (cornea), herpes labialis, peri-orbital, peri-oral,
peri-nasal
skin eruptions, keratoconjunctivitis, retinitis, oesophagitis, pneumonitis,
hepatitis,
meningitis, adult and neonatal encephalitis, myelitis, erythema multiforme.
HSV-2
infection is associated with genital herpes with clinical manifestations
ranging
from mild genital inflammation to vesicular lesions and ulceration, genital
and
other cutaneous lesions, oesophagitis, pneumonitis, hepatitis, meningitis,
adult
and neonatal encephalitis, myelitis, erythema multiforme. Herpes varicella
zoster
virus is associated with chicken pox and shingles.
The copper silicate is preferably applied at a site of visible HV infection.
For
example, where a lesion exists or where it is apparent that a lesion will soon
appear. In this regard, prior to the appearance of a cold sore, there may be
localized reddening, tingling, itchiness and/or swelling that is indicative of
HV
infection. Thus, the present invention-also provides for the use of copper
silicate
for preparing a medicament for treating the clinical manifestations of HV
infections such as cold sores (predominantly caused by HSV-1) or the sores
associated with genital herpes (predominantly caused by HSV-2).
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Surprisingly, it has been found that the copper silicate has superior anti-
viral
activity against HSV-1 relative to other copper salts such as copper sulfate.
It is
also expected that the copper silicate products will have greater efficacy
relative
to other anti-virals including topicals for treating cold sores.
The copper silicate may be administered in a variety of ways but is preferably
topically administered.
For the purposes of the present invention, the term "topical" and variants
such as
"topically" means application to a localized area of the body and/or to the
surface
of a body part and includes administration to the lips or mouth area, vagina
(such
as intra-vaginally) and surrounding areas, penis and surrounding areas and to
mucous membranes.
Topical administration may be achieved in any one of a number of ways.
Preferably, the copper silicate is administered by painting, wiping, rubbing,
dabbing or spraying. Alternatively, topical administration may be achieved by
applying the copper silicate using a means for dispensing an effective amount
of
copper silicate. Dispensing means can be varied and include slow release
carriers and materials impregnated with copper silicate such as plasters,
patches,
bandages, cotton wool and gauze. Preferably, the dispensing means is adapted
to deliver the copper silicate over a predetermined time and/or at a
predetermined
dose.
The amount of copper silicate administered in the method of the present
invention
will be sufficient to effectively treat the infection and thus will
necessarily vary
depending at least on the severity, type and location of the infection, the
strength
of the copper silicate and the manner in which individual patients respond to
the
treatment. Preferably, a copper silicate composition containing about 0.1 -10%
wt copper is administered. The precise dose administered for a particular
disorder may be determined by suitable qualified medical practitioner.
Similarly, the frequency with which, and the duration for which, the copper
silicate
is applied will be sufficient to effectively treat or prevent the infection
and thus will
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also vary depending at least on the severity and type of infection, the
strength of
the composition and the manner in which individual patients respond to the
treatment. Preferably, the copper silicate is applied at least one-, two,
three or up
to 5-10 times a day for at least 1 to 6 days or 1, 3, 6 or 12 weeks.
Alternatively, it
- may be applied once every 2 - 14 days for as long as necessary to treat or
prevent infection. The dosage regime for treating a particular disorder may be
determined by suitable qualified medical practitioner.
Thus, the present invention also provides a method of treating or preventing
HV
infection in a subject, the method comprising the step of repeatedly topically
administering an effective amount of copper silicate.
In some situations, it may be desirable to combine the copper silicate with
other
agents such as other anti-virals. Thus, the present invention also provides a
method of treating or preventing HV infection in a subject, the method
comprising
the step(s) of topically administering an effective amount of copper silicate
and (ii)
administering to said subject an effective amount of at least one other anti-
viral
agent.
The other anti-viral agent may be varied and include systemic or topical
agents or
an agent selected from the list comprising: Aciclovir, Valaciclovir,
Penciclovir,
Famciclovir, Ganciclovir.
The copper silicate may also be combined with anaesthetics. Thus, the present
invention also provides a method of treating or preventing HV infection in a
subject, the method comprising the step(s) of topically administering an
effective
amount of copper silicate and (ii) administering to said subject an effective
amount of at least one anaesthetic.
Preferably, the anaesthetic is a local anaesthetic. Even more preferably the
local
anaesthetic is of the ester type such as cocaine, procaine, tetracaine
(amethocaine) and chloroprocaine, or the amide class such as lidocaine,
prilocaine, mepivacaine, ropivacaine, levobupivacaine and bupivacaine.
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The present invention also provides for the use of copper silicate for
preparing a
medicament for treating or preventing HV infection or a disease or disorder
caused by HV.
Topical Formulations
The present ' invention also provides a composition adapted for topical
administration to a site of HV infection comprising an anti-viral effective
amount of
copper silicate.
The form of the topical composition of the present invention may also be
varied
provided it retains its anti-viral properties. Preferably, the composition is
a
solution. However, the composition may also be in solid form provided it is
properly formulated. In this regard, the composition could comprise copper
silicate in the form of a micronized solid.
When the copper silicate is provided in the form of a solution, it preferably
is
provided as an aqueous acidified solution. Acidified solutions are
particularly
preferred because copper silicate is more soluble and stable at acidic pH.
Particularly preferred pHs are 3-6, 4-6 and 5-6. In one example, the copper
silicate is prepared according to the methods described and claimed in US
patent
5,474,972.
The composition adapted for topical administration may be in the form of any
one
of the following: solution, lotion, suspension, emulsion, cream, gel,
ointment,
liniment and salve. Particularly preferred forms are ointments, creams or gels
as
these are more amenable to administration at the sites of HV infection.
Ointments generally are prepared using either (1) an oleaginous base, i.e.,
one
consisting of fixed oils or hydrocarbons, such as white petroleum or mineral
oil, or
(2) an absorbent base, i.e., one consisting of an anhydrous substance or
substances that can absorb water, for example anhydrous lanolin. Customarily,
following formation of the base, whether oleaginous or absorbent, the active
ingredient is added to an amount affording the desired concentration.
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Creams are oil/water emulsions. They consist of an oil phase (internal phase),
comprising typically fixed oils, hydrocarbons and the like, waxes, petroleum,
mineral oil and the like and an aqueous phase (continuous phase), comprising
water and any water-soluble substances, such as added salts. The two phases
are stabilised by use of an emulsifying agent, for example, a surface active
agent,
such as sodium lauryl sulfate; hydrophilic colloids, such as acacia colloidal
clays,
veegum and the like. For the purposes of the present invention, the copper
silicate may be added to the water phase prior to formation of the emulsion,
in an
amount to achieve the desired concentration.
Gels comprise a base selected from an oleaginous base, water,, or an emulsion-
suspension base. To the base is added a gelling agent that forms a matrix in
the
base, increasing its viscosity. Examples of gelling agents are hydroxypropyl
cellulose, acrylic acid polymers and the like. For the purposes of the present
invention the copper silicate may be added to the formulation at the desired
concentration at a point preceding addition of the gelling agent.
Excipients that can be incorporated into the gel, ointments and lotions of the
present invention include: isopropyl myristate NF, trolamine NF, SD alcohol 40
(20%), white petrolatum USP, lanolin alcohols NF, mineral oil USP, polyvinyl
alcohol gel, cetostearyl alcohol NF, lactic acid USP, sodium hydroxide USP,
polysorbate 60 USNF, Cetyl alcohol USP, Mono- & Di- glycerides USNF, titanium
dioxide calcium stearate, dextran, polyoxyl 40 stearate, methylparaben,
propylene
glycol, sodium lauryl sulfate, polyethylene glycol (PEG) base, synthetic
beeswax
(B wax), calcium acetate, purified water USP and similar products. These
excipients serve a variety of functions as carriers, vehicles, diluents,
binders,
preservatives, buffers, pH adjusters, emulsifiers and other formulating aids.
Dosage forms of the invention for use in treating female genital
manifestations of
HSV infections, especially HSV-2, are prepared in dosage forms for vaginal
insertion including vaginal suppositories, gels, creams and tablets. These
preparations optionally may include one or more of the following suitable and
pharmaceutically acceptable excipients, including but not limited to:
isopropyl
myristate NF, mineral oil USP, stearyl alcohol NF, benzoic acid USP, pegoxyl 7
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stearate, methylparaben, propylparaben, propylene glycol, butylated
hydroxyanisole, coconut or palm kernel oil triglycerides, polysorbate 60 or
polysorbate 8, peglicol 5, PEG-100 stearate and sorbitan monostearate, calcium
lactate, hydroxypropyl methylcellulose, polysaccharide carrageenan, corn
starch,
lactose, calcium lactate, silicon dioxide and purified Water USP.
The topical formulations of the present invention may also be adapted to be
delivered to the eye(s) of a subject. In this regard, the eye drop dosage form
of
the invention will optionally include one or more suitable and
pharmaceutically
acceptable inactive excipients, including but not limited to: preservatives
from a
group including benzalkonium chloride, methylparaben, edetate disodium,
thimersol, chlorbutanol; buffers from a group including sodium citrate,
potassium
chloride, magnesium chloride, sodium acetate, citric acid, sodium lactate;
vehicles
from a group including polyvinyl alcohol, hydroxy methylcellulose, cetyl
alcohol,
carboxymethylcellulose, hydroxy-propylenemethyl cellulose; pH adjusters from a
group including sulfuric acid, hydrochloric acid, sodium hydroxide, monosodium
or
disodium phosphate; purified water USP; poloxamer 407 or 188, polysorbate 80;
polyoxyethylene polyoxypropylene compound; mineral oil USP. .
The compositions of the present invention may be produced by dissolving or
combining the copper silicate in an aqueous or non-aqueous carrier. In
general,
any liquid, cream, or gel, or similar substance that does not appreciably
react with
the copper silicate or any other active ingredient that may be introduced and
which is non-irritating is suitable.
The composition of the present invention may further comprise an auxiliary
agent
such as any one or more of: preservatives, stabilizers, emulsifiers, wetting
agents,
fragrances, colouring agents, odour controllers and thickeners such as natural
gums. However, it has been found that the copper silicate compositions of the
present invention do not require the addition of preservatives to maintain an
acceptable shelf-life.
The concentration of the copper in the composition may be varied depending at
least on the severity and type of infection that the composition is to be used
to
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treat or prevent. However, preferably, the concentration of the copper is
approximately 0.01 - 10%. More preferably, the copper concentration is to a
final
concentration of approximately 0.1 - 5 % or 0.2 - 3%. In one particular form,
the
concentration of copper is to a final concentration of about 0.22%.
General
Those skilled in the art will appreciate that the invention described herein
is
susceptible to variations and modifications other than those specifically
described. It is to be understood that the invention includes all such
variations
and modifications. The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in the specification,
individually or collectively and any and all combinations or any two or more
of the
steps or features.
The present invention is not to be limited in scope by the specific
embodiments
described herein, which are intended for the purpose of exemplification only.
Functionally equivalent products, compositions and methods are clearly within
the
scope of the invention as described herein.
The entire disclosures of all publications (including patents, patent
applications,
journal articles, laboratory manuals, books, or other documents) cited herein
are
hereby incorporated by reference. No admission is made that any of the
references constitute prior art or are part of the common general knowledge of
those working in the field to which this invention relates.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood
to imply the inclusion of a stated integer or group of integers but not the
exclusion
of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the
detailed
description of the invention and apply throughout. Unless otherwise defined,
all
other scientific and technical terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which the invention
belongs.
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The present invention will now be described with reference to the following
examples. The description of the examples is in no way to limit the generality
of
the preceding description.
Examples
General Materials/Methods
Copper silicate is made in situ by a combination of copper sulfate, and sodium
silicate in an acidic solution. This solution can then be formulated further
to
solutions, creams, gels, etc as appropriate. As a result of the "in situ"
production
of the copper silicate, the contents of the *copper silicate formulations
listed here
do not specifically contain "copper silicate" as one of the starting
ingredients.
Nonetheless, the copper silicate is present due to the reaction of the copper
sulfate and the sodium silicate under the specified conditions.
Copper Silicate Solution (Formulation A)
' In redient %wt
Deionised Water 96.68
Copper sulfate pentah drate ' 1.14
Acetic acid (90%) 0.93
Sodium silicate solution 1.20
Sodium Alkyl ether sulfate 0.05
Note: When used in the examples Formulation A was applied at pH 7 conditions
Copper Silicate Lotion (Formulation B)
Ingredient %wt
Deionised Water 74.16
Copper sulfate pentah drate 0.87
Acetic acid (90%) 0.72
Sodium silicate solution 0.92
Triethanolamine 1.10
Carbopol Ultrez 10 1.89
Glycerine 9.97
Alcohol 6.98
Sodium hydroxide solution 18% 3.39
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Copper Sulfate / Acetate Solution (Formulation C)
Ingredient %wt
Deionised Water 97.93
Copper sulfate pentahydrate 1.14
Acetic acid (90%) 0.93
Copper Silicate Cream (Formulation D)
This cream formulation is made in two steps, first by production of a
concentrated
copper silicate solution (I) that is then immediately on formulated into the
final
copper silicate cream.
Concentrated Copper Silicate Solution (I)
Material Unit Formula
(% by wt)
Sodium silicate solution 4.60
Purified Water 87.81
Copper sulfate pentahydrate 4.35
Acetic acid 3.24
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Copper Silicate Cream (Formulation D)
Material Quantity
(% w/w)
Watei Phase
Concentrated Copper silicate solution 20.00
Purified water 41.20
Propylene glycol 9.00
Sodium hydroxide q.s.
Oil Plzase
Polysorbate 60 7.40
Cetyl alcohol 7.40
Mono- & Di- Glycerides 7.40
Polyoxyl 40 stearate 7.10
Titanium dioxide 0.50
Example 1- Anti-viral activity of copper silicate (Formulation A) compared
to copper sulfate
Materials/Methods
HSV-1 was exposed, separately to Formulation A and a copper sulfate solution
(each over a range of 175 - 1400 ppm Cu) for 30 min. The HSV/copper mixtures
were diluted and layered onto the cell monolayer. After 1 hour the mixture was
washed away and the cells incubated to allow plaques to form. Plaque numbers
were reported as a percentage of the control:
Results
Copper silicate Formulation A began to kill HSV-1 at 600 ppm Cu and at 700 ppm
the kill rate was >90% (Figure 1). By comparison, under these conditions,
copper
sulfate was still ineffective at 1400 ppm Cu (Figure 1).
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Example 2. Anti-viral activity of copper silicate (Formulation A) assessed by
exposure time.
Methods and Materials
The clinical isolate of HSV 1 was incubated with copper silicate (Formulation
A)
dilutions in the same way as example 1. After 30 min, 60 min and 120 min the
mixture was washed away and the cells incubated to allow plaques to form.
Plaque numbers were reported as a percentage of the control.
Results
Using copper silicate, when the exposure times were increased to 60 min,
maximum kills of 99.9% were achieved with 700 ppm Cu and 99.999% with 800
ppm Cu (Figure 2).
Example 3 - Comparison of anti-viral activity of two copper formulations
Materials/Methods .
A formulation similar to Formulation A but without the silicate component
(Formulation C - effectively a mixture of copper sulfate and acetic acid in
solution) was made and tested against Formuiation A. The concentration used in
the testing of both formulations was 700ppm Cu and the exposure times were 0-
120 min in 30 min intervals.
Results
As the exposure time increased the percentage of virus particles killed by
both
formulations increased (Figure 3). The average percentage kill for Formulation
A
at 30 min is 80% compared to 50% for Formulation C. By 60 min copper silicate
Formulation A achieved 99% kill, while Formulation C achieved 90% kill (Figure
3). At 120 min exposure Formulation A achieved >99.99% kill, which is a 2 log
greater percentage kill than that seen for Formulation C (Figure 3).
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Example 4- Anti-viral activity of copper silicate (Formulation B)
Materials/Methods
HSV-1 was exposed to Formulation B at various copper concentrations for 60 min
in a similar protocol to that used for Example 1.
Results
A concentration of 1980 ppm Cu achieved a 99.9% kill (Figure 4). At 1100 ppm
Cu and an exposure time of 60 min a 90% kill was achieved (Figure 4). This
increased to 99.99% kill with 120 min exposure time.
Example 5 - in vitro comparison of copper silicate and copper sulfate
Materials/Methods
(i) Preparation of Vero Cells
Vero cells were grown to passage 25 and frozen with DMSO (dimethylsulfoxide).
Reserve stocks were frozen at passage 24 to allow for extra stocks to be
rapidly
grown if needed. The cells were frozen and. random samples were tested for
viability. The stocks were shown to have good viability. The cells were used
for
the plaque assay at passage 27.
HSV1 and HSV2 as well as acyclovir resistant HSV2 (HSV2 ACR) were obtained
from ECACC (European Collection of Cell Cultures) and grown up to produce 50
mL of each virus in a cell free medium. The final concentration of the virus
in the
initial stock aliquots were as follows:
1.6 x 10' pfu/mL for HSV1
6.1 x 105 pfu/mL for HSV2
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2.7 x 105 pfu/mL for HSV2 ACR (stock 2: 6 x 105 pfu/mL for HSV2 ACR)
(ii) Copper solutions
1. Copper silicate solution (- 0.28% w/w as Cu)
Ingredient % b wt
Sodium silicate solution 1.15
Water 96.95
Copper sulfate pentah drate 1.09
Acetic acid 0.81
2. Copper sulfate solution 0.28% w/w as Cu)
Ingredient % b wt
Copper sulfate pentahydrate 1.09
Water 98.91
Solutions of copper silicate and copper sulfate were supplied at a
concentration of
2,800 ppm copper and a pH of approximately 4. The copper solutions required
dilution to a maximum working concentration of 1,000 ppm and neutralisation of
the acidity. Dilution and neutralisation were achieved by using Tris
(hydroxymethyl) buffer (0.012g/L) and 0.425M NaOH. Neutralization produced
distinct precipitates for copper silicate and copper sulfate, which were kept
in
suspension for the treatment of virus.
(iii) Plaque Assay (method)
The viral particles for HSV1, HSV2, and HSV2ACR were thawed and mixed at
appropriate pfu/ml with the copper solutions at final copper concentrations of
750, 400, 200 100, 50, 25, and 0 ppm of copper to produce between 10 and 250
pfu per 24 well plate after dilution. The copper solutions and the viruses
were
incubated together for 30 minutes. The copper treated HSV was then diluted
1:500 to avoid toxicity on the Vero cells and plated on 24 well plates at
concentrations between 10 to 250 pfu/well. The 24 well plates contained a
monolayer of Vero cells in a supporting media. The resulting 500 fold dilution
of
the copper solutions took the concentration of copper down to 1.5, 0.8, 0.4,
0.2,
0.1, 0.05 and 0 ppm in 24 well plates.
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Dimethyl cellulose (DMC) was added to the wells to help identify plaque
formation. The plates were then incubated for 3 days to allow HSV plaque
formation. Following incubation the fluid over the Vero cells was washed off
and
the Vero cells stained with crystal violet in 4% paraformaidehyde to
inactivate the
virus and identify defects in the Vero cell monolayer caused by focal HSV
replication (virus plaque). Plaques were counted with 5 fold digital
magnification
of each well after scanning of stained 24 well plates. There were 7 solutions
for
each viral strain and each solution was duplicated 4 times on 24 well plates.
Each plaque assay was repeated at least 8 times.
(iv) Cell toxicity (Method)
The cytotoxic effect of copper compounds was tested on Vero cells prior to
plaque reduction assays using the CeIlTiter-BIueTM (Promega) Cell viability
assay,
followed by colorimetric analysis in 96 well plates.
Toxicity of high concentration Cu solutions was tested in 2 independent
experiments. Cu treated virus solutions were tested for cytotoxicity in 96
well
plates in parallel to each plaque assay.
Results
(i) Plaque Assays
Antiviral activity against HSV1 of soluble copper silicate became evident at
25
ppm Cu with a kill rate of 48% and at 400 ppm the kill rate was 100 % (Figure
5)
following an exposure time of 30 minutes. By comparison, under these
conditions, the kill rate of copper sulfate was only 9% at 25 ppm Cu and only
achieved a kill rate of 100% at 750 ppm Cu.
Antiviral activity against HSV2 of soluble copper silicate became evident at
25
ppm Cu with a kill rate of 57% and at 400 ppm the kill rate was 100 % (Figure
6)
following an exposure time of 30 minutes. By comparison, under these
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conditions, the kill rate of copper sulfate was only 35% at 25 ppm Cu and
copper
sulfate did NOT achieve a kill rate of 100% at the Cu concentrations tested.
Antiviral activity against HSV2 ACR of soluble copper silicate became evident
at
25 ppm Cu with a high kill rate of 84% and at 400 ppm the kill rate was 100 %
(Figure 7) following an exposure time of 30 minutes. However, by comparison,
under these conditions, the kill rate of copper sulphate was almost identical
with
82% at 25 ppm Cu. Copper sulphate did achieve a kill rate of 100% at 750 ppm
Cu.
Exposure times up to 3 days lead to 100% HSV virus kill rates at Cu
concentrations of 25 ppm and lower (results not shown).
(ii) Cell Toxicity
Toxicity of soluble copper silicate became evident (less than 100% cell
vialbility)
at 0.0125 ppm Cu (Figure 8) following an exposure time of 30 minutes. Under
these conditions, copper sulphate was toxic at 0.00125 ppm Cu and more toxic
than copper silicate throughout.
Cu toxicity interfering with plaque assays (less than 50% cell viability)
could be
observed at Cu concentrations down to 1.5 ppm (750 ppm 1:500 diluted) after 3
days of exposure (= duration of the plaque assay). 1:500 diluted Cu solutions
prior to plaque assay showed minimal cytotoxicity allowing for read-out of
plaque
assays at concentrations of 2 to 1.5 ppm (1000 ppm to 750 ppm Cu-virus
solutions).
