Title of Invention

A METHOD FOR PREPARING AN ARTHROSPIRA-BASED COMPOSITION

Abstract The present invention concerns a composition comprising physiologically stressed Arthrospira maxima for use as a biocide and/or therapeutic. The invention also concerns a method for preventing or treating an infection or infestation of a subject by an organism, wherein the method comprises the step of administering to the subject an effective amount of a composition comprising physiologically stressed Arthrospira.
Full Text WO 2006/047830 PCT/AU2005/001693
TITLE OF THE INVENTION
Arthrospira-based Compositions and Uses Thereof
TECHNICAL FIELD
The present invention relates to a composition comprising Arthrospira and the
use of the composition as a biocide and/or therapeutic.
BACKGROUND ART
Arthrospira (formerly known as Spirulina) is a cyanobacteria that is grown primarily
for use as a food and/or protein source. Therapeutic uses for Arthrospira have also been
described, including use as an anti-viral agent, anti-cancer agent, reducer of cholesterol,
reducer of diabetes, reducer of hypertension and immunomodulator. See, for example, the
references cited at the Spirulina Source.com website at
http://www.spirulinasource.com/library.html.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an Arthrospira composition for
use as a biocide and/or therapeutic.
Physiologically Stressed Arthrospira has Biocidal Activity
The present inventors have discovered that Arthrospira, when physiologically
stressed, produces at least one type of bioactive agent that is active against fungi, bacteria
and presumably viruses. Arthrospira can be physiologically stressed, for example, by
depriving the organism of nutrients or light, or by dehydrating/drying the organism. A sign
of an Arthrospira filament (trachoma) being "physiologically stressed" is when it enters
an anabiotic state, in which state the filament rounds up and a thick mucoid coating is
produced. The filament and/or thick mucoid coating is believed by the inventors to
contain one or more bioactive agents that protect the organism against predators. One
or more of the bioactives may break down the cell wall or exoskeleton of a predatory
organism. One or more of the bioactive agents may be, for example, a lytic or modifying
agent such as a chitinase, chitosanase or chitin deacetylase, that digests chitin, a chitin
derivative (such as chitosan) or other cell wall polymer having, for example, N-
acetylglucosamine or D-glucosamine as a polymer subunit. Peptidoglycan of Gram-positive
bacteria is one such polymer, comprising N-acetylglucosamine and N-acetylmuramic acid.
According to a first aspect of the present invention, there is provided a
composition for digesting or modifying chitin, a chitin derivative or a polymer having
N-acetyl-D-glucosamine as a polymer subunit, said composition comprising
physiologically stressed Arthrospira.

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According to a second aspect of the present invention, there is provided a
method for digesting or modifying chitin, a chitin derivative or a polymer having N-
acetyl-D-glucosamine as a polymer subunit, said method comprising the step of
contacting chitin, chitin derivative or a polymer having N-acetyl-D-glucosamine as a
polymer subunit with a composition comprising physiologically stressed Arthrospira.
The chitin, chitin derivative or polymer can be in any suitable form. The chitin,
chitin derivative or polymer can be in a substantially purified form or can be part of an
organism such the cell wall of a fungus or Gram-positive bacteria, or part of the exoskeleton
of an insect.
According to a third aspect of the present invention, there is provided a method for
identifying a bioactive agent from Arthrospira, said method comprising the steps of:
(I) combining a composition comprising physiologically stressed Arthrospira with at
least one test substrate modifiable by a bioactive agent from Arthrospira', and
(II) assaying for modification of the test substrate.
Any suitable type of test substrate can be used. Assaying for modification of the test
substrate can be carried out in any suitable way. High throughput screening, however, is
preferred.
Preferably, the bioactive agent is a chitinase, chitosanase or chitin deacetylase and
the test substrate is chitin, a chitin derivative or a polymer having N-acetyl-D-glucosamine
as a polymer subunit. Preferably, respirometry is used to assay modification (ie.
degradation) of the chitin, chitin derivative or other polymer.
The method can further comprise the step of purifying the bioactive agent.
Purification can be carried out in any suitable way. The method can also comprise the steps
of cloning and expressing the gene/s of the bioactive agent. These steps can be carried out in
any suitable way. For instance, the steps can be as described in Maniatis, et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, the entire contents of
which are incorporated herein by cross reference.
Use of Physiologically Stressed Arthrospira as a Biocide and/or Therapeutic
Chitin, chitin derivatives and glucosamine containing polymers are found
throughout nature, usually as a component of a cell wall or exoskeleton of, for
instance: algae, fungi, protozoans, ciliates, coelenterates, bryozoans, molluscs,
annelids, arthropods, branchiopods, phoronida, pogonophora, and bacteria (Gram-
positive bacteria and Rhizobia).

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Hence, physiologically stressed Arthrospira having lytic or modifying activity
against chitin, chitin derivatives and glucosamine containing polymers is potentially
capable of being used as a biocide against a broad range of organisms. "Biocide" as
used herein refers to either the killing of an organism or inhibition of the growth or
replication of an organism (ie. the organism may not actually be killed). It is to be
appreciated that some applications of the biocide will be for therapeutic purposes.
According to a fourth aspect of the present invention, there is provided a
composition for use as a biocide and/or therapeutic, said composition comprising an
effective amount of physiologically stressed Arthrospira.
According to a fifth aspect of the present invention, there is provided a
method for preventing or treating an infection or infestation of a subject by an
organism, said method comprising the step of administering to the subject an
effective amount of a composition comprising physiologically stressed Arthrospira.
Preferably, the method comprises the step of identifying a subject in need of
such treatment or requiring such prevention.
There is also provided the use of physiologically stressed Arthrospira in the
preparation of a medicament for the prevention or treatment of an infection or
infestation of a subject by an organism.
Surprisingly, the inventors have found that taking Arthrospira from its growth
environment may be enough for physiological stressing to occur and for inducing expression
of one or more bioactive agents having biocidal activity. That is, steps for physiologically
stressing Arthrospira as taught in the specifications of New Zealand Patents No. 336620 and
336619 need not be carried out and the Arthrospira need not be further processed
(disrupted, explosively decompressed, potentiated, milled, rehydrated in sodium bicarbonate
or freeze dried) in order for there to be biocidal activity.
Furthermore, all commercially available preparations (tablets, capsules and powders)
of Arthrospira that have thus far been tested by the inventors have been found to have
activity against a substrate, and that activity is believed to be indicative of biocidal activity.
Such preparations are usually prepared by harvesting the Arthrospira, washing and drying
the Arthrospira, without a step for specifically stressing the organism. In fact, commercial
growers of Arthrospira tend to minimise stressing of the organism as much as possible as the
thick mucoid coating tends to clog machinery for processing the organism.
For any one or more of the compositions described herein, the Arthrospira can be
any suitable species, or mixture of species or Arthrospira variants including, but not limited

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to, A. maxima. It is to be pointed out that Arthrospira and Spirulina are two separate genera.
The separation between these two genera has been repeatedly affirmed on the basis of many
characteristics including: helicity and trichome size, cell wall structure and pore pattern, gas
vacuoles, thylakoid pattern, trichome motility and fragmentation, GC content,
oligonucleotide catalogue of 16S rRNA, and mutations in the cpcB-cpcA locus. See, for
instance, J. F. Manen and J. Falquet (2002) "The cpcB-cpcA locus as a tool for the genetic
characterization of the genus Arthrospira (Cyanobacteria): evidence for horizontal transfer".
International Journal of Systematic and Evolutionary Microbiology, Vol 52, 861-867; and,
Chapter 1: Morphology, Ultrastructure and Taxonomy of Arthrospira (Spirulina) Maxima
and Arthrospira (Spirulina) Platensis by Luisa Tomaselli, of Spirulina platensis
(Arthrospira)'. Physiology, Cell-biology and Biotechnology. Taylor and Francis. Avigad
Vonshak (Ed) 1997. The compositions described in this specification exclude species of the
Spirulina genus.
The composition can comprise intact Arthrospira filaments, segments of the
filaments, disrupted/lysed segments or extracts/fractions thereof. The composition can
comprise Arthrospira that is alive or that is not alive. Preferably, the composition
comprises disrupted Arthrospira filaments and segments.
The Arthrospira-based composition can be prepared in any suitable way, provided
that the biocidal activity is not compromised. Normally, this would involve the steps of: (I)
growing Arthrospira in any suitable way; (II) harvesting the grown Arthrospira in any
suitable way; and (III) drying the Arthrospira in any suitable way. Optional steps include:
additionally physiologically stressing the Arthrospira in any suitable way; disrupting the
filaments and segments in any suitable way, sterilising the organism in any suitable way,
decolourising the Arthrospira in any suitable way, removing or degrading the chlorophyll in
any suitable way, and milling the dried Arthrospira.
Some of the above steps are described, for example, in the specifications of New
Zealand Patents No. 336620 and 336619, the entire contents of which are incorporated
herein by cross reference. For example, stressing can be carried out at the time of harvest by
either depriving the Arthrospira of essential nutrients (nutrient diminution) or light for a
period sufficient to stress it but not kill it by management of the growing conditions.
Stressing can also be by partial desiccation or by harvesting the Arthrospira and keeping it
alive in damp conditions until stressed but not dead. For example, Arthrospira can be spray
dried to form a fine powder of disrupted cells. Spray drying may take place, for example, at
50-190°C for a few seconds. For example, drying can be carried out in any suitable way,

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including by cyclonic drying, heat pump drying, heat tube drying, refractory drying or
thermal drying under about 67°C for short periods of time. For example, filaments can be
disrupted using milling or explosive decompression as described in New Zealand Patents
No. 328013 and No. 328740, the entire contents of which are incorporated herein by cross
reference.
The concentration of Arthrospira in the composition can vary depending on the
application, eg. anywhere from about 0.01% to about 100%. The composition can include
any suitable solvent, carrier, base, excipient, filler, binder, plasticiser, emulsifier, stabiliser,
lubricant, buffering agent, emollient, solubilising agent, suspending agent, thickener,
fragrance, colourant and preservative.
Depending on the intended use, the composition can further comprise one or more
actives not derived from Arthrospira, such as antimicrobials (e.g. bactericides or
antifungals), therapeutic agents (e.g. wound healers, steroids) or general nutrients (e.g.
amino acids, vitamins). Such actives are well known in the art.
The composition can be in any suitable form. The composition can be administered
or applied in any suitable way. The composition can be a liquid, gel or solid, or a mixture
thereof. The composition can be a liquid culture of Arthrospira which is applied as a spray.
Alternatively, the composition can be a powder which is evenly distributed over a target
site by shake or puffer application. The infected or infested subject can be dipped
into a bath containing a liquid form of the composition. The composition and method
of application or administration can be as described in the specifications of New
Zealand Patents No. 336620 and 336619.
For therapeutic use, the composition can be administered or applied, for example,
orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or vaginally.
The composition can be a tablet, solution, aerosol, spray, cream, ointment, lotion, emulsion,
gel or powder. The composition can be in the form of an Arthrospira-impregnated
bandage, dressing, adhesive plaster, suppository, pessary or poultice.
Preferably, the biocide is active against fungi, Gram-positive bacteria,
protozoa, viruses, crustaceans, acari and insects, some of which are addressed in
more detail below.
According to a sixth aspect of the present invention, there is provided a method for
preparing an Arthrospira-based composition for use as a biocide and/or therapeutic, said
method comprising the steps of:
(1) physiologically stressing Arthrospira; and

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(2) combining the stressed Arthrospira of (1) with a suitable carrier, solvent, base or
excipient.
According to a seventh aspect of the present invention, there is provided a method for
preparing an Arthrospira-based composition for use as a biocide and/or therapeutic, said
method comprising the steps:
(1) physiologically stressing Arthrospira by removing up to about 80% of liquid within
which the Arthrospira is being grown;
(2) washing the stressed Arthrospira in order to remove contaminants;
(3) drying the washed Arthrospira; and
(4) combining the washed Arthrospira with a suitable carrier, solvent, base or excipient.
Either of the above methods can be used to prepare one or more of the compositions
described herein.
Preferably, the above methods exclude specific steps of explosive decompression
and/or freeze drying the physiologically stressing Arthrospira prior to adding the carrier,
solvent, base or excipient
Use of Physiologically Stressed Arthrospira as an Antifungal
According to an eighth aspect of the present invention, there is provided a
composition for use as an antifungal, said composition comprising an effective amount
of physiologically stressed Arthrospira.
According to a ninth aspect of the present invention, there is provided a
method for preventing or treating a fungal infection or infestation of a subject, said
method comprising the step of administering to the subject an effective amount of a
composition comprising physiologically stressed Arthrospira.
Preferably, the method comprises the step of identifying a subject in need of
such treatment or requiring such prevention.
There is also provided the use of physiologically stressed Arthrospira in the
preparation of a medicament for the prevention or treatment of a fungal infection or
infestation of a subject.
Physiologically stressed Arthrospira may be used to treat at least the following types
of fungi: Zygomycotina; Ascomycotina; Fungi Imperfecti; Malassezia; Microsporum;
Trichophyton;and Epidermophyton.
In a first embodiment, the subject is a human or an animal such as, mammal or vertebrate.
In human subjects, the composition can be used to treat:

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- tinea (e.g. athlete's foot, jock itch, ringworm, deep nail bed infection) which is
caused chiefly by species of Microsporum, Trichophyton, Candida and
Epidermophyton;
- thrush, which is caused by Candida',
- dandruff, which is caused by Malassezia;
- tropical mycetoma - a granular spreading growth in the skin, muscle tissue and
lymph nodes - which is caused by Madurella;
- sporothricosis - a granular spreading growth in skin and lymph nodes - which is
caused by Sporothrix schenkii; and
- histoplasmosis - a chronic pneumonia with spreading systemic infection - which is
caused by Histoplasma capsulation.
In animal subjects, the composition can be used to treat, for example, ring worm,
hoof infections {Candida, Malassezia), dermatitis and folliculitis {Microsporum,
Trichophyton, Alternaria, Fusarium). Topical fungal conditions in animals are usually
caused by the same fungi as in humans.
In a second embodiment, the subject is an agricultural or horticultural product such as
a plant, flower, fruit, vegetable, cereal, grain, pulse, mushroom spawn, pasture or lawn. The
composition can be used to treat, for example, head blight and crown rot {Fusarium), facial
eczema {Pithomyces chartaruni), botrytis {Botrytis cineria), leaf spot {Septoria, Alteria,
Bipolaris), powdery mildew {Sphaerotheca macularis, Erysiphe, Sphaerotheca pannosd),
brown rot {Moniliniajruiticola) and leaf/stem rust {Puccinia).
In a third embodiment, the subject is soil, timber, building material or a building.
The composition can be used, for instance, in or on buildings for the treatment of mould, and
on building materials and power poles for the treatment of fungal wood rot.
Surprisingly, the present inventors have found that physiologically stressed
Arthrospira interacts synergistically with monographed anti-fungal actives (fungi-static
agents) to provide superior broad spectrum antifungal activity. The inventors have found
that stressed Arthrospira can inhibit the growth of fungi, kill fungi and prevent regrowth of
fungi.
According to a tenth aspect of the present invention, there is provided an antifungal
composition comprising a synergistic combination of physiologically stressed Arthrospira
and at least one fungi-static agent.
According to an eleventh aspect of the present invention, there is provided a
method for preventing or treating a fungal infection or infestation of a subject, said

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method comprising the step of administering to the subject an effective amount of a
composition comprising a synergistic combination of physiologically stressed
Arthrospira and at least one fungi-static agent.
Preferably, the method comprises the step of identifying a subject in need of
such treatment or requiring such prevention.
There is also provided the use of physiologically stressed Arthrospira in
combination with at least one fungi-static agent in the preparation of a medicament for
the prevention or treatment of a fungal infection or infestation of a subject.
Any suitable fungi-static agent (monographed active) can be used, such as
terbinafine, bifonazole, clotrimazole, miconazole, econazole, ketoconazole or tolnaftate.
Any suitable concentration of monographed active can be used and will depend on the
intended use for the composition.
Use of Physiologically Stressed Arthrospira as an Antibacterial
Physiologically stressed Arthrospira has been found by the inventors to be an
effective antibacterial against Gram-positive bacteria. The mode of antibacterial action is not
clearly understood. It is possible that cell walls of Gram-positive bacteria are lysed or
otherwise modified by one or more bioactives from Arthrospira. The cell walls contain
peptidoglycan (comprising N-acetylglucosamine and N-acetylmuramic acid) and
peptidoglycan may be digestible by one or more of the bioactives.
According to a twelfth aspect of the present invention, there is provided a
composition for use as an antibacterial, said composition comprising an effective
amount of physiologically stressed Arthrospira.
According to a thirteenth aspect of the present invention, there is provided a
method for preventing or treating a bacterial infection or infestation of a subject,
said method comprising the step of administering to the subject an effective amount
of a composition comprising physiologically stressed Arthrospira.
Preferably, the method comprises the step of identifying a subject in need of
such treatment or requiring such prevention.
There is also provided the use of physiologically stressed Arthrospira in the
preparation of a medicament for the prevention or treatment of a bacterial infection
or infestation of a subject.
Preferably, the bacteria is Gram-positive bacteria and may be, for example, Bacilli,
Clostridia, Staphylococci or Pneumococci. The bacteria is preferably Propionibacterium
acne, the causative agent of acne.

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In a first embodiment, the subject is a human or an animal such as a mammal or
vertebrate. In human subjects, the composition can be used to treat acne, dermatitis, ulcers
or wounds caused by or infected with Gram-positive bacteria. The composition can be used
to treat internal infections, such as those in the airways, in the mouth, in the digestive and in
genitourinary tracts (e.g. strep throat, middle ear infections).
In a second embodiment, the subject is an agricultural or horticultural product such
as a plant, flower, fruit, vegetable, cereal, grain, pulse or mushroom spawn.
In a third embodiment, the subject is, for example, soil, a man-made structure, or a
waterway in which bacteria can grow.
Use of Physiologically Stressed Arthrospira as a Pesticide
According to a fourteenth aspect of the present invention, there is provided a
composition for use as a pesticide, said composition comprising an effective amount of
physiologically stressed Arthrospira.
According to a fifteenth aspect of the present invention, there is provided a
method for suppressing or removing a pest, said method comprising administering or
applying an effective amount of a composition comprising physiologically stressed
Arthrospira.
The pesticide can be directed to pests such as crustacean and acari. The
pesticide can be directed to insects such as flies (eg. Simulidae - black flies), wasps,
mosquitoes and termites. The pesticide can be used to stop the transmission of
parasites, such as those causing malaria, and other insects-borne diseases.
In a first embodiment, the composition can be formulated as an insect repellent or
bait.
In a second embodiment, the composition can be applied to an agricultural or
horticultural product such as a plant, flower, fruit, vegetable, cereal, grain, pulse or
mushroom spawn.
hi a third embodiment, the composition can be applied to, for example, soil, a man-
made structure, a building, building material, a stream, a lake or wetlands.
Use of Arthrospira as a Therapeutic
When using physiologically stressed Arthrospira as a biocide and/or therapeutic on
skin, the inventors have found that one or more other substituents of Arthrospira interact
synergistically with the one or more bioactive agents to promote healing of the skin. These
other substituents are likely to include: beta-carotene, which provides nourishment to the

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skin; phycocyanin, which is an anti-inflammatory; other proteins; and other nutrients,
including vitamins, minerals, trace elements, anti-oxidants, essential oils and carbohydrates.
First, the stressed Arthrospira inhibits or kills the microbe - be it a fungus, bacteria or
virus - and then other substituents of Arthrospira repair the damage caused by the microbe.
The present inventors have found that Arthrospira has therapeutic qualities even
when not physiologically stressed, such as in the treatment of skin conditions and in skin
repair.
According to a sixteenth aspect of the present invention, there is provided a
composition for repairing or preventing a defect of the skin of a mammal, said
composition comprising an effective amount of Arthrospira.
Preferably, the composition comprises physiologically stressed Arthrospira.
According to a seventeenth aspect of the present invention, there is provided a method
of repairing or preventing a defect of the skin of a mammal, said method comprising
the application of an effective amount of a composition comprising Arthrospira.
Preferably, the composition comprises physiologically stressed Arthrospira.
Preferably, the method comprises the step of identifying a mammal in need of
such treatment or requiring such prevention.
There is also provided the use of Arthrospira, preferably physiologically
stressed Arthrospira, in the preparation of a medicament for repairing or preventing
a defect of the skin of a mammal.
The compositions according to the sixteenth and seventeenth aspects of the invention
can have one or more ingredients or properties as described for the compositions according
to the other aspects of the invention that have been defined.
The compositions according to the sixteenth and seventeenth aspects of the invention
are preferably prepared by any of the methods described above.
The skin defect can be, for example, a pit, acne damage, rosacea, a reddened area, a
crack, a burn, a blister, psoriasis, eczema, scaling, wrinkles, a papule, a stomatitis, a lesion, a
pustule, a wound, cradle cap, diaper rash, an ulcer, a cold sore, shaving rash, chicken pox,
dermatitis, cracked heels and elbows. The composition can be used to treat burns, insect and
animal bites, and to remove inflammation of the skin. The composition can be used to reduce
itchiness of the skin. The composition can be used to repair scar tissue, sun-damaged skin, and
dry and scaly skin that has lost its elasticity.
Other aspects and embodiments of the invention will become apparent from the
following detailed description thereof.

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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows activity of physiologically stressed Arthrospira (AMYCOT® and
unprocessed powder) on chitin as assessed by respirometry;
Figure 2 shows antifungal activity of AMYCOT® on C. albicans;
Figure 3 shows antibacterial activity of AMYCOT® on P. acne as assessed by
respirometry;
Figure 4 shows the antifungal activity of different commercial preparations of
Arthrospira (Spirulina) on T. rubrum;
Figure 5 shows antifungal activity of a commercial preparation (Life Stream) of
Arthrospira on A. niger;
Figure 6 shows antifungal activity of AMYCOT® on M. canis;
Figure 7 shows antifungal activity of AMYCOT® onF. graminearum;
Figure 8 shows antifungal activity of AMYCOT® on M furfur;
Figure 9a shows antifungal activity of shampoo AMYCOT® on T. rubrum;
Figure 9b shows antifungal activity of shampoo AMYCOT® on C. albicans;
Figure 10 shows antifungal activity of AMYCOT® on P. chartarum;
Figure 11 shows antifungal activity of AMYCOT® on B. cinerea;
Figure 12 shows T. rubrum inhibitory results using AMYCOT® and various
monographed anti-fungal actives;
Figure 13 shows T. mentagrophytes inhibitory results using AMYCOT® and various
monographed actives;
Figure 14 shows C. albicans inhibitory results using AMYCOT® and various
monographed actives;
Figure 15 shows C. albicans inhibitory results using a monographed active;
Figure 16 shows T. mentagrophytes inhibitory results using AMYCOT® in
combination with a monographed active and various monographed actives;
Figure 17 shows C. albicans inhibitory results using AMYCOT® in combination
with a monographed active and various monographed actives;
Figure 18 shows T. mentagrophytes inhibitory results using AMYCOT® in
combination with monographed actives;
Figure 19 shows T. mentagrophytes inhibitory results using AMYCOT® in
combination with monographed actives;
Figure 20 shows T. mentagrophytes inhibitory results using AMYCOT® in
combination with a monographed active and various monographed actives;

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Figure 21 shows C. albicans inhibitory results using AMYCOT® in combination with a
monographed active and various monographed actives;
Figure 22 shows T. mentagrophytes fungicidal results using AMYCOT® in
combination with monographed actives;
Figure 23 shows T. rubrum fungicidal results using AMYCOT® in combination with
monographed actives;
Figure 24 shows T. mentagrophytes fungicidal results using AMYCOT® in
combination with monographed actives;
Figure 25 shows T. mentagrophytes fungicidal results using AMYCOT® in
combination with monographed actives;
Figure 26 shows T. mentagrophytes fungicidal results using AMYCOT® in
combination with monographed actives;
Figure 27 shows C. albicans fungicidal results using AMYCOT® in combination
with a monographed active and various monographed actives;
Figure 28 shows T. rubrum fungicidal results using AMYCOT® and monographed
actives;
Figure 29 shows activity of physiologically stressed Arthrospira (AMYCOT®) on
chitin at different temperatures as assessed by respirometry;
Figure 30 shows activity of physiologically stressed Arthrospira (AMYCOT®) on
chitin at various pH conditions as assessed by respirometry;
Figure 31 shows antifungal activity of Arthrospira on T. mentagrophytes;
Figure 32 shows antifungal activity of Arthrospira on T. rubrum;
Figure 33 shows antifungal activity of Arthrospira on M. fructicola; and
Figure 34 shows antifungal activity of "potentiated AMYCOT®" and "original
AMYCOT®" on Alternaria sp.
BEST MODE AND OTHER MODES OF CARRYING OUT THE INVENTION
By way of illustration only, preferred embodiments of the present invention are
described in detail, with reference to the following series of examples.
Example 1 - Preparation of Physiologically Stressed Arthrospira
A culture of Arthrospira maxima (obtainable from Biovite Australia Pty Ltd) was
stressed by nutrient diminution or partial desiccation. The Arthrospira was dewatered
and dried to form an "unprocessed" powder. Dried Arthrospira was then explosively
decompressed (potentiated) using the method described in New Zealand Patents No. 328013
and No. 328740. The Arthrospira was re-hydrated, decolourised, dried and milled to form a

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dry "premix powder". The premix powder was then dispersed in a suitable carrier (eg.
commercially available aqueous British Pharmacopoeia cream or water). The premix powder
is referred to as "AMYCOT® premix powder".
Example 2 - Characterisation of Activity of Stressed Arthrospira
The results of respirometry suggest that physiologically stressed Arthrospira is
capable of modifying or lysing substrates comprising chitin, chitosan and/or N-acetyl-D-
glucosamine.
Chitin Substrate
Standard amounts of AMYCOT® premix powder and the unprocessed powder were
separately reacted with four-unit polymer BHD chitin (Sigma, catalogue number C7170) in a
Warburg Respirometer using standard techniques. Each composition was mixed together
with chitin and the quantity of gas evolved was measured. The reaction was monitored for
one-hour following equilibration for 30 minutes. The same composition, but pre-boiled in a
water bath for five minutes, did not produce any gas which suggested that the activity could
be enzymic and could be due to one or more types of denaturable proteins.
The results are plotted in Figure 1. The rate of activity (presumably enzymatic
activity) of unprocessed powder was 694.9 ml/g cellular material/g substrate/hour. The rate
of activity of AMYCOT® premix powder was 729.65 ml/g cellular material /g
substrate/hour.
Respirometry was also used to identify the optimum temperature for the activity.
AMYCOT® premix powder (0.02 g and 0.06 g) was incubated with chitin (0.02 g) at 20°C,
30°C, 33°C and 40°C. AMYCOT® premix powder in a cream based (0.05 g) was also
incubated with chitin (0.02 g) at those temperatures. The results are plotted in Figure 29.
The optimum temperature for the activity in each case was determined to be 33°C.
Respirometry was also used to identify the optimum pH for the activity.
AMYCOT® premix powder (0.02 g) was suspended in buffers of varying pH (60-70%
ethanol, 30-40% water, 0-0.05% phenolphthalein, 0-0.03% bromothymol blue, 0-0.02%
methyl red, sodium salt, 0-0.01% sodium hydroxide and 0-0.01% methyl orange, sodium salt
at pH 5, 7,9 and 11) and was incubated with chitin (0.02 g) at 33°C. The results are plotted
in Figure 30. The optimum pH for activity was determined to be 6.
These results suggest that the bioactive agent or agents should have activity when
topically applied to skin as skin has a similar temperature and pH.

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N-acetyl-D-glucosamine and Chitosan Substrates
AMYCOT® premix powder (0.02g) was separately incubated with N-acetyl-D-
glucosamine (0.02g, Sigma catalogue number A8625-5G) and chitosan (0.02g, Sigma
catalogue number C3646) substrates in a Warburg Respirometer at 33°C. Each reaction was
monitored for one-hour following equilibration for 30 minutes.
The results for AMYCOT® plus N-acetyl-D-glucosamine are presented in the table
below.

These results suggest that physiologically stressed Arthrospira has one or more
bioactive agents that lyse or modify chitin, chitosan and/or N-acetyl-D-glucosamine, or
possibly another type of polymer having N-acetyl-D-glucosamine as a polymer subunit. The
bioactive agent could be, for instance, a chitinase, chitosanase or chitin deacetylase.

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Example 3 - Antifungal and Antibacterial Activity of Stressed Arthrospira
The biocidal activity of AMYCOT® premix powder was tested in vitro on live fungi
and Gram-positive bacteria.
PCT/AU2005/001693
Live cultures of target pathogens were sourced from the American Type Culture
Collection (ATCC), the Australian Collection of Microorganisms (ACM) and Sullivan
Niccolaides. The cultures that were tested are shown in Table 4.

A uniform lawn of each pathogen was grown on a dish containing Potato Dextrose
Agar (PDA) medium. After 3-4 days of incubation, four wells were cut in the PDA medium
with a sterile 8 mm diameter cork borer. Measured amounts of AMYCOT® premix powder
and control creams/powders were placed in each well. The dishes were cultured at
conditions appropriate for the respective pathogen and the dishes were inspected daily.
After 2-4 days two concentric zones around the well were evident:
1. A smaller transparent circular "clearance" zone around a well. This is an area
where fungi have been destroyed, leaving only residual dry cytoplasmic matter (as seen by
light and electron microscopy).
2. A larger semitransparent "affected" zone extending from the perimeter of the
clearance zone. The affected zone is recognised by the lesser height of the lawn, its lesser
density and greater transparency compared with unaffected lawn. This is an area where
fungi cell walls have been largely destroyed, but that some are still present, often as rounded
cytoplasmic spheres (as seen by light and electron microscopy).
Measurement of the zones is taken from the outside perimeter of the well to the
outside perimeter of the zone in each case. This measurement is referred to as the zonal
distance (ZD). The radius of the zones is a non-linear measure of the efficacy of the biocidal
agent/s against the pathogen in vitro.

WO 2006/047830 K PCT/AU2005/001693
The in vitro control results are shown in Table 5.

Figure 2 shows clearance of Canada albicans (ACM 4574) using AMYCOT®,
which is evidence of the efficacy of one or more biocidal agents against the condition thrush.
AMYCOT® premix powder is at the 6 o'clock position and a "human trial cream" having
12.5% w/w AMYCOT® in an aqueous cream is at the 12 o'clock position (ZD of 9 mm).
In the case of the anaerobic acne-causing bacteria Propionibacterium acne, no
clearance was obtained when tested under anaerobic conditions. An explanation for this is
provided in Example 4.
Example 4 - Antibacterial Activity of Stressed Arthrospira on Propionibacterium acne
Respirometry showed that AMYCOT® has antibacterial activity against P. acne.
AMYCOT® premix powder (0.02g) and 5g "human trial cream" comprising 12.5%
w/w AMYCOT® were separately reacted with a broth of the anaerobic acne-causing
organism P. acne (ACM No. 5109) in a Warburg Respirometer in the presence or absence of
oxygen. The results were compared with those of a control comprising P. acne broth devoid

WO 2006/047830 '^ PCT/AU2005/001693
of AMYCOT®. In the absence of oxygen, no gas was liberated. In the presence of oxygen,
gas was liberated and each composition had antibacterial activity.
The results for AMYCOT® and human trial cream in the presence of oxygen are
plotted in Figure 3. The (presumably enzymatic) rate of AMYCOT® premix powder was
626.00 ml/g cellular material/g substrate/hour. The rate of human trial cream was 12.00 ml/g
cellular material/g substrate/hour.
The results showed that in order for the composition to work as an antibacterial on P.
acne in vivo, the presence of oxygen is required. Therefore, at least one of the biocidal
agents may be an oxidase.
The inventors suspect that one or more of the biocidal agents against fungi may lyse
or modify the cellular wall of fungi. Such an agent may be a chitinase, chitosanase or chitm
deacetylase.
Although P. acne and other Gram-positive bacteria are not believed to have chitin or
chitosan in their cellular walls, it is suspected that one or more biocidal agents lyse or
modify N-acetyl-D-glucosamine/peptidoglycans in the bacterial cell wall.
Example 5 - Different Commercial Sources of Arthrospira have Antifungal Activity
Commercial preparations of dried Arthrospira (powders, tablets and capsules) were
sourced from different suppliers in different jurisdictions and tested for antifungal activity.
The powders are typically prepared by harvesting Arthrospira, washing the harvested
Arthrospira, and drying the washed Arthrospira. Some suppliers mill the Arthrospira whilst
wet. Preparation does not entail the step of specifically stressing Arthrospira post-harvest
such that the Arthrospira enter the anabiotic state. In fact, as mentioned before, commercial
growers of Arthrospira tend to minimise stressing of the organism as much as possible as the
thick mucoid coating tends to clog machinery for processing the organism.
Arthrospira (Spirulina) powders were obtained from the following suppliers:
1. China Spirulina - Jiangsu Cibainian Nutrition Food Co., Ltd. China
2. Febico-Far East Biotech Co. Ltd. Taiwan
3. Life Stream/Earthrise - DIC. California USA
4. Pacifica - Cyanotech. Hawaii USA
5. Siam Algae Co. - DIC. Thailand
6. Spirin -Yunnan Spirin Co. Ltd. China
7. Synergy - DIC. China
Each powder was mixed in an aqueous cream using 12.5% w/w concentration. Each
cream was then decolourised. A uniform lawn of T. rubrum was grown on PDA medium in

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a dish. After 3-4 days of incubation, wells were cut in the PDA medium with a sterile cork
borer. Measured amounts of each cream were placed in a respective well, the dishes were
then incubated for 2-4 days.
Figure 4 shows the antifungal activities of 12.5% w/w Siam Algae Co. (3 o'clock
position), 12.5% w/w Life Stream (6 o'clock position), and 12.5% w/w China Spirulina (9
o'clock position).
The results showed that all commercial powders tested had some antifungal activity.
Respirometry confirmed that all commercial powders tested also had activity (presumably
enzymatic) when incubated with chitin (results not shown).
One powder (Life Stream) was randomly selected for blending with an over-the-counter
aqueous cream at a concentration of 12.5% w/w without further processing and tested for
antifungal activity against Aspergillus niger. The cream produced a clearance of 4 mm, as
seen in Figure 5 at the 12, 3, 6 and 9 o'clock positions. This indicated that possibly all
commercial preparations of dried Arthrospira were likely to have some level of antifungal
activity, without requiring an additional stressing step or farther processing step such as
filament disruption.
Example 6 - Activity of Stressed Arthrospira Against Fungal Diseases of Mammals
The antifungal activity of AMYCOT® was tested in vitro against different fungal
diseases of mammals. The following pathogens were sourced from the Department of
Primary Industries, Department of Agriculture, ACM and nurseries:
● Candida albicans
● Microsporum canis
● Trichophyton mentagrophytes
● Trichophyton rubrum
● Epidermophytonfloccosum
● Fusarium graminearum
● Alternaria sp.
● Malassezia furfur
A uniform lawn of each pathogen was grown on PDA medium. After 1-2 days of
incubation, wells were cut in the PDA medium with a sterile 8 mm diameter cork borer.
Measured amounts of AMYCOT® were placed in each well. The plates were cultured at an
appropriate temperature depending on the pathogen. The welled plates were inspected daily.
The clearance was measured after 2-3 days. Measurement of the zone is taken from the
perimeter of the well to the outside perimeter of the zone.

WO 2006/047830 ' PCT/AU2005/001693
This zone was void of live fungal cells proving efficacy of AMYCOT® against the
target pathogens.
12.5% w/w AMYCOT® cream produced a clearance of 8 mm of the pathogen T.
rubrum. T. rubrum is a causal agent of hoof infections and ring worm.
12.5% w/w AMYCOT® cream produced a clearance of 15 mm of the pathogen T.
mentagrophytes. T. mentagrophytes is a causal agent of hoof infections and ring worm.
Figure 6 shows a well of 12.5% w/w AMYCOT® cream produced a clearance of 3
mm of the pathogen, M. cards. M. canis is a causal agent of hoof infections and ring worm.
12.5% w/w AMYCOT® cream produced a clearance of 3 mm of the pathogen E.
floccosum. E. floccosum is a casual agent of hoof infections and ring worm.
12.5% w/w AMYCOT® cream produced a clearance of 4.5 mm of the pathogen C.
albicans. C. albicans is a causal agent of ring worm and dermatitis.
Figure 7 shows a well of 12.5% w/w AMYCOT® cream produced a clearance of 12
mm of the pathogen F. graminearum. F. graminearum is a causal agent of hoof infections.
Figure 8 shows that wells of 12.5% w/w AMYCOT® cream produced clearances of
M. furfur. M. furfur is a causal agent of dandruff.
Figure 9a shows a well of 5% w/w AMYCOT® shampoo produced a clearance of 3
mm of the pathogen T. rubrum.
Figure 9b shows a well of 5% w/w AMYCOT® shampoo produced a clearance of 4
mm of the pathogen C. albicans.
Example 7 - Activity of Stressed Arthrospira Against Fungal Diseases of Plants
The antifungal activity of AMYCOT® was tested in vitro against different fungal
diseases of plants. The following pathogens were sourced from the Department of Primary
Industries, Department of Agriculture, ACM and nurseries:
● Fusarium graminearum
● Pithomyces Chartarum
● Botrytis cinerea
● Alternaria sp.
A uniform lawn of each pathogen was grown on PDA medium. After 1-2 days of
incubation, wells were cut in the PDA medium with a sterile 8 mm diameter cork borer.
Measured amounts of AMYCOT® were placed in each well. The plates were cultured at an
appropriate temperature depending on the pathogen. The welled plates were inspected daily.
The clearance was measured after 2-3 days. Measurement of the zone was taken
from the perimeter of the well to the outside perimeter of the zone.

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The zone was void of live fungal cells proving efficacy of AMYCOT® against the
target pathogens.
12.5% w/w AMYCOT® cream produced a clearance of 13 mm of the pathogen F
graminearum. F. graminearum is a causal agent of head blight and crown rot in small grain.
Figure 10 shows a well of 12.5% w/w AMYCOT® cream produced a clearance of 10
mm of the pathogen P. chartarum. P. chartarum is a causal agent of facial eczema in sheep
cattle.
12.5% w/w AMYCOT® cream produced a clearance of 8 mm of the pathogen B.
cinerea. B. cinerea is a causal agent of botrytis in fruit.
12.5% w/w AMYCOT® cream produced a clearance of 9 mm of the pathogen
Alternaria sp, the casual agent of leaf spot in fruit.
Example 8 - Testing of AMYCOT® as a Spray on Plant Fungal Pathogens
AMYCOT® in the form of an antifungal spray was tested in vitro against a common
plant pathogen, Botrytis cinerea. 5% w/w AMYCOT® powder was hydrated with water, but
was only sparingly soluble.
A uniform lawn of B. cinerea was grown on PDA medium in a dish. The right-hand
side of the dish was then sprayed with the spray and allowed to sit at room temperature for
24 hours. The left-hand side of the dish was not sprayed. Figure 11 shows that the sprayed
side of the dish had dead fungi. The spray was effective in eliminating botrytis and
inhibiting further sporulation.
Example 9 -In Vitro Antimicrobial Tests with Stressed Arthrospira and Monographed
Actives
The antifungal and antibacterial activities of various over-the-counter fungal and
antibacterial creams (including Lamisil™, Dakta Gold™, Canesten™, Tinaderm™,
Daktarin™, Tripod Labs™, Resolve Tinea™, Resolve Balm™, Resolve Plus™, Clearasil™,
Benzac W™) and AMYCOT® were tested in this and other examples.
In this example, the antifungal and antibacterial activities of various over-the-counter
creams and AMYCOT® were tested by an independent laboratory (ConMac Laboratory
Services) against the target pathogens Trichophyton mentagrophytes, Epidermophyton
floccosum, Trichophyton rubrum andPropionibacterium acne.
The results are shown in Table 6.

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The results show that only AMYCOT® was active against each pathogen. The only

Example 10 - In Vitro Tests with Monographed Actives and Combinations with
AMYCOT®
Antifungals such as miconazole, tolnaftate, bifonazole and clotrimazole work by
inhibiting specific protein production, preventing normal functions such as reproduction.
The antifungal terbinafine works by preventing the formation of vital sterols.
The first aim of these in vitro tests was to determine the efficacies of various over-
the-counter creams or their actives as fungal inhibitors and to compare the efficacy of
AMYCOT®. The second aim was to determine the efficacy of AMYCOT® in combination
with one or more of the over-the-counter creams or their actives as fungal inhibitors.
Sabouraud's liquid medium was inoculated with the chosen pathogen. The broths
were then incubated for one week at 28°C. A sterile swab was dipped into a broth of the

WO 2006/047830 PCT/AU2005/001693
pathogen, and a PDA medium plate was inoculated by streaking the plate at right angles,
insuring that the plate had an even lawn.
Monographed actives not already in a cream base were mixed with a respective
cream base. A select cream was placed onto a 10 mm diameter filter paper disc and the disc
was placed onto a newly inoculated plate. Up to four different discs were placed on a plate.
The plate was then incubated for 24-48 hours at the appropriate temperature depending on
the pathogen. Photos were then taken at the completion of the 24-48 hours.
The inhibitory test results are shown in Figures 12-21 and in Tables 7-16 below.


WO 2006/047830 ^ PCT/AU2005/001693


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The monographed actives in Figure 21 are in an aqueous cream base as opposed to a
spirit base which is normally used.
In the in vitro tests miconazole and clotrimazole were seen as being the most
effective inhibitors of pathological fungi, (see Figures 12-21). Figures 16 and 17 and Tables
11 and 12 establish that miconazole is an effective fungal inhibitor. AMYCOT® also acts as
an inhibitor, but is only approximately 40% as effective as clotrimazole and miconazole (see
Figures 12-14). Figures 18-21 and Tables 13-16 suggest that the combinations of
AMYCOT® plus miconazole and AMYCOT® plus clotrimazole are the most effective
fungal inhibitors in vitro when compared to terbinafine and tolnaftate.
The third aim of these in vitro tests was to determine the efficacies of various over-
the-counter creams or their actives as fungicides and to compare the efficacy of
AMYCOT®. The fourth aim was to determine the efficacy of AMYCOT® in combination
with monographed actives.
The fungicidal test involved growing a lawn of the pathogen on PDA medium plates
over a 2-3 day period and then exposing the mature pathogen to the antirungals. This was
done by cutting wells into the lawned agar, and removing the agar and filling the wells with
cream.
The fungicidal test results are shown in Figures 22-28 and Tables 17-23 below.


WO 2006/047830 ^ PCT/AU2005/001693


2$>
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The monographed actives in Figure 28 are in an aqueous cream base as opposed to a
spirit base which is normally used.
The results support that the present over-the-counter antifungals and monographed
actives are fungi-static agents and not fungicides. Figures 22-28 and Tables 17-23
demonstrate that AMYCOT® is an efficacious fungicide, even when combined with
monographed actives.
The combination of AMYCOT® with either miconazole or clotrimazole did not
greatly reduce the efficacies of either. The combination increased the inhibitory effect (see
Figures 17-21). This created a very effective broad spectrum product.
The combination of AMYCOT® with clotrimazole slightly inhibited the antifungal
activity of AMYCOT® (see Figures 22-28), but the combination of AMYCOT® with
miconazole had no great detrimental effects on the inhibitory or the fungicidal activity of the
combination cream (see Figures 17-26).
The combination cream of AMYCOT® and miconazole was more efficacious as a
broad spectrum product than either due to the inhibitory nature of miconazole and both the
inhibitory and fungicidal activities of AMYCOT®.
The combination cream acted as an inhibitor and then "held its ground", while
clearances achieved with miconazole alone became encroached by regrowth as in the case of
the pathogen T. mentagrophytes (see Figure 20).
It was also found that the combination cream AMYCOT® and 2% miconazole
restricted regrowth of the pathogen after a clearance was made around the wells.
Example 11 - Trials of Arthrospira on Various Skin Conditions
A subject had advanced athlete's foot, with deep lesions between the toes. The
subject reported that itching caused by the fungus stopped within 15 minutes of first
applying 12.5% w/w AMYCOT® cream. Inflammation reduced and in some cases
disappeared completely in 12 hours. The lesions began closing within 24 hours and
completely healed within 4-5 days.
These observations were repeated in the case of acne with subjects recording that the
associated inflammation disappeared and that their skin returned to normal colour and
became soft and pliable like new skin. The pustules also dried out
It is the belief of the inventors that one or more biocidal agents (eg. chitinase,
chitosanase, chitin deacetylase) interact synergistically with other components of
Arthrospira to give rise to the therapeutic effects. The itching in fungal infections is an

WO 2006/047830 PCT/AU2005/001693
indication of the release of digestive enzymes or metabolites from the fungi. Once normal
fungal metabolism is disrupted as the cell wall is disrupted, itching ceases.
Similar results were obtained when using the cream for conditions unrelated to
fungus, on dry and cracked heels and elbows, rosacea, eczema, sun damaged skin and
psoriasis. Subjects reported almost immediate relief from itching in dermatitis.
Example 12 - Antifungal Activity of Stressed Arthrospira Using "Unprocessed" Powder
Example 5 supported that commercial preparations of dried Arthrospira were likely
to have some level of antifungal activity without requiring an additional stressing step or
post-harvest processing step such as filament disruption. Example 12 further supports this
finding.
A dried preparation of Arthrospira maxima was sourced from a commercial grower.
The preparation was blended with an over-the-counter aqueous cream at a concentration of
12.5% w/w without further processing, and was tested for antifungal activity against T.
mentagrophytes, T. rubrum and M.fructicola (as described in Example 5).
As seen in Figure 31, the cream produced a clearance against T. mentagrophytes at
the 12,3,6 and 9 o'clock positions.
As seen in Figure 32, the cream produced a clearance against 71 rubrum at the 6
o'clock position.
As seen in Figure 33, the cream produced a clearance against M. fructicola at the 6
o'clock position.
These findings support that post-harvest steps such as explosive decompression, dry
freezing, decolourisation and milling need not be carried out in order to produce Arthrospira
having fungicidal activity.
Example 13 - Antifungal Activity of Arthrospira Not Explosively Decompressed
This example further supports that the post-harvesting step of explosive
decompression (potentiation) is not required in order to produce Arthrospira having
fungicidal activity.
A dry preparation of Arthrospira maxima was sourced from a commercial grower. In
one instance, dried Arthrospira was disrupted by explosive decompression (potentiated)
using the method described in New Zealand Patents No. 328013 and No. 328740. The
Arthrospira was re-hydrated, decolourised, dried and milled to form a dry "premix powder".
The premix powder was then suspended in a suitable carrier (12.5%) and will be referred to
as "potentiated AMYCOT®".

WO 2006/047830 2& PCT/AU2005/001693
In another instance, the preparation excluded the explosive decompression method
described in New Zealand Patents No. 328013 and No. 328740, and this preparation will be
referred to as "original AMYCOT®".
As seen in Figure 34, both "original AMYCOT®" and "potentiated AMYCOT®"
produced almost equal clearances against Alternaria sp. at the 12 and 7 o'clock positions,
respectively. Hence, explosive decompression is not an essential post-harvest step for
producing Arthrospira having fungicidal activity.
It will be further appreciated that many changes can be made to the compositions,
methods of use and preparation exemplified above without departing from the broad ambit
and scope of the invention.
The term "comprise" and variants of the term such as "comprises" or "comprising"
are used herein to denote the inclusion of a stated integer or stated integers but not to exclude
any other integer or any other integers, unless in the context or usage an exclusive
interpretation of the term is required.
Any reference to publications cited herein is not an admission that the disclosures
constitute common general knowledge in Australia.

PCT7AU2005/001693
Received 9 February 2007
29
CLAIMS
1. A method for preparing an Arthrospira-based composition for topical use on
a subject, said method comprising the steps of:
(1) physiologically stressing Arthrospira but not freeze drying the Arthrospira; and
(2) combining a fungicidally or bactericidally effective amount of the stressed Arthrospira of
(1) with a carrier, solvent, base or excipient suitable for topical administration to the subject.

2. An Arthrospira-based composition when prepared by the method of claim 1.
3. A method for preparing an Arthrospira-based composition for topical use as a
fungicide or bactericide on a subject, said method comprising the steps of:

(1) physiologically stressing Arthrospira by removing up to about 80% of liquid within
which the Arthrospira is being grown;
(2) washing the stressed Arthrospira in order to remove contaminants;
(3) drying the washed Arthrospira; and
(4) combining a fungicidally or bactericidally effective amount of the washed Arthrospira
with a carrier, solvent, base or excipient suitable for topical administration to the subject,
wherein the Arthrospira has not been freeze dried.

4. A topical Arthrospira-based composition when prepared by the method of
claim 3.
5. A topical fungicidal composition comprising a fungicidally effective
amount of physiologically stressed Arthrospira and a carrier, solvent, base or excipient
suitable for topical administration to the subject, wherein the Arthrospira has not been freeze
dried.
6. A method for preventing or treating a topical fungal infection or
infestation of a subject, said method comprising the step of topically administering
to the subject a fungicidally effective amount of a composition comprising
physiologically stressed Arthrospira and a carrier, solvent, base or excipient suitable for
topical administration to the subject, wherein the Arthrospira has not been freeze dried.
7. The method of claim 6 further comprising the step of initially
identifying a subject in need of such treatment or requiring such prevention.
8. The method of claim 6, wherein the subject is a human, an animal, an
agricultural or horticultural product, soil or a man-made structure.
9. A method for preparing a topical fungicidal composition, said method
comprising the steps of:
(1) physiologically stressing Arthrospira; and
Amended Sheet
IPEA/AU

PCT/AU2005/00165
Received 9 February 20C
30
(2) combining a fungicidally effective amount of the stressed Arthrospira of (1) with a
carrier, solvent, base or excipient suitable for topical administration, to a subject, wherein the
Arthrospira has not been freeze dried.
10. A topical fungicidal composition comprising a synergistic combination of a
fungicidally effective amount of physiologically stressed Arthrospira and at least one fungi-
static agent and a carrier, solvent, base or excipient suitable for topical administration to a
subject, wherein the Arthrospira has not been freeze dried
11. The topical fungicidal composition of claim 10, wherein the fungi-static
agent is selected from the group consisting of terbinafine, bifonazole, clotrimazole,
miconazole, econazole, ketoconazole and tolnaftate.
12. A method for preventing or treating a topical fungal infection or
infestation of a subject, said method comprising the step of administering topically
to the subject a composition comprising a fungicidally effective amount of
physiologically stressed Arthrospira and at least one fungi-static agent, and a carrier,
solvent, base or excipient suitable for topical administration to the subject, wherein the
Arthrospira has not been freeze dried.
13. The method of claim 12, wherein the fungi-static agent is selected from the
group consisting of terbinafine, bifonazole, clotrimazole, miconazole, econazole,
ketoconazole and tolnaftate.
14. A method for preparing a topical fungicidal composition, said method
comprising the steps of:

(1) physiologically stressing Arthrospira; and
(2) combining a fungicidally effective amount of the stressed Arthrospira of (1) with at least
one fungi-static agent and a carrier, solvent, base or excipient suitable for topical
administration to a subject, wherein the Arthrospira has not been freeze dried.

15. A topical bactericidal composition comprising a bactericidally effective
amount of physiologically stressed Arthrospira and a carrier, solvent, base or excipient
suitable for topical administration to a subject, wherein the Arthrospira has not been freeze
dried
16. A method for preventing or treating a topical bacterial infection or
infestation of a subject, said method comprising the step of topically administering
to the subject a composition comprising a bactericidally effective amount of
physiologically stressed Arthrospira and a carrier, solvent, base or excipient suitable for
topical administration to the subject, wherein the Arthrospira has not been freeze dried
Amended Sheet
EPEA/AU

PCT/AU2005/001693
Received 9 February 2007
31
17. The method of claim 16 further comprising the step of initially
identifying a subject in need of such treatment or requiring such prevention.
18. The method of claim 16, wherein the bacterial infection is caused by
Propionibacterium acne.
19. The method of claim 16, wherein the subject is human.
20. A method for preparing a topical bactericidal composition, said method
comprising the steps of:

(1) physiologically stressing Arthrospira; and
(2) combining a bactericidally effective amount of the stressed Arthrospira of (1) with a
carrier, solvent, base or excipient suitable for topical administration to a subject, wherein the
Arthrospira has not been freeze dried.

21. A topical composition for repairing or preventing a defect of the skin of
a mammal, said composition comprising a bactericidally or fungicidally effective amount
of physiologically stressed Arthrospira and a carrier, solvent, base or excipient suitable
for topical administration to the mammal, wherein the Arthrospira has not been freeze dried.
22. A method of repairing or preventing a defect of the skin of a mammal,
said method comprising the step of topically administering to the mammal a
composition comprising a bactericidally or fungicidally effective amount of
physiologically stressed Arthrospira and a carrier, solvent, base or excipient suitable for
topical administration to the mammal, wherein the Arthrospira has not been freeze dried.
23. The method of claim 22 further comprising the step of initially
identifying a mammal in need of such treatment or requiring such prevention.
24. The method of claim 22, wherein the skin defect is selected from the group
consisting of a pit, acne damage, rosacea, a reddened area, a crack, a burn, a blister, psoriasis,
eczema, scaling, wrinkles, a papule, a stomatitis, a lesion, a pustule, a wound, cradle cap,
diaper rash, an ulcer, a cold sore, shaving rash, chicken pox, dermatitis, and cracked heels and
elbows.
25. The method of claim 1, wherein the Arthrospira is A. maxima.

The present invention concerns a composition comprising physiologically
stressed Arthrospira maxima for use as a biocide and/or therapeutic. The invention
also concerns a method for preventing or treating an infection or infestation of a
subject by an organism, wherein the method comprises the step of administering to
the subject an effective amount of a composition comprising physiologically stressed
Arthrospira.

Documents:

02006-kolnp-2007-abstract.pdf

02006-kolnp-2007-claims.pdf

02006-kolnp-2007-correspondence others 1.1.pdf

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02006-kolnp-2007-form 1.pdf

02006-kolnp-2007-form 2.pdf

02006-kolnp-2007-form 3.pdf

02006-kolnp-2007-form 5.pdf

02006-kolnp-2007-international exm report.pdf

02006-kolnp-2007-international publication.pdf

02006-kolnp-2007-international search report.pdf

02006-kolnp-2007-pct request form.pdf

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2006-KOLNP-2007-(05-06-2013)-ANNEXURE TO FORM 3.pdf

2006-KOLNP-2007-(05-06-2013)-CORRESPONDENCE.pdf

2006-KOLNP-2007-(07-12-2012)-ANNEXURE TO FORM 3.pdf

2006-KOLNP-2007-(07-12-2012)-CORRESPONDENCE.pdf

2006-KOLNP-2007-(08-11-2011)-ABSTRACT.pdf

2006-KOLNP-2007-(08-11-2011)-CLAIMS.pdf

2006-KOLNP-2007-(08-11-2011)-DESCRIPTION (COMPLETE).pdf

2006-KOLNP-2007-(08-11-2011)-DRAWINGS.pdf

2006-KOLNP-2007-(08-11-2011)-EXAMINATION REPORT REPLY RECIEVED.pdf

2006-KOLNP-2007-(08-11-2011)-FORM 1.pdf

2006-KOLNP-2007-(08-11-2011)-FORM 2.pdf

2006-KOLNP-2007-(08-11-2011)-FORM 3.pdf

2006-KOLNP-2007-(08-11-2011)-FORM 5.pdf

2006-KOLNP-2007-(08-11-2011)-OTHERS PCT FORM.pdf

2006-KOLNP-2007-(08-11-2011)-OTHERS.pdf

2006-KOLNP-2007-(09-12-2013)-ANNEXURE TO FORM 3.pdf

2006-KOLNP-2007-(09-12-2013)-CORRESPONDENCE.pdf

2006-KOLNP-2007-(22-01-2015)-ANNEXURE TO FORM 3.pdf

2006-KOLNP-2007-(22-01-2015)-ASSIGNMENT.pdf

2006-KOLNP-2007-(22-01-2015)-CLAIMS.pdf

2006-KOLNP-2007-(22-01-2015)-CORRESPONDENCE.pdf

2006-KOLNP-2007-(22-01-2015)-DESCRIPTION (COMPLETE).pdf

2006-KOLNP-2007-(22-01-2015)-FORM-1.pdf

2006-KOLNP-2007-(22-01-2015)-FORM-2.pdf

2006-KOLNP-2007-(22-01-2015)-FORM-5.pdf

2006-KOLNP-2007-(22-01-2015)-PETITION UNDER RULE 137.pdf

2006-KOLNP-2007-(23-12-2011)-CORRESPONDENCE.pdf

2006-KOLNP-2007-(23-12-2011)-OTHER PATENT DOCUMENT.pdf

2006-KOLNP-2007-(27-07-2012)-ANNEXURE TO FORM 3.pdf

2006-KOLNP-2007-(27-07-2012)-CORRESPONDENCE.pdf

2006-KOLNP-2007-CORRESPONDENCE 1.1.pdf

2006-KOLNP-2007-CORRESPONDENCE 1.2.pdf

2006-KOLNP-2007-CORRESPONDENCE OTHERS-1.2.pdf

2006-KOLNP-2007-FORM 3 1.1.pdf

2006-kolnp-2007-form-18.pdf

2006-KOLNP-2007-PA.pdf

2006-KOLNP-2007-PETITION UNDER SECTION 8(1).pdf


Patent Number 265625
Indian Patent Application Number 2006/KOLNP/2007
PG Journal Number 10/2015
Publication Date 06-Mar-2015
Grant Date 02-Mar-2015
Date of Filing 04-Jun-2007
Name of Patentee BIOVITE AUSTRALIA PTY LTD.
Applicant Address 1/45-47 TOWNSHIP DRIVE, WEST BURLEIGH, QUEENSLAND
Inventors:
# Inventor's Name Inventor's Address
1 DUNCAN, KELVIN WINSTON 28B LODGE PLACE, AVON-HEAD, CHRISTCHURCH
2 BROWN, ASHLEY MICHAEL 1/17 LEONARD AVENUE, SURFERS PARADISE, QUEENSLAND 4217
3 JOHNSTON, PETER OWEN 44 MEADOW PEAK, 85 PALM MEADOWS DRIVE, CARRARA, QUEENSLAND 4211
PCT International Classification Number C12N 1/00, 9/36
PCT International Application Number PCT/AU2005/001693
PCT International Filing date 2005-11-03
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2004906317 2004-11-03 Australia