U.S. patent application number 11/664098 was filed with the patent office on 2008-02-28 for apparatus for treatment of dermatological conditions.
Invention is credited to Peter Luebcke.
Application Number | 20080051680 11/664098 |
Document ID | / |
Family ID | 33443702 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051680 |
Kind Code |
A1 |
Luebcke; Peter |
February 28, 2008 |
Apparatus for Treatment of Dermatological Conditions
Abstract
This invention relates to ultrasound delivery apparatus
comprising flexible arrays of transducers and to methods and
topical compositions for the treatment of skin, in particular for
the treatment of cosmetic skin conditions and to improve the
appearance of sun damaged and/or aged skin; the invention further
relates to the use of such apparatus and compositions in methods of
treating skin, which methods may incorporate the application of
ultrasound. The composition may comprise one or more anti-gfycation
agent, one or more anti-oxidant, a dermatologically acceptable
excipient and optionally one or more substance capable of inducing
expression of a molecular chaperone.
Inventors: |
Luebcke; Peter; (Meldreth,
GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
33443702 |
Appl. No.: |
11/664098 |
Filed: |
October 11, 2005 |
PCT Filed: |
October 11, 2005 |
PCT NO: |
PCT/GB05/50181 |
371 Date: |
March 29, 2007 |
Current U.S.
Class: |
601/2 ; 600/459;
604/289; 604/304 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 3/04 20180101; A61P 43/00 20180101; A61N 7/00 20130101; A61N
2007/0008 20130101; A61N 2007/0078 20130101; A61N 2007/0073
20130101 |
Class at
Publication: |
601/002 ;
600/459; 604/289; 604/304 |
International
Class: |
A61H 1/00 20060101
A61H001/00; A61F 13/00 20060101 A61F013/00; A61M 35/00 20060101
A61M035/00; A61N 7/00 20060101 A61N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 11, 2004 |
GB |
0422525.6 |
Claims
1-74. (canceled)
75. An apparatus for application of ultrasound to the skin
comprising a plurality of ultrasound transducer elements arranged
as an array in a flexible material in spaced configuration, wherein
the ultrasound transducer elements are capable of delivering
ultrasound at low and/or high frequency to an area of the skin.
76. An apparatus according to claim 75, wherein the ultrasound
transducer elements are capable of delivering low and high
frequency ultrasound simultaneously or sequentially.
77. An apparatus according to claim 75, wherein the transducer
elements are dual frequency transducer elements capable of
delivering low and high frequency ultrasound sequentially or
simultaneously.
78. An apparatus according to claim 77, wherein the dual frequency
transducer elements are capable of delivering low and high
frequency ultrasound along a common axis.
79. A dual frequency transducer element comprising a high frequency
transducer element and a low frequency transducer element.
80. A composition comprising one or more anti-glycation agent, one
or more anti-oxidant, a dermatologically acceptable excipient and
optionally one or more substance capable of inducing expression of
a molecular chaperone.
81. A method for treatment of the skin, comprising applying to the
skin a composition according to claim 80.
82. The use of a composition according to claim 80 in the
manufacture of a composition for the treatment of a skin
condition.
83. A dressing comprising a composition according to claim 80.
84. A kit comprising a composition according to claim 80 and a
device comprising an ultrasound source and/or an applicator for
applying ultrasound to the skin and/or for applying the composition
to the skin.
Description
TECHNICAL FIELD
[0001] This invention relates to ultrasound delivery apparatus,
methods and topical compositions for the treatment of skin, in
particular for the treatment of cosmetic skin conditions and to
improve the appearance of sun damaged and/or aged skin; the
invention further relates to the use of such apparatus and
compositions in methods of treating skin, which methods may
incorporate the application of ultrasound.
BACKGROUND OF THE INVENTION
[0002] The skin is a potential route for delivery of pharmaceutical
or cosmetically active agents to the body. However, the skin is not
generally thought of as an efficient delivery route, due to the low
permeability of the stratum corneum and the epidermis in general.
Traditionally, topical application of pharmaceutical therapeutic
agents has been targeted at localized dermatological sites. More
recently, transdermal techniques have been used for systemic
targeting especially as this route bypasses the hepatic circulation
where degradation of the active agent may occur.
[0003] Ultrasound can be used to deliver molecules to within the
skin. When ultrasound is used in this context it is termed
"sonophoresis". Ultrasound applied to the skin has two main
effects. First, cavitation results from the rapidly oscillating
pressure field, causing bubble formation and collapse, which
mechanically creates channels through the stratum corneum. The
second effect is the direct heating of the material through which
the sound waves are travelling, due to attenuation of the acoustic
energy through reflection, absorption and dispersion. In skin, this
occurs up to four times more than other tissues due to its
heterogeneity. Heating is known to disrupt the lipid bilayer system
in the stratum corneum also contributing to the enhanced
permeability of the epidermis. Several factors can affect the
heating capacity of ultrasound, including:
[0004] (i) applying ultrasound in continuous rather than pulsed
mode,
[0005] (ii) prolonging the exposure time,
[0006] (iii) focusing the ultrasound rather than using unfocused
application,
[0007] (iv) avoidance of using aqueous gels which are used to
decrease the degree of reflection,
[0008] (v) applying the ultrasound at higher power densities,
[0009] (vi) application of ultrasound to tissues immediately
adjacent to bone.
[0010] With ultrasound, diffusion of low molecular weight molecules
has been shown to increase by 2-5000 times across isolated
epidermis in vitro and by up to 1700 times in theoretical studies.
Even large molecule drugs such as insulin and heparin have been
delivered effectively when using 15 minutes of 20 kHz US. One in
vitro study found that poly-L-lysine molecules of up to 51 kDa
could be delivered with ultrasound at 20 kHz and intensities in the
range of 2 to 50 W/cm.sup.2. By way of explaining this increase in
permeation, some studies have reported an increase in the number of
pores rather an increase in the individual pore diameters (28.+-.12
.ANG.). However, the term `sonomacroporation` has been adopted for
specific ultrasound that actually causes larger pore formation.
[0011] The permeability of the skin is increased by disruption of
the intercellular lipids through heating and/or mechanical stress,
and through the increase in porosity. Temperature rises of
6.degree. C. (1 MHz, 0.25 W/cm.sup.2) to 500C (20 kHz, 10-30
W/cm.sup.2) have been reported, but rises as little as 11.degree.
C. (1 MHz, 2 W/cm.sup.2) have been shown to cause skin damage.
Continuous mode ultrasound at an intensity of 1 W/cm.sup.2 raises
the temperature of tissue at a depth of 3 cm to 40.degree. C. in 10
minutes.
[0012] For smaller molecules, such as mannitol, enhancement of
permeation through the skin occurs when ultrasound is applied as a
pre-treatment or simultaneously with application of the molecule;
whereas for large molecules such as insulin, enhancement of
permeation has only been recorded during application of
ultrasound.
[0013] Ultrasound can be used to improve transdermal drug delivery.
WO 99/34857 discloses transdermal drug delivery of various active
agents using a power density of less than 20 W/cm.sup.2, preferably
less than 10 W/cm.sup.2; the frequency used being less than 2.5
MHz, preferably less than 2 MHz, preferably less than 1 MHz, most
preferably 20-100 kHz; Experimental data in vivo on rats was
generated using a frequency of 20 kHz and a power density of 1 or
1.5 or 7 W/cm.sup.2.
[0014] U.S. Pat. No. 4,767,402, describes transdermal drug delivery
using ultrasound at a power density of 0-3 W/cm.sup.2, preferably
0.5-1.5 MHz, and recommends that as the power density is reduced,
the frequency should also be reduced. A power density of
1-2W/cm.sup.2 at frequency 870 kHz is exemplified.
[0015] Cosmetic treatments that aim to improve skin quality are
also hindered by the barrier function of the epidermis and in
particular the outer stratum corneum. The epidermis provides a
significant mechanical and chemical barrier to solute transfer due
to the cornified cell/lipid bilayer. Also, there is significant
enzymatic activity in the epidermis and dermis, which provides a
biochemical defence to neutralise applied xenobiotics and which is
comparable to that of the liver in terms of activity per unit
volume. Additionally, the molecular weight of active substances is
known to be important in determining their propensity to diffuse
across the skin. Diffusion of substances of molecular weight around
500 Da and above is known to be inefficient. Methods and apparatus
involving ultrasound have been described for use in cosmetic of the
skin and in medical treatments.
[0016] U.S. Pat. No. 6,113,559 discloses a method and apparatus of
reducing wrinkles by application of a focused ultrasound beam
(ultrasound power density 100-500 W/cm.sup.2, frequency 1-500 MHz)
to a region of skin, so that the energy delivered to the dermis
layer is sufficient to heat the tissue in order to stimulate or
irritate the dermis layer, causing a change in the dermis layer
that confers a change in smoothness of the epidermis layer.
[0017] Ultrasound therapy for the treatment of cellulite is well
known and the application of ultrasonic wave energy has generally
proven effective in breaking down subcutaneous fatty tissue. As an
example, EP 0 695 559, relates to multifunctional equipment for
treatments of cellulite, which can include emitters of ultrasonic
vibrations for application to, for example, the thighs of a
patient's body. However, suitable power densities and frequencies
are not discussed. GB 2303552 discloses ultrasound apparatus useful
for the non-invasive reduction of cellulite. The ultrasound devices
are used for the ultrasonic treatment of cellulite at a
predetermined frequency of about 3.3 MHz and a typical power
density of 2.8 W/cm.sup.2, with 50% of the energy being absorbed
within a depth of from 1.27 cm to 2.54 cm below the skin
surface.
[0018] U.S. Pat. No. 6,030,374 discloses a method for enhancing
transport of an active agent through the skin by exposing skin to
ultrasound and applying an active agent to the skin by injection.
The active agent may be used to reduce the appearance of cellulite.
For lower frequency ultrasound, an ultrasound frequency between 25
kHz and 3 MHz at a power density of 0.5-2.0 W/cm.sup.2 is used; for
higher frequency ultrasound, an ultrasound frequency between 3 MHz
and 16 MHz at a power density of 0.2-1.0 W/cm.sup.2 is used.
[0019] U.S. Pat. No. 5,665,053 relates to an endermology body
massager having ultrasound generators that are selectively
controlled by the operator. The very low frequency long wave
ultrasound disclosed, 10 to 40 kHz, is in the range generally
recognised as being disruptive ultrasound, which may be damaging to
cells, and thus for safety reasons this is not suitable for general
use except at very low power levels.
[0020] U.S. Pat. No. 5,507,790 discloses apparatus for focusing
ultrasound energy such that the temperature of a site within the
patient's subcutaneous adipose tissue layer is raised to between
40.0 and 41.5.degree. C., to accelerate local fat tissue lipolysis
reaction rates. The apparatus includes an ultrasonic transducer
which supplies ultrasound energy of an undisclosed frequency and at
an undisclosed power density to a focusing element.
[0021] WO 99/56829 discloses ultrasound bandages and ultrasound
transducer array bandages which are said to be useful to accelerate
the healing of wounds by positioning the ultrasound bandages and
ultrasound transducer array bandages adjacent to a wound and
generating ultrasonic pulses.
[0022] WO 99148621 describes large-area flexible piezoelectric
composite transducer elements and large-area arrays of such
transducer elements have sufficient flexibility to conform to the
contours of the human anatomy, e.g., the hip, spine.
[0023] To be effective, treatment for cosmetic skin conditions,
such as skin ageing and sun damage, must deliver actives to at
least the depth of the upper (papillary) dermis and therefore must
employ a mechanism to overcome this effective physical and
biochemical barrier, even when it has deteriorated with age.
[0024] The deterioration of human skin due to natural or
`intrinsic` ageing is characterised by a number of symptoms. Such
symptoms include a thinning of both the epidermis and the dermis, a
flattening of the junction between them, poor wound healing,
thermoregulation and immune function along with a deterioration of
associated mechanical properties such as tear resistance,
elasticity and barrier function. The visible appearance also
deteriorates giving a rougher, lined and dry appearance along with
uneven pigmentation. In most cases skin ageing is of little medical
importance except in such cases as impaired wound healing which
allows infection and dysfunction.
[0025] Visible deterioration in skin with age is due to a
combination of several changes which happen more or less
concurrently. This deterioration can be accelerated by lifestyle
choices such as smoking and sunbathing. The visibly apparent
changes include: sagging skin, rough skin texture, dyspigmentation,
dull complexion and a general loss of radiance. Wrinkling, or
rhytide formation, is probably the symptom most commonly associated
with skin ageing and is known to be caused by a change in the type
and distribution of matrix proteins and proteoglycans. Similarly,
functions of the skin that decline with age include: cell
replacement, immune recognition, sensory perception, injury
response, vascular responsiveness, vitamin D production, barrier
function, thermoregulation, sebum production, chemical clearance,
sweat production and mechanical protection. There may also be
changes in pH (from 4.5 to 5).
[0026] Ageing skin is characterised by decreased epidermal
thickness and proliferation along with the flattening of the rete
ridge pattern. The apparent thinning may be linked to increased
apoptosis in the basal and spinous layers, in conjunction with
impaired cell proliferation of the basal layer. Senescent skin
thins, becomes less elastic and has reduced barrier function. This
is because the dermis contains a reduced cellular content with
stiff, inflexible matrix proteins and a diminished number of
capillary loops. The overlying epidermis consequently suffers
because the dermal-epidermal junction (DEJ) flattens, resulting in
a reduced contact surface area as there are fewer capillary loops
in proximity to the DEJ. The exchange of nutrients and metabolites
between the two layers decreases and the communication needed to
maintain layer integrity in response to changes in external
environment conditions is impaired.
[0027] The skin is not only subjected to intrinsic or chronological
ageing processes, but also environmental or extrinsic ones. For
example, factors such as diet, pollution and smoking are known to
affect the rate of skin ageing. However one factor stands out as
the most potent `gerontogen`: sunlight. It has been suggested that
approximately 80% of facial ageing is due to sun exposure.
[0028] Collagen, elastin and other intra- and extracellular
proteins of the skin are affected resulting in solar elastosis, the
build-up of localised elastic tissue in fibrous bundles throughout
the dermis.
[0029] The UV component of sunlight has also been linked to the
reduction in cellular population of the epidermis (keratinocytes)
and dermis (fibroblasts). It has been suggested that this is due to
the increase in programmed cell death or apoptosis. The epidermis
and the dermis are known to become increasingly acellular with age,
which supports this hypothesis. Despite the epidermis influencing
the dry and rough appearance of the skin, it is the dermis that
dictates the degree of surface smoothness. Reduction and/or a
redistribution of matrix proteins and high water-binding
proteoglycans largely govern the appearance of wrinkles and general
surface smoothness. Similarly, scarring of the skin is due to
abnormal protein content, conformation and distribution via the
formation of granulation tissue following trauma, again primarily a
dermal rather than an epidermal problem.
[0030] Typical symptoms of photoageing include coarseness,
wrinkling, irregular pigmentation, telangiectasia, scaliness and a
variety of benign, premalignant and malignant neoplasms.
Photoageing is predominant in fair-skinned Caucasians who have a
history of sun-exposure and occurs most severely on the face, neck
and extensor surfaces of the upper extremities. Elastosis,
recognised as the pebbly goose flesh seen on the neck and upper
chest, is due to nodular aggregations of altered elastin fibres in
the dermis. A proliferation of increasingly thickened and tangled
elastin fibres has been observed in the papillary and reticular
dermis of sun-exposed skin. Even in mildly sun-damaged skin, a 5-20
fold increase in elastin fibre diameter has been found, with slight
changes in the fibrillar structure and an alteration of the normal
architecture, giving a disrupted and "moth-eaten" appearance.
[0031] Overall, photodamage is manifested by the progressive injury
to dermal fibroblasts with quantitative and qualitative alterations
to the supporting extracellular matrix. As solar energy passes
through the skin and is absorbed a gradient of damage occurs, the
most damage being seen in the outer papillary dermis, with less to
the deeper reticular dermis.
[0032] Intrinsic (chronological) aging is characterised by atrophy
of skin with loss of elasticity and reduced metabolic activity.
Specifically, the stratum corneum remains unchanged, but the
epidermis thins overall, with a flattening of the dermal-epidermal
junction resulting in increased fragility of the skin. Dermal
thickness and dermal vascularity are decreased; this is accompanied
by a decrease in the number and the biosynthetic activity of dermal
fibroblasts. This later change is manifested by delayed wound
healing. Increasing age also has the effect of reducing the
response of keratinocytes and fibroblasts to growth factors.
[0033] At the molecular and ultrastructural level, there are
changes in elasticity and other changes in matrix proteins. As
regards elasticity, there is a reduction in the extracellular
protein fibrillin which is a major component of microfibril bundles
that connect the dermal-epidermal junction to the papillary dermis.
These bundles, often called oxytalan fibres, essentially provide an
elastic connection between the epidermis and dermis. Previously
considered to be synthesised only by fibroblasts, the fibres
present at the dermal-epidermal junction have been shown to be
synthesised by keratinocytes. The concentration of fibrillin in
photoaged skin has been found to be decreased and has proved to be
a useful biomarker for photoageing as it is known to be connected
with wrinkle formation. Fibrillin concentration is also reduced in
skin that has been subjected to tensile stress and exhibits stretch
marks (striae distensae).
[0034] In vivo proteins are post-translationally modified by a
non-enzymatic reaction (Maillard reaction) between proteins (both
intra- and extracellularly) and sugars. This reaction is known
either as glycation, or glycosylation, and is well recognized to
play an important part in protein turnover, tissue remodelling,
diabetes and ageing. In skin, this process is exacerbated by UV,
with dermal glycation often increasing significantly after 35
years. Glycation of proteins occurs when reducing sugars such as
glucose and fructose, or their reactive intermediates such as
glyoxal, react with the amino groups of long half-life proteins
such as collagen (t.sub.1/2=15 years in human skin) and elastin in
the dermis. As a result of this process, cytotoxic Advanced
Glycation End-products (AGEs) (AGEs) accumulate.
[0035] An increase in glycation has been seen in skin previously
irradiated with UV. A well-known biomarker for protein glycation,
carboxymethyllysine (CML), has been shown to be present
predominantly in areas of solar elastosis in the dermis and
generally at higher concentrations in photoaged skin, suggesting
that UV-induced oxidation may accelerate the formation AGEs in
photoaged skin.
[0036] The build-up of AGEs has several effects. Advanced glycation
end product-modified proteins are endogenous sensitizers of
photo-oxidative cell damage in human skin by UVA-induced generation
of reactive oxygen species (ROS) contributing to photoageing and
photocarcinogenesis. ROS generation has also been linked to early
and late stages of AGE formation with a direct link with the rate
of ROS generation which in turn increases matrix metalloproteinase
expression with a consequent decrease in healthy digestible matrix.
There is also cross-linking of extra-cellular proteins which causes
deterioration of the structural mechanical properties of the
protein and reduces their susceptibility to the body's natural
enzymes, such as matrix metalloproteinases (MMPs), which normally
ensure a regular, healthy protein turnover. Cross-linking AGEs
include species such as pentosidine. Non-cross-linking AGEs include
species such as CML. Glycation also decreases water accessibility
of proteins making them more heat stable and less likely to be
thermally denatured.
[0037] The body has a host of physiological mechanisms that defend
against deleterious protein modifications, including
protein-digesting enzymes. Timely proteolysis removes damaged
proteins before they undergo oxidative damage and cross-linking.
Therefore, rapid effective proteolysis is essentially an anti-aging
mechanism. It has been mentioned already that proteins such as
collagen and elastin, which have been post-translationally modified
through UV-induced glycation, are more resistant to digestion by
endogenous enzymes (e.g. metalloproteinases). This, coupled with
the increase in expression of such enzymes, further reduces the
ratio of healthy digestible matrix proteins to modified deleterious
proteins.
[0038] Not only are native proteins turned over by endogenous
enzymes such as collagenase and elastase, but other systems are
present both intra- and extra-cellularly to deal with ageing and/or
denatured/stressed proteins. One such mechanism employs molecular
chaperones. Increasing age is associated with a reduced capacity to
maintain homeostasis in all physiological systems and this may
result, in part at least, from a parallel and progressive decline
in the ability to produce heat shock proteins. An attenuated heat
shock protein response may contribute to increased susceptibility
to environmental challenges in aged individuals.
[0039] Heat Shock Proteins (HSPs), also known as stress proteins,
are thought to act as molecular chaperones by assisting with
protein synthesis, transport, folding and degradation. They are a
group of proteins that are present in all cells, in all life forms.
They are induced when a cell undergoes environmental stress, heat,
cold, or oxygen deprivation. HSPs are also present in cells under
perfectly normal conditions and have been linked to modulation of
contraction and relaxation responses in vascular smooth muscle;
they play an important role in protein folding and function, even
in the absence of stress.
[0040] The formation of Advanced Glycation End-products causes
protein unfolding irreversible cross-linking and other chemical
modifications. HSPs are known to promote refolding/maintenance of
conformation and also the rapid degradation of irreversibly-damaged
proteins. Small heat shock proteins, such as .alpha.-crystallin,
are known to protect eye lens proteins from glycation induced
changes. Small heat shock proteins (sHSPs) are known to have common
`crystallin` core that appears to be responsible for the catalytic
activity of these chaperones. It has been suggested that a greater
understanding of .alpha.-crystallin/sHsp chaperone action will have
implications for the development of therapeutics to treat and
prevent cataract.
[0041] The heat shock protein family includes the 8-kD ubiquitin
(known in connection with the ubiquitin-proteasome protein
degradation pathway), 32-kD heme oxygenase-1 (connected to UVA
induced oxidative stress) and HSP-47, a known collagen chaperone.
HSP-27 has been found in human skin and has been suggested to play
a protective role in inflammatory diseases due to its links with
interleukin-1 and tumour necrosis factor-.alpha.. This, along with
the understanding that HSP-27 expression is closely linked with
epidermal keratinocyte differentiation suggests that heat shock
proteins such as HSP-27 play a role in skin protection and possibly
in the UV-sunburn inflammation cycle. In contrast to other cells
and organ systems, epidermal keratinocytes are known to express
HSP-72 constitutively, i.e. without exposure to previous stress.
The heat shock protein HSP47 has been shown to be important as a
molecular chaperone for procollagen synthesis in human fibroblasts.
HSP47 synthesis is reduced in aged and photo-aged skin.
[0042] HSP expression following exposure to UV has been linked with
increased resistance to UV-induced cell death. Non-toxic inducers
of HSPs may protect against the immediate and long-term effects of
UV exposure. Studies have shown that prior exposure of cells to red
and infra-red (IR) light protects them against subsequent exposure
to UV light. Similarly, IR pre-treatment of cells also protects
cells against subsequent lethal (51.degree. C.) applied heat
stress.
[0043] The well-known protective effect of HSPs from environmental
stress is not constant with age. The HSP response to stress is
attenuated with age, probably at the transcriptional level.
Repetitive mild heat shock (RMHS) of human skin fibroblasts has
been found to reduce the rate of age-related changes. One study has
connected the age-related decrease in the ability of human
fibroblasts to reduce the accumulation of glycated proteins with a
parallel reduction in the ability to express HSP70, as human
fibroblasts exposed to RMHS exhibited increased HSP70 expression
and reduced accumulation of glycated protein accumulation. The
beneficial effects of RMHS have been attributed to increased
proteasomal activity, increased ability to decompose
H.sub.2O.sub.2, reduced accumulation of lipofuscin and an enhanced
resistance to UVA radiation.
[0044] Temperature rises of 3-5.degree. C. above baseline in muscle
have been shown to cause the induction of HSPs. Induction of HSPs
by 30 mins of pulsed ultrasound applied at normal body temperature
has been demonstrated in the rat embryo, showing that the heat
shock response is not specific to heat but can occur in response to
mechanical stress. Similarly, chick embryos exposed to ultrasound,
without any significant thermal contribution, have shown heightened
synthesis of HSP72 suggesting that the mechanical stimulus can
induce a stress response. It was also concluded that to produce a
`full biological effect, stress must be constant for approximately
10 s or more over any time interval during exposure`. It is
possible that cumulative effects can stimulate HSP production as
has been found when mild heat shock was repeated over 3 days
causing significantly elevated muscle HSP levels.
[0045] Certain substances have an effect on HSP expression. For
example, salicin has been shown to reduce the necessary degree of
temperature rise from 42.degree. C. to 39.degree. C. to elicit HSP
expression and to reduce the degree of subsequent UV-induced damage
in cultured human fibroblasts and keratinocytes. Known irritants
such sodium lauryl sulphate (SLS) also induce HSP expression. HSP27
upregulation due to SLS application to excised human skin has been
used as a method of determining cellular stress due to chemical
irritancy. In a similar study, however, SLS induced expression of
HSP27 in human epidermis was suppressed by topical application of
vitamin C.
[0046] The substance zinc-L-carnosine, known also as Polaprezinc
commercially, has been shown to induce HSP72 (stress-induced HSP70)
expression in gastric mucosa protecting cells from applied stress
through chemical irritancy. As a control, ZnSO.sub.4 and carnosine
were also tested and found not to elicit the same response. Known
as an anti-ulcer drug, zinc-L-carnosine's wound-healing action has
been linked to its proliferative response in non-endothelial cells
such as fibroblasts.
[0047] The influence of aspirin on HSP70 expression in intact rats
subjected to heat stress has been investigated. Rats were injected
intraperitoneally either with aspirin (100 mg/kg), or vehicle
alone, 60 min prior to their placement at 37.degree. C. or room
temperature for 30 min. The combination of aspirin with heat
treatment resulted in 3 to 4 fold higher levels of HSP70 mRNA
relative to those seen with heat treatment alone.
[0048] The role of HSP-72 and -70 in conferring resistance to
aspirin attack of the rat gastric mucosa has been investigated;
expression of these HSPs was elevated following chronic exposure to
aspirin.
[0049] Analgesics such as aspirin, ibuprofen and paracetamol are
known to protect against cataract. This action has been attributed
to the inhibition of sugar-induced cross-linking in small HSPs such
as .alpha.-crystallin. Enzymes that protect against cataract are
prone to glycation-induced inactivation, but aspirin has been shown
to protect against this.
[0050] Similarly, acetyl-L-carnitine has been recognised as a
potential chaperone-protecting agent due to its abilities to
acetylate potential glycation sites of small HSPs and
correspondingly protect them from glycation-mediated protein
damage.
[0051] Small heat shock proteins (sHSPs) and Clusterin are
molecular chaperones that share many functional similarities
despite their lack of significant sequence similarity. Small heat
shock proteins are ubiquitous intracellular proteins whereas
clusterin is generally found extracellularly. Both chaperones
prevent the amorphous aggregation and precipitation of target
proteins under stress conditions such as elevated temperature,
reduction and oxidation. Transcription of both HSPs and clusterin
are mediated by the transcription factor HSF-1. However, clusterin
has been shown to be much more efficient than certain sHSPs, such
as .alpha.-crystallin, in preventing the precipitation from
solution of stressed target proteins.
[0052] Clusterin is expressed as a 75-80 kDa heterodimeric protein
that is heavily glycated such that 30% of its mass is comprised of
sugar. Whereas the chaperone activity of small heat shock proteins
such as .alpha.-crystallin is reduced significantly at lower pH,
the activity of clusterin is enhanced at lower pH. This has
important implications for sites of tissue damage or inflammation
where local acidosis (pH<6) occurs. Another similarity that
clusterin shares with sHSPs is the ability to regulate apoptosis.
Over-expression of clusterin can protect cells from a variety of
agents (e.g. TNF-.alpha. and UV irradiation) that otherwise induce
apoptosis. It has been suggested that clusterin may interact with
stressed cell surface proteins to inhibit pro-apoptotic signal
transduction or prevent inappropriate interactions of intracellular
proteins during stress.
[0053] Many topical skin preparations are available for the
treatment of medical skin conditions and for the treatment of
cosmetic skin conditions, in particular skin ageing and sun damage.
In many instances these preparations are ineffective, with only
minimal or short lived efficacy. There is thus a desire for new
preparations effective in the treatment of skin conditions.
Furthermore, the present invention addresses the problems of
achieving efficient delivery to the skin of such novel
preparations.
DISCLOSURE OF INVENTION
[0054] The invention provides an apparatus for application of
ultrasound to the skin comprising a plurality of ultrasound
transducer elements arranged as an array in a flexible material in
spaced configuration, wherein the ultrasound transducer elements
are capable of delivering ultrasound at low and/or high frequency
to an area of the skin.
[0055] According to this aspect of the invention, an ultrasound
array can be incorporated into a mask, patch or patches that can be
applied to the skin to supply ultrasound.
[0056] The mask or patch is preferably shaped to conform to at
least part of the surface of the face. Thus the flexible array of
ultrasound transducer elements can be formed in a circular or any
other simple or complex shape, especially those optimised to
conform to bodily shapes and features, especially the parts of the
face and neck. The array should be sufficiently flexible to allow
bending to a curvature of 3-4 cm radius, preferably to allow
bending to shape around doubly curved surfaces as well as singly
curved surfaces.
[0057] Application of ultrasound using an apparatus according to
the invention can be used as a pre-treatment before application of
a composition of the invention, or a composition of the invention
can be applied to the skin, either directly or via material
impregnated with the composition, e.g. a pad, such as a gel pad,
and then the ultrasound delivered via the flexible array. The
flexible ultrasound array can be coupled to a thin (2-3 mm),
disposable gel pad that contains the composition and couples the
ultrasound energy. Suitably the flexible array can be affixed,
directly or indirectly (e.g. via a pad) to the skin for the
duration of the treatment.
[0058] The transducer elements of the array are preferably
hermetically sealed, e.g. contained within a waterproof flexible
material capable of electrical performance even when
adhered/coupled to aqueous formulations.
[0059] In the apparatus, it is preferred that the flexible material
is at least approximately acoustically matched, to one or
preferably both of the transducer elements, to inhibit generation
of reflections in the material that might divert or otherwise
dissipate the ultrasound waves. The flexible material may comprise
a polymeric material selected from thermoplastics, thermosets,
rubbers or mixtures thereof. The flexible acoustically matched
material will ordinarily be formed from a polymeric material, and
optionally, a filler. The polymeric material should have good
compatibility with the components of the transducer element,
biocompatibility and flexibility. Suitable polymeric materials
include thermoplastics such as high density polyethylenes,
polymethyl methacrylates, polypropylenes, polybutylene
terephthalates, polycarbonates, polyurethanes such as CA 118 and CA
128 available from Morton Chemical and estane polyester, and the
like; thermosets such as epoxies such as Spurr epoxy and Stycast
80, Stycast 1365-65 and the like; and rubbers such as silicone
rubbers such as dispersion 236 available from Dow Corning and
RTV-141 available from Rhone-Poulenc, Inc. and the like. If
desired, the acoustic impedance of the polymeric materials may be
increased by the incorporation of one or more fillers. Suitable
fillers include PZT, tungsten, alumina, silica glass, tungsten
carbide, titanium, glass powder and the like with glass powder
being preferred. The size of the filler particles should be in the
range of about 0.1 to about 50 microns and preferably from about
0.5 to about 5 microns. The amount of filler employed will be that
amount necessary to impart the desired acoustic impedance.
Normally, from about 2 to about 50 percent filler by volume and
preferably from about 5 to about 30 volume percent filler is
employed. A preferred polymeric material is silicone rubber.
[0060] Typically the transducer elements will be individually
connected to a ultrasound generator, such that the ultrasound
transducer elements are capable of delivering low and high
frequency ultrasound simultaneously or sequentially.
[0061] An apparatus according to the invention can comprise a
flexible array having a set of high frequency transducer elements
and a set of low frequency transducer elements respectively capable
of delivering high and low frequency ultrasound. The high and low
frequency transducers may be alternated, or otherwise arranged in a
pattern, for example a substantially regular arrangement of the two
types of transducers. In other embodiments the high and low
frequency elements may be mounted together, e.g. on top of one
another, in particular coaxially. In this aspect the transducer
elements may be dual frequency transducer elements capable of
delivering low and high frequency ultrasound sequentially or
simultaneously, along a single axis. Dual frequency transducers may
be arranged in a pattern, for example a substantially regular
arrangement of dual frequency transducers. The transducers may be
of circular or other regular or irregular shape. Transducers
elements suitably comprise transducer materials known in the art,
e.g. piezoceramics, PVDF, and/or piezoelectric materials such as
PZT powders commercially available from Morgan Matroc, Inc.,
ceramic, single crystal relaxor ferroelectric, lead zirconate
titanate Pb (Zr, Ti) 03, lead metaniobate Pb (Nb206), modified lead
titanate PbTi3 such as (Pb, Ca)Ti03 and (Pb, Sm)Ti03, barium
titanateBaTiO3, PMN-PT(1-x) Pb(Mg''3Nb2/3) 03-xPbTi03,
PZN-PT/BTNb2/3)03-x(yPbTiO3-(1-y)PbZrO3)Pb(Zn1/3Nb2/3)O3-xPbTiO3-BaTiO3,(-
1-x)Pb(Zn1/3, and the like.
[0062] In an apparatus according to the invention, transducer
elements can be capable of delivering the low frequency component
in pulsed mode and the high frequency component in continuous mode,
or more preferably capable of delivering the low frequency
ultrasound component in continuous mode and the high frequency
ultrasound component in pulsed mode. The pulsed mode can be
controllable, such that it is variable, to provide variable pulsing
regimes, for example 2 ms on, 8 ms off (20% duty cycle).
[0063] In an apparatus according to the invention, suitably the
transducer elements are capable of delivering a low ultrasound
frequency of from 20 to 500 kHz, preferably .about.50 kHz and/or a
high ultrasound frequency of from 0.5 to 3.5 MHz, preferably
.about.1 MHz up to 3 MHz. The spatial average power density of the
low frequency ultrasound energy is suitably from 20 to 500
mW/cm.sup.2. The spatial average power density of the high
frequency ultrasound energy is suitably from 0.5 to 3
W/cm.sup.2.
[0064] In a second aspect, the invention provides a dual frequency
transducer element comprising a high frequency transducer element
and a low frequency transducer element, preferably the high and low
frequency transducer elements are co-axially mounted and may be
mechanically and electrically connected, In a preferred embodiment
the high frequency transducer element comprises a piezo ceramic
material and the low frequency transducer element comprises PVDF.
The high and low frequency transducer elements can be bonded
together, optionally with a spacer element in between, which may be
a metal spacer element.
[0065] An apparatus according to the invention may comprise an
array of dual frequency transducer elements as described
herein.
[0066] The ultrasound array can be programmed to deliver a desired
sequence of high and/or low ultrasound frequencies, in pulsed or
continuous mode, in set patterns, thereby avoiding problems of over
or under exposure of the skin the ultrasound, which can cause
over-heating of the skin. An apparatus of the invention is
controllable such that low and high frequencies are capable of
being driven so that the ultrasound field moves across the array in
a preset pattern and at a preset speed, for example 2-3 seconds
from left to right across the full width (e.g. 5-10 cm) of the
array then 2-3 seconds back again, i.e. 4-6 seconds cycle time; or
into the centre of the array and then out again, especially if the
array has circular shaped geometry. The pattern can be varied
within the same treatment session, e.g. left to right then up and
down. Ideally the high and low frequencies are applied so that each
frequency covers the area being treated as evenly as possible. The
flexible array is preferably configured such that ultrasound is not
applied to the eye and such that the transducers will be sited and
controlled so that the possibility of over exposure of skin which
is in proximity to bone to ultrasound (e.g. cheek bones or the
orbit of the eye) is minimised. This can be achieved by application
of ultrasound in pulsed mode and for example by a delivering
ultrasound in a pre-determined phased array sequence. Use of a
mask, patch or patches to apply ultrasound is particularly suitable
for home use.
[0067] The apparatus may comprise a power and control unit, which
is suitably of an appropriate size to enable it to be held in the
hand. The unit is preferably provided in a waterproof wipe-clean
casing. Power may be supplied from batteries, e.g. rechargeable
batteries, to allow use away from mains supply. The unit is
preferably provided with controls to allow the user to select
settings for a desired treatment, these may include pre-set levels
to enable the user to select settings for different uses, e.g. for
anti-ageing treatments, cellulite treatment or for scar reduction,
the various settings being based on different frequency and
amplitude/power settings. Suitably the control unit may include a
maximum time cut-out to prevent over-exposure, e.g. 10 minutes. A
memory function may be provided, e.g. to record date and/or
duration of treatment
[0068] In a particularly preferred embodiment the invention relates
to an ultrasonic treatment system comprising a plurality of
transducer elements (15) arranged as an array (2) and held in
proximity to each other by compliant material (4), which is
suitably silicone rubber (FIGS. 1a, b and c).
[0069] Each element (15) may comprise two components, a high
frequency transducer element, e.g. a piezo ceramic disc element (5)
and a low frequency transducer element, e.g. a pvdf element (7)
positioned so that the positive polarised electrode of each element
is mechanically and electrically connected at interface (9). The
upper surface (30) of the PZT element (5) and the lower surface
(31) of the pvdf element (7) are connected together electrically
(FIG. 1(d)). Each element (1) is individually connected to a power
source described in FIG. 3 via spring connectors (8) attached to
juxta-positioned contacts (3) on flexibly mounted plate (6) FIG.
1a. The transducer array may then be connected to an ultrasound
generator via connectors (11).
[0070] FIGS. 2a and 2b show a particular form of the transducer
element in which PZT disc (12) is conductively attached to metal
element (13) which in turn is conductively attached to a pvdf
material (24) via metal ring (23) and insulating spacer ring (22).
The common HT connection (9) is achieved via conductive ring (21).
Alternate drive frequencies of 50 kHz and 1 MHz are generated
either by individual circuits in system FIG. 3B or via DDS chip in
FIG. 3A. The combined transducer is thus alternatively energised in
burst of 50 kHz and 1 MHz sine wave pulses. The length and ratio of
activation signals may be processor controlled or derived from a
sensor control related to measured characteristics of the target
tissue.
[0071] In FIG. 2a, element (13) may be formed as a focussing device
by shaping the lower surface with a shaped, focussing profile, e.g.
a concave profile, thus imparting similar properties to the
geometrically compliant pvdf film.
[0072] In a third aspect, the present invention provides a
composition comprising one or more anti-glycation agent, one or
more anti-oxidants, a dermatologically acceptable excipient or
excipients and optionally one or more substance capable of inducing
expression of a molecular chaperone.
[0073] Compositions of the invention are useful in the treatment of
cosmetic skin conditions, in particular acting to improve the
appearance of ageing skin, especially by ameliorating the effects
of sun damage. Usually, the or each anti-glycation agent is present
at from about 0.5 to 5%, preferably from about 1 to 3% w/w of the
composition.
[0074] Suitably, in some embodiments of compositions of the
invention, the anti-glycation agent(s) also has anti-oxidant
activity.
[0075] Preferred anti-glycation agents for incorporation into
compositions include one or more of a histidine containing
dipeptide, alanyl-L-histidine (L-carnosine) or a peptidomimetic
thereof, N-acetylcysteine, aminoguanidine, d-penicillamine,
acetylsalicyclic acid (aspirin), paracetamol, indomethacin and
ibuprofen and/or a functional homolog; derivative or prodrug
thereof.
[0076] Histidine-containing natural dipeptides, such as L-carnosine
(.beta.-alanyl-L-histidine, or "carnosine") are known to be
effective against different oxygen-derived free radicals, and also
lipoperoxyl radicals. Carnosine, present at high concentrations in
skeletal muscle tissue, can delay senescence and provoke cellular
rejuvenation in cultured human fibroblasts. The mechanism by which
such a simple molecule induces these effects is not known despite
carnosine's well documented anti-oxidant and oxygen free-radical
scavenging activities. In addition to the prophylactic actions of
carnosine, it may also directly participate in the
inactivation/disposal of aged proteins possibly by direct reaction
with the carbonyl groups on proteins. The possible fates of these
carnosinylated proteins include the formation of inert lipofuscin,
proteolysis via the proteasome system and exocytosis following
interaction with receptors
[0077] It is believed that carnosine may tag glycated proteins for
removal. Protein turnover relies on hydration for thermal
denaturation and glycated proteins are known to have higher
enthalpies of denaturation obviously rendering them less
degradable. `Carnosinylation` of glycated proteins, it has been
suggested, may increase the water accessible surface of such
proteins and therefore promote hydration and unfolding during
thermal denaturation. This theory has been borne out by observing
lower .DELTA.H and .DELTA.G denaturation for carnosinylated
glycated proteins.
[0078] Carnosine acts as an anti-glycation agent, it inhibits
carbonyl attack by methylglyoxal (MG) and by the AGE carboxymethyl
lysine (CML). Carnosine itself has been shown to be readily
glycated by a variety of sugars forming non-mutagenic adducts and
its protective role has been attributed to effect of preventing
glycation of crystalline superoxide dismutase (SOD) and catalase.
Carnosine has been found to offer a superior efficacy and toxicity
profile when compared to the anti-glycation agent aminoguanidine,
thus carnosine is a preferred anti-glycation agent.
[0079] Carnosine exhibits Mn.sup.+ chelation and ROS scavenging
properties, but these alone cannot adequately explain the effect it
has in rejuvenating senescent fibroblasts. One study has attributed
its properties to the reaction of carnosine with carbonyl groups on
glycated/oxidised proteins and other molecules; this reaction,
termed `carnosinylation,` inhibits cross-linking of glycoxidised
proteins to normal macromolecules; and carnosinylation could affect
the fate of glycoxidised polypeptides. Studies on rat embryonic
fibroblasts demonstrated that L-carnosine sustains the retention of
cell morphology even during a nutritional insult for five weeks.
Also, L-carnosine significantly reduces the formation of
8-hydroxy-deoxyguanosine (8-OH dG) in the cells after four weeks of
continuous culture. Thus it could be inferred that the
anti-senescent effect of L-carnosine is probably linked to its
inhibition of formation of intracellular 8-OH dG during oxidative
stress. Carnosine also extends cultured human fibroblast life-span,
kills transformed cells, protects cells against aldehydes and an
amyloid peptide fragment and inhibits, in vitro, protein glycation
and DNA/protein cross-linking. Fibroblasts retain a juvenile
appearance in the presence of carnosine, and revert to a senescent
phenotype when carnosine is removed.
[0080] In addition to anti-glycation anti-oxidant activity,
carnosine also has an anti-inflammatory action. Denatured protein
at the site of inflammation is more susceptible to glycation, hence
the anti inflammatory effect may enhance the inhibition of
glycation.
[0081] Carnosine is water soluble and this suggests that it may
represent the aqueous phase counterpart to lipid-soluble
antioxidants such as .alpha.-tocopherol which act to protect cell
membranes. Carnosine, and carnosine-related compounds (CRCs)
(imidazole, histidine, anserine), and ergothioneine were found to
be equally efficient in singlet oxygen quenching. During generation
of hydroxyl radicals from hydrogen peroxide in the Fenton reaction,
carnosine was found to be more effective than the CRCs tested.
However, the following rank order of efficiency of
carnosine-related compounds has been demonstrated while measuring
the oxidation of human serum lipoproteins:
acetylcarnosine<acetylanserine<homocarnosine=ophidine<carnosine&-
lt;anserine whereas carnosine's component amino acids, histidine
and alanine, have shown little or no inhibitory action against
lipid or protein oxidation. Natural levels of carnosine decrease
with age in parallel with the activities of other antioxidant
systems such as superoxide dismutase (SOD) system. Additionally,
carnosine itself can protect against peroxyl radical fragmentation
of protein in Cu,Zn-SOD which would otherwise inactivate the
enzyme. Carnosine is well known for its singlet oxygen quenching
activity.
[0082] Carnosine has been shown to complex Cu.sup.2+ dimerically,
this may explain why carnosine reduces free radical production, as
metal complexing will reduce available levels of Cu.sup.2+ and
Fe.sup.2+ which would otherwise be coordinatively bonded by AGEs in
proteins (the imidazole ring of carnosine can be compared with that
of the many different imidazole containing AGE X-links) leading to
hydroxyl and other reactive oxygen species production in situ.
Carnosine also interferes with iron/ascorbate induced phospholipid
oxidation.
[0083] Carnosine produces dose-dependent vascular relaxation
(vasodilation) that is independent of endothelium. Interestingly,
in the same study, carnosine's component amino acids L-histidine
and alanine have been found to produce no effect and dose dependent
vasoconstriction respectively.
[0084] Carnosine is hydrolysed physiologically into its component
amino acids: histidine and .beta.-alanine. .beta.-alanine is
believed to have be involved in the promotion of collagen
synthesis. Histidine is known for its anti-inflammatory properties,
its ability to scavenge single oxygen and interfere with redox
reactions involving iron and other metal ions.
[0085] Carnosine has been shown to improve the rates of wound
healing when given as part of a complete enteral formula, but has
not to date been reported to be used topically in wound healing
preparations.
[0086] CRCs such as the carnosine pro-drug N-acetyl-L-carnosine
(NAC) undergo hydrolysis yielding carnosine in situ. NAC has been
shown to treat oxidative stress in ocular disorders such as
cataracts and glaucoma.
[0087] Other carnosine homologs include homocarnosine and anserine
which protect Cu,Zn-SOD from inactivation and prevent release of
Cu.sup.2+. Many carnosine homologs are produced by the enzyme
carnosine synthetase.
[0088] Functional homologs, derivatives and pro-drugs of carnosine
that may be incorporated into compositions according to the
invention include one or more of .beta.-alanylhistamine
(carcinine), N-acetyl-.beta.-alanylhistamine (N-acetyl carcinine),
L-prolyl histamine, and/or n-acetyl-L-carnosine.
[0089] Decarboxylation of L-carnosine provides a derivative with
increased resistance to hydrolytic enzymes. Carnosine
peptidomimetics (functional homologs) are known, which have free
radical scavenging and lipid hydroperoxide deactivating properties
similar to or even better than the natural carnosine peptide.
[0090] Two carnosine peptidomimetics (functional homologs)
N-acetyl-p-alanylhistamine and L-prolylhistamine are highly
effective inhibitors of lipid hydroperoxide-mediated cross-linking
of a protein. In vivo, N-acetyl-.beta.-alanylhistamine has been
shown to protect skin enzymes from UV-induced degradation.
[0091] A composition according to the invention comprises one or
more anti-oxidant(s), preferably selected from the group
comprising: arginine, ascorbic acid, a prodrug or derivative of
ascorbic acid, ascorbyl palmitate, magnesium ascorbyl phosphate,
trisodium ascorbyl phosphate, anserine, carnosine, opidine,
homocarnosine and/or acetylanserine. Generally, the or each
anti-oxidant is present at from about 0.5 to 5%, preferably from
about 1 to 3% w/w of the composition.
[0092] Arginine is a powerful antioxidant and a very effective
sacrificial target for Maillard type protein cross-linking
reactions. Both arginine and lysine have been shown to be effective
inhibitors of glycation, but arginine especially tends to form AGEs
itself. It is known that the number and diameter of capillary loops
close to the dermal-epidermal junction (DEJ) is reduced with age.
The supply of nutrients and removal of by-products from metabolism
and other cellular processes is consequently impaired. L-arginine
acts as a vasodilator due to enzyme-catalysed formation of nitric
oxide (NO) in situ. The formation of nitric oxide (NO) from
L-arginine is now recognized as a ubiquitous biochemical pathway
involved in the regulation of the cardiovascular, central, and
peripheral nervous systems, as well as in other homeostatic
mechanisms.
[0093] Ascorbic acid (vitamin C, AA) is an essential nutrient
involved in many physiological functions. It readily (yet
reversibly) undergoes two consecutive, one-electron oxidation
processes to form the ascorbate radical, a relatively unreactive
free radical, and is therefore considered an excellent reducing
agent. In living organisms, ascorbic acid can protect tissues and
cells against oxidative damage by free radicals and reactive
oxygen-derived species. AA is known to exert a strong UVA
protecting ability in studies on eye lens proteins including X-ray
irradiation.
[0094] Unfortunately, in some situations, ascorbic acid in solution
can undergo oxidation and produce dehydro-L-ascorbic acid as well
as many degradation products, which can result in browning of
compositions containing ascorbic acid. Several factors can
accelerate ascorbic acid degradation such as high storage
temperatures, light, high pH values and the presence of dissolved
oxygen, although the reaction mechanism of ascorbic acid with an
oxygen molecule has not yet been fully elucidated. Moreover, the
reaction of ascorbic acid with oxygen is strongly catalysed by
metal ions, particularly cupric and ferric ions. To avoid
degradation, the ascorbic acid component of a composition can be
provided separately and mixed into the other components of the
composition shortly before use. A stable prodrug or derivative of
ascorbic acid can be included in the composition as an alternative,
or in addition to, ascorbic acid.
[0095] Ascorbyl palmitate is a fat-soluble derivative of vitamin C
widely used in skin care products. It is non-irritating and more
stable than ascorbic acid. Furthermore, ascorbyl palmitate is a
fat-soluble antioxidant and is at least as effective as vitamin E
in protecting the skin from lipid peroxidation (a key type of free
radical damage in the skin).
[0096] Magnesium ascorbyl phosphate is a water-soluble derivative
of vitamin C. It is non-irritating and more stable than vitamin C.
Most importantly, magnesium ascorbyl phosphate appears to have the
same potential as vitamin C to boost skin collagen synthesis but is
effective at significantly lower concentrations. Most vitamin C
formulas are highly acidic and therefore produce exfoliation, so
magnesium ascorbyl phosphate is a preferred ascorbic acid
derivative for use in compositions, particularly those for
individuals with sensitive skin and those wishing to avoid
exfoliating effects.
[0097] Trisodium ascorbyl phosphate (Stay-C.RTM. 50) is the sodium
salt of the monophosphate ester of ascorbic acid. It is a
pro-vitamin, with greater stability in aqueous solution than
ascorbic acid. Phosphatases in the skin act on trisodium ascorbyl
phosphate to release ascorbic acid.
[0098] Compositions according to the invention may contain one or
more substances capable of inducing expression of a molecular
chaperone, particularly useful are substances capable of inducing
expression of a heat shock protein, clusterin and/or alpha
crystallin. The one or more substance capable of inducing
expression of a molecular chaperone can be acetyl salicylic acid,
salicylic acid, zinc ions, a zinc salt, zinc sulphate, and/or
zinc-L-carnosine. Usually, a zinc containing agent is present at
from about 0.1 to 1%, preferably from about 0.25 to 0.75%, most
preferably around 0.5% w/w of the composition. When acetyl
salicylic acid or salicylic acid is present in the composition a
suitable concentration is from about 0.5 to 2.5 %, preferably from
about 1 to 1.5% w/w of the composition.
[0099] A composition according to the invention may further
comprise one or more anti-apoptotic substance, preferably selected
from the group comprising nicotinoamide, L-carnitine,
acetyl-L-carnitine, N-acetyl-cysteine and/or L-carnosine. The or a
anti-apoptotic substance is usually present at a concentration of
from about 0.5 to 5%, preferably 1 to 3% of the composition.
[0100] In a fourth aspect, the present invention provides a
composition comprising one or more substance capable of inducing
expression of a molecular chaperone and a dermatologically
acceptable excipient.
[0101] A composition according to the invention may further
comprise one or more ingredient selected from the group comprising
one or more vitamins, one or more small peptide(s), and/or one or
more amino acid(s) or a derivative or prodrug thereof.
[0102] Vitamins that may be incorporated into compositions of the
invention include vitamin B compounds such as thiamine (vitamin
B1), e.g. as thiamine pyrophosphate, such as benfotiamine;
pyridoxamine (vitamin B6), vitamin A and/or E, or a derivative or
prodrug thereof
[0103] Pyridoxamine (B6) has been shown to effectively inhibit AGE
and lipoxidation product formation, and in particular blocks
formation of methylglyoxal-lysine dimer by itself forming
methylglyoxal-pyridoxamine dimer. Pyridoxamine (B6) and thiamine
pyrophosphate (B1) have both been shown to be effective
post-Amadori inhibitors of AGE formation with B6 effecting a
measurable decrease in rate of AGE formation and final AGE levels
and B1 effecting a measurable decrease in final AGE levels only.
Both compounds show far greater potency in post-Amadori inhibition
of AGE formation than aminoguanidine. Thiamine derivatives such as
benfotiamine (lipid-soluble prodrug of thiamine) have been
identified as potential therapeutic agents in the inhibition of
intracellular glycation in the treatment of vascular diabetic
complications and have been shown to inhibit imidazolone-type AGE
accumulation.
[0104] The composition may comprise one or more small peptide(s)
suitably as a dipeptide, tripeptide and/or tetrapeptide, and/or one
or more amino acid(s), e.g. proline, lysine, histidine, alanine, or
a derivative or prodrug thereof.
[0105] A composition according to the invention may further
comprise one or more polysaccharide, which may be one or more
proteoglycan, such as a glycosaminoglycan.
[0106] The one or more glycosaminoglycan employed can be a low
and/or high molecular weight hyaluronan, chondriotin sulphate,
dermatan sulphate and/or one or more derivative(s) thereof.
[0107] In addition to the need to deglycate matrix proteins and
increase the vascular function of the dermis, an important effect
of compositions according to the present invention is the
re-establishment of the proteoglycan content and distribution.
Proteoglycans (PGs) are important for providing the `smooth`
turgidity of skin due to hydration and are also important as
intercellular reservoirs for growth factors and other cytokines.
PGs are synthesized by the dermal fibroblasts and have a close
relationship with growth factors such as basic fibroblast growth
factor (b-FGF). The N-terminal binding domain of collagen is
affected by glycation and consequently the quantity and location of
PGs in the dermis are affected by AGE accumulation. For example,
heparan sulfate proteoglycans (HSPGs) promote cellular
proliferation through interaction with FGF-2.
[0108] Some GAGs, especially Hyaluronic acid, have been shown to be
decreasingly present in ageing skin. Even though the
mucopolysaccharides only constitute 0.1-0.3% of the dry weight of
the skin, any decrease can be easily understood to influence the
skin turgor as the molecules bind water in the dermis up to 1000
times the volume of the molecule itself. Additionally, these
substances are known to influence migration, growth and
differentiation of connective tissue cells in some instances.
[0109] Hyaluronic Acid or Hyaluronan ("HA") is a long-chained
polysaccharide that is a major constituent surrounding cells in
most animal tissues. HA is attracted to and adheres to specific
receptors on cell membranes which can be found in increasing
numbers at sites of damage and disease in the body with a
significant amount on the skin. This means that drugs can
potentially be targeted to and held at the site where the drug is
needed. The safety profile of HA, its ability to carry drug and its
potential targeting characteristics make it an excellent vehicle
for topical drug delivery. Drugs can be covatently attached to HA
or contained within the X-linked networks of derivatives of HA.
[0110] Hyaluronan has been used for decades in cosmetics,
viscosurgery and viscosupplementation without immunological
reactions or any other side-effects. It is present naturally at
high concentrations in connective tissues such as skin and
cartilage, in the vitreous body of the eye and in synovial fluid.
Mostly it is bound to cells and proteins but some HA is present in
the interstitial fluid. HA is a polysaccharide consisting of
alternating units of glucuronic acid and N-acetylglucosamine. The
carboxyl groups present are largely ionised at the pH of the skin
(generally around pH 4.5 to 5.5) and it is therefore highly
hydrophilic. The water binding properties and polymeric molecular
size of HA predispose HA to forming viscoelastic gels which have
potential for surface retention, acting as a reservoir for
therapeutic agents. Despite the hydrophilic properties of HA, it
can penetrate normal epidermis and accumulate extracellularly in
the dermis before disposal via known metabolic pathways.
[0111] In preferred embodiments, a composition according to the
invention will comprise a low and high molecular weight hyaluronan
and/or one or more derivative(s) thereof. Low molecular weight
hyaluronan characteristically has a molecular weight of less than
1.times.10.sup.6 Da, whereas a high molecular weight hyaluronan
generally has molecular weight of greater than 1.times.10.sup.6
Da.
[0112] HA forms a viscoelastic, smooth, lubricating film when
applied to the surface of the skin, thus externally applied HA not
only has a beneficial effect on the skin, but also can be used to
enhance the viscosity of a composition so that on application to
the skin it remains in contact with the skin in a gel-like layer.
This is particularly beneficial when ultrasound is to be applied to
skin treated with a composition according to the invention.
[0113] The HA molecule can be derivatised via modification of the
acetamido, the reducing end group but most commonly the hydroxy and
carboxylate groups. The glycosidic bond is also readily hydrolysed
to create shorter chains or oligosaccharides. HA-drug adducts have
been synthesised for controlled delivery applications and
HA-protein adducts as biomaterials and cell substrates.
[0114] HA exists as Na-HA at physiological pH. It has a complete
lack of immunogenicity which makes it an ideal building block for
biomaterials and drug delivery systems. HA effects a controlled and
sustained release of drugs through the skin by the formation of a
reservoir of the drug around the basement membrane. Neither NaCMC
(at a weight or Theologically equivalent concentration) nor
chondroitin sulphate (at a weight equivalent concentration) exert
the controlled release effect seen for the hyaluronan formulation
in full thickness skin, thus HA is a particularly preferred
glucosaminoglycan for the purposes of the present invention. The
enhancement of percutaneous absorption by HA is believed to be
partly due to its ability to hydrate the skin so disrupting the
compact cell/lipid layers. HA is superior to other GAGs in this
respect due to its high water binding capacity.
[0115] Tetrasaccharides of HA have been found to exert an
anti-apoptotic effect as they up-regulate HSP72 expression under
conditions of stress and suppress cell death. High molecular weight
HA polysaccharides are generally space filling molecules with
anti-angiogenic, anti-inflammatory and immunosuppressive activity.
Lower molecular weight fragments (6-20 kDa) are angiogenic,
inflammatory and immunostimulatory.
[0116] HA has been shown to be depolymerised by Maillard reaction
products (glucose-lysine) via a free-radical mechanism. Hydroxyl
radicals depolymerise HA and have been linked to inflammation in
arthritis and the breakdown of synovial fluid. High (but not low)
molecular weight HA and derivatives of HA such as BEHA act as
antioxidants scavenging reactive oxygen species (ROS) such as
O.sub.2'.sup.z,900 and OH, which otherwise would impair the
migratory and proliferative properties of dermal fibroblasts
thereby prolonging inflammation and delaying wound healing.
[0117] Hyaluronans have the ability to increase proteoglycan
synthesis, stimulate tissue inhibitor of metalloproteinase-1, has
the ability to stimulate collagen remodelling, to enhance cell
migration, stimulates wound healing by upregulating the expression
of transforming growth factor-.beta..
[0118] Low-molecular weight HA (.about.300 kDa) is available from
Sigma, Poole, Dorset (isolated from bovine vitreous humor). High
molecular weight HA is available from ConvaTec, Flintshire, UK
(isolated from human umbilical cord). Commercially available HA
preparations are given in tables 1a, 1b and 1c. TABLE-US-00001
TABLE 1a Examples of NaHA used in the clinical treatment of
osteoarthritis Trade name Molecular weight of NaHA* Manufacturer
Artz .RTM. .sup. 600,000-1,200,000 Seikagaku (Japan) Hyalgan .RTM.
500,000-730,000 Fidia (Italy) Synvisc .RTM. mildly cross-linked HA
Biomatrix (USA)
[0119] TABLE-US-00002 TABLE 1b Examples of NaHA used in
ophthalmologic surgery Trade name Molecular weight of NaHA*
Manufacture Opegan .RTM. .sup. 600,000-1,200,000 Seikagaku (Japan)
OpeganHi .RTM. 1,900,000-3,900,000 Seikagaku (Japan) Healon .RTM.
1,900,000-3,900,000 Pharmacia-Upjohn (Sweden) Opelead .RTM.
1,530,000-2,130,000 Shiseido (Japan) *Molecular weight of active
ingredient
[0120] Other HAs include NIF-NaHA marketed under the name of
Healon.RTM. for medical and Hylartil.RTM. for veterinary use; Hylan
A (elastoviscous fluid) and Hylan B (viscoelastic gel) developed by
Biomatrix Inc. TABLE-US-00003 TABLE 1c Trade Name Generic Molecular
Weight Elasticity Complex viscosity Polysaccharide (manufacturer)
Name (.times.10.sup.6 Da) (%) @3 Hz (Pa s @ 0.02 Hz) concentration
(mg/ml) Hyalgan .RTM. Hyaluronan 0.5-0.65 26 <0.1 10 (Fidia)
Artz .RTM. Hyaluronan 0.75 33 0.3 10 (Seikagaku) Orthovisc .RTM.
Hyaluronan 1.5 66 42 15 (Anika) Synvisc .RTM. Hylan 6 88 213 8
Biomatrix SkyePharma 0.6 (Solareze diclofenac gel)
[0121] A dermatogically acceptable excipient or excipients suitable
for use in a composition according to the invention include water,
a water/ethanol mixture (e.g. up to 25%, preferably up to 20%
ethanol in the composition % w/w), a viscous gel or emulsion, an
aqueous gel, a hydrogel, a water-based emulsion in the form of a
cream or application, an oil-in-water emulsion in the form of a
cream or application, or a jelly.
[0122] Generally, a composition which is to be applied in
conjunction with ultrasound treatment (where the composition is
applied prior to, during ultrasound treatment, or shortly after an
ultrasound pre-treatment), will have a viscous nature, so that a
layer of the composition can be spread on the skin and will remain
in place on the skin until it is removed, e.g. by wiping the
composition away with tissue or cotton wool, or by rinsing the
formulation off.
[0123] A composition according to the invention may comprise a
film-forming ingredient. One or more ingredient selected from: a
sun block, humectant, pigment, foundation or concealer pigment,
fake tan pigment or composition may be included in a composition
according to the invention.
[0124] A composition according to the invention is preferably at a
pH close to the pH of skin, e.g. at a pH of from pH 4 to pH 6, or
pH 4.5 to pH 5.5.
[0125] In a fifth aspect the present invention provides a method
for treatment of the skin, comprising applying to the skin a
composition according to the invention.
[0126] Preferably the method is a method of cosmetic treatment of
cosmetic skin conditions. However the invention also encompasses
the treatment of medical skin conditions, in which instances the
method is a method of medical treatment.
[0127] A method for treatment of the skin may further comprise
application of ultrasound directly or indirectly to an area of skin
to which the composition has been applied, or as a pre-treatment to
an area of skin to which the composition is to be applied.
[0128] In preferred methods of the invention application of
ultrasound is performed at low and/or high frequency directly or
indirectly to an area of the skin where the composition has been
applied, or is to be applied.
[0129] Low and high frequency ultrasound can be applied
simultaneously, sequentially or separately, e.g. sequentially as
several alternating single applications of low and high frequency
or, separately where a series of applications of low frequency is
alternated with a series of applications of high frequency. Low
frequency ultrasound is believed to be useful to facilitate
delivery of molecules to the skin (a process termed
"sonophoresis"). High frequency ultrasound has a lesser
sonophoretic effect than low frequency, but it also has many other
effects beneficial to the skin in that it stimulates fibroblast
proliferation, stimulates collagen and other extracellular matrix
(ECM) component formation (e.g. fibrillin), stimulates blood
supply, renews the elastic quality of ECM which stiffen with age,
stimulates the expression of Heat Shock Proteins
(HSPs--intracellular molecular chaperones) in fibroblasts (dermis)
and keratinocytes (epidermis) through thermal and mechanical
stimulation.
[0130] In a preferred method, low and high frequency ultrasound is
applied simultaneously.
[0131] In methods of the invention involving application of low and
high frequency ultrasound, the low frequency component of the
ultrasound is preferably applied in continuous mode and the high
frequency component is preferably applied in pulsed mode.
[0132] The term "ultrasound" describes sound frequencies of 20 kHz
and above, a low ultrasound frequency is from 20 to 500 kHz, the
spatial average power density of the low frequency ultrasound
energy being from 20 to 500 mW/cm.sup.2; a high ultrasound
frequency is from 500 kHz (0.5 MHz) to 3.5 MHz, the spatial average
power density of the high frequency ultrasound energy being from
0.5 to 3 W/cm.sup.2.
[0133] In methods of the invention generally the beam is not
focussed and is diverging. The target rise in skin temperature is
up to 42 or 43.degree. C., but preferably no higher. Single areas
of skin are insonated for a minimum of 5 minutes, preferably 10
minutes, or a time between 5 and 12 minutes.
[0134] In methods of the invention, ultrasound can be applied using
a hand-held applicator, optionally adapted for application of a
composition according to the invention to the skin. For example, a
cartridge/dispenser can be attached to the ultrasound head such
that the formulation is gradually released as the head is moved
around the skin surface, the cartridge may contain a pre-set amount
of formulation. Different cartridges with different formulations
can be attached depending on the skin condition being treated, e.g.
different cartridges may contain different compositions for
anti-ageing treatments, the treatment of scars, stretch-marked skin
or cellulite. The ultrasound is applied by gently massaging the
ultrasound applicator on the skin in a circular or linear stroking
movement.
[0135] Ultrasound can be applied as a pre-treatment, before
application of a composition of the invention. Alternatively or
additionally, a layer of the composition or a material impregnated
with the composition can be applied to the skin during or prior to
the application of ultrasound. When using an applicator, the
applicator is moved across the skin so that no single area is
over-exposed to ultrasound, which could cause overheating. The
combination of ultrasound and the mechanical stimulus afforded by
the massaging action helps to stimulate the skin to encourage
renewal and repair.
[0136] Ultrasound can be applied by applied by immersion of the
area to be treated in an ultrasound bath, e.g. directly by
immersing bare skin in a composition of the invention, or by
coating the skin with the composition, wrapping the part to be
treated, and immersion in a liquid that can transmit ultrasound
energy.
[0137] As described above, an apparatus for application of
ultrasound to the skin may comprise a plurality of ultrasound
transducer elements arranged as an array in a flexible material in
spaced configuration, wherein the ultrasound transducer elements
are capable of delivering ultrasound at low and/or high frequency
to an area of the skin.
[0138] Skin treatment using these method can be performed in a
beauty clinic or in a medical clinic such as a hospital clinic, or
in a doctor's surgery.
[0139] A circular area of skin 2 to 3 cm in diameter should be
subjected to ultrasound for a minimum of 5 minutes, preferably 10
minutes or a time between 5 and 12 minutes.
[0140] Where skin treatments are to be carried out in a beauty
clinic, or a medical clinic, such as in a hospital or doctors
surgery, a suitable ultrasound device for use in methods of the
invention is an ultrasound array incorporated into a mask or patch
as described herein; or a device which can generate low and high
frequency outputs and has a handset designed to give combinations
of output frequencies and energies, such as the Duo Son.TM. unit
(Orthosonics, Devon UK). The Duo Son.TM. unit can be used either at
the current specification (described in Table 2), or at a slightly
altered specification dictated by optimisation of frequency,
wattage and duty cycle parameters to enhance actives delivery and
cell/protein stimulation. Such adjustments remain within the
guidelines for CE marking of the unit.
[0141] The effectiveness of ultrasound (US), or sonophoresis for
delivery of molecules to the skin, is due to two aspects of its
action on the skin. Firstly, cavitation results from the rapidly
oscillating pressure field causing bubble formation and collapse
which mechanically creates channels through the stratum corneum.
The second effect is actual direct heating of material through
which the sound waves are travelling due to attenuation of the
acoustic energy through reflection, absorption and dispersion. This
occurs in skin up to four times more than in other tissues due to
its heterogeneity. Heating is known to disrupt the lipid bilayer
system in the stratum corneum also contributing to the enhanced
permeability of the epidermis. TABLE-US-00004 TABLE 2 Power Supply
Input 90 V 260 V ac (50/60 Hz) Output 24 V, 1.25 A dc Battery life
15 mins to 2 hours depending upon mode selection Other Modes LF
only, LF + HF (pulsed) Weight 2 kg Dimensions 200 .times. 250
.times. 70 mm (w .times. d .times. h) Class BF Display Liquid
Crystal Display Standard 1EC 61689 (96) High Frequency Low
Frequency 45 kHz 1 MHz Effective beam radiating 16.3 cm.sup.2 0.38
cm.sup.2 area Beam non-uniformity <6 <6 ratio Beam Type
Diverging diverging Power Settings 0.15 W, 0.4 W, 0.75 W 0.07 W,
0.14 W Maximum Intensity 100 m W/cm.sup.2 1 W/cm.sup.2 (420
mW/cm.sup.2 pulse averaged) Mode of Operation Continuous pulsed 20%
duty cycle
[0142] Essentially the skin's permeability is increased by
disruption of the intercellular lipids through a combination of
heating and/or mechanical stress and through the increase in
porosity. Temperature rises to 50.degree. C. (20 kHz, 10-30
W/cm.sup.2) have been reported, but rises as little as 11.degree.
C. (1 MHz, 2 W/cm.sup.2) have been shown to cause skin damage.
Continuous mode ultrasound at an intensity of 1 W/cm.sup.2 raises
the temperature of tissue at a depth of 3 cm to 40.degree. C. in 10
minutes. It has been suggested that tissue must reach a temperature
of 40-45.degree. C. for at least 5 minutes to be therapeutically
beneficial. Tissue temperatures have been shown to increase at a
rate of 0.860C/min when exposed to 1 W/cm.sup.2-1 MHz
ultrasound.
[0143] Ultrasound is known to act to degrade polymers and has been
used to increase in the release of incorporated drugs from these
polymers. When ultrasound is applied to compositions of the
invention comprising polymers such as polysaccharides, preferably
proteoglycans, in particular glucosaminoglycans, such as HA, the
ultrasound will degrade (depolymerise) the polymer. This has a
number of effects, when HA is present in the composition,
ultrasound generates shorter chain HA polymers that are more
readily able to penetrate the skin. In addition to employing
ultrasound to facilitate permeation of active substances, it is
important that the effective residence time of such actives at the
site of action is maximised. The microvasculature enables
substances to be removed from the site of permeation for breakdown
in the liver. Optimisation of the residence time is necessary to
permit the active substance to have the desired effect. When
composition of the invention comprise polymers such as
polysaccharides or proteoglycans, in particular glucosaminoglycans,
such as HA, ultrasound treatment will assist in formation of a
depot of the polymer (e.g. HA) within the skin which can act as a
reservoir for slow release of actives providing an extended
duration of effect.
[0144] Ultrasound also stimulates liberation of the active
ingredients of the composition. Ultrasound will also act to
depolymerise polymers polysaccharides or proteoglycans, in
particular glucosaminoglycans, such as HA that have penetrated the
skin. When HA and ascorbic acid are present in a composition,
depolymerisation of the HA will be encouraged by ascorbic acid in
which will react with atmospheric oxygen and stimulate
depolymerisation.
[0145] A composition of the invention can be applied in conjunction
with ultrasound, but this is not essential, a composition of the
invention may also be applied to the skin without the application
of ultrasound. In a preferred method for treatment of skin, a first
composition of the invention is applied in conjunction with
ultrasound (either with an ultrasound pre-treatment of the skin to
which the composition is applied, or with ultrasound treatment of
skin during or after application of the composition to the skin) on
a weekly, fortnightly or monthly basis. Following the ultrasound
treatment of the skin, a second composition of the invention may be
applied, e.g. on a daily or 12 hourly basis without the need for
ultrasound. The first and second compositions can be identical or
may differ in composition, but will both be compositions according
to the invention.
[0146] The present invention further provides a dressing comprising
a composition according to the invention, which may be presented in
the form of a plaster, patch, gel patch bandage or foam.
[0147] Also provided is a kit comprising a composition according to
the invention and optionally, a device comprising an ultrasound
source and/or optionally an applicator for applying ultrasound to
the skin and/or for applying the composition to the skin. A kit
according to the invention is suitable for performing a method of
the invention as described herein. A kit may further comprise
instructions for use of the kit.
[0148] The invention provides the use of a composition according to
the invention in the treatment of a cosmetic skin condition. Also
provided is the use of composition according to the invention in
the manufacture of a cosmetic composition for the treatment of a
cosmetic skin condition e.g. selected from the group: scarring, sun
damaged skin, ageing skin, wrinkles, coarseness, irregular
pigmentation, telangiectasias, elastosis, cellulite, orange peel
appearance of skin; dry skin conditions, scaliness, acne, stretch
marks; rashes, chapping, inflamed skin; blemishes, rosacea, acne
ice-pick scars, hypertrophic and keloid scars, and hairloss.
[0149] The term "cosmetic skin conditions", as used herein,
includes signs of skin ageing which include, but are not limited
to, all outward visibly and tactilely perceptible manifestations as
well as any other macro or micro effects due to skin ageing. Such
signs may be induced or caused by intrinsic or extrinsic factors,
e.g., chronological ageing and/or environmental damage (e.g.,
sunlight, UV, smoke, ozone, pollutants, stress, etc.). These signs
may result from processes which include, but are not limited to,
the development of textural discontinuities such as wrinkles,
including both fine superficial wrinkles and coarse deep wrinkles,
skin lines, facial frown lines, expression lines, rhytides,
dermatoheliosis, photodamage, premature skin ageing, crevices,
bumps, pits, large pores (e.g., associated with adnexal structures
such as sweat gland ducts, sebaceous glands, or hair follicles),
"orange peel" skin appearance, dryness, scaliness, flakiness and/or
other forms of skin unevenness or roughness; excess skin oil
problems such as over-production of sebum, oiliness, facial shine,
foundation breakthrough; abnormal desquamation (or exfoliation) or
abnormal epidermal differentiation (e.g., abnormal skin turnover)
such as scaliness, flakiness, keratosis, hyperkeratinization;
inadequate skin moisturization (or hydration) such as caused by
skin barrier damage, environmental dryness; loss of skin elasticity
(loss and/or inactivation of functional skin elastin) such as
elastosis, sagging (including puffiness in the eye area and jowls),
loss of skin firmness, loss of skin tightness, loss of skin recoil
from deformation; non-melanin skin discoloration such as under-eye
circles, botching (e.g., uneven red coloration due to, e.g.,
rosacea), sallowness (pale colour), discoloration caused by
telangiectasia; melanin-related hyperpigmented (or unevenly
pigmented) skin regions; post-inflarmmatory hyperpigmentation such
as that which occurs following an inflammatory event (e. g., an
acne lesion, in-grown hair, insect/spider bite or sting, scratch,
cut, wound, abrasion, and the like); atrophy such as, but not
limited to, that associated with ageing or steroid use; other
histological or microscopic alterations in skin components such as
ground substance (e.g., hyaluronic acid, glycosaminoglycans, etc.),
collagen breakdown and structural alterations or abnormalities
(e.g., changes in the stratum corneum, dermis, epidermis, the skin
vascular system such as telangiectasia); tissue responses to insult
such as itch or pruritus; and alterations to underlying tissues
(e.g., subcutaneous fat, cellulite, muscles, trabeculae, septae,
and the like), especially those proximate to the skin.
[0150] The invention also provides the use of a composition
according to the invention in a medical treatment or as a
medicament. Thus a composition of the invention is provided for use
as a medicament. Further provided is the use of a composition
according to the invention in the manufacture of a medicament for
the treatment of a medical skin condition, e.g. selected from the
group: scarring, sunburn, wounds, cuts, bruises, burns, burn
scarring, eczema, dermatitis, dry skin conditions, urticaria,
psoriasis.
LIST OF FIGURES
[0151] FIG. 1(a)-(c) show an array of transducer elements; FIG.
1(d) shows an individual transducer element with dual frequency
capability.
[0152] FIG. 2(a)-(b) show a transducer element.
[0153] FIG. 3A shows a single DDS chip for generation of
alternative high and low frequencies, whereas in FIG. 3B high and
low frequencies are generated in individual circuits.
[0154] FIG. 4 shows the effect of Topical 1 formulation on
fibrillin expression proximal to the dermal-epidermal junction.
[0155] FIG. 5 shows the effect of Topical 1 formulation on MMP-1
expression in the epidermis.
[0156] FIG. 6 shows the of Topical 1 formulation on RAR.alpha.
expression in the epidermis.
[0157] FIG. 7 shows the effect of Topical 1 formulation on HSP72
expression in the epidermis.
[0158] FIG. 8 shows the effect of Topical 1 formulation on
fibrillin expression proximal to the dermal-epidermal junction
following ultrasound treatment.
[0159] FIG. 9 shows the effect of Topical 1 formulation on
epidermal MMP-1 expression following ultrasound treatment.
[0160] FIG. 10 shows the effect of Topical 1 formulation on
epidermal HSP72 expression following ultrasound treatment.
[0161] FIG. 11 shows the effect of Topical 1 formulation on dermal
clusterin expression following ultrasound treatment.
EXAMPLES
Example 1
Topical Formulation "Topical 1"
[0162] Topical 1 is a composition according to the invention
containing the following components: TABLE-US-00005 TABLE 3
Component Amount % w/w Hyaluronic acid (0.8-3 MDa mol wt range) 1%
Hyaluronic acid (1.5-1.8 MDa mol wt range) 3% Ethanol 20% Ascorbic
acid 3% Trisodium ascorbyl phosphate (STAY C-50) 1% Carnosine 3%
N-Acetyl Carnosine 1% Arginine 3% Sterile distilled water to
100%
Example 2
Patch Tests
[0163] The standard four-day patch test assay was extended to a
seven-day period to examine the effect of the Topical 1 formulation
under occlusion or delivered by ultrasound on the induction of
fibrillin expression as a mechanism by which clinical signs of
photoageing may be improved.
[0164] Subjects with clinical evidence of photoageing of forearm
skin had the following products applied under occlusion to the
dorsum of the forearm.
[0165] (1) 0.025% t-RA made up in a vehicle comprising 67.2%
ethanol 30% propylene glycol and 0.05% butyl hydroxytoluene
(preservative) to 100% with deionised water (positive control),
[0166] (2) Vehicle alone as above,
[0167] (3) Topical 1 formulation.
[0168] Topical 1 was delivered with and without use of an
ultrasound device.
[0169] The positive control, topical application of 0.025%
all-trans RA with occlusion was applied within the standard
four-day time frame to minimise potentially deleterious side
effects.
[0170] Fibrillin is a major constituent of the elastin network in
the papillary dermis and plays an important role in securing the
epidermis to the underlying dermis. Topical application of
all-trans retinoic acid (t-RA) as a positive control ameliorates
the clinical signs of photoageing. Previous work has demonstrated
that topical application of t-RA under occlusion for four days (the
four-day patch test) produces a significant increase in fibrillin
mRNA and protein, predictive of those seen after several weeks of
non-occluded topical application providing a rapid and convenient
means of assessing the action of new therapeutic interventions upon
the skin.
[0171] Ageing can also be monitored by examination of other
molecules within the skin. Previous work has identified alterations
in the expression of the enzyme matrix metalloproteinase 1 (MMP-1;
essential for extracellular matrix remodelling).sup.3 and in the
nuclear retinoid receptor retinoic acid receptor a (RARa; necessary
for modulating the effects of vitamin A and its derivatives).sup.4.
Two molecular chaperones were assessed to identify pathways for
eliminating abnormal proteins; these were heat shock protein-72
(HSP72).sup.5 and the extracellular chaperone, clustering.
[0172] The study was an open clinical study; assessment of biopsies
was randomised and blinded. The study was carried out on 10
subjects, aged 40-80 years, who were judged to have moderate to
severe photoaged forearm skin.
[0173] The inclusion criteria applied were:
[0174] Aged 40 to 80 years; willing to submit to examination of
photoaged forearm skin; willing to wear test patches on forearm for
up to 4 days; willing to submit to 3 mm punch biopsies from each of
the four test sites (total of 4 biopsies); no disease state that
would impair evaluation of the test sites; not on systemic drugs;
no topical or systemic retinoids within the past 6 months and 12
months respectively prior to entry to the study; no topical
steroids or other topical drugs two weeks; signed informed
consent.
[0175] The exclusion criteria applied were:
[0176] History of use of experimental drug or experimental device
in the 30 days prior to entry into the study; history of keloid
scars; pregnancy or breastfeeding.
[0177] The study procedure involves taking of history and a
physical examination; clinical grading of the severity of extensor
forearm photoageing on a 0 to 9 scale (0=no damage; 9=maximum
photodamage); written, witnessed, informed consent is obtained.
Those subjects admitted to the study have photodamage grade of 6 or
greater.
[0178] Materials and Methods
[0179] Patch-Test Protocol
[0180] Six healthy but photoaged female volunteers were recruited
(age range 52-79 years). Test substances were applied separately
under standard 6 mm diameter Finn chambers to the extensor aspect
of the forearm: these were Topical 1 formulation (20
.mu.l/chamber), vehicle base (20 .mu.l/chamber) and 0.025%
all-trans RA (Retin-A.RTM. cream, Janssen-Cilag Ltd., 20
.mu.l/chamber). In addition, a baseline untreated biopsy was taken
as a reference point. Formulations were applied to clean skin on
days 1 and 4 of the assay. All-trans RA was applied to an untreated
site on day 4. On day 8, Finn chambers were removed and 3 mm punch
biopsies were obtained under 1% lignocaine anaesthesia from each of
the test site. Biopsies were embedded in OCT compound
(Tissue-Tek.RTM., Miles, Ind., USA) and snap frozen in liquid
nitrogen. Biopsy sites were sutured with 1.times.4/o ethilon and
subjects instructed to return between 7-10 days for suture
removal.
[0181] Ultrasound Protocol
[0182] Ten healthy but photoaged volunteers were recruited (men: 2;
female: 8; age range 40-79 years). Topical 1 formulation was
applied to extensor forearms alone and with the aid of an
ultrasound device (Duo Son.TM. unit, Orthosonics, Devon UK). Low
frequency (45 kHz, 100 mW/cm.sup.2, continuous) and high frequency
(1 MHz, 1 W/cm.sup.2 pulsed average, pulsed 20% cycle) were applied
simultaneously for a period of ten minutes, during which time the
ultrasound delivery head was moved over the skin. Treatment
occurred on days 1, 4 and 7 of the assay. On day 8, 3 mm punch
biopsies were removed from each treatment site as described
previously.
[0183] Slide Preparation
[0184] Frozen sections were prepared at a thickness of 10 .mu.m
(OTF cryostat, Bright Instruments Ltd.) and mounted onto
gelatin-coated slides prior to histological analysis.
[0185] Immunohistochemistry
[0186] A number of extracellular matrix (ECM) molecules known to be
reduced in photoaged skin were assayed by immunohistochemistry to
detail the potential effects of the Topical 1 formulation and its
method of delivery. The primary marker of outcome was the
distribution of fibrillin-rich microfibrils proximal to the
dermal-epidermal junction (DEJ). Also assessed were the number of
epidermal keratinocytes expressing MMP-1 and RARa. Molecular
chaperones, HSP72 and clusterin were examined to assess potential
repair mechanisms.
[0187] For each analysis, the marker was identified in each of
three sections (i.e., 3 sections/treatment/patient).
[0188] Sections were optimally fixed. Following hydration in
tris-buffered saline (TBS; 100 mM Tris, 150 mM NaCl), sections were
solublised by addition of 0.5% Triton.RTM.-X 100 (10 minutes).
Following washing, endogenous peroxidase activity was abolished by
incubation with an excess of hydrogen peroxide in methanol (30
minutes). Sections were blocked prior to application of primary
antibody (overnight incubation at 4.degree. C.). Negative controls
were concurrently incubated with either block alone or control
mouse serum. Following incubation, sections were stringently washed
with TBS prior to application of an appropriate biotinylated
secondary antibody. This was further conjugated to the enzyme
horseradish peroxidase using a commercially available kit following
the manufacturers instructions (ABC Elite System, Vector
Laboratory, Peterborough UK). Antibody was localised using Vector
SG.RTM. as chromogen (10 minute incubation, washing in TBS quenched
this reaction. Sections were counterstained using Nuclear Fast Red
and finally dehydrated through serial alcohols, cleared and
permanently mounted. TABLE-US-00006 TABLE 4 Marker Host Clone
Fixation Dilution Fibrillin-rich Mouse NeoMarkers; 11C1.3 4% PFA
1:100 microfibrils IgG MMP-1 Mouse Oncogene; 41-1E5 4% PFA 1:100
IgG RAR.alpha. Rabbit Santa Cruz acetone 1:100 IgG Biotechnologies
HSP72 Mouse Stressgen 4% PFA 1:100 IgG Biotechnologies Clusterin
Rabbit Santa Cruz 4% PFA 1:100 IgG Biotechnologies
[0189] Quantification
[0190] Sections were randomised, blinded and examined on a Nikon
OPTIPHOT microscope (Tokyo, Japan), For assessment of ECM
components, the degree of immunostaining was assessed on a 5 point
semi-quantitative scale where 0=no staining and 4=maximal staining.
Four sections (including control) were examined per subject per
site. The degree of immunostaining was scored for three high power
fields per section, and the average score calculated for each
site/test area,
[0191] For cell-associated staining, the numbers of positive
epidermal keratinocytes were assessed per high powered field, and
the average score calculated for each site/test area.
[0192] Differences in the distribution between the test sites, and
after application of test substances for varying periods of time,
were assessed for significance using the repeated measures analysis
of variance test (ANOVA). To assess whether delivery methods
affected outcome measures, data was tested using paired Student's
t-tests, Both models were tested using SPSS+ software (v11.5, SPSS
Inc., Ill. USA) with significance taken at the 95% confidence
level.
[0193] Results
[0194] Erythema
[0195] All volunteers tolerated the patch test protocol well.
Furthermore, all-trans RA produced marked erythema at the site of
application. Erythema was not observed using the Topical 1
formulation.
[0196] Patch-Test Protocol
[0197] Fibrillin-1 IHC
[0198] Application of all-trans RA (our "gold" standard) produced
deposition of fibrillin-1 proximal to the DEJ in 3/6 volunteers.
The Topical 1 formulation resulted in increased fibrillin-1
deposition in 416 volunteers tested but to a lower level than that
generally observed using all-trans RA (FIG. 4). TABLE-US-00007
TABLE 5 Fibrillin-1 staining Treatment Mean Std Deviation Baseline
1.1111 0.4608 vehicle 1.3241 0.6050 Topical 1 formulation 1.3704
0.9930 All-trans RA 1.8241 0.7985 p > 0.05, non significant
[0199] MMP-1 IHC
[0200] MMP-1 staining was observed in both epidermis and dermis.
Overall, topical application of all-trans RA for 4-d under
occlusion reduced MMP-1 expression in epidermal keratinocytes,
although this did not reach significance. Application of the
Topical 1 formulation resulted in a similar level of reduction
(FIG. 5). TABLE-US-00008 TABLE 6 MMP-1 staining Treatment Mean Std
Deviation Baseline 12.1650 6.4340 vehicle 7.9890 3.2898 Topical 1
formulation 5.0093 4.2616 All-trans RA 5.3333 4.3165 p > 0.05,
non significant
[0201] RAR.alpha. IHC
[0202] RAR.alpha. staining was observed in primarily in the
epidermis. Overall, topical application of all-trans RA for 4-d
under occlusion significantly reduced RAR.alpha. expression in
epidermal keratinocytes. Application of the Topical 1 formulation
resulted in a similar level of reduction although this did not
reach significance in this data set (FIG. 6). TABLE-US-00009 TABLE
7 RAR.alpha. staining Treatment Mean Std Deviation Baseline 16.8370
6.7277 Vehicle 10.1111 4.1407 Topical 1 formulation 8.3611 5.1287
All-trans RA 6.0463 5.1090 p > 0.05, non significant
[0203] HSP72
[0204] HSP72 is found mainly in the epidermis where strong staining
was observed. Treatment with all-trans RA significantly increased
the expression of epidermal HSP72 as did the Topical 1 formulation
(p=0.005 and 0.012 respectively; FIG. 7). TABLE-US-00010 TABLE 8
HSP72 staining Treatment Mean Std Deviation Baseline 56.18 8.55
Vehicle 63.61 4.83 Topical 1 formulation 74.08* 17.49 All-trans RA
77.03** 15.44 *p = 0.012 **p = 0.005
[0205] Ultrasound Protocol
[0206] Fibrillin-1
[0207] The Topical 1 formulation was further applied to photoaged
extensor forearm by basic topical application without occlusion or
via an ultrasound device. In all those studied (n=10) delivery by
ultrasound resulted in significantly increased fibrillin deposition
proximal to the dermal-epidermal junction (FIG. 8). TABLE-US-00011
TABLE 9 Fibrillin-1 staining Treatment Mean Std Deviation Topical
1.8272 0.8698 Ultrasound 2.6065* 1.0052 *p = 0.033
[0208] MMP-1
[0209] MMP-1 staining was observed in both epidermis and dermis.
There was a slight, non-significant, reduction in MMP-1 expression
in epidermal keratinocytes following ultrasound treatment (FIG. 9).
TABLE-US-00012 TABLE 10 MMP-1 staining Treatment Mean Std Deviation
Topical 114.71 22.03 Ultrasound 107.46 31.96 p > 0.05, non
significant
[0210] HSP72
[0211] HSP72 showed a slight, but non-significant, increase in
epidermal expression following ultrasound treatment (FIG. 10).
TABLE-US-00013 TABLE 11 HSP72 staining Treatment Mean Std Deviation
Topical 56.31 16.58 Ultrasound 67.57 16.41 p > 0.05, non
significant
[0212] Clusterin
[0213] Clusterin also showed a slight, but non-significant,
increase in dermal expression following ultrasound treatment (FIG.
11). TABLE-US-00014 TABLE 12 Clusterin staining Treatment Mean
Deviation Topical 1.80 0.98 Ultrasound 1.95 1.00 p > 0.05, non
significant
SUMMARY
[0214] Application of the Topical 1 formulation using an ultrasound
device significantly increased fibrillin deposition proximal to the
dermal-epidermal junction of photoaged skin.
[0215] HSP72 expression was modulated by both the Topical 1
formulation and all-trans RA. Furthermore, ultrasound delivery of
Topical 1 formulation also resulted in increased HSP72 expression.
It is possible that increased heat shock protein expression may be
beneficial to the skin, by `priming` cells to cope with
environmental stressors (hormesis). Hence, increased HSP72
expression by epidermal keratinocytes may be involved in cellular
repair processes following damage.
[0216] The data suggests that there is reduction in MMP-1 and
RAR.alpha. expression in the epidermis of photoaged skin following
application of this formulation, this supports its suitability for
use in the treatment of aged skin.
[0217] Additionally, the following non-quantitative observations
were made during the course of the trial relating to the
effectiveness of ultrasound-based skin treatments. Although not
directly comparable due to the protocol structuring of the trial,
Topical 1 formulation applied with ultrasound appeared to induce a
similar level of fibrillin to the retinoid formulation. Topical 1
formulation only and Topical 1 formulation applied with ultrasound
did not induce erythema or epidermal hyperplasia, unlike the
retinoid formulation. The degree of scarring at biopsy sites
previously treated with ultrasound was noticeably lower than
scarring at all other sites
REFERENCES
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A, Kielty C M. Fibrillin-rich microfibrils are reduced in photoaged
skin: Distribution at the dermo-epidermal junction. J Invest
Dermatol, 112: 782-787, 1999
[0219] 2. Watson R E B, Craven N M, Kang S, Jones C J P, Kielty C
M, Griffiths C E M. A short term screening protocol, using
fibrillin-1 as a receptor molecule for photoageing repair agents. J
Invest DermatolI 116:672-678, 2001
[0220] 3. Brennan M, Bhatti H, Nerusu K C, Bhagavathula N, Kang S,
Fisher G J, Varani J, Voorhees J J. Matrix metalloproteinase-1 is
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UV-irradiated human skin. Photochem Photobiol, 78:43-48, 2003
[0221] 4. Watson R E B, Ratnayaka J A, Brooke R C, Yee-Sit-Yu S,
Ancian P, Griffiths C E M. Retinoic acid receptor alpha expression
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