Note: Descriptions are shown in the official language in which they were submitted.
WO 95tlO283 ~ 1 ~7 o ~ PCT/AU94/00619
"TREATMENT OF MEDICAL DISORDERS ASSOCIATED
WITH FREE iRADlCAL FORMATION"
5 FIELD OF THE INVENTION
The present invention reiates to the treatment of medical disorders arising
from biochemical and celluiar damage resulting from the action of free radicals
in animals, including humans.
BACKGROUND OF THE INVENTION
A free radical is any species capable of independent existence that
contains one or more unpaired electrons. Such species show paramagnetism
15 and may be highly reactive. The formation and reactions of free radicals havebeen reviewed generally by Halliwell and Gutteridge (1984), while Slater t1984)
has provided a discussion of cell injuries that are mediated by free radical
intermediates, as well as general concepts relevant to free radical processes and
to rnechanisms of protection against such types of injury.
Some biological processes generate more or less stable intermediates that
contain an unpaired electron, which can either be donated, or paired with an
additional electron from the surroundings. Such intermediates are called free
radicals, and they may be the products of various enzymatic and non-enzymatic
25 reactions, some of which are vital for body functions, e.g. reduction of
ribonucleoside diphosphates for DNA synthesis and the generation of
prostaglandins in the prostaglandin synthase reaction. The latter is essential for
inflammatory response following cell injury, and a number of other functions.
Most organisms contain chemical antioxidants such as a-tocopherol (vitamin E),
30 ascorbic acid and different radical and peroxide-inactivating enzymes, e.g.
superoxide dismutase, catalase and glutathione peroxidase, which act as
endogenous protecting agents against free radical damage.
WO 95/10283 59 ~ - 2 - PCTIAU94/00619
Free radicals of various types are becoming increasingly associated with
a broad range of conditions and diseases such as ischaemic or reperfusion injury,
atherosclerosis, thrombosis and embolism, allergic/inflammatory conditions such
as bronchial asthma, rheumatoid arthritis, conditions related to Alzheimer's
5 disease, parkinson's disease and ageing, cataract, diabetes, neoplasms and
toxicity of anti-neoplastic or immunosuppressive agents and chemicals. One
possible explanation for these conditions and diseases is that, for unknown
reasons, the endogenous protecting agents against radical damage are not
sufficiently active to protect the tissue against radical damage. Lipid peroxidation
10 caused by excess generation of radicals may constitute one significant damaging
pathway in the above conditions and diseases. Administration of additional
antioxidants, which inhibit radical reactions, e.g. Iipid peroxidation, would thus
provide a way of preventing or curing the above conditions and diseases.
Free radicals cause cellular damage by different mechanisms such as
oxidation of thiol groups of proteins, DNA strand breaks and lipid peroxidation.The properties of free radicals include: high reactivity with a consequent
extremely short life span, self perpetuating and diverse chemical reactivity, low
chemical specificity and the ability to be generated both in vivo and in vitro.
The main sources of free radicals are cellular, and environmental. Free
radical production may also be increased during vascular disease states, e.g.
inflammation and during unfavourable metabolic conditions such as hypoxia and
tissue ischaemia. Free radicals have also been proposed to cause oxidative
25 damage to biological molecules involved in the development of many severe
disorders of humans, including atherosclerosis, cancer and parasitic viral
infections.
Free radical molecular species include, but are by no means limited to,
30 hydroxyl, peroxyl, hypochlorite, superoxide and alkoxy radicals, and reactivemolecules such as hydrogen peroxide. They also include singlet oxygens which
WO 95tlO283 9 ~ PCT/AU94/00619
- 3 -
are not free radicals but are certainly reactive and capable of causing cellulardarnage.
As a result of the relative instability of free radicals and their potential to
5 damage cells and tissues, there are both enzymes and small-molecular-weight
molecules with antioxidant capabilities that are able to protect against the adverse
effects of free radical reactions. There is therefore a critical balance between free
radical generation and antioxidant defences in cells and tissues. Two types of
antioxidants can be identified, namely biological and non-enzymatic biological
10 antioxidants. In the former class are the well defined enzymes such as
superoxide dismutase, catalase and selenium glutathione peroxidase. Non-
enzymatic biological antioxidants include tocopherols, carotenoids, bilirubin,
ascorbic acid, uric acid and metal-binding proteins as typical examples.
The damaging effects of sunlight on the skin are well known. In addition
to the major short term hazard of erythema (i.e. sunburn) primarily caused by UVradiation, there are also long term hazards associated with UV radiation exposure
including malignant changes in the skin surface (i.e. skin cancer) and prematureageing of the skin.
In International Patent Application No. PCT/US91/02400 (WO 91/16035)
it is pointed out that ultraviolet light induces inflammation of the skin and harmful
photochemical reactions therein. During exposure and as repair of the UV
damage takes place, super-oxide (2-) radicals are formed in the skin. UV
25 irradiation also causes some microvascular damage in the skin, leading to local
haemorrhage and "leakage" of blood cells into the dermis Iron from the
haemoglobin accumulates in the extra-cellular matrix of the tissue as Fe+2 and
Fe+3. It is known that iron catalytically participates in the conversion of
superoxide radicals to hydroxyl radicais, a species which is known to be very
30 damaging to tissue. Another process which is damaging to tissue is membrane
lipid peroxidation, which is also accelerated by iron. (See Halliwell and
Gutteridge, 1985.)
59 ~ PCT/AU94/00619
In tissue injured by physical means, infection, or a toxic compound, cell
destruction may occur accompanied by the release of iron "transit pools". This
free iron can then be used to shown to react with H2O2 to form hydroxyl free
radicals (OH') according to the Fenton reaction, since Fe2' has been shown to
react with H2O2 in vivo, as follows:
Fe2+ + 2 ~ Fe2+ - 2 + Fe3+ + 2-
2O2- + 2H+ H2O2 + 2
H2O2 + Fe2+, OH' + OH' + Fe3+ (Fenton reaction)
(Halliwell et al. 1986)
In addition to their role in UV radiation induced tissue damage, oxygen
radicals are known to be capable of reversibly or irreversibly damaging
compounds of all biochemical classes, including nucleic acids, proteins and free15 amino acids, lipids and lipoproteins, carbohydrates, and connective tissue
macromolecules. These compounds may have an impact on such cell activities
as membrane function, metabolism, and gene expression. (See Halliwell et al.
1985, 1986.)
Clinical conditions in which oxygen radicals are thought to be involved
include those concerning multiorgan involvement, including inflammatory-immune
injury such as glomerulonephritis (idiopathic, membranous), vasculitis (hepatitis
B virus, drugs), autoimmune disease; ischaemia-reflow states; drug and toxin-
induced reactions; iron overload such as idiopathic haemochromatosis, dietary
iron overload (red wine, beer brewed in iron pots), th~i~ssemia and other chronic
anaemias; nutritional deficiencies, such as Kwashiorkor, vitamin E deficiency;
alcohol; radiation injury; ageing, such as disorders of "premature ageing",
immune deficiency of age; cancer and amyloid diseases. Additional conditions
in which oxygen radicals are thought to be involved include those concerning
primary single organ involvement including erythrocyte related conditions, such
as phenylhydrazine, primaquine, lead poisoning, protoporphyrin photo-oxidation,
malaria, sickle-cell anaemia, fauvism, Fanconi anaemia; lung related conditions
w095~l02g3 2 1 705 9 ~ PCTIAU94/00619
- 5 -
such as cigarette-smoking effects, emphysema, hyperoxia, bronchopulmonary
dysplasia, oxidant pollutantsS acute respiratory distress syndrome, mineral dustpneumoconiosis, bleomycin toxicity, paraquat toxicity; heart and cardiovascular
system related conditions, such as alcohol cardiomyopathy, Keshan disease
5 (selenium deficiency), atherosclerosis, doxorubicin toxicity; kidney related
conditions, such as nephrotic antiglomeruiar basement membrane disease,
aminoglycoside nephrotoxicity, heavy metal nephrotoxicity, renal grant rejection;
gastrointestinal tract related conditions, such as endotoxin liver injury, carbon
tetrachloride liver injury, diabetogenic action of alloxan, free-fatty-acid-induced
10 pancreatitis, nonsteroidal-anti-inflammatory-drug induced lesions; joint
abnormalities, such as rheumatoid arthritis; brain related conditions, such as
hyperbaric oxygen, neurotoxins, senile dementia, Parkinson disease-MPTP,
hypertensive cerebrovascular injury, cerebral trauma, neuronal ceroid
lipofuscinoses, allergic encephalomyelitis and other demyelinating diseases,
15 ataxia-telangiectasia syndrome, potentiation of traumatic injury, aluminium
overload, a-~B-lipoproteinaemia; eye related conditions, such as solar radiation,
thermal injury, porphyria, contact dermatitis, photosensitive dyes, and bloom
syndrome. (See Cross et al. , 1987).
Free radicals are therefore potentially dangerous in living systems, and it
is biologically advantageous for cells to control the amount of radicals allowed to
accumulate. The chelating effect of iron bound to protein, such as in transferring,
renders the iron almost completely inactive in accelerating lipid peroxidation and
hydroxyl radical formation. Other protective mechanisms include catalase, which
25 exists to remove H202 within cells, converting it to H20. Glutathione peroxidase
also reduces H202 to H20 through the involvement of glutathione, itself an
antioxidant that may quench radicals, as do other molecules such as ascorbate,
urate, glucose and metallothioniens (Halliwell et al., 1986).
In view of the potential danger of free radicals in living systems, a great
deal of attention has been given to compounds which act as free radical
scavengers, the use of which will regulate biochemical and cellular damage
WO 95/10283 2 ~ 7 5 9 ~ PCT/AU94/00619
- 6 -
resulting from free radical reactions in living systems (see, for example,
International Patent Applications No. PCT/US91/02400 above, and No.
PCT/US91/02398 - WO/9116034, both of which disclose chelator compositions
for use as free radical scavengers, particularly in protecting against photodamage
5 of the skin).
SUMMARY OF THE INVENTION
It has now been found that compounds within the class of bile alcohols and
10 their derivatives are effective free radical scavengers, particularly hydroxyl radical
scavengers.
The bile alcohols are a group of polyhydroxylated steroids derived
structurally from 5a-cholestane or 5,~-cholestane (also known as coprostane).
15 They include, for example, the compounds known as 5~-scymnol (3a, 7a, 12a,
24, 26, 27-hexahydroxy-5,~-cholestane) and 5a-cyprinol (3a, 7a, 12a, 26, 27-
pentahydroxy-5a-cholestane).
Scymnol is an unusual bile alcohol in that the terminal methyl groups, C26
20 and C27 together with C24, are present as alcohols. The natural bile salt occurs
as the 27-sulphate sodium salt and was first isolated by Hammerstein in 1898
from the gall bladders of the shark Scymnus borealis .
OH OH
OH i~ H OH if ~A~ CH2CH
r'~~~ CH20H ~ CH20SO,H
30HO ~ ' ~U HO ~ ~J .~
5~-Scymnol 5~-Scymnol sulphate
WO95/10283 2 ~ 70 S ~ 1 PCT/AU9~/00619
- 7 -
According to the present invention, there is provided a method for the
treatment of biochemical damage or a medical disorder associated with free
radical formation in a patient which comprises administering to the patient an
effective amount of a bile alcohol or a derivative thereof.
The present invention also extends to a composition for use in the
treatment of biochemical damage or a medical disorder associated with free
radical formation in a patient, which comprises an effective amount of a bile
alcohol or a derivative thereof, together with a pharmaceutically acceptable carrier
10 or diluent.
Whilst the present invention is principally directed to the treatment of
human patients, it is to be understood that in its broadest aspect the inventionalso extends to the treatment of non-human patients, especially non-human
15 mammals.
The medical disorders associated with free radical formation which may be
treated in accordance with the present invention include those described above
arising from biochemical damage to nucleic acids, proteins and free amino acids,20 lipids and lipoproteins, carbohydrates and connective tissue macromolecules.
They include, in particular, the results of photodamage to the skin following UVirradiation so that in one particular aspect, the present invention includes a
method and composition as described above for the treatment of photodamage
to the skin and/or hair of a patient associated with free radical formation resulting
25 from UV irradiation.
Inflammatory disorders of the skin, including erythema associated with
exposure to solar or ultraviolet radiation, sunburn, eczema, dermatoses, inflamed
lesions of acne and other inflammatory skin conditions, may also be associated
30 with free radical formation, and accordingly, may be treated in accordance with
the present invention.
PCT/~U94/006 19
WO95/10283 ~ q ,~ - 8 -
Accordingly, in yet another aspect, the present invention includes a method
and a composition as described above for the treatment of inflammatory disordersor conditions of the skin of a patient.
Throughout this specification and the claims which follow, unless the
context requires otherwise, the word "comprise", or variations such as
"comprises" or "comprising", will be understood to imply the inclusion of a stated
integer or group of integers but not the exclusion of any other integer or group of
integers.
DETAILED DESCRIPTION OF THE INVENTION
The bile alcohols which may be used in the method or composition of this
invention include any of the group of polyhydroxylated steroids derived structurally
15 from 5a-cholestane or 5a-cholestane (or coprostane).
Preferred bile alcohols are compounds in this group which include at least
one hydroxyl group, preferably two or three hydroxyl groups, at positions 24, 25,
26 and/or 27 of the cholestane molecule. Most preferred are bile alcohols with
hydroxyl groups at positions 26 and 27 of the cholestane molecule.
Particularly preferred bile alcohols for use in accordance with the present
invention include:
5a-scymnol (3a, 7a, 12a, 24R, 26, 27-hexahydroxy-5a-cholestane)
5,~-scymnol (3a, 7a, 12a, 24R, 26, 27-hexahydroxy-5a-cholestane)
5a-cyprinol (3a, 7a, 12a, 26, 27-pentahydroxy-5a-cholestane)
5,~-cyprinol (3a, 7a, 12a, 26, 27-pentahydroxy-5,~-cholestane)
5a-bufol (3a, 7a, 12a, 25, 26-pentahydroxy-5a-cholestane)
5~B-bufol (3a, 7a, 12a, 25, 26-pentahydroxy-5,~-cholestane)
5a-chimaerol (3a, 7a, 12a, 24, 26-pentahydroxy-5a-cholestane)
5,~-chimaerol (3a, 7a, 12a, 24, 26-pentahydroxy-5~B-cholestane)
5a-ranol (3a, 7a, 12a, 24R, 26-pentahydroxy-27-nor-5a-cholestane)
2 1 7 0 5 9 1 PCT/AU9~/00619
WO 95/102~3
_ 9
5~-ranol (3a, 7~, 12a, 24R, 26-pentahydroxy-27-nor-5,a-cholestane)
5a-dermophol (3a, 7a, 12a, 25, 26, 27-hexahydroxy-5a-cholestane)
5,~-dermophol (3a, 7a, 12a, 25, 26, 27-hexahydroxy-5,~-cholestane).
The derivatives of bile alcohols which may be used in accordance with the
present invention include esters with inorganic acids such as sulphuric acid or
organic acids such as acetic acid, propionic acid and butyric acid.
Particularly preferred bile alcohol derivatives for use in accordance with the
present invention include:
5,~-scymnol sulphate
5a-cyprinol sulphate.
In accordance with this invention, the bile alcohols or derivatives will
15 norrnally be administered orally, rectally, dermally or topically, or by injection, in
the form of pharmaceutical preparations comprising an effective amount of the
active substance in a pharmaceutically acceptable dosage form.
Suitable pharmaceutically acceptable dosage forms are well known and are
20 described, by way of example, in Remington's Pharmaceutical Sciences, 18th
Edition, Mack Publishing Company, Pennsylvania, USA. The dosage form may
be a solid, semisolid or liquid preparation. Usually the active substance will
constitute between 0.1 and 99% by weight of the preparation, more specifically
between 0.5 and 20% by weight for preparations intended for injection and
25 between 0.2 and 50% by weight for preparations suitable for oral administration.
Derrnal or topical administration would normally utilise 0.1-10% by weight, morespecifically 0.5-5% by weight, of the active substance in a suitable dermal or
topical carrier or vehicle.
To produce pharmaceutical preparations containing the active substance
in the form of dosage units for oral application, the selected compound may be
mixed with a solid excipient, e.g. Iactose, saccharose, sorbitol, mannitol, starches
' q '\ PCT/AU94/00619
WO 95/10283 2. ~ 7 ~ 3
- 10 -
such as potato starch, corn starch or amylopectin, cellulose derivatives, a binder
such as gelatine or polyvinylpyrrolidone, and a lubricant such as magnesium
stearate, calcium stearate, polyethylene glycol, waxes, paraffin, and the like, and
then compressed into tablets. If coated tablets are required, the cores, prepared
5 as described above, may be coated with a concentrated sugar solution which maycontain e.g. gum arabic, gelatine, talcum, titanium dioxide, and the like.
Alternatively, the tablet can be coated with a polymer known to the man skilled
in the art, dissolved in a readily volatile organic solvent or mixture of organic
solvents. Dyestuffs may be added to these coatings in order to readily distinguish
10 between tablets containing different active substances or different amounts of the
active substances.
For the preparation of soft gelatine capsules, the active substance may be
admixed with e.g. a vegetable oil or polyethylene glycol. Hard gelatine capsules15 may contain granules of the active substance using either the abovementioned
excipients for tablets e.g. Iactose, saccharose, sorbitol, mannitol, starches (e.g.
potato starch, corn starch or amylopectin), cellulose derivatives or gelatine. Also
liquids or semisolids of the drug can be filled into hard gelatine capsules.
Dosage units for rectal application can be solutions or suspensions or can
be prepared in the form of suppositories comprising the active substance in
admixture with a neutral fatty base, or gelatine rectal capsules comprising the
active substance in admixture with vegetable oil or paraffin oil.
Liquid preparations for oral application may be in the form of syrups or
suspensions, for example solutions containing from about 0.2% to about 20% by
weight of the active substance herein described, the balance being sugar and
mixture of ethanol, water, glycerol and propylene glycol. Optionally, such liquid
preparations may contain colouring agents, flavouring agents, saccharine and
30 carboxymethyl-cellulose as a thickening agent or other excipients known to the
man in the art.
WO 95/102~3 2 1 7 0 5 9 1 PCT/AU94/006 19
.
- 11 -
Preparations for parenteral applications by injection can be prepared in an
aqueous solution or suspension of the active substance, preferably in a
concentration of from about 0.5% to about 10% by weight. These preparations
may also contain stabilising agents and/or buffering agents and may conveniently5 be provided in various dosage unit ampoules.
Where the active substance of this invention is to be used for the treatment
of photodamage to the skin and/or hair of a patient associated with free radicalformation resulting from UV radiation, the active substance is preferably
10 formulated so as to be administered topically. In such formulations, an effective
amount of the active substance is incorporated into a suitable carrier material as
a topical pharmaceutical/cosmetic composition which may be made up in a variety
of product types including, for example, lotions, creams, oils, gels, sticks, sprays,
ointments, pastes, mousses and cosmetics.
Suitable topical pharmaceutical/cosmetic carrier materials for such
compositions are also well known and are described by way of example in
International Patent Application No. PCT/US91/02400. As described therein, in
addition to the active substance and suitable carrier material, such topical
20 pharmaceutical/cosmetic compositions may also include one or more penetrationenhancing agent(s), and/or anti-inflammatory agent(s), as well as sunscreen or
sunblock agent(s) to enhance protection of the skin against the effects of UV
irradiation.
In work leading to the present invention it has been shown that the bile
alcohols, particularly 5~-scymnol and 5a-cyprinol, and their derivatives,
particularly 5,t~-scymnol sulphate and 5a-cyprinol sulphate, are surprisingly
effective free radical scavengers, particularly of hydroxyl radicals.
One test system used to determine the scavenging potential of these
compounds is a simple, non-biological free radical generating and detection
system. It is the frequently used thiobarbituric acid (TBA) reaction, most
PCT/AU9~/006 19
WO 95/10283 ~9! ~ - 12 -
commonly used for the detection of lipid peroxidation in biological systems. Themethod however has been utilised due to its ability to assess iron-dependent free
radical damage to carbohydrates (Gutteridge, 1981 ) and hydroxyl radical
scavenging capabilities of systems (Moorhouse et al., 1987).
Deoxyribose is utilised as the target for hydroxyl radical attack which
breaks down to produce a three carbon compound malondialdehyde (MDA) which
forms a characteristic chromogenic adduct with two molecules of TBA (Gutteridge,1981). The three test compounds used as measures of comparison were the
10 known free radical scavengers, promethazine, mannitol and dimethyl sulphoxide(DMSO). Mannitol has been utilised as a model scavenger widely by Moorhouse
et al. (1987) however its scavenging ability is somewhat less than that of
promethazine, a known hepatoprotector against carbon tetrachloride free radical
attack. Moorhouse et al., (1987) have also found that dimethyl sulphoxide is a
15 potent free radical scavenger. Through the use of these compounds, a qualitative
analysis of the abilities of test compounds as free radical scavengers can be
ascertained.
Further details of these tests, and of the activity of the test compounds as
20 free radical scavengers, are set out in the Examples below.
As noted above, free radicals may be generated on the surface of skin by
the action of the environment, particularly ultraviolet radiation (UVR). In the
absence of a sunscreen there is nothing to attenuate the effect of UVR and so
25 the potential for free radical formation must be highest in the outermost layers of
the skin. Topical application of antioxidants and free radical scavengers has
been shown to provide a wide range of benefits to living skin. In most cases it
has not been shown that the protectant molecule has penetrated to the viable
tissues, but the half life of a free radical is so short that it is diffficult to imagine
30 any other mechanism (notwithstanding that free radicals can set up a chain ofreactions). Oxygen free radicals and other reactive oxygen species have been
shown to be involved in UVR damage to the skin. Although the predominant
WO95/102~3 ~ 1 7 ~5 9 1 PCT/AU94/00619
damage is due to the direct action of UVB on DNA, at wavelengths above 327nm
(UVA) the predominant effect is due to the action of UVR on other molecules,
generating free radicals that then affect DNA. Peroxidation by UVR of the lipid
causes damage to cell membranes at the subcellular level, leading to cell
S damage. The adverse effects of photosensitisation (erythema and oedema) have
been minimised by topical application of free radical scavengers. Free radical
scavengers also suppress the formation of erythema, sunburn cells, peroxidation
of lipids, increased production of ornithine decarboxylase, promotion and initiation
of skin cancer, and premature aging of the skin. It has been reported that
10 antioxidant therapy with Vitamin E produced a favourable effect on facial skin
melanoderma in women; melanoderma is associated with abnormally high
concentrations of lipid peroxide caused by UVR. It has also been shown that
injection of superoxide dismutase, a free radical scavenger that occurs naturally
in the skin, significantly prevented the depletion of Langerhans cells in the skin
15 of guinea pigs that had been exposed to UVR (Langerhans cells are an important
part of the skin's immune system), and that a garlic extract which contains
several antioxidants was able to reduce the immunosuppressive effects of UVR
on hairless mice.
There is a considerable interest in finding a sunscreen for hair. The
benefits that are sought are to prevent loss of colour, particularly on hair which
has been artificially coloured; and to prevent damage (weathering) to the
structure of the hair which leads to split ends, loss of hair cuticles, roughness and
general weakening and loss of condition. These adverse effects are mainly
caused by exposure to UVR and are mediated by free radical formation.
Manufacturers of hair products have tried to use conventional sunscreen actives
(e.g. cinnamates and PABA derivatives) to protect hair, but to be very effectivethey must be deposited in such large amounts that they leave the hair greasy andgenerally in an unnatural state. A free radical scavenger such as a bile alcoholor a derivative thereof may be effective in much smaller amounts, and so not
adversely affect the other hair properties, and may favourably influence the
greasiness of skin and hair. Perception of greasiness is a function of the total
PCT/AU94/00619
WO 95/10283 "1'~ 1 ~5
- 14 -
quantity of sebum and of its viscous properties. The latter are a function of the
degree of crystallinity of the sebum and will be influenced by peroxidative
processes against which free radical scavengers can protect.
Acne is a chronic inflammatory disease of the pilosebaceous units which
is most prevalent in teenage years. It is characterised by the formation of bothinflammatory and non inflammatory lesions which affect primarily the face, neck
and trunk. Four major factors are involved in the pathogenesis: increased sebum
production, an abnormality of the microbial flora, cornification of the
pilosebaceous duct and the production of inflammation. Topical therapy is usually
effective for the management of mild to moderate acne, however patients with
severe acne usually require oral and topical treatment. Benzoyl peroxide is wellestablished as a topical agent in the treatment of acne as it is an effective
keratolytic, comedolytic, antimicrobial and anti-inflammatory agent. It has now
lS been shown that the bile alcohols and derivatives are effective anti-inflammatory
agents when applied topically to the skin.
The following Examples illustrate the free radical scavenging activity of the
bile alcohols and derivatives thereof in accordance with this invention.
EXAMPLE 1
A. METHOD
The method used for this investigation is essentially that described by
Gutteridge (1981). A combination of ascorbic acid (0.1 mM), ferrous ammonium
sulphate (0.22 mM) and EDTA (0.23 mM), added in this order, produced a
hydroxyl radical generating system which after being sparged in nitrogen was
added to a solution of deoxyribose (1.0 mM) in phosphate buffer (20 mM, pH 7.4).
To the test system 120 ,ul of the hydroxyl radical scavenger was added and
30 to the control an equal volume of water. A blank tube was set up for each runcontaining water and scavenger without deoxyribose. Each test and control were
prepared in triplicate.
WO 95/10283 2 1 7 o ~ 9 ~ PCTtAU94/00619
.
- 15 -
Hydroxyl radical attack proceeded for 30 minutes at 37C after which time
the solution pH was reduced by addition of trichloroacetic acid (TCA (1 ml of
2.8% w/v) and TBA (1 ml of 1% w/v in 0.05 M NaOH). Upon heating at low pH
the chromogen was determined by its absorbance at 532 nm.
Ascorbate is almost invariably contaminated with iron salts and is able to
initiate the reaction alone, hence its inclusion in the iron salt solution avoided
premature initiation of the reaction during preincubation. In order to avoid false
results through iron contamination, all glassware used in the procedure was
10 washed with concentrated hydrochloric acid and rinsed twice with milli Q water.
All solutions were also made up with milli Q water.
B. RESULTS
Table 1 shows that 5,~-scymnol gave very high levels of protection against
15 deoxyribose damage as did 5a-cyprinol. The sulphated bile salts were not as
protective, giving levels of protection similar to promethazine and DMSO.
Figure 1 shows the combined inhibition studies from 3 experiments for
the scavengers. Rate constants for 5,~-scymnol and 5,~-scymnol sulphate were
20 determined from their respective slopes from Lineweaver-Burk plots and
calculated to be 0.267 M~1 S~' and 0.195 M-'S-' respectively. The rate constant
for mannitol was determined in the same manner as the bile salts and calculated
to be 0.49 M-'S-'.
At low molecular concentrations, cholic acid appeared to be a very
effective scavenger (Figure 2), intermediate in its effect to that of 5,a-scymnol and
5~-scymnol sulphate. As the scavenger concentration increased however, unlike
the effect shown by 5,a-scymnol, scavenging ability of cholic acid was lost.
In order to determine whether the action of 5,~-scymnol was indeed that of
a radical scavenger and not due to the prior formation of a colourless scymnol-
TBA adduct, 5,~-scymnol was added to the reaction mixture after incubation but
wo 95/10283 ~ 5 9 PCT/AU9~/00619
- 16 -
prior to the addition of TBA. The results of this run replicated that of the control
species indicating that a chromogen was not formed between 5,~-scymnol and
TBA.
5 TABLE 1 Effect of hydroxyl radical scavengers on damage by systems
generating hydroxyl radicals.
Scavenger (10mM) % Inhibition % Inhibition
(with EDTA) (without EDTA)
5,~-scymnol 97 i 2 64 + 5
5,~-scymnol sulphate 72 i 3 42 i 4
5a-cyprinol 86 i 4
5a-cyprinol sulphate 74 i 6
Promethazine 69 i 3
Dimethyl sulphoxide 69 i 1
Mannitol 28 i 2
Cholic acid 57 i 6
The hydroxyl radicals were generated via the Fenton reaction with
deoxyribose used as the target for radical attack. The reaction was run for 30
minutes at 37C. Results are expressed as the average of three experiments
20 S.E.M.
C. DISCUSSION
The system used in this investigation demonstrated the great ability with
which 5,~-scymnol can protect against hydroxyl radical damage to an extent
25 greater than that of mannitol, promethazine and DMSO, which are well known and
effective free radical scavengers.
~ 1 7 PCT/AU94/00619
WO 95/10283 ~ I / 0 5 9 1
- 17-
5,~-Scymnol and 5,a-scymnol sulphate were iess effective in scavenging
hydroxyl radicals when EDTA was removed as a chelating agent from the system.
This indicates the ineffectiveness of the bile salts as chelators and the importance
of chelators in biological systems.
The apparent radical scavenging ability for hydroxyl radicals shown by 5~-
scymnol may be attributed to the hydroxy methyl groups at C26 and C27. The
precursor, cholic acid which does not possess the unique 1,3 diol structure was
assayed for comparison. Cholic acid has a terminal carboxylic acid at C24,
10 therefore it can be suggested that the effectiveness of 5,~-scymnol as a
scavenger is due to the C26 and C27 hydroxy methyl groups, since the remainder
of the molecule has an identical 5,~-steroid structure to that of cholic acid.
The in vifro studies here establish evidence that both 5,~-scymnol and 5,a-
15 scymnol sulphate, and 5a-cyprinol and 5a-cyprinol sulphate, are hydroxy radical
scavengers with significant potential in vivo, with the effectiveness possibly being
attributed to the C26 and C27 hydroxy methyl groups. They both demonstrate
second order rate constants of significant orders of magnitude greater than
mannitol, a known radical scavenger (Moorhouse et al., 1987).
E~CAMPLE 2
The compounds 5,~-scymnol and its 27-sulphate ester have been tested
25 usir~g in vitro systems for their ability to function as a biological antioxidant
against a number of reactive oxygen species (ROS) which are normal byproducts
of rnetabolism and play a major role in free-radical induced toxicity.
The tests were designed to evaluate the antioxidant capacity of the test
30 compounds and to compare their effectiveness with the lipid- and water-soluble
forms of vitamin E as appropriate against the following ROS: hydroxyl,
superoxide, lipid- and water-soluble peroxyl radicals, and hydrogen peroxide.
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Trolox [6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid] is a
hydrophilic analogue of a-tocopherol with antioxidant activity more active than
BHA, BHT, nordihydroguaiaretic acid (NDGA) and propyl gallate (PG).
There is a wide range of ROS which can arise physiologically from a
variety of sources. Compounds such as the bile alcohols and their derivatives
can be tested using in vitro systems for their ability to function as biologicalantioxidants against a number of ROS which are formed naturally in vivo or
following an external event such as injury, infection or the like. The study wasdesigned to evaluate the antioxidant capacit,v of the test compounds and to
compare their efficiencies with lipid- and water-soluble forms of vitamin E and
vitamin C as appropriate against the following ROS:
ROS TEST SYSTEM
15 A Superoxide radical Hypoxanthine/xanthine oxidase production of
urate. Comparison with Trolox and vitamin C.
B Hydroxyl radicals Fenton reaction-induced thiobarbituric acid
reaction products (TBARs) in liposomes.
Comparison with Trolox and vitamin C.
C Hydrogen peroxide Horseradish peroxidase-induced oxidation of
guaiacol by measurement of the guaiacol-
peroxidase reaction.
D Microsomal lipid peroxidation Microsomes were isolated from rat livers
and their peroxidation in the presence of iron
ions and ascorbic acid was measured by
thiobarbituric acid (TBA).
E Peroxyl radical scavenging Theabilityofthecompoundstoscavenge
alkylperoxyl radicals generated in the aqueous
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phase by the controlled thermolysis of 2,2'-azo-
bis-(2-amidinopropane)dihydrochloride(ABAP) .
Table 2 below summarises the observations made concerning the activity
5 of the test compounds in quenching ROS in these test systems.
TABLE 2
Compounds
Test Trolox 5a-Scymnol 5~-Scymnol
sulphate
A Superoxide radicals strong moderate weak
B Hydroxyl radicals strong strong moderate
C Hydrogen peroxide strong moderate- weak
strong
D Microsomal lipid strong moderate weak
peroxidation
E Peroxyl radical strong moderate- weak
scavenging weak
The ~ssessment to quench radicals is Strong +++
Moderate ++
Weak +
These results indicate that in all cases tested, 5~-scymnol exhibits a
moderate ability to act as an antioxidant against the reactive oxygen species
(ROS) which are implicated in free radical induced toxicity.
EXAMPLE 3
The aim of this study was thus to compare the efficacy and skin tolerance
of topical 5,~-scymnol sulphate with 5% benzoyl peroxide in the treatment of mild
to moderate acne.
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METHODS
A total of 70 patients with mild to moderate acne entered this prospective
double blind trial which compared two topical medicaments. Mild to moderate
5 acne was defined as primarily open and closed comedones and some superficial
inflamed lesions (papules not pustules). 44 male and 26 female were included
according to certain criteria, i.e. they were older than 12 years, free from
intercurrent disease, and not taking systemic antibiotics, corticosteroids, retinoids,
anticonvulsants or androgens in the 30 days prior to starting the trial. No topical
10 acne therapy was allowed in the two weeks before the trial. Female patients
were not to have commenced or ceased the oral contraceptive pill in the six
months before the trial, and males were to be without beards and moustaches.
The patients were randomised into two treatment arms; 5% benzoyl peroxide
(n=35) and 5,~-scymnol sulphate (n=35).
5,a-scymnol sulphate (Isolutrol) was supplied as a solution with a
concentration of 0.15gm/100ml (purified by McFarlane Marketing (Aust.) Pty.Ltd.).
The benzoyl peroxide was a commercially available 5% water-based solution. All
treatments were pre-packed in identical numbered packages.
The trial was designed as a double blind study and the patients were
instructed not to describe to the invesligator any characteristics of the product
such as colour, smell or consistency. An initial baseline assessment was carriedout and patients were reassessed at two, four, eight and twelve weeks. The
25 severity of each patient's acne was determined using the "counting technique"described by Burke and Cunliffe (1984). All patient assessments were carried oulby the same investigator.
The parameters used to assess the relative efficacy of each treatment
30 were changes in the number of superficial inflamed lesions, as well as non-
inflamed lesions (open and closed comedones) over the duration of the trial. An
assessment of skin tolerance was also conducted at each review with respect to
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burning and stinging, erythema, scaiing, pruritus and dryness. These were
graded from 0-nil, 1-mild, 2-moderate to 3-severe, and the patient was asked to
comment on any adverse effects experienced.
5 Statistical Analysis.
Reductions in the number of superficially inflamed and non-inflamed
lesions between baseline and final review were statistically analysed using a
paired Student's t-test. The grades assessing skin tolerability for both groups at
each review were analysed using an unpaired Student's t-test. A p value of less
10 than 0.05 was regarded as significant.
RESULTS
The mean age of patients was 18.6 years (range 13-35 years). There was
15 no significant difference between the two study groups with respect to age, sex,
duration of acne, or baseline assessment of facial erythema, pruritus, burning and
stinging, dryness, and scaling.
Baseline counts of superficial inflamed lesions were 23.9i3.0 in the
20 isolutrol group and 33.3+4.9 in the benzoyl peroxide group (mean i SEM) and
there was no stali~lically significant difference between the two groups at
baseline. Both isolutrol and benzoyl peroxide were effective in reducing the
number of superficially inflamed lesions throughout the trial (see Figure 3). This
reduction between baseline and final review was statistically significant in both the
25 isolutrol and the benzoyl peroxide group (p~0.001).
Baseline counts of non-inflamed lesions were 23.4i5.0 in the isolutrol
group and 25.5+4.5 in the benzoyl peroxide group (mean + SEM) and there was
no statistically significant difference between the two groups at baseline. Benzoyl
30 peroxide was effective in reducing the number of non-inflamed lesions throughout
the trial (p~0.001). The reduction in non-inflamed lesions between baseline and
final review was not significant for the isolutrol group (p=0.06, see Figure 4).
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Clinical assessment for skin dryness, erythema, pruritus and scaling
showed a significant difference between the isolutrol group and the benzoyl
peroxide groups at two weeks (p~0.05) and four weeks (p<0.05), eight weeks
(p~0.05) and 12 weeks tp~0.05), the benzoyl peroxide showed increased dryness,
5 erythema, pruritus and scaling. Clinical assessment for skin burning showed a
significant difference between the isolutrol group and the benzoyl peroxide group
at two weeks (p~0.05), the benzoyl peroxide showing increased skin burning.
There was no significant difference between the treatment groups for the clinical
assessment of skin burning at four, eight or 12 weeks. Overall, 94% of benzoyl
10 peroxide treated patients reported unwanted effects during the trial, whereas only
34% of isolutrol treated patients reported such problems (see Table 3).
TABLE 3 The percentage of patients reporting side effects during the trial
within each treatment group.
Benzoyl Isolutrol
peroxide
Burning 34% 0.3%
Erythema 49% 9.0%
Scaling 72% 9.0%
Pruritus 43% 14%
Dryness 83% 31%
DISCUSSION
The results of this study reveal that Isolutrol is an effective treatment for
25 the reduction in the severity of acne. Isolutrol is as effective as benzoyl peroxide
in reducing the numbers of inflamed lesions however, unlike benzoyl peroxide,
Isolutrol is not capable of significantly reducing the numbers of non-inflamed
lesions. It is possible that this difference may be due to the different modes of
action and the particular effficacy of Isolutrol in reducing the number of inflamed
30 lesions indicates that Isolutrol has anti-inflammatory properties in addition to
reducing hyperseborrhoea.
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Clinical assessment of side-effects showed that Isolutrol was more
acceptable than benzoyl peroxide in the first month of treatment. Dryness,
pruritus, burning, erythema and scaling are well known irritant effects of benzoyl
peroxide and were problems commonly experienced by patients using benzoyl
5 peroxide in this trial. Thus Isolutrol may prove to be a useful adjunct to the treatment regime of patients unable to tolerate benzoyl peroxide.
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REFERENCES:
1. Burke, B.M. and Cunliffe, W.J. (1984). Br. J. Dermatol. 111, 83-92.
2. Cross, C.E. ef al. (1987). Annals of Internal Medicine, 107(4), 526-545.
3. Gutteridge, J. (1981). FEBS Leff. 128, 343-346.
4. Halliwell, B. and Gutteridge, J.M.C. (1984). Biochem. J., 219, 1-14.
5. Halliwell, B. and Gutteridge, J.M.C. (1985). "Free Radicals in Biology and
Medicine", Clarendon Press, Oxford, England, p.147.
6. Halliwell, B. and Gutteridge, J.M.C. (1986). Arch. Biochem. Biophys. 246,
501-514.
7. Moorhouse, C. et al. (1987), FEBS Leff. 213, 23-28.
8. Slater, T.F. (1984). Biochem. J. 222, 1-15.
