U.S. patent application number 10/084518 was filed with the patent office on 2002-12-05 for super-oxidized water, preparation and use thereof as sterilizers and medicaments.
This patent application is currently assigned to Sterilox Medical (Europe) Limited. Invention is credited to Selkon, Joe B..
Application Number | 20020182262 10/084518 |
Document ID | / |
Family ID | 10859664 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020182262 |
Kind Code |
A1 |
Selkon, Joe B. |
December 5, 2002 |
Super-oxidized water, preparation and use thereof as sterilizers
and medicaments
Abstract
Super-oxidized water based on hypochlorous acid, such as is
obtained by the electrochemical treatment of a saline solution, may
be used in the treatment of leg ulcers or other open wounds.
Preferably, the pH of the super-oxidized water is in a range of 4
to 7, and the water has a redox potential of >950 mV.
Medicaments based on the super-oxidized water may be in liquid or
gel form. The super-oxidized water is able to control the microbial
population within the wound and at the same time permit cell
proliferation.
Inventors: |
Selkon, Joe B.; (Oxford,
GB) |
Correspondence
Address: |
AKIN, GUMP, STRAUSS, HAUER & FELD, L.L.P.
ONE COMMERCE SQUARE
2005 MARKET STREET, SUITE 2200
PHILADELPHIA
PA
19103
US
|
Assignee: |
Sterilox Medical (Europe)
Limited
|
Family ID: |
10859664 |
Appl. No.: |
10/084518 |
Filed: |
February 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10084518 |
Feb 25, 2002 |
|
|
|
PCT/GB00/03264 |
Aug 23, 2000 |
|
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Current U.S.
Class: |
424/600 |
Current CPC
Class: |
A61K 33/40 20130101;
C02F 2303/04 20130101; C02F 1/4674 20130101; C02F 2103/026
20130101; C02F 2209/06 20130101; A61K 33/00 20130101; C02F 2209/04
20130101; Y10S 514/887 20130101; A61P 17/02 20180101; Y10S 514/944
20130101; Y10S 514/886 20130101; Y10S 514/925 20130101; A61P 31/04
20180101 |
Class at
Publication: |
424/600 |
International
Class: |
A61K 033/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 23, 1999 |
GB |
GB 9919951.5 |
Claims
I claim:
1. A medicament for the treatment of open wounds, the medicament
comprising super-oxidized water based on hypochlorous acid.
2. The medicament according to claim 1, wherein the super-oxidized
water is in liquid form for application to the wound by bathing or
spraying.
3. The medicament according to claim 1, wherein the super-oxidized
water is in gel form for topical application to the wound.
4. The medicament according to claim 1, wherein the super-oxidized
water or a preparation derived therefrom has a pH of 4 to 7.
5. The medicament according to claim 4, wherein the pH is in a
range of about 4.0 to 6.5.
6. The medicament according to claim 5, wherein the pH is in a
range of about 4.0 to 6.2.
7. The medicament according to claim 6, wherein the pH is in a
range of about 4.3 to 6.2.
8. The medicament according to claim 7, wherein the pH is about
5.4.
9. The medicament according to claim 1, wherein the super-oxidized
water has a redox potential of >950 mV.
10. The medicament according to claim 9, wherein the super-oxidized
water has a redox potential of about 1000 mV.
11. The medicament according to claim 1, wherein the super-oxidized
water or a preparation derived therefrom has a biocide rate (D
Value) of approximately 1 log reduction unit of bacillus subtilis
spores in less than 1 minute with a 9:1 super-oxidized
water:innoculum mix.
12. The medicament according to claim 1, wherein the super-oxidized
water is diluted to an extent that it does not inhibit cell
proliferation in use.
13. The medicament according to claim 12, wherein the
super-oxidized water is diluted to an extent that it promotes cell
proliferation in use.
14. The medicament according to claim 1, wherein the super-oxidized
water comprises an output solution obtained by electrochemical
treatment of a saline solution.
15. The medicament according to claim 14, wherein the
super-oxidized water has a pH adjusted to a desired level by using
an alkaline solution output from an electrochemical cell in which
the saline solution is treated.
16. The medicament according to claim 14, wherein the output
solution further comprises a phosphate buffer to adjust a pH of the
solution to a desired level.
17. A super-oxidized water based on hypochlorous acid for use in
treatment of a human or animal body.
18. The super-oxidized water according to claim 17 having a pH of 4
to 7.
19. The super-oxidized water according to claim 18 having a pH in a
range of about 4.0 to 6.5.
20. The super-oxidized water according to claim 19 having a pH in a
range of about 4.0 to 6.2.
21. The super-oxidized water according to claim 20 having a pH in a
range of about 4.3 to 6.2.
22. The super-oxidized water according to claim 21 having a pH of
about 5.4.
23. The super-oxidized water according to claim 17, having a redox
potential of >950 mV.
24. The super-oxidized water according to claim 23 having a redox
potential of about 1000 mV.
25. The super-oxidized water according to claim 17, having a
biocide rate (D Value) of approximately 1 log unit reduction of
bacillus subtilis spores in less than 1 minute with a 9:1
super-oxidized water:innoculum mix.
26. A method for treatment of a human or animal body, comprising
administering to the human or animal body a super-oxidized water
based on hypochlorous acid.
27. The method according to claim 26, comprising administering
super-oxidized water as a medicament for treatment of leg ulcers or
other open wounds.
28. The method according to claim 26, comprising administering the
super-oxidized water as a medicament for controlling microbial
population and permitting cell growth.
29. The method according to claim 26, wherein the super-oxidized
water or a preparation derived therefrom has a pH of 4 to 7, a
redox potential of >950 mV, and a biocide rate (D Value) of
approximately 1 log unit reduction of bacillus subtilis spores in
less than 1 minute with a 9:1 super-oxidized water:innoculum
mi.
30. A method of preparing a medicament according to 1, comprising
passing a saline solution through an electrochemical cell having
electrodes separated by a semipermeable membrane, operating the
cell in such manner as to produce super-oxidized water based on
hypochlorous acid, and incorporating the super-oxidized water in a
carrier preparation at a sufficient concentration to impart
biocidal properties and allow cell proliferation when applied to a
human or animal body.
31. The method according to claim 30, further comprising
controlling a pH of the super-oxidized water generated in the cell
by a buffering action within the cell involving re-feeding alkaline
solution also generated in the cell.
32. A method of treating a leg ulcer or other open wound on a human
or animal body, comprising applying to the wound a medicament
according to claim 1.
33. The method according to claim 32, wherein the step of applying
the medicament comprises immersing the wound in a hydrobath
containing the medicament.
34. A hydrobath preparation for the treatment of leg ulcers or
other open wounds comprising super-oxidized water based on
hypochlorous acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/GB00/03264, filed Aug. 23, 2000, which was
published in the English language on Mar. 1, 2001, under
International Publication No. WO 01/13926 A2, and the disclosure of
which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to mixtures of oxidants which are
referred to in this specification as "super-oxidized water," a term
which is known in the art.
[0003] Super-oxidized water may be used as a sterilizing,
disinfecting and biocidal solution. One form of super-oxidized
water is produced by the applicant under the trademark
STERILOX.RTM.. This STERILOX super-oxidized water is generated at
the point of use, for example in a hospital, by passing saline
solution over coated titanium electrodes separated by a
semi-permeable ceramic membrane at a current of about 6 to 9 Amps.
An apparatus having coated titanium electrodes separated by a
semi-permeable ceramic membrane is disclosed in the specifications
of UK Patent Nos. 2253860 and 2274113. The basic structure of the
apparatus is disclosed in GB2253860 and can be used to produce the
STERILOX super-oxidized water.
[0004] STERILOX super-oxidized water contains a mixture of
oxidizing species, predominantly hypochlorous acid (HOCl) and
sodium hypochlorite. The STERILOX super-oxidized water has a pH of
5-7 and an oxidation reduction potential (redox) of around 1000 mV.
The high redox potential allows for the quick and efficient
destruction of microbes (bacteria, viruses, fungi and spores).
Hypochlorous acid and hypochlorite are in equilibrium and the
position of the equilibrium is determined solely by the pH.
[0005] Applicant has found that the resultant super-oxidized water
is non-hazardous, non-irritating and non-sensitizing to the skin,
non-irritating to the eyes, not harmful if swallowed and shows no
evidence of mutagenic activity.
[0006] It is considered that hypochlorous acid exerts its biocidal
effect by attacking the surface and plasma membrane proteins,
impairing transport of solutes and the salt balance of bacterial
cells (Pieterson et al, Water SA, 22(1): 43-48 (1996)). However, it
is believed that HOCl does not enter freely into eukaryotic cells,
which may explain the selectivity of hypochlorous solutions.
[0007] The STERILOX process produces an extremely effective
sterilizing, cold, non-toxic solution, which is free from highly
toxic chemicals and acts against a wide variety of bacteria, fungi,
viruses and spores. The generation of STERILOX solutions requires
only water, electricity and pure, vacuum-dried crystalline salt.
Applicant considers that the STERILOX super-oxidized water will be
suitable for a broad range of applications in both medical and
non-medical environments, such as the preservation of poultry and
fish and general agricultural and petrochemical uses, the breaking
down of bacterial biofilm, water treatment and general disinfection
in medical and veterinary applications. The STERILOX super-oxidized
water has been found to be particularly useful for the disinfection
of endoscopes which are sensitive to other cold disinfectants, such
as peracetic acid, which are commonly used.
[0008] While glutaraldehyde may be used as a reliable disinfecting
agent of flexible fiber-optic endoscopes and other heat-sensitive
instruments, although being widely practiced in many hospitals, its
use can cause asthma and dermatitis in healthcare staff as a result
of exposure to glutaraldehyde fumes, hence a predilection to the
use of peracetic acid and the relatively recent move towards the
use of STERILOX super-oxidized water in such applications.
[0009] STERILOX super-oxidized water has been tested and is the
subject of two scientific papers by Selkon et al, Journal of
Hospital Infection, 41: 59-70 (1999) and Shetty et al, Journal of
Hospital Infection, 41: 101-105(1999). In these studies, freshly
produced STERILOX super-oxidized water was found to be highly
active against Mycobacterium tuberculosis, Mycobacterium
avium-intracellulare, Mycobacterium chelonae, Escherichia coli
(including type 0157), Enterococcus faecalis, Pseudomonas
aeruginosa, Bacillus subtilis var niger spores,
methicillin-resistant Staphylococcus aureus, Candida albicans,
poliovirus type 2 and human immunodeficiency virus HIV-1.
[0010] There has been a recent upsurge in interest in the use of
super-oxidized water as a disinfectant, because of its rapid and
highly biocidal activity against a wide range of bacteria. Tanaka
et al, Journal of Hospital Infection. 34: 43-49 (1996), report the
electrolysis of a saline solution to produce a super-oxidized water
with a highly acidic pH of 2.3-2.7, which limits its suitability
for many applications, particularly the disinfection of endoscopes.
The acidic pH of the super-oxidized water produced by the method
described by Tanaka et al. also precludes its use in other medical
indications.
[0011] Having carried out trials in a large number of applications,
including those mentioned above, Applicant turned its attention to
the use of STERILOX super-oxidized water as a disinfectant of
mammalian tissue, in particular the treatment of open wounds such
as leg ulcers.
[0012] An article by Cherry in The Prescriber (May 1996) entitled
"GP guide to the care of patients with leg ulcers" states "leg
ulcers are notoriously difficult to treat successfully and can
seriously reduce the patient's quality of life." Indeed, according
to Cherry, "epidemiological studies have shown that at any given
time there are approximately 100,000 patients in the UK that have
leg ulceration. In treating these patients it has been estimated
that over .English Pound.39 million per year alone is spent on
materials used in their ulcer care."
[0013] There are two types of leg ulcers: arterial and venous.
Arterial ulcers, which are much harder to treat, are caused by
ischaemia, while venous ulcers are caused by blood stasis in the
veins.
[0014] There are many proposals for the management of ulcers, all
of which have varying degrees of success. Successful ulcer
management is very much dependent on the rigid adherence to a
program of treatment in combination with effective disinfection of
the wound, which reduces bacterial infection and promotes the
regeneration of dermal fibroblasts and keratinocytes in the bed of
the ulcer which are essential for healing of the wound and the
growth of new tissue. If the bacterial growth is not controlled,
the wound cannot heal.
[0015] The most useful treatment for venous ulcers is the use of
compression bandages together with elevation of the leg(s). This
mimics the pumping action of the calf muscles which return the
blood back to the body and maximizes the removal of blood from the
leg(s). In conjunction with this, other treatment strategies
include the use of topical treatments such as GRANUFLEX.RTM. to aid
granulation and skin repair, alginates to clean the wound of
debris, dry inert dressings to protect the wound (but which do not
promote healing), and bacteriostatic or bactericidal ointments to
reduce the infection. While antibiotics have been used to reduce
infection in the past, nowadays this is not a treatment of choice
due to the increased risk of antibiotic resistance.
[0016] While potassium permanganate (KMnO.sub.4) is an oxidant
which has stood the test of time in the treatment of leg ulcers, it
still nevertheless has the disadvantages of irritating and injuring
newly grown skin and causing skin discoloration. Known
hypochlorites, such as EUSOL (Edinburgh University Solution of
Lime) and Daikin's solution, rely on a high concentration of
hypochlorite ions for their disinfectant properties. In fact, these
compounds are no longer recommended for use due to their irritant
and painful effects and impairment of cell growth which outweigh
their therapeutic value, resulting in these preparations falling
out of use. Attempts have been made to reduce the alkaline effect
of the high hypochlorite ion content of these solutions, e.g. by
the use of suitable buffers, but have been found to be ineffective
in such circumstances.
[0017] All this has militated against the use of preparations
including hypochlorites for the treatment of leg ulcers. However,
the success in disinfection and sterilization of endoscopes and the
known non-irritant effects of the STERILOX super-oxidized water,
have led the Applicant to re-address the treatment of open wounds
such as leg ulcers.
BRIEF SUMMARY OF THE INVENTION
[0018] As a first step, in vitro tests were carried out on single
layers of cultured human dermal fibroblast cells and keratinocyte
cells to ascertain whether or not super-oxidized water had any
effect on the viability of the cells. The cells were incubated
under sterile conditions in a super-oxidized water based on
hypochlorous acid and including sodium hypochlorite and other
oxidized chlorine species, having a pH range from 4 to 7 and a
redox potential of around 1000 mV.
[0019] Surprisingly, Applicant found that there is an optimum pH at
which cell growth is not inhibited despite there being other pH
levels within the range at which the viability of the cells is
impaired. Indeed, in view of the findings of Tanaka et al,
Applicant expected that the lower pH range would be more
effective.
[0020] The next step was crucial, because the applicant then had to
ascertain whether or not the in vitro results could not only be
replicated in vivo but also that there would be a sufficient
biocidal effect to counteract any bacterial activity which would
prejudice the viability of new cells. Accordingly, a clinical trial
was carried out on a patient with chronic venous leg ulcers using
freshly produced super-oxidized water based on hypochlorous acid
having a pH of 5.4.
[0021] The patient did not experience any pain, and in fact
commented that the treatment was comfortable and had a soothing
effect. There was a positive effect on the bacterial flora as well
as the clinical appearance of the wounds. No adverse effect was
observed on the surrounding skin which, in a number of patients
with long-standing ulcers, is often sensitive.
[0022] Applicant believes that the effects which have been observed
can be explained by the low concentration of oxidized (free
available) chlorine present in a super-oxidized water based on
hypochlorous acid. This is in contrast to commonly known
hypochlorite solutions which owe their biocidal activity to a high
concentration of free chlorine (including hypochlorite) as
indicated by their characteristic smell.
[0023] It could be said that Applicant has discovered a principle,
which is that a balance between the biocidal effect and
non-inhibition of cell growth enables the microbial population
present in a wound to be controlled such as to allow cell growth to
occur.
[0024] In order to carry this principle into effect and from one
aspect of the invention, there is provided a super-oxidized water
which is based on hypochlorous acid, acts as a biocide and allows
cell growth. Expressed in another way, the present invention
resides in a super-oxidized water in which the biocidal effect is
due to hypochlorous acid and the non-inhibitory effect on cell
growth is dependent on the level of the pH of the hypochlorous acid
solution.
[0025] From a still further aspect of the invention, in a
super-oxidized water based on hypochlorous acid and having a pH of
4 to 7, the solution has a pH selected in a range of about 4.3 to
6.2.
[0026] In a further aspect of the invention, there is provided, for
use in the treatment of, or medicament for, skin ulcers or other
open wounds, a super-oxidized water or other formulation, such as a
gel, which is based on hypochlorous acid, acts as a biocide and
allows cell growth. The super-oxidized water or other preparation
based on hypochlorous acid may have a pH of 4 to 7, with the pH
preferably being selected in a range of about 4.0 to 6.5, and more
preferably in a range of about 4.0 to 6.2.
[0027] In any of the aspects of the invention defined above, the
super-oxidized water has a biocide rate (D Value) of approximately
1 log unit reduction unit of bacillus subtilis spores in less than
1 minute with a 9:1 super-oxidized water:innoculum mix. A biocide
rate of about 3.4 seconds may be achieved and is particularly
preferred.
[0028] The super-oxidized water based on hypochlorous acid may be
obtained by the electrolysis of a saline solution. Accordingly,
from another aspect of the invention there is provided a method of
obtaining a super-oxidized water based on hypochlorous acid, the
method comprising passing the saline solution through an
electrochemical cell having electrodes separated by a
semi-permeable membrane and operating in such manner as to produce
a super-oxidized water based on hypochlorous acid which acts as a
biocide and allows cell growth.
[0029] In a preferred method of carrying out the invention, the
super-oxidized water is obtained and the pH level of the solution
is adjusted by a buffering action, which involves the use of an
alkaline feed from the cell. By using an alkaline solution in this
way, the addition of conventional buffers and the consequent
expense is avoided.
[0030] While the pH of the super-oxidized water is preferably
adjusted to be in a range of about 4.3 to 6.5, Applicant has found
that, in the case of wounds, such as leg ulcers, it is advantageous
if the pH is adjusted to about 5.4.
[0031] From a still further aspect, the invention resides in a
method of producing a medicament containing super-oxidized water as
defined above for use in the treatment of leg ulcers or other open
wounds.
[0032] The invention also comprehends, in a super-oxidized water or
other formulation having a pH of 4 to 7, the selection of a pH in a
range of about 4.3 to 6.5, and preferably about 5.4, that is used
for the treatment of leg ulcers or other open wounds.
[0033] By means of any of the aspects of the invention defined
above, the disinfection of wounds such as leg ulcers is readily
achieved without irritation and pain, the spread of infection is
reduced, and cell growth and regeneration are facilitated, thereby
enhancing healing. An added advantage is that there is no
resistance or tolerance developed to disinfection which would occur
with antibiotics.
[0034] Furthermore, the super-oxidized water of the invention lends
itself readily to the treatment being carried out in many ways,
although it has been found that a hydrobath in which the leg is
immersed is soothing and generally pleasant for the patient.
However, it should be appreciated that preparations other than
solutions may be used, for example gels.
[0035] From yet another aspect, the invention also resides in a
method of treating a human or animal body having a leg ulcer or
other open wound using any of the super-oxidized water based on
hypochlorous acid herein referred to above and any of the methods
defined hereinabove.
[0036] Applicant believes that the present invention, in any or all
of its aspects is a breakthrough and will revolutionize the
treatment of open wounds, in particular leg ulcers, in a way which
has been found to be impossible hitherto.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0037] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiments which are presently preferred. It should be understood,
however, that the invention is not limited to the precise
arrangements and instrumentalities shown. In the drawings:
[0038] FIG. 1a is a bar graph illustrating the effect on dermal
fibroblast proliferation, measured by absorption assay at 3 and 6
days, of various concentrations super-oxidized water; and
[0039] FIG. 1b is a bar graph similar to FIG. 1a, showing the
results of the absorption assay at 6 days.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In order that the invention may be more readily understood,
reference will now be made, by way of example, to the accompanying
examples, in which:
[0041] Example 1 describes an in vitro study, which investigated
the effect of super-oxidized water based on hypochlorous acid on
the proliferation of cultured human dermal fibroblast cells.
[0042] Example 2 describes an in vitro study, which investigated
the cytotoxic effect of super-oxidized water based on hypochlorous
acid on cultured human dermal fibroblast cells.
[0043] Example 3 describes an in vitro study, which investigated
the effect of super-oxidized water based on hypochlorous acid on
the proliferation of cultured human keratinocyte cells.
[0044] Example 4 describes a clinical trial of super-oxidized water
on a patient with chronic leg ulcers.
EXAMPLE 1
[0045] Fibroblasts are flattened, irregular-shaped, connective
tissue cells which are ubiquitous in fibrous connective tissue.
They secrete components of the extracellular matrix, including
collagen, and play an important role in tissue regeneration.
[0046] Three in vitro trials of super-oxidized water based on
hypochlorous acid were carried out on single-layer cell cultures of
human dermal fibroblast (HDF) cells to ascertain whether the
super-oxidized water affected HDF cell proliferation. A range of
dilutions of super-oxidized water at different pH levels was
investigated.
[0047] Method
[0048] The super-oxidized water used in the trials was the product
of the electrolysis of an aqueous saline solution passed over a
mixture of proprietary catalysts on titanium electrodes to give a
mixture of oxidizing species, particularly hypochlorous acid (HOCl)
at a concentration of about 144 mg/l to 400 mg/l available free
chlorine (Cl). The super-oxidized water was produced as required
for each test; the apparatus (supplied by Sterilox Medical Limited,
Abingdon, UK) was operated to give a final solution redox potential
of >950 mV as recommended by the company. Appropriate dilutions
of the super-oxidized water were made, and the pH of the final
solution was adjusted using a phosphate buffer.
[0049] For the proliferation assay, HDF cells were seeded in normal
(10%) fetal calf serum (FCS) and Dulbecco's Modified Eagle Medium
(DMEM) at 1.5.times.10.sup.3 cells/well. After 24 hours the medium
was changed to low (0.4%) FCS/DMEM. After a further 48 hours
incubation super-oxidized water at varying concentrations was added
to the cells. The viability of the cells was observed, using a
standard absorption assay, 3 and 6 days after the application of
super-oxidized water.
[0050] Results
[0051] i) Trial 1: HDF cells were incubated with super-oxidized
water in a range of dilutions at a pH of 4.3. The dilutions used
were: 1, 1/3, 1/7, 1/14, 1/28, 1/56, 1/112, 1/224, 1/448, 1/896,
1/1792 and 0.
[0052] As shown in the accompanying graphs, on both day 3 (FIG. 1a)
and day 6 (FIG. 1b) super-oxidized water dilutions of 1/28 or less
significantly inhibited HDF proliferation or killed the cells.
Slight inhibition of proliferation was seen at a dilution of 1/56.
Dilutions of 1/112 to 1/448 showed no effect on proliferation,
while the 1/896 dilution showed some cell proliferation and the
dilution of 1/1792 showed significant proliferation of HDF
cells.
[0053] These results show that high concentrations of
super-oxidized water significantly inhibit HDF proliferation,
probably because of the acidity, and therefore toxicity, of the
super-oxidized water. The high level of proliferation seen with a
concentration of super-oxidized water at 1/1792 may be attributed
to other factors.
[0054] ii) Trial 2: Using the same conditions as Trial 1, HDF cells
were incubated with super-oxidized water in a range of dilutions at
a pH of 6.2. The dilutions used were: 1/20, 1/40, 1/60, 1/80,
1/100, 1/120, 1/1000, 1/1500, 1/2000, 1/3000, 1/4000 and 0.
[0055] No stimulation of proliferation was seen and indeed,
inhibition of HDF growth was seen with cells incubated with a
dilution of super-oxidized water of 1/20. However, the higher
dilution of 1/40 showed no cell toxicity.
[0056] This trial was repeated with the HDF cells seeded in
super-oxidized water at dilutions of 1, 1/4, 1/8, 1/12, 1/16, 1/20,
1/24, 1/28 and 1/32. After both day 3 and day 6, cell damage or
inhibition of proliferation was seen at dilutions of 1/20 and
below. However, dilution of more than 1/20 showed no damage or
inhibition or proliferation.
[0057] In conclusion, while the more alkaline pH appears to be less
toxic to HDF cells, proliferation of HDF cells is not seen at this
pH.
[0058] iii) Trial 3: In this trial, two plates of cells were grown
always in normal growth medium (10% FCS/DMEM). One plate of cells
was treated as described in Trial 1 but after three days of
incubation with the super-oxidized water, the growth medium was
changed from 0.4% FCS/DMEM to 10% FCS/DMEM in order to observe the
recovery of the cells.
[0059] HDF cells were incubated at 31.degree. C. with
super-oxidized water in a range of dilutions at a pH of 5.4. The
dilutions used were: 1/10, 1/20, 1/40, 1/60, 1/80, 1/100, 1/150,
1/1000, 1/1500, 1/2000, 1/4000 and 0.
[0060] On day 3 and day 6 cell proliferation was seen in cells
incubated with super-oxidized water at a dilution of 1/20 or higher
in either 0.4% or 10% FCS/DMEM. Some levels of proliferation had
reached statistical significance. A dilution of 1/10 super-oxidized
water inhibited cell growth in HDF cells grown in 0.4% FCS/DMEM but
not in 10% FCS/DMEM.
[0061] After 3 days incubation in 0.4% FCS/DMEM with or without
super-oxidized water, the medium was changed to 10% FCS/DMEM. The
cells that had been incubated with super-oxidized water showed the
same ability to recover from depression of cell growth seen while
growing in 0.4% FCS/DMEM as control cells.
[0062] This trial was repeated with the HDF cells seeded in
super-oxidized water at dilutions of 1/7, 1/10, 1/15, 1/20, 1/40,
1/60, 1/100, 1/500, 1/1000, 1/2000, 1/4000 and 0.
[0063] Again, on both day 3 and day 6, cell proliferation was seen
with HDF cells grown in 0.4% FCS/DMEM with the difference seen
being statistically significant at most dilutions. No inhibition of
cell growth was seen, even at the dilution of 1/7 super-oxidized
water. Stimulation of cell growth was also seen in cells grown in
10% FCS/DMEM in the presence of super-oxidized water. However, the
levels of proliferation did not reach statistical significance.
Where cell growth had been impaired by incubation with
super-oxidized water, recovery was seen, confirming the
observations from the first set of experiments.
[0064] In summary, HDF cells incubated with super-oxidized water at
pH 5.4 showed no inhibition of cell growth, even in the presence of
a 1/7 dilution of super-oxidized water.
CONCLUSION
[0065] The presence of super-oxidized water at a pH of 5.4 does not
inhibit HDF cell growth in vitro.
EXAMPLE 2
[0066] Three in vitro trials were carried out to investigate the
cytotoxic effect of super-oxidized water based on hypochlorous acid
on HDF cells.
[0067] Method
[0068] The super-oxidized water based on hypochlorous acid was
identical to that described in Example 1.
[0069] HDF cells were seeded in 10% FCS/DMEM at 5.times.103
cells/well. After incubation at 31.degree. C. for 72 hours,
dilutions of super-oxidized water were prepared in HBSS and added
to the cells. The viability of the cells was ascertained by a
standard absorption assay at time intervals of 15 minutes up to one
hour from the addition of the super-oxidized water.
[0070] Results
[0071] i) Trial 1: HDF cells were incubated with super-oxidized
water in a range of dilutions at a pH of 4.3. The dilutions used
were: 1, 1/3, 1/7, 1/14, 1/28, 1/56, 1/112, 1/224, 1/448, 1/896,
1/1792 and 0.
[0072] No effect on cell viability was seen in the presence of
super-oxidized water at dilutions of 1/28 or more at any of the
time points. A dilution of 1/14 induced mild damage to the cells
while dilutions of 1/7 and less killed the cells.
[0073] ii) Trial 2: Using the same conditions as trial 1, HDF cells
were incubated with super-oxidized water in a range of dilutions at
a pH of 6.2. The dilutions used were: 1, 1/4, 1/8, 1/12, 1/16,
1/20, 1/24, 1/28, 1/32, 1/36, 1/40 and 0.
[0074] No significant effect was seen on the viability of HDF cells
in the presence of super-oxidized water at dilutions of 1/20 or
more at any time point. However, dilution of 1/16 or less induced
cell damage.
[0075] iii) Trial 3: Using the same conditions as Trials 1 and 2,
HDF cells were incubated with super-oxidized water in a range of
dilutions at a pH of 4.0. The dilutions used were: 1, 1/4, 1/8,
1/12, 1/16, 1/20, 1/24, 1/28, 1/50, 1/100, 1/200 and 0.
[0076] Dilutions of super-oxidized water at 1/24 and 1/20 induced
slight damage to the cells while dilution of 1/16 or less induced
cell death.
CONCLUSION
[0077] The results of these trials support the results shown in
Example 1 in that, while super-oxidized water at pH 4.0 to 4.3 and
pH 6.2 induce damage to cultured HDF cells, greater cytotoxic
effects are seen at the lower pH.
EXAMPLE 3
[0078] In view of the results described in Examples 1 and 2, two in
vitro trials were carried out to investigate the effect
super-oxidized water on human keratinocyte (HK) cell proliferation.
The super-oxidized water used in these trials was identical to that
described in Example 1. Keratinocytes are epidermal skin cells that
synthesize keratin and, together with dermal fibroblasts, are
essential for skin healing.
[0079] Trial 1
[0080] HK cells (subcultured, P2, FS, 7 years) were seeded at
8.times.10.sup.3 cells/well and incubated at 31.degree. C. in
CLONETICS.RTM. (Biowhittaker, US) serum-free medium with complete
supplements, hereinafter referred to as keratinocyte growth medium
(KGM), in four 24-well plates. After 24 hours incubation the medium
in plates 1 and 2 was changed to CLONETICS.RTM. (Biowhittaker, US)
serum-free medium without complete supplements, hereinafter
referred to as keratinocyte basal medium (KBM).
[0081] After a further 48 hours incubation super-oxidized water
diluted in KBM at pH 5.4 was added to plates 1 and 2, and
super-oxidized water diluted in KGM was added to plates 3 and 4.
The dilutions of super-oxidized water were: 1/10, 1/20, 1/50,
1/100, 1/150, and 0. The pH of the final super-oxidized water
solution was adjusted using a phosphate buffer.
[0082] After incubation for a further 3 days a standard absorption
assay was carried out on plate 3 to observe the viability and
growth of the cells. The absorption assay was carried out on plate
4 after a still further two days. Since the cells incubated in KBM
did not grow well, plates 1 and 2 were discarded.
[0083] The absorption assay showed that, on both day 3 and day 5,
cell proliferation had occurred in the presence of all dilutions of
super-oxidized water. At day 5, the level of cell proliferation had
reached a significant level compared to cell growth in the absence
of super-oxidized water.
[0084] Trial 2
[0085] In view of the fact that the HK cells did not grow in KBM
and showed significant proliferation in the presence of
super-oxidized water in KGM, it was decided to use KBM with lower
concentrations of supplements as a holding medium, with or without
super-oxidized water.
[0086] HK cells (thawed, P2, FS, 7 years) were seeded to six
96-well plates at 3.times.10.sup.3 cells/well in KGM. After
incubation for 24 hours, the medium in plates 1 and 2 was changed
to KBM with 20% supplements, and the medium in plates 3 and 4 was
changed to KBM with 50% supplements.
[0087] After incubation for a further 24 hours, super-oxidized
water diluted in KBM with 20% supplements was added to plates 1 and
2, super-oxidized water diluted in KBM with 50% supplements was
added to plates 3 and 4, and plates 5 and 6 received super-oxidized
water diluted in complete KGM. The dilutions of super-oxidized
water were: 1/7, 1/10, 1/15, 1/20, 1/40, 1/60, 1/100, 1/500,
1/1000, 1/2000, 1/4000 and 0.
[0088] The cells were incubated for a further 3 days in the
presence of super-oxidized water, after which time, a standard
absorption assay was carried out on plates 1, 3 and 5 to ascertain
the viability of the cells, and the medium in plates 2, 4 and 6 was
changed to KGM. Plates 2, 4 and 6 were assayed after a further 48
hours of incubation.
[0089] Stimulation of cell proliferation on both day 3 and day 5
was seen in all percentages of KGM supplements. However, the level
of stimulation was not significantly different when compared to
control cell growth. No cytotoxicity was seen even at the low
dilution of 1/7 super-oxidized water.
CONCLUSION
[0090] Dermal keratinocytes cultured in the presence of KGM and
super-oxidized water showed enhanced cell proliferation, and no
cytotoxicity was seen in the presence of super-oxidized water.
EXAMPLE 4
[0091] A preliminary clinical evaluation of super-oxidized water
based on hypochlorous acid was carried out on one patient with
chronic venous ulcers on both left and right legs. The aim of the
trial was to determine whether the bacterial status of the ulcers
is altered and the bed of the ulcer improved by treatment with
super-oxidized water.
[0092] Method
[0093] The patient's legs were immersed in 40 liters of
super-oxidized water in a hydrobath for fifteen minutes before
being allowed to dry. An intermediate assessment without treatment
was carried out after one week.
[0094] A second treatment with super-oxidized water was repeated
after two weeks in which the patient was subjected to three
fifteen-minute washes at approximately three-hour intervals.
Post-treatment clinical evaluation was carried out one and several
days after the second treatment.
[0095] Semi-quantitative microbiological analysis of the leg ulcers
was carried out on swabs taken before and fifteen minutes after
treatment with super-oxidized water.
[0096] Results
[0097] After the first treatment, the patient reported that the
treatment was comfortable and free from pain. The appearances of
the ulcers on both legs were markedly improved when assessed five
hours after treatment.
[0098] As shown in Table 1, quantitative microbiology showed a
reduction in the number of colony-forming units in the order of
10.sup.2 in the right leg ulceration and a reduction in the order
of 10.sup.4 on the left leg.
1TABLE 1 semi-quantitative microbiological analysis of leg ulcers
before and after treatment with super-oxidized water based on
hypochlorous acid. Figures quoted indicate the number of
colony-forming units (cfu) per ml. Right Leg Left Leg Pre-treatment
1 .times. 10.sup.7 1 .times. 10.sup.7 Post-treatment 1 .times.
10.sup.5 1 .times. 10.sup.3
[0099] The patient was seen one week after the first treatment and
was treated with conventional therapy, including potassium
permanganate. The effect of these on the appearance of the leg
ulcers following treatment did not appear to be as striking as that
seen with super-oxidized water.
[0100] A second treatment with super-oxidized water was repeated a
further week later, and similar beneficial results were obtained.
In between the treatment periods the ulcers had become sloughy on
both legs. Immediately after the first wash, the ulcer bed was
whitish due to effervescence. A cleansing effect was seen after the
later two washes, and a marked improvement was seen with the state
of the ulcer 18 hours after the first wash.
[0101] The patient reported no discomfort to the treatment, the
solution in the bath was soothing, and the skin felt a bit tight
afterwards. The patient commented that the tightness started to be
felt once cold air was accessible to the skin.
[0102] Referring to Table 2, quantitative microbiology showed a
reduction in the number of colony-forming units in the order of
10.sup.2 in the right leg ulceration and a reduction in the order
of 10.sup.4 on the left leg.
2TABLE 2 semi-quantitative microbiological analysis of leg ulcers
before and after treatment with super-oxidized water based on
hypochlorous acid. Figures quoted indicate the number of
colony-forming units (cfu) per ml. Right leg organisms Left leg
organisms (cfu/ml) found (cfu/ml) found pre-treatment 1 1.9 .times.
10.sup.8 Coliforms 4.5 .times. 10.sup.6 Coliforms Proteus spp
Proteus spp skin flora skin flora post-treatment 1 1.2 .times.
10.sup.5 Coliforms 4.5 .times. 10.sup.4 skin flora skin flora
.beta.-haemolytic Proteus spp streptococci Coliforms Proteus ssp
pre-treatment 2 3.0 .times. 10.sup.3 Coliforms 1.5 .times. 10.sup.4
coliforms Proteus spp skin flora skin flora post-treatment 2 1
.times. 10.sup.3 Coliforms <10 no growth Proteus spp skin flora
pre-treatment 3 <10 no growth 6.3 .times. 10.sup.3 Coliforms
.beta.-haemolytic streptococci skin flora post-treatment 3 3.0
.times. 10.sup.2 Coliforms 3.0 .times. 10.sup.3 .beta.-haemolytic
skin flora streptococci Proteus spp post 24 hours 2.7 .times.
10.sup.2 Coliforms 1.5 .times. 10.sup.6 .beta.-haemolytic Proteus
spp streptococci skin flora Coliforms Proteus spp
CONCLUSION
[0103] The main objectives of the clinical study were to examine
patient comfort and safety, as well as the efficiency of a
treatment of super-oxidized water based on hypochlorous acid.
[0104] The use of antiseptics for cleansing wounds is
controversial, particularly with reference to the degree of pain
associated with this kind of treatment. This patient did not
experience pain and, in fact, commented on a soothing effect. There
was a positive effect on the bacterial flora as well as the
clinical appearance of the wounds. There was no adverse effect on
the surrounding skin which, in a number of patients with
long-standing ulcers, is often sensitive.
[0105] While the invention has been described with reference to the
examples in relation to the treatment of leg ulcers, it should be
appreciated that the invention has considerably wider
applicability. For example, the invention has applicability to
burns, to organ transplants in relation to which current practice
is to disinfect organs with antibiotics for two weeks before they
are used in a patient, to disinfection of valve-replacements, and
to surface wounds, open wounds and plural cavity infections which
are exhibiting drug-resistance.
[0106] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications within the spirit and scope of the present invention
as defined by the appended claims.
* * * * *