DERMAL COMPOSITIONS CONTAINING UNNATURAL
HYGROSCOPIC AMINO ACIDS
The present application relates to substances and compositions suitable to enhance hydration
and moisturisation of the skin.
Xerosis, or dry skin, is a common condition experienced by most people at some point in
their life. Seasonal xerosis is common during the cold, dry winter months, and evidence
shows that xerosis becomes more prevalent with age (Whit-Chii, 201 1). Many inflammatory
skin conditions, such as atopic dermatitis, irritant contact dermatitis, and psoriasis, cause
localised areas of xerotic skin. In addition, some patients have hereditary disorders, such as
ichthyosis, resulting in clironic dry skin.
An important role of natural moisturising factor (NMF) is to maintain adequate skin
hydration. Adequate hydration of the stratum corneum serves three major functions: (1) it
maintains plasticity of the skin, protecting it from damage; (2) it allows hydrolytic enzymes
to function in the process of desquamation (Rawlings, 1994), and (3) it contributes to
optimum stratum corneum barrier function.
NMF is composed principally of free amino acids, and various derivatives of these amino
acids such as sodium pyrrolidone carboxylic acid (pyroglutamate, 2-oxo-pyrrolidone
carboxylic acid, or PCA), urocanic acid (a natural absorber of ultraviolet [UV] light),
inorganic salts, sugars, and lactic acid and urea (Table 2) (Clar, 1 1) . Inorganic salts so far
associated with NMF include the chlorides, phosphates, and citrates of sodium, potassium,
calcium, and magnesium. NMF is packaged within the corneocytes, making up
approximately 10 percent of the corneocyte mass and 20 percent to 30 percent of the dry
weight of the stratum corneum.
NMF components are highly efficient humectants that attract and bind water from the
atmosphere, or from deeper skin layers, drawing it into the corneocytes. This process can
occur even at a relative humidity as low as 50 percent, allowing the corneocytes to maintain
an adequate level of water in low-humidity environments. The water absorption is so
efficient that NMF essentially dissolves within the water that it has absorbed (Rawlings,
1994). Hydrated NMF, particularly the neutral and basic amino acids, forms ionic
interactions with keratin fibres, reducing the intermolecular forces between the fibres and,
thus, increasing the elasticity of the stratum corneum. This elasticity serves to make the skin
appear healthy and supple and to help prevent cracking or flaking due to mechanical stress. In
addition, NMF allows the corneocyte cells to balance the osmotic pressure exerted by the
intracellular "cement" surrounding them,
Keeping the solute concentrations balanced is important for preventing both excessive water
influx, as seen in the wrinkled skin after a long bath, and water efflux, which would cause the
corneocytes to shrink.
Traditionally, the stratum corneum is thought of as nonviable tissue. While this is technically
true, the stratum corneum is a dynamic structure in which numerous enzymes still function,
and these enzymes require a certain amount of free, or liquid, water to perform. NMF-bound
water provides much of this necessary water, and many of these enzymes are involved in the
process of desquamation, breaking the various bonds and forces holding the corneocytes
together in the most superficial layers of the skin. Research shows the activity of these
desquamatory enzymes is affected by water levels within the tissue (Harding, 2000).
Reduction in, or the lack of, NMF has been correlated with various stratum corneum
abnormalities that manifest clinically as areas of dry skin with scaling, flaking, or even
fissuring and cracking. These conditions include atopic dermatitis, psoriasis, ichthyosis
vulgaris, and xerosis. In atopic dermatitis, it has been shown that the amounts of NMF in the
skin are often reduced (Palmer, 2006) while, in psoriatic skin and ichthyosis, NMF is
essentially absent (Harding, 2000). Reduced NMF levels are also seen in more common skin
conditions such as xerosis. Routine soap washing of the skin has been shown to remove
NMF from the superficial layers of the stratum corneum. In fact, the outermost layers
typically show reduced NMF levels, largely due to bathing or exposure to UV light. In
addition, aging appears to dramatically reduce the amino acid content in the stratum corneum.
Studies have shown a significant correlation between the hydration of the skin and its amino
acid content (Horri, 1989). All of these conditions show characteristics of abnormal
desquamation, with the accumulation of corneocytes resulting in the visible dryness,
roughness, scaling, and flaking properties of dry skin (Harding, 2000).
The source of NMF was the subject of intensive research for a considerable time. Numerous
studies on urocanic acid and PCA established that these compounds were derived from amino
acids in the stratum corneum, which had been assumed to contain no active enzymes, as
noted above. As a result of this research, it is now recognised that, while the stratum corneum
is biologically dead, it is biochemically very active. Analysis of the amino acid composition
of the stratum corneum eventually led to the discovery that NMF components were
breakdown products resulting from the proteolysis of the filaggrin protein (Scott, 1982).
Filaggrin is a large, histidiiie rich protein localised in newly formed corneocytes, present in
the corneocyte layer above the granular layer. The function of filaggrin is to aggregate
filaments, and specifically to align epidermal and inner root sheath keratin filaments into
highly ordered linear arrays, or macrofibrils.
Filaggrin has a high-molecular-weight precursor, profilaggrin, which originates in the
keratohyalin granules of the granular layer. As the granular cells differentiate into cornified
cells, profilaggrin is dephosphorylated and degraded into the highly basic, lower molecular
weight filaggrin. It is at this stage that filaggrin works to aggregate filaments, catalysing the
formation of disulphide bonds between the keratin fibres. These aggregated fibres form part
of the envelope surrounding the cells entering the stratum corneum, allowing them to
maintain the extremely flattened shape characteristic of corneocytes (Scott, 1982).
Filaggrin is subject to almost immediate proteolytic and degradative attack, once the keratin
fibres have been formed. One of the first steps in this degradation process is the conversion of
the filaggrin arginine residues to citriilline residues. This process increases the acidity of the
filaggrin molecule, resulting in the loosening of the filaggrin/keratin complex and increasing
the access of proteolytic enzymes. At this point, the filaggrin molecules are completely
degraded into their respective amino acids and derivatives, which go to make up 70 to 100
percent of the free amino acids and their derivatives present in the stratum corneum (Scott,
1982).
The conversion of filaggrin to NMF occurs as the corneocytes are moving to the more
superficial layers of the stratum corneum. The timing and exact depth in the stratum corneum
of filaggrin processing is dependent on the water activity within the corneocyte and the
external relative humidity. In a humid environment, where there are no drying effects, the
hydrolysis of filaggrin occurs almost at the outermost surface. In low humidity, proteolysis
occurs at deeper layers where NMF works to prevent desiccation of the skin (Harding, 2000).
It has been demonstrated that occlusive patches applied to the skin can prevent filaggrin
degradation altogether.
Conversion of filaggrin to NMF is also controlled by the water activity within the corneocyte,
and only occurs within a narrow range - if the water activity is too high, filaggrin is stable,
while if it is too low, the hydrolytic enzymes will be unable to function and degrade the
filaggrin (Harding, 2000). Thus, the hydration status of the skin influences the degradation
process of filaggrin.
Importantly, the creation of NMF creates substantial osmotic pressure within the corneocyte.
Therefore, the degradation process does not occur until the corneocytes have matured and
strengthened and migrated toward the more superficial layers of the stratum corneum, where
the surrounding lipids and other extracellular components balance the resulting osmotic
pressure (Harding, 2000).
NMF is generally considered to comprise the water-extractable material released by 30
minutes water treatment of acetone/ ether treated stratum corneum (Jokura, 995). The water
extractable material is considered to be the total natural moisturising factor found within the
stratum corneum. Typically, the composition of NMF is approximately: amino acids 48.3%;
PCA 10.2%; uric acid 2.1%; lactic acid 10.1%; citric acid 7.9%; other organic acids 2%; urea
14%, and inorganic ions 5.2%. The inorganic ions which account for 5% of NMF include
potassium, sodium and calcium. The calcium ions and potassium ions are important in
terminal differentiation of epidermis and disappear after barrier perturbation, whereas the
magnesium ions accelerate skin barrier recovery in the stratum corneum (Nakagawa, 2004).
Sodium pyrrolidone carboxylic acid (PCA) and lactic acid are both highly hydroscopic and
act as efficient humectants, with both accounting for approximately 10% of NMF. The largest
percentage of NMF is the amino acids at 48%, with neutral amino acids accounting for
34.5%, acidic amino acids contributing 5% and basic amino acids making up the remaining
8%.
Serine is the largest free amino acid found within NMF and accounts for 36% of all free
amino acids found in NMF, Glycine is the second largest free amino acid at 22%, followed
by alanine attributing for 13% of free amino acids in NMF. Histidine (8%), ornithine (7%),
citrulline (6%), arginine (6%), and proline (2%) are all also present within NMF.
While the importance of NMF in skin hydration has been understood by some skin
researchers since the 1960s, and the relationship of NMF to filaggrin processing determined
in the 1980s, the full significance of the association was only appreciated with the recent
identification of filaggrin loss-of-function mutations.
Inherited loss-of-function mutations in the filaggrin gene (FLG) have been shown to cause
nioderate-to-severe ichthyosis vulgaris, and to predispose patients to atopic dermatitis,
including early-onset atopic eczema that recurs or persists into adulthood. In atopic
dermatitis, the levels of PCA, urocanic acid, and histidine have been shown to be correlated
with the FLG genotype, being reduced in patients carrying various FLG mutations. Multiple
mutations in the FLG gene have been identified; just two of these variants are carried by
approximately nine percent of people of European origin, suggesting a prevalence of filaggrin
mutations in certain populations. Patients carrying loss-of-function filaggrin mutations have
significantly reduced levels of NMF in the stratum corneum at all depths. In addition, carriers
of filaggrin imitations exhibit increased transepidermal water loss compared with noncarriers.
Filaggrin proteolysis abnormalities can occur in response to environmental factors. As noted
above, low humidity impairs the ability of hydrolytic enzymes to break down filaggrin into
NMF, thus generating skin surface dryness. In addition, UV radiation has been shown to
impair the natural breakdown of filaggrin to its NMF components. Further, NMF levels in the
skin decline with age, and this decline has been attributed to the decreased synthesis of
profilaggrin, and a decline in barrier function, in the elderly.
As noted above, approximately one-third of water contained within the stratum corneum is
bound, with the remainder being free water. Increasing the level of free water has no effect
on the elasticity of the stratum corneum, and it is NMF-bound water that provides the skin
with its elastic qualities. Replacing or replenishing the supply of NMF in the skin through
the external application of moisturisers containing NMF has proven to be a successful
approach for the treatment of xerotic skin (Weber, 2012).
Several NMF components have been used for decades in moisturising vehicles, without
knowledge of why they have any effect. For example, urea has been included in moisturising
creams as far back as 1943 (Harding, 2000). However, skin urea levels, which are now
known to be reduced in patients with atopic dermatitis, and in elderly skin, were not
measured in normal and atopic patients until 1966. Topical application of urea, or its
precursor, arginine, has been shown to correct urea deficits. Lactate was first reported to be
used in a moisturiser as a treatment for ichthyosis in 1946. It has been shown to improve and
prevent the reappearance of symptoms of dry skin compared with lactate-free moisturisers.
L-Iactic acid and D,L-lactic acid appear to work by stimulating the synthesis of ceramides in
the stratum corneum. PCA is the most prevalent single component of NMF, and has been
shown to be reduced in the outermost layers of the skin as a consequence of soap washing
and/or age. Topical application of PCA has been widely reported to alleviate the symptoms
of dry skin (Harding, 2000).
Within the skin, water can move into the atmosphere from the stratum corneum by passive
diffusion. This normal movement of water is known as transepidermal water loss (TEWL).
This results as there is no absolute barrier to water permeation. In a healthy epidermis, the
water content should be around 40% at the stratum granulosum/ stratum corneum interface
and 15%-25% at the skin's surface. Visible scaling of the skin occurs when the water content
is around 10% or lower.
Practical Dermatology (July 2012, 24 - 26) mentions a tyrosine derivative, without disclosing
the nature of the derivative, which had been found to significantly build volume in the dermal
matrix over a relatively short period of time when applied topically. No further information
is provided.
t has now, surprisingly, been found that unnatural, hygroscopic amino acids are useful to
hydrate and otherwise enhance the moisture retention and uptake properties of skin and
keratinaceous structures. These properties also enable these amino acids to serve as
penetration enhancers that can act synergistically with other penetration enhancers.
Thus, in a first aspect, the present invention provides an unnatural, hygroscopic amino acid
for use in enliancing hydration and/or the moisture retention and/or uptake properties of an
external keratinaceous structure of an animal. A preferred such structure is the skin, but the
amino acids of the invention may be used on nails, horn, hair and the eyes.
The present invention also provides the use of an unnatural, hygroscopic amino acid to
enhance hydration and/or the moisture retention and/or uptake properties of an external
keratinaceous structure of an animal.
The present invention further provides a method for enhancing the hydration and/or moisture
retention and/or moisture uptake properties of an external keratinaceous structure of an
animal, comprising administering an effective amount of an unnatural, hygroscopic amino
acid to said skin.
A surprising finding is that individual amino acids of the present invention are capable of
enhancing permeation, or percutaneous absorption, of substantially lipophilic drugs, with
increasing effects observed for increasing lipophilicity. Conventional penetration enhancers
of the art show the biggest effect on those drags that do not penetrate the skin well.
However, the enhancement effect of the amino acids of the present invention seems to
increase with log P, such that the effect is not as great for those drugs with more hydrophilic
properties, but increases with increasing lipophilicity.
As used herein, the term rug' refers to any pharmacologically active agent that it may be
desired to administer topically or transdermally.
Thus, there is further provided the use of an unnatural amino acid as a penetration enhancer
for a d ug intended for topical administration. Exemplary such drugs include steroids and
other molecules that are retained in the stratum corneum, and which stick thereto or bind to
keratin, and those with a log P > 3, but the penetration enhancing effect is applicable to all
drugs for topical administration, with the preference being for those with a more lipophilic
than hydrophilic nature.
Three preferred drug and amino acid combinations are: metronidazole and N-hydroxyserine;
diclofenac diethylamine (DDEA) and N-hydroxyglycine; and acyclovir and L-homoserine.
However, in general, the preferred amino acids of the present invention are a so preferred as
penetration enhancers, with advantageous effects being observed for most amino acids of the
invention, especially those having an O/C ratio of at least 0.7. An O/C ratio of 1 or more is
advantageous.
Preferred amino acids for use as penetration enhancers include N-hydroxyserine, Nhydroxyglycine,
L-homoserine and a-hydroxyglycine.
Other suitable drugs are as follows:
Type Of Drue
Local antipruritics Crotamiton
Doxepin hydrochloride
Mesulphen
Polidocanol
Amethocaine (Hydrochloride in solutions
1 LOCAL ANAESTHETICS
or creams, base in gels or ointments)
Amylocaine (Hydrochloride)
Benzocaine
Bucricaine (hydrochloride)
Butacaine Sulphate
Butyl Aminobenzoate Picrate
Cincocaine (base, hydrochloride or
benzoate)
Dimethisoquin Hydrochloride
Dyclocaine Hydrochloride
Ethyl Chloride
Lidocaine
Lignocaine
Myrtecaine
Oxethazaine (Oxetacaine)
Prilocaine
Propanocaine Hydrochloride
Tetracaine
Antihistamines Antazoline
Chlorcyclizine Hydrochloride
Dimethindene Maleate
Diphenhydramine
Histapyrrodine
Isothipendyl Hydrochloride
epyra ine
Mepyramine Maleate
Tolpropamine Hydrochloride
Tripelennamine Hydrochloride
Triprolidine Hydrochloride
Corticosteroids Alclometasone dipropionate
Beclomethasone dipropionate
Betamethasone valerate
Clobetasol propionate
Clobetasone butyrate
Desoximetasone
Diflucortolone valerate
Fludroxycortide/Flurandrenolone
Fluocinolone acetonide
Hydrocortisone
Hydrocortisone acetate
Hydrocortisone butyrate
Topical preparations for psoriasis Calcipotriol
Coal tar
Dithranol
5-Fluoiiracil
Ciclosporin
Fumeric acid
Lonapalene
Methotrexate
Methoxsalen
Salicylic acid
Tacalcitol
Tazarotene
Topical preparations for acne Azelaic acid
Benzoyl peroxide
Dithiosalicylic acid
Motretinide
Resorcinol
Topical antibacterials for acne Clindamycin
Erythromycin
'Dermatological drugs' ecapler n (Diabetic skin ulcers)
Bentoquatum (prevents allergic contact
dermatitis caused by poison ivy)
Gamolenic acid
Glycolic acid (Photodamaged skin)
Hydroquinone/Mequinol (Depigmenting
agents)
Ichthammol
Keluamid (seborrhoeic dermatitis)
Lithium succinate
Monobenzone (vitiligo)
Polyphloioghicinol Phosphate (Treatment
of wounds and pruritic skin disorders)
Sodium pidolate (humectant, applied as
cream/lotion for dry skin disorders)
Sulphur (mild antifungal/antiseptic)
Sulphurated Lime (For acne, scabies,
seborrhoeic dermatitus)
Sulphurated Potash (Acne)
Minoxidil (hair growth)
Topical retinoids and related preparations Adapalene
for acne Isotretinoin
Polyprenoic acid
Tretinoin
Other topical preparations for acne Nicotinamide
Topical antibacterials Amphomycin
Bacitracin/Bacitracin Zinc
Bekanamycin Sulphate
Chloramphenicol
Chlorquinaldol
Chlortetracycline
Framycetin sulphate
Fusidic Acid
Halquinol
Mupirocin
Mupirocin
Neomycin sulphate
Polymyxins (Polymyxin B Sulphate)
Silver sulphadiazine (sulfadiazine)
Sulphaniiamide
Sulphasomidine
Sulphathiazole (sulfathiazole) Sodium
Topical antifungals (Benzoyl peroxide)
Amorolfme
Benzoic acid
Bifonazole
BromochloiOsalicylaniiide
Buclosamide
Butenafine Hydrochloride
Chlormidazole Hydrochloride
Chlorphenesin
Ciclopirox Olamine
Clotrimazole
Croconazoie Hydrochloride
Eberconazole
Econazole nitrate
Fenticlor
Fenticonazole Nitrate
Flutrimazole
Halopiogiii
Ketoconazole
Mepartricin
Miconazole nitrate
Naftifine Hydrochloride
Natamycin
Neticonazole Hydrochloride
Nystatin
Omoconazole Nitrate
Oxiconazole Nitrate
Pyrrolnitrin
Sertaconazole Nitrate
Sodium Propionate
Sulbentine
Sulconazole nitrate
Sulconazole Nitrate
Terbinafine
Tioconazole
Tolciclate
Tolnaftate
Triacetin
Undecenoates/Undecanoic Acid
Antiviral preparations 1-Docosanol
Aciclovir
Brivudine
Edoxudine
Ibacitabine
Idoxuridine
Idoxuridine in dimethyl sulfoxide
Imiquimod
Penciclovir
Vidarabine
Parasinoidal preparations Benzyl benzoate
Carbaryl
Malathion
Permethrin
Phenothrin
Preparations for minor cuts and abrasions Cetrimide
Collodion
Magnesium sulphate
Proflavine
Topical circulatory preparations Heparinoid
Antiperspirants Aluminium chloride
Glycopyrronium bromide
Transdermal drugs Scopolamine
Buprenorphine
Granisetron
Nicotine
Rivastigmine
Nitroglycerin
Methylphenidate
Clonidine
Testosterone
Norethindrone acetate
Fentanyl
Selegiline
Ethinyl estradiol
Norelgestromin
Rotigotine
Estradiol
Oxybutynin
While skin is generally referred to hereinbelow, it will be understood that this term includes
reference to any other keratinaceous structure, such as nail, as well as other external
membranes, such as the cornea, unless otherwise apparent from the context.
As used herein, an unnatural amino acid is one that is either not synthesised by the host of the
skin to be treated, or which is not associated with a dedicated host t NA therefor. It is an
advantage of such amino acids, especially those not synthesised by the host, that they are less
subject to catabolism, such as by naturally occurring enzymes, so that they are retained in the
skin for longer than naturally occurring amino acids, so that any moisturising or permeation
enhancing effect may be prolonged.
The terms 'moisturising', 'moisture retention', and 'moisture uptake', and related terms, are
used herein interchangeably when illustrating the present invention, and reference to one
includes reference to the others, unless otherwise apparent from the context. Individually, the
terms have specific meanings. The term 'moisturising' is an inclusive term, and indicates
substances or conditions that lead to the balancing, or progress towards balancing, of
moisture levels in dry skin. Enhanced 'moisture retention' indicates a reduced propensity of
skin to allow water to escape, and 'moisture retention' indicates the propensity of skin to
retain water. 'Moisture uptake' is the property of skin to absorb water from the environment,
such as humid air. The term 'hydration' includes both the level of water in the skin as well as
the process of water uptake into the skin, such as in moisture uptake, supra.
As used herein, the term 'hygroscopic' indicates an amino acid that is capable of absorbing
and retaining moisture from the atmosphere at a relative humidity (RH) of <50%, and
preferably 40%, or less, at 32°C.
For a compound to be hygroscopic it must be able to form a non-bonding association with
water. Magnesium sulphate is very hygroscopic and forms non-bonding interactions between
the oxygen atom in water and the magnesium atoms. Further investigation into what makes a
compound hygroscopic lead to work performed on soil samples and aerosols. The most
abundant free amino acids found within aerosols are glycine, serine and alanine. These are
also the three most abundant free amino acids found within NMF, The hygroscopic
properties of humic materials in atmospheric aerosol experiments lead to investigation
between the hygroscopic properties observed and the chemical structure of humic substances
(Sasaki, 2007). Through this work it was determined that compounds with a higher oxygen
to carbon ratio (O/C) generally exhibited greater hygroscopic properties (Sasaki, 2007). For
example, L-serine has 3 oxygen atoms and 3 carbon atoms, so that it has an O/C = 1.0.
Amino acids of the present invention are able to deliquesce at 32°C. Preferably, amino acids
of the present invention have a deliquescence relative humidity (DRH) of no greater than
80% at 32°C. Preferred amino acids of the present invention have a DRH of no greater than
80% at 32°C and an O/C ratio of at least 0.7.
Naturally occurring amino acids are the L-amino acids in animals, and the preferred animals
to be treated with the present invention are the mammals. Preferred mammals are those that
have exposed, or hairless skin, whether wholly or in part, and particularly preferred are
humans.
The unnatural amino acids will generally be D-amino acids, but L-amino acids not
synthesised in the animal to be treated can include unusual L-amino acids, such as
a-hydroxyglycine and L-homoserine.
Where amino acids of the invention are novel, then these are provided as aspects and
embodiments of the invention.
The amino acids of the invention are any molecule that comprises a COOH group linked via
one or two, and preferably one, carbon atom to an imide or, more preferably, an amine group.
While it is preferred that the amino acids of the invention are in their free, zwitterionic form,
they may also be provided in salt form in solution, or as ion pairs.
The amino acids of the invention may be applied to the skin in any suitable form, such as
cream, lotion, gel, unguent, ointment, mousse, foam, solution, injection, suspension, colloidal
system or spray (propellant or pump), either in a carrier comprising an aqueous component,
such as one that may act as a solvent for the amino acid, or in a carrier comprising an organic
vehicle capable of dissolving or entraining the amino acid. Such forms may alternatively, or
further, include one or more drugs for topical administration, and may further comprise any
additional substances, such as film forming agents, antimicrobials, antioxidants, stabilisers,
emulsifiers, sterlilants, thickeners, and colourants.
The application form may comprise the one amino acid of the invention, or may contain two
or more amino acids of the invention. Regardless, the administration form may further
comprise one or more additional moisturiser ingredients as taught in the art, and may
comprise further amino acids, such as natural, hygroscopic amino acids, or natural and
unnatural amino acids that are not as hygroscopic as the amino acids of the invention. A
preferred naturally occurring amino acid is L-homoserine.
In one aspect, it is preferred to mimic NMF, by using one or more amino acid ingredients of
NMF, preferably in amounts and/or ratios approximating those found in NMF. Amounts and
proportions of amino acids in NMF are as detailed above.
It is further preferred to mimic NMF by including one or more non-amino acid ingredients of
NMF, preferably in amounts and/or ratios approximating those found in NMF. These
preferably include one or more salts, especially the sodium and potassium salts.
It is preferred that all ingredients of any administration form are pharmaceutically acceptable.
Preferred amino acids of the present invention have an O/C ratio of at least 1. A more
preferred ratio is at least 1.5 : 1, and a ratio of 2 : 1, and also higher than 2 : 1 is preferred.
The following compounds are preferred compounds of the present invention:
N-hydroxyserine N -hydroxyglycine 2-(aminooxy)-2-hydroxyacetic acid
afpha- 2-hydroxy-2- 2-(aminooxy)acetic acid
hydroxyglycine (hydroxyamino) acetic
acid
It will be appreciated that compositions comprising the amino acids of the present invention
may be applied in preventative, or prophylactic capacity, particularly in cold weather, such as
winter.
Compositions of the present invention may also be used for the treatment or prophylaxis of
such conditions as inflammatory skin disease, atopic dermatitis, eczema, ichthyoses (dry skin
conditions), winter xeroses, localised lichenifications, and eczematous episodes.
Compositions of the present invention are useful in the treatment of wounds, especially of
topical membranes, preferably of the skin,
It will be appreciated that compositions of the present invention are particularly useful as
cosmetic formulations. Such formulations may be for the enhancement of skin appearance,
such as wrinkle treatment by plumping the skin, and skin elasticity, where the amino acids
may be used in conjunction with collagen treatments, for example. Used on the nails, the
cosmetic treatment may be used to soften the nails for cutting, or to hydrate the nails to help
prevent chipping. In hair treatments, hydration may be used to increase suppleness of the
hair and to help to prevent splitting.
It will be appreciated that the amino acids of the present invention may also be used in other
applications that benefit from their skin moisturising properties. Thus, the present invention
envisages the use of the amino acids of the invention as excipients in topical formulations.
This may be to counteract the dehydrating effect of other excipients, such as efhanol, or
simply as an emollient, or anti-dehydrating agent, or to enable enhanced drug absoiption.
There is provided the use of amino acids of the present invention as anti-inflammatory
agents, especially where a contributory factor in said inflammation is GM-CSF. Particularly
preferred amino acids for such use are N-hydroxyserine, L-homoserine, N-hydroxyglycine,
and combinations containing one or more thereof.
There is provided the use of amino acids of the present invention as anti-irritants.
Particularly preferred amino acids for such use are N-hydroxyserine, L-homoserine, Nhydroxyglycine,
and combinations containing one or more thereof.
The amino acids of the invention may find use as, or in, emollients.
The amino acids of the invention may be used in eye drops or other ocular formulation for the
treatment of dry eye.
The amino acids of the invention may also be used to moisturise thickened skin, so as to
facilitate callus removal, for example.
The amino acids of the invention may be used to help with nail and/or hoof softening.
The amino acids of the invention may also be used in conjunction with hair removal
techniques.
Given the hydrative nature of compositions of the invention, they also find use in face packs.
The amino acids of the invention may also be used to hydrate the skin in the treatment of skin
conditions including psoriasis, warts and verrucae, thereby permitting more effective drug
delivery to the target site.
It will be appreciated that the amino acids of the invention may be applied to the skin before
application of a barrier preparation, especially where the aim is to prevent drying out, such as
desiccation, of the skin.
In general, the amino acids of the present invention find use in cosmetic preparations,
especially those that may cause skin drying, and in those intended to enhance skin health and
appearance, such as skin moisturisers and anti-wrinkle creams. Hair products, such as hair
conditioners, also benefit.
The following synthetic route illustrates how amino acids of the present invention may be
conjugated. Such conjugates may optionally be used in addition to, or in place of, amino
acids of the invention in any uses as indicated herein, save where the skilled physician
decides otherwise, or wherein a conjugate is less preferred, such as for reasons of speed of
uptake into the stratum corneum for example.
HO-W -Ser-i-Ser -HomoSer-OH
The invention will now be further illustrated with respect to the accompanying drawings, in
which:
Figure 1 shows an experimental set up for measuring the RH of a saturated solution;
Figure 2 shows the average percentage weight increase after 24h at 40% RH, compared to
water against the DRH (deliquescence relative humidity) for the same compound;
Figure 3 shows the average percentage weight increase after 24h at 40% RH, following 24h
of treatment with 1.33M amino acid solution, for each of the test amino acids;
Figure 4 shows the cumulative permeation of diclofenac through epidermal membrane over a
48 h experimental period; each bar represents the average permeation + SEM, n=5-6;
Figure 5 shows the cumulative permeation of diclofenac through epidermal membrane after
48 h; each bar represents the average permeation + SEM, n=5-6;
Figure 6 shows cumulative permeation of metronidazole through epidermal membrane over a
48 h experimental period; each bar represents the average permeation + SEM, n=5-6;
Figure 7 shows cumulative permeation of metronidazole through epidermal membrane after
48 h; each bar represents the average permeation + SEM, n=5-6;
Figure 8 shows cumulative permeation of Acyclovir through epidermal membrane over a 48
h experimental period; each bar represents the average permeation + SEM, n=5-6;
Figure 9 shows cumulative permeation of Acyclovir through epidermal membrane after 48 h;
each bar represents the average permeation + SEM, n=5-6;
Figure 10 shows the average percentage weight increase after 24h at 75% RH, following 24h
of treatment with 1.33M amino acid solution, for each of the test amino acids;
Figure 1 shows the plot for drug absorption enhancement factor for 3 drugs;
Figure 12 shows viability of RHE tissues post 42 minute exposure and 42 post exposure
incubation; each point represents the mean tissue viability with error bars representing the
range; =4;
Figure 13 shows viability of RHE tissues post 24 h exposure; each point represents the mean
tissue viability with error bars representing the range; =3;
Figure 4 shows tissue viability after exposure to solutions of 10% w/v N-H-G, 1% w/v N-HG,
10% w/v L-Serine and 10% w/v glycine at t= 2, 6 and 24 h and positive control ( 1 % w/v
Triton™ X-100, t= 3 and 7 h); each time point represents the mean tissue viability and error
bars represent the range, n=3;
Figure 15 shows release of GM-CSF from psoriasis tissues; each point represents the average
cumulative release of GM-CSF over 6 day with measurements made at 2, 4, and 6 days with
error bars representing the range n=2-3; and
Figure 16 shows total release of GM-CSF. Each bar represents the total release of GM-CSF
over 6 day from both psoriasis model and control RHE tissues; error bars represent the range
n=2-3.
The present invention will now be illustrated by the following, non-limiting Examples.
Example 1
Experimental
5-Bromopental:
A solution of ethyl 5-broniopentanoate (3.9295g, 18mmol) in anliydrous DCM (lOOmL) was
stirred under a nitrogen atmosphere at -78°C, to which DIBAL-H (31mL, 31mmol, 1.7equiv,
1M in hexanes) was added. The resultant orange solution was left stirring under a nitrogen
atmosphere at -78°C, whilst the reaction was followed by TLC. After 8h of stirring, the
reaction was quenched with the addition of HC1 (1M, 50mL) and water (lOOmL). The
colourless mixture was removed from the nitrogen atmosphere and left stirring over night to
gradually return to room temperature. The mixture was extracted with DCM (2 x 50mL),
dried over MgS0 4, filtered and concentrated in vacuo to afford the crude title product as a
pale yellow oil (2.766g, 80%, 21mmol), which is used without further purification. Rf = 0.30
diethyl ether/ hexane (2/5).
'H MR (400MHZ, CDC13) 1.76-1.83 (2H, m, J=12.0Hz, H-3), 1.87-1.94 (2H, , J=12.0Hz,
H-2), 2.48-2.52 (2H, t, J=8.0Hz, H-4), 3.41-3.44 (2H, t, J=8.0Hz, H-l), 9.79 (1H, t, J=4.0Hz,
H-5) ppm.
3C NMR (400MHz, CDC13) 20.6 (C-3), 31.9 (C-2), 33.0 (C-4), 42.9 (C-l), 201.8 (C-5) ppm.
ra (FT R CCC13, KBr plates)/ cm 1 2938 (C-H), 2725 (C-H aldehyde), 1721 (C=0), 1437
(C-H), 1390 (N-O), 1253, 1042, 913, 743.
(E)-Ethyl-7-bromohept-2-enoate:
To a stirred suspension of crude 5-bromopental (2.766g, 16mmol) in DCM (lOOmL) was
added (carbethoxymethylene)triphenylphosphorane (8.098g, 23mmol, 1.4 equiv.) in one
portion. The mixture was left stirring at room temperature for 22h before being quenched
with saturated ammonium chloride (50mL). The mixture was extracted with DCM (2 x
50mL) and the combined organic extracts were washed with water (50mL). The organic layer
was dried over MgSC4, filtered and concentrated in vacuo to afford a crude yellow liquid
(10.320g). The crude product was then purified by column chromatography on silica gel
eluting with diethyl ether/ hexane (1/3) to afford the title product (2.723g, 72%, l l.Smmol);
R = 0.39 diethyl ether/ hexane (1/3);
dH (400MHz, CDC13) 1.27-.132 (3H, t, J=8.0Hz H-7), 1.55-1.60 (2H m, J=8.0Hz, H-3), 2.02-
1.06 (2H, m, J= 16.0Hz, H-2), 2.27-2.29 (2H, t, J=16.0Hz, H-4), 4.16-4.22 (2H, q, J=32.0Hz,
H-l), 4.38-4.42 (2H, t, J=l 6.0Hz, H-6) ppm.
c (400MHz, CDCI3) 14.28 (C-9), 26.52 (C-3), 31.20 (C-2), 32.02 (C-4), 33.26 (C-8), 60.25
(C-l), 121 .90 (C-6), 148.14 (C-5), 166.55 (C-7)ppm.
v (FT IR, CCC13, K r plates)/ cm 1 2938 (C-H), 1714 (CO), 1654 (C=C), 1445, 1367 (NO),
1266, 1185, 1134 (C-O), 1095, 1039, 979 (C-H), 913 (C-H), 848, 742.
(E)-EthyI-7-nitrohept -2-enoate:
A solution of ethyl-7-bromohept-2-enoate (2.6486g, 11.3mmol) in DMF (13mL) was stirred
at 0°C, to which sodium nitrite (1.1657g, 16.9mniol, 1.5 equiv.) was added in one portion.
The resultant solution was left stirring at 0°C, whilst the reaction was followed by TLC.
After 8h, TLC showed majority consumption of starting material. The reaction was added to
ice cold water (20mL) and extracted with diethyl ether (25mL). The organic layer was then
washed with brine (saturated 25mL). The organic extract was dried over MgS0 , filtered and
concentrated in vacuo to give a yellow liquid (1.9390g). The crude product was then purified
by column chromatography on silica gel eluting with diethyl ether/ hexane (l/4)to afford the
title product (0.5283g, 23%, 2.6mmol);
Rf = 0.29 diethyl ether/ hexane (1/4);
dH (400MHz, CDCI3) 1. 7- ,131 (3H, t, J=8.0Hz H-9), 1.54-1.62 (2H, quin, J=l 6.0Hz, H-3),
2.00-2.08 (2H, quin, J= 16.0Hz, H-2), 2.25-2.30 (2H, q, J=16.0Hz, H-4), 4.16-4.22 (2H, q,
J=16.0Hz, H-8), 4.38-4.42 (2H, t, J=8.0Hz, H-l), 5.83-5.85 (1H, d, J=12.0Hz, H-6), 6.88-
6.95 (1H, dt, J=8.0Hz, J'=l 6.0Hz) ppm.
c (400MHz, CDCI3) 14.28 (C-9), 26.52 (C-3), 31.20 (C-2), 32.02 (C-4), 33.26 (C-8), 60.25
(C-l), 121.90 (C-6), 148.14 (C-5), 166.55 (C-7)ppm.
v (FT IR, CCC13, KBr plates)/ cm 1 2936 (C-H), 2865, 1716 (C=0), 1644 ( 0 ), 1445 (CH,
1415, 1388, 1288 (C=C), 1229, 1170 (C-O), 1134, 1096, 1033, 912 (=C-H), 823, 734,
649.
tert-butyl (2-chIoro-2-oxoethyl)carbamate
Boc-Gly (2.833g, 16.2mmol) was dissolved in anhydrous DCM (30mL) under a nitrogen
atmosphere at room temperature. The Boc-Gly solution was transferred to a solution of oxalyl
chloride (3.10mL, 2.2 equiv, 35.6mmol) in anhydrous DMF (3 drops). The resultant solution
was left stirring at room temperature under a nitrogen atmosphere, whilst the reaction was
followed by TLC. After 3.5h, TLC showed consumption of starting material. The mixture
was concentrated i vacuo to yield the acid chloride (3.4634g, 1 1%, 18mmol).
Rf = 0.15 diethyl ether/ exane (4/1);
1H NMR (400MHz, CDC13) 1.49 (9H, s, H-6), 3.69 (2H s, H-2), 5.95 (1H, br s, H-3) ppm.
C MR (400MHz, CDC13) 39.77 (C-6), 66.77 (C-2), 106.35 (C-5), 138.34 (C-4), 183.29
(C-l) ppm.
v a (FT IR, CCC13, KBr plates)/ 2989 (C-H), 1766 (C=0), 1742 (C=0), 1375, 1314, 1264,
1189, 1158, 1009 (C-N), 919, 852, 747 cm' .
tert-butyl (2-((4R,5S)-4-methyl-2-oxo-5-phenyloxazolidin -3-yl)-2-oxoethyl)carbamate:
10
5
LiH DS (2.5474g, lSiranol, 1.2 equiv) was added a cooled solution of (4R, 5S)-(+)-4-
methyl-5-phenyl-2-oxazolidone (2.2104g, 12mmol) in anhydrous THF (lOmL) at -78°C. The
mixture was stirred for 30mins before the addition of Boc-Gly-Cl (2.4634g, l.Sequiv,
18mmol) in anhydrous THF (lOmL) before being warmed to room temperature. After 14h of
stirring at room temperature, the reaction was quenched with the addition of NH C 1 (saturated
30mL) as TLC showed consumption of the acid chloride. The mixture was extracted with
ethyl acetate (50mL). The organic layer was further extracted with sodium bicarbonate (sat,
50mL). The organic layer was dried over MgS0 4, filtered and concentrated in vacuo to afford
a deep blood red liquid (2.2964g). The crude product was left to stand overnight at room
temperature to yield a brown/ red solid. A solution of diethyl ether/ hexane (4/1) was added
to the solid and filtered to yield the pure brown solid of the title product (1.2702g, 32%,
3.8mmol).
R = 0,43 diethyl ether/ hexane (4/1);
Mpt =106-1 11°C.
1H NMR (400MHz, CDC13) 0.00 (9H, s, H-10), 0.82 (3H d, J=6Hz, H-5), 1.55 (2H, s, H-6),
4.20 (1H, quin, J=8Hz, H-4), 4.92 (1H, br s, H-7), 5.72 (1H, d, J=8Hz, H-2), 7.52-7.26 (5H,
m, H-l) ppm,
,3C MR (400MHz, CDC13)0.06 (C-10), 3.66 (C-5), 7.66 (C-6), 47.92 (C-4), 51.53 (C-2),
54.57 (C-9), 126- 128 (C-l), 169.22 (C-3), 179.35 (C-8), 183.05 (C-5) ppm.
vmax (FT IR, CCCI3, KBr plates)/ 3200 ( -H), 1754 (C=0), 1500 (C=0 amide) , 9 3 (C=C
bending), 744 cm 1.
(4R,5S)-3 -(2-br0moacetyl)-4-methyl-5-phenyloxazoIi(lm-2- one:
n-BuLi (lmL, lOmmol, 1.1 equiv.) was added to a stirred solution of the oxazolidiii-2-one
(1.5930g, 9mmol, 1.0 equiv.) in dry THF (8mL) at -78°C under nitrogen. After 15 in, the
bromoacetyl bromide (0.57mL, 12mmol, 1.3 equiv.) was added dropwise as a solution in
THF (9.6mL) and stirred at -78°C for 15 min before being wanned to ambient temperature,
After 2 h, the reaction was quenched with NH4CI (saturated 5mL) and acetic acid (5mL),
extracted with EtOAc (20mL), washed with NaHC0 (saturated 20mL) and brine (saturated
20mL), dried over MgSO^ and concentrated in vacuo. The crude product was purified by
flash column chromatography on silica gel eluting with EtOAc/ hexane (3/17) to afford the
title product (0.2593g, 10%, 0.9mmol),
Rf 0.41 EtOAc/ hexane (3/17); 1H NMR (400MHz, CDC13) 0.93-0.94(3H, d J=4.0Hz, H-
5), 4.51-4.59 (2H, q, J=20.0Hz, H~7), 4.77-4.83 (1H, q, J=12.0Hz, H-4), 5.74-5.76 (1H, d,
J=8Hz, H-2), 7.26-7.46 (5H, m, H-l) ppm.
C NMR (400MHz, CDC13)14.29 (C-5), 28.23 (C-7), 55.24 (C-4), 79.53 (C-2), 125.64 (C-l),
129.03 (C-l), 134.59 (C-3), 150.20 (C-6) ppm.
ma (FT IR, CCC13 KBr plates)/ 2964 (C-H), 2917, 2849, 1776 (C=0), 1700 (C=0), 1 96
(Ph), 1455, 1415, 1340 (C~N), 7, 1260, 1217, 1196 (C-O), 1169, 1146, 1120, 1089, 1066,
1037, 1001, 990, 970 9 11, 883, 803, 765, 730, 700, 661, 639, 6 8 cm' .
(4R,5S)-3-(dibenzylglycyl)-4-methyl-5-phenyIoxazoIidin-2-one:
Dibenzylamine (0.485mL, 3.0mmol, 2.2 equiv.) was added to a stirred solution of the N-acyloxazolidin-
2-one (0.408 lg, 1.37mmol, 1.0 equiv.) in anhydrous DCM (ImL) at rt. The
reaction mixture stirred for 18 h under nitrogen. The resulting mixture is partitioned between
DCM (lOmL) and water (lOmL). The aqueous layer was washed with DCM (lOmL). The
combined organic layers are washed with water (lOmL), dried over MgS0 4 and concentrated
in vacuo. The crude product was purified by flash column chromatography on silica gel
eluting with EtOAc/ hexane (3/17) to afford the title product (0.38844g, 73%, l.Ommol).
Rf = 0.30 EtOAc/ hexane (3/17);
NMR (400MHz, CDC13) 0.87-0.89(3H, d, J=8.0Hz, H-5), 3.83-3.91 (2H, q, J=20.0Hz, H-
7), 3.95 (2H, s, H-8), 4.64-4.70 (1H, q, J=12.0Hz, H-4), 5.61-5.63 (1H, d, J=8Hz, H-2), 7.23-
7.42 (15H, m, H-l) ppm.
3C NMR (400MHz, CDC13) 14.67 (C-5), 54.56 (C-7), 55.24 (C-8), 58.18 (C-4), 79.50 (C-2),
125.64-129.03 (C-l), 167.23 (C-3), 184.49 (C-6) ppm.
vm (FT IR, CCCI KBr plates)/ 3027 (=C-H), 2360, 2341, 1790 (Ph), 1705 (C=0) >
1494(Ph), 1454 (C-H), 1367, 1344 (C-N), 1245, 1218, 1197 (C-O), 1150, 1120, 1089, 1067,
1028, 984, 954, 917, 791, 766, 747, 698, 668, 643 cm"1.
2-Amino-2-hydroxyacetic acid:
Ammonium acetate (9.4725g, 0.1 mol, 2 equiv.) in ice-cold water (IOmL) was added to a
stirred solution of glyoxylic acid nionohydrate (4.601 5g, 0.05mol, 1 equiv.) in ice cold water
(IOmL). A white precipitate appeared within minutes of stirring. After 45 minutes of stirring
and 2h standing at 0°C, the reaction mixture was filtered. The white precipitate was washed
with water (20mL) and methanol (2 x IOmL) to yield the crude title product (4.6951g,
0.05 ImoL 103%). The crude product (4.1697g) was dried by a high vacuum pump for 5
hours to afford the title product (4.057 lg, 97%).
d (400MHz, D20 ) 4.93 (1H, s, H-l) ppm.
c (400MHz, D 0)26.81 (C-l), 175.83 (C-2) ppm.
Vm (FT IR, CCC13, KBr plates)/ cm 1 3056 (OH, C-H), 1637 (C=0), 1561 (N-H), 1449,
1394, 1352, 1305 (C-O), 194, 1129, 1071 (C-N), 881, 827, 616, 564, 536.
/V-Hydroxyglycine
Glyoxylic acid (1.630g, 22mmol) and NH30H.HC1 (1.1540g, 22mmol) are stirred in water
(lOOmL) at room temperature. 1M NaOH was used to raise the pH to 5 before the addition of
sodium cyanohydridoborate (3.306g, 52mmol, 2.5 equiv). The reaction was left to stir for 48h
before 1M HC1 was added drop wise to lower the pH to 1. The mixture was filtered and the
filtrate was concentrated in vacuo. Water was added to the white residue and again the
mixture was concentrated in vacuo to yield a white solid (7.7289g). The white residue was
taken up in water and passed through amberlite 200C sodium. The product was removed
from the amberlite by addition of 2% ammonia solution. The filtrate is concentrated in vacuo
and recrystallised from hot ethanol to yield the yellow title product (0.6562g, 33%, 7.2mmol).
Melting point=137-139°C.
d (400MHz, D 0 ) 1.18 (1H, br s, N-H), 3.62 (2H, br s, H-l) ppm.
8 (400MHz, D 0 ) 17.72 (C-l), 185.00 (C-2) ppm.
Benzaldoxime
Benzaldehyde (2.00mL, 20mmol) was stirred in a mixture of ice: water: ethanol (2:1:1,
20mL) at room temperature. Hydroxylamine hydrochloride (1.3860g, 20mmol) was added to
the stirred suspension followed by 50% aqueous sodium hydroxide (4mL, 40mmol), while
keeping the temperature below 30°C. After stirring for lhour at room temperature, the
mixture was extracted with diethyl ether (2 x 25mL). The aqueous extract was acidified to pH
6 using cone. H , while keeping the temperature below 30°C, before again being extracted
with diethyl ether (2 x 25mL). The combined organic extracts were dried over MgS0 ,
filtered and concentrated i vacuo to yield a colourless liquid (2.4246g, 100%, 20mmol).
(400MHz, CDCI3) 7.38 (2H, m, H-5, H-3), 7.58 (2H, m, H-2, H-6), 7.88 (1H, t J=l,4Hz, H-
4), 8.15 (1H, s, H-7)ppm. C (400MHz, CDC13) 127.0 (C-5 and C-3), 128.8 (C-6 and C-2),
130.0 (C-4), 131.8 (C-l), 150.4 (C-7) ppm. (FT IR, CCC13 KBr plates)/ cm 1 3280, 3061
(C-H), 3028 (C-H), 2985 (C~H), 2898, 2358, 2341, 1896, 1810, 1697 (C=N), 1631 (N-H),
1598 (C-C), 1577 (C=C), 1492 (N-O), 1443, 1303, 1288, 1209, 1176, 158, 1102, 1074, 946,
868, 752, 702, 644. /z (FTMS + ESI) found 122.0597 ([M+H] +, 100%) C7H NO requires
122.0600.
2-bromo-3-hydroxypropanoic acid
1
Potassium bromide (350.634g, 2.94mol) and L-serine (100. Og, 0.95mol) were stirred in 2.5
aqueous sulphuric acid (1.8L) at 0°C. Sodium nitrite (92. Og, 1.33mol) was added slowly to
ensure the temperature remained below 5°C, Half the sodium nitrite was added over the first
8h, the mixture was left stirring over night before the remaining sodium nitrite was added
over the next 30mins. After stirring for 2.5 hours at 0°C, the mixture was extracted with
diethyl ether (3 x 250mL). The organic extracts are combined, dried over MgS0 , filtered and
concentrated in vacuo. The residue was then further concentrated on the high vac over 8h to
yield the title product (154.6969g, 96%, 0.915mol). H (400MHz, CDC13) 3.97-4.09 (2H,
ddd, J=5.2Hz, J'=12.1Hz, J"=25.8Hz, H-2), 4.40 (1H, t, J=5.3Hz, H-3) ppm. C (400MHz,
CDC13) 43.8 (C-2), 63.7 (C-3), 172.8 (C-4) ppm. vm (FT IR, CCCI3, KBr plates)/ cm 3407
(O-H), 2931 (C-H), 2355, 1722, 1615 (C=0), 1452, 1398, 1290, 1269, 1245, 1191 (C-O),
1160 (C-O), 1068, 1026, 909.
Ethyl 2-bromo-3-hydroxypropanoate
1
Concentrated sulphuric acid (2.8mL, 0.003mL per mmol) was added slowly to a stirred
mixture of 2-bromo-3-hydroxypiOpanioc acid (154.6969g, 0.92 ol) in absolute ethanol
( 1.83L, 2mL per mmol) before heating under reflux for 1.5h. The mixture was cooled and ice
cold water (1.83L) was added and extracted with diethyl ether (2 x I.8111L). The organic
extracts are combined and washed with ice cold water (1.8L), 5%M aqueous sodium
carbonate (2 x 1.8L) and saturated brine (1.8L),. The organic extract was dried over
magnesium sulphate, filtered and concentrated in vacuo to yield the title product (179.27g,
99.5%, 0.91mol). d (400MHz, CDC13) 1.30 (3H, t, J=6.8Hz, H-6), 2.41 (1H, br s, H-l),
3.92-4.07 (2H, ddd, J=7.6Hz, J'=12.0Hz, J"=40.0Hz, H-2),4.25 (2H, J=7.2Hz, H-5), 4.30
(1H, t, J=1.6Hz, H-3) ppm. C (400MHz, CDC13) 13.92 (C-6), 44.61 (C-3), 62.43 (C-5),
63.85 (C-2), 166.7 (C-4) ppm. vma (FT R CCC13, KBr plates)/ cm 1 3434 (O-H), 2987 (CH),
2939 (C~H), 1736 (C=0), 1464, 1373, 1298, 1269, 1244, 1184, 1152, 1098 (C-O), 1080
(C-O), 1041 (C-O), 951, 857, 797, 678, 615.
N-hydroxyglycine (alternative synthesis)
Sodium (0.4626g, 0.02mol) was added to a stirred mixture of benzaldoxime (2.3999g,
0.02mol) in absolute ethanol (40mL). Ethyl bromoacetate (2.44mL, 0.022mol, 1.1 equiv.)
was added and the mixture was stirred until the pH reached 7 which took 3h. The mixture
was filtered and the solid was washed with chloroform (2 x 40mL). The combined filtrate
was concentrated in vacuo. The residue was taken up in diethyl ether (50mL) and placed in
the fridge overnight. The mixture was filtered, washed with cold diethyl ether and the solid
was dried under suction (3.1580g). The nitrone (1.5g) solid was stirred in cone HC1 (20mL)
and heated under reflux for 0.5h. The mixture was concentrated in vacuo. The residue was
taken up in water and the pH was raised to 6 using ammonium hydroxide solution. The
mixture was cooled in the fridge for 48h, filtered and the solid was recrystallized from hot
aqueous ethanol (75%) to yield (0.8095g, 8.90mmol, 76%). Melting point = 137-139°C. v la
(FT R, CCC13, KBr plates)/ cm- 1 3375, 3234, 3094, 2908, 1645, 1592, 1549, 1508, 1406,
1305, 1214, 178.
V-hydroxyserine
Sodium (0.4600g, 0.02mol) was added to a stirred mixture of benzaldoxime (2.4201g,
0.02mol) in absolute ethanol (40mL). Ethyl 2-bromo-3-hydroxypropanoate (4.3 34g,
0.022mol, 1.1 equiv.) was added and the mixture was stirred until the pH reached 7 which
took 3h. The mixture was filtered and the solid was washed with chloroform (2 x 40mL). The
combined filtrate was concentrated in vacuo. The residue was taken up in diethyl ether
(50mL) and placed in the fridge overnight. The mixture was filtered, washed with cold
diethyl ether and the solid was dried under suction (2.413 lg). The nitrone (1.5g) solid was
stirred in cone HC1 (20mL) and heated under reflux for 0.5 The mixture was concentrated
in vacuo. The residue was taken up in water (lOmL) and the pH was raised to 6 using
ammonium hydroxide solution. The mixture was cooled in the fridge for 48h, filtered and the
solid was recrystallized from hot aqueous ethanol (75%). Acetone was added to encourage
the title product to drop out of solution to yield the title product (0.6724g, 5.56mmol, 88%).
Melting point = 159-163°C. H (400MHz, CDCi3) 3.12 (1H, , H-8), 3.56 (1H, m, H-8), 4.59
(1H, m, H-3) ppm . vm (FT , CCC13, K r plates)/ cm 1 3308 (O-H), 3032 (COOH), 2908
(CH), 1648 (N-H), 1591 (C=0), 1540 (N-O), 151 1, 1402, 1306, 1229, 1174.
Ethyl 2-bromo-2-hydroxyacetate
N-bromosuccimide ( 1.5343g, 65mmol) was stirred in carbon tetrachloride (80mL) at 80°C.
Dibenzyoyl peroxide (0.0521 g, 0.2mmol) in ethyl glycolate (6.15mL, 65mmol) was added
drop wise to the mixture before heating under reflux for 30 minutes, by which time the
exothermic reaction had subsided. The mixture was cooled to room temperature, filtered and
concentrated in vacuo. The residue was left until a precipitate formed, which took around 3
days. The mixture was filtered and washed with dichloromethane to yield a colourless solid
(0.8544g, 4.7mmol, 7%). Melting point = 107-1 10°C. dH (400MHz, CDC1 ) 1.07 (3H, t,
J=3.9Hz, H-5), 2.66 (1H, br s, H-l), 3.44 (2H, q, J-7.1Hz, H-4), 4.01 (1H, s, H-2) ppm. C
(400MHz, CDC13) 16.7 (C-5), 57.3 (C-4), 86.0 (C-2), 172.9 (C-3) ppm. v li (FT IR, CCCl3
KBr plates)/ cm 3479 (O-H), 3406 (C-H), 1697 (C=0), 1614, 1439 (C-H), 1393, 1228 (CO),
191 (C-O), 1083 (C-O), 904, 808, 776, 719.
Example 2
Experiment summary:
This experiment was to measure the deliquescence relative humidity (DRH) caused by a
saturated amino acid solution in a sealed vessel at 32°C; the lower the DRH, the greater the
water holding capacity of the solution measured. Deliquescence relative humidity is the
relative humidity at which the compound deliquesces, i.e. the relative humidity in which the
compound absorbs so much water that it dissolves within the absorbed water.
Equipment:
A diagrammatic representation of the equipment set-up is shown in Figure 1, which shows an
experimental set up for measuring the RH of a saturated solution.
The apparatus is maintained at 32°C. The temperature of 32°C was chosen for being the
temperature of outer stratum corneum. As the amino acids are to eventually be delivered to
the outer layer of the skin, then the water holding capacity of these compounds at 32°C is
important.
Methodology
The saturated solution is prepared as above by adding the amino acid to water (ImL, 32°C),
whilst swirling. The total amino acid is weighed before adding to the water and the remaining
amino acid so that the mass of compound used can be determined. The saturated solution
(ImL, 32°C) is then transferred into the vial, again because of partial molar volume causing a
change in volume by the addition of the amino acid to the water.
The temperature and %RH (% Relative Humidity) shown on the thermo-hygrometer are
recorded every 30 minutes until the %RH remains constant. The temperature confirms that
the temperature experienced by the sample is 32°C.
The results at 24 hours are summarised in Table 1, in which S.D. is standard deviation.
Table 1
It can be seen that a-hydroxyglycine and L-homoserine, in these tests, demonstrated
particularly advantageous levels of relative humidity. More particularly, amino acids of the
present invention advantageously have a DRH of no greater than 80% at 32°C. In addition,
amino acids of the present invention can also be seen to have an O/C ratio of at least 0.7.
From the above Table, from an average of 3 experiments, the relative humidity of water after
24hr was 100%, as expected, serving as a control. L and D-serine had similar %RH, as
expected, as they are enantiomers. Hydroxyglycine has an RH of 79.7%, better than the
serines. L-homoserine had an RH at 24 hrs of 71.4%, thereby showing an excellent
deliquescent relative humidity (DRH) measure.
Example 3
Snake study
Protocol
• All experiments were performed in triplicate (i.e. three separate solutions, three pieces
of skin, one per solution). The skin used was from the dorsal side of Elaphe guttata,
the American com snake
A 1.33M concentration solution of each amino acid was made by stirring the amino
acid (glycine 0.1008 g L-serine 0.1395 g, D-serine 0.1400 g, L-homoserine 0.1582 g,
a-hydroxy- glycine 0.1213 g) in water ( L).
« Snake skin, from the top section (dorsal side) of the same donor, was cut in to ~lcm 2
pieces. Each piece was weighed and placed directly into one the of the amino acid
solutions, one piece of snake skin per solution.
• After 1 hour, the snake skin was removed from the solution and blotted dry on filter
paper. The weight of the snake skin was recorded before the same snake skin was
returned to the amino acid solution.
• After an additional 23 hours (24hours in total), the snake skin was again blotted dry
on filter paper and weighed.
• The snake skin, now having absorbed the test amino acid, was then placed in a
vacuum desiccator over silica (0% RH) for 48 hours before being weighed. The skin
was now dry, and contained the test amino acid.
» The snake skin was then placed in a desiccator at 40% RH (controlled using a
saturated solution of zinc nitrate), in order to establish how much water the skin was
able to absorb from the atmosphere at this level of humidity.
Each skin sample was then reweighed.
Accompanying Figure 2 shows the average percentage weight increase after 24h at 40% RH,
compared to water against the DRH (deliquescence relative humidity) for the same
compound. Figure 2 shows the DRH at 24 hrs after the skin was saturated with test amino
acid, dried, and then exposed to 40% humidity.
Accompanying Figure 3 shows the average percentage weight increase after 24h at 40% RH,
following 24h of treatment with 1.33M amino acid solution, for each of the test amino acids.
Again, it can be seen that a-hydroxyglycine and L-homoserine, in these tests, demonstrated
particularly advantageous properties,
Protocol
• A 10% w/w concentration solution of each compound is made by stirring the
compound (lOOmg) in water (lmL). Three separate solutions of each compound are
made separately so that the experiment can be carried out in triplicate simultaneously.
Where commercial creams are used, a sample of the cream (lOOmg) is placed into
vial.
Snake skin, from the top section of the same donor, is cut in to 1cm pieces. Each
piece is weighed and placed directly into one of the compound solutions. One piece of
snake skin per solution. The snake skin is placed directly onto the top of the solution
so that only one side conies into contact with the solution. Care needs to be taken
when placing the snake skin into the solution so that the skin lays flat on the solution
surface.
• After 1 hour, the snake is removed from the solution and blotted dry on filter paper.
The weight of the snake skin is recorded before the same snake skin is returned to the
same compound solution.
• After an additional 23 hours (24hours in total), the snake skin is again blotted dry on
filter paper and weighed.
The snake skin is then placed in a vacuum desiccator over silica (0% relative humidity
(RH)) for 48 hours before being weighed. The silica should be dried for 24h before
being placed into the vacuum desiccator.
• The snake skin is then placed into a desiccator at a certain RH (40% RH is controlled
using a saturated solution of zinc nitrate; 70% RH is controlled by a 1:1 NaC :
Na2C0 3 saturated solution; 100% RH is controlled using water). Each chamber should
be prepared 48h prior to the snake skin sample being placed in to the chamber and the
RH checked using a hygrometer.
The accompying figure 10 shows the result the average percentage weight increase after 24h
at 70% RH, following 24h of treatment with 10% w/w amino acid solution, for each of the
test amino acids.
Example 4
The effect of pre-treatment of skin with amino acid formulations of the present invention to
enhance the permeation of three compounds with different physicochemical properties was
investigated. The three compounds were:
Acyclovir
Metronidazole and
Diclofenac diethylamine
Methods
Acyclovir (ACV), metronidazole and diclofenac diethylamine (DDEA) were analysed using
HPLC, using of a Waters Alliance Separations Module and Waters detector. The temperature
of the column and samples were maintained at 45 ± 2 °C and 5.0 ± 2 C respectively. A
Kinetix™ C-18 (Phenomenex, USA) column ( 50 mm x 4.6 mm 5 mih particle size) with a
guard column (Phenomenex USA, C-18 4.0 x 3.0 mm) was employed as the stationary phase.
The mobile phase was three part; mobile phase A, water; mobile phase B, methanol; and
mobile phase C, 10 mM ammonium phosphate buffer pH 2.5. The mobile phase was run
using a gradient flow (Table 2) with a flow rate of 0.8 mL/min. Samples were run for 12
minutes with an injection volume of 10 L . Acyclovir, metronidazole and DDEA were
processed at a wavelength of 276 nm, with approximate retention times of 4.7, 6.5, and 8.7
min, respectively. Calibration curves were constructed from a series of standards prepared by
serial dilution in conjunction with separately prepared quality controls. Standards and QC's
were diluted with receiver fluid (phosphate buffered saline). Data were recorded and analysed
using Empower Pro Software.
Table 2, Flow gradient for analytical method.
Solutions for pre-treatment of N-hydroxyserine, L-homoserine, N-hydroxyglycine and a
combination of N-hydroxyserine, L-homoserine, N-hydroxyglycine in a 1:1:1 ratio were
prepared in deionised water at 10 % w/v.
Donor solutions of ACV, Metronidazole and DDEA were prepared by saturating the solvent
system (50:50, PEG-400: water) with the API for ca. 16 h. The donor solutions were
centrifuged prior to dosing in the in vitro permeation experiments.
Human epidermal membrane was prepared from skin post cosmetic reduction surgery
(abdominoplasty), Full thickness skin was defrosted at ambient temperature until malleable.
The subcutaneous fat was removed mechanically by blunt dissection. Upon removal of the
fat, skin was immersed in hot deionised water (60 ± 3 °C) for 45 s. The epidermal membrane
(comprising the Stratum co ewn and epidermis) was removed from the underlying dermis
using a gloved finger and the dermis was discarded. The epidermal membrane was then
floated (Stratum corneum side up) in deionised water onto filter paper. Excess water was
removed from the surface and the tissue was mounted in Franz type diffusion cells. Each cell
had an average surface area approximately 0.60 cm2 and a volume of approximately 2.0 mL.
The temperature of the water bath was set to maintain the surface temperature of the skin at
32 °C to represent skin in vivo.
Three experiments were performed, one for each API (Acyclovir, metronidazole and DDEA).
During each experiment a total of 32 cells per API were prepared. The integrity of the
epidermal membrane was assessed using electrical resistance to ensure the epidermal
membrane was intact. The cells were each pre-treated with 100 of the 10 % w/v solutions
of N-Hydroxyserine (n=6), L-homoserine (n=6), N-hydroxyglycine (n=6) and the
combination pre-treatment solution (n=6) overnight (ca. 16 h), n=6 cells were not pre-treated.
In addition a blank (no pre-treatment) and a placebo cell (pre-treated with the combination
pre-treatment solution) were also prepared to assess any potential interference with the
analytical assay. Following the pre-treatment period the pre-treatment solution was removed
from the surface of the epidermal membrane and the surface was dried. The lower receptor
chamber was filled with receiver fluid (phosphate buffered saline) and the cells were dosed
with a 6 mg dose (i.e. 0 mg/cm2) of each API saturated donor solution using a pre-calibrated
positive displacement pipette with exception of the blank and placebo cells. Samples of
receiver fluid (200 L) were removed from the sampling arm at 0, 1, 2, 4, 6, 24, 30 and 48 h
time points. After each sample was removed, an equal volume of pre-warmed receiver fluid
was replaced. Samples were analysed using HPLC and the level of each API that had
permeated was quantified.
Results
Diclofenac permeation was observed to be notably higher from all cells that had been pretreated
with the amino acids of the invention (Figures 4 and 5). The highest amount of
diclofenac permeated was observed following pre-treatment of the epidermal membrane with
N-hydroxy glycine where at t=24 h, a maximal enliancement n diclofenac permeation was
observed at 18.79 times that of the epidermal membrane that had no pre-treatment.
Figure 4 shows the cumulative permeation of diclofenac through epidermal membrane over a
48 h experimental period. Each bar represents the average permeation + SEM, n=5-6.
Figure 5 shows the cumulative permeation of diclofenac through epidermal membrane after
48 h . Each bar represents the average permeation + SEM, n=5-6.
Table 3. Mean cumulative permeation of diclofenac following no pre-treatment and pre-treatment with novel amino acid solutions. Enhancement
ratio (ER) refers to the enhancement compared to diclofenac permeation with no pre-treatment.
no pre- pre-treatment N pre-treatment L- pre-treatment N- pre-treatment 1:1:1
treatment hydroxyserine homoserine hydroxyglycine combination
Mean Mean Mean Mean
Time (h) Mean DDEA
DDEA DDEA DDEA DDEA
permeated Mea ER Mean ER Mea ER Mean ER
permeated permeated permeated permeated
^g/cm 2)
g cm2) g cm2) cm ) g c 2)
1.0 0.00 0.00 n a 0.00 a 0.00 n/a 0.00 n/a
2.0 0.00 0.19 a 0.00 n/a 0.00 n/a 0.00 n/a
4.0 0.05 0.83 16.05 0.00 0.00 0.39 7.63 0.00 0.00
6.0 0.15 1.53 9.97 0.14 0.93 1.03 6.72 0.04 0.27
24.0 1.31 11.83 9.01 2.93 2.23 24.67 18.79 8.23 6.27
30.0 1.64 13.99 8.51 4.27 2.60 25.84 15.72 12.07 7.34
48.0 3.17 20.14 6.35 8.21 2.59 36.87 1.63 23.19 7.31
In Table 4, metronidazole permeation was observed to be notably higher from all cells that
had been pre-treated with the amino acids of the invention following the t=2 h time point
(Figures 6 and 7). The highest amount of metronidazole permeated was observed following
pre-treatment of the epidermal membrane with N-hydroxyserine where at t=30 h, a maximal
enhancement in metronidazole permeation was observed at 14.72 times that of the epidermal
membrane that had no pre-treatment.
Figure 6 shows cumulative permeation of metronidazole through epidermal membrane over a
48 h experimental period. Each bar represents the average permeation + SEM, n~ 5-6.
Figure 7 shows cumulative permeation of metronidazole through epidermal membrane after
48 h. Each bar represents the average permeation + SEM, n=5-6.
Table 4. Mean cumulative permeation of metronidazole following no pre-treatment and pre-treatment with novel amino acid solutions.
Enhancement ratio (ER) refers to the enhancement com ared to metronidazole permeation with no pre-treatment.
In Table 5, Acyclovir permeation was observed in the receiver fluid earlier in cells that had
been pre-treated with the amino acids of the invention (Figures 8 and 9). The highest amount
of Acyclovir permeated was observed following pre-treatment of the epidermal membrane
with L-homoserine where at t=48 h, a maximal enhancement in Acyclovir permeation was
observed at 11,72 times that of the epidermal membrane that had no pre-treatment.
Enhancement ratios prior to t=48 h could not be calculated due to the lack of Acyclovir
permeation from the untreated skin prior to this point,
Figure 8 shows cumulative permeation of Acyclovir through epidermal membrane over a 48
h experimental period. Each bar represents the average permeation + SEM, n=5-6.
Figure 9 shows cumulative permeation of Acyclovir through epidermal membrane after 48 h.
Each bar represents the average permeation + SEM, n=5-6.
Table 5. Mean cumulative permeation of Acyclovir following no pre-treatment and pre-treatment with novel amino acid solutions. Enhancement
ratio (ER) refers to the enhancement compared to Acyclovir permeation with no pre-treatment.
NC - Not calculated
Summary
Pre-treatment of the epidermal membrane with amino acids of the present invention enhances
drug permeation thereover.
Different amino acids of the invention have differing enliancement effects depending on the
physicochemical properties of the drugs.
The plot for drug absorption enhancement factor is shown in Figure 11. From this it can
clearly be seen that the enhancement increases with increasing Log P. In this regard, the
enhancement factor and Log P are as follows:
ER Log
Diclofenac diethylamine 19 0.85
Metronidazole 15 -0.02
ACV 12 -1.56
Example 5
Water uptake into snake skin using mixtures and combinations:
Combinations with common formulations and bases
Using the protocol described in Example 3 with the only difference being that the snake skin
was finally rehydrated over a solution at ~75%RH (prepared using 1:1 NaCl: Na2C0 3
saturated solution). All samples at 10% (except hyaluronic acid where one sample was used
at 1% and vehicles were tested alone). The results are shown in Figure 0 .
From the results, it can be seen that a combination of an amino acid of the present invention
with Oilatum is particularly beneficial in its moisturising ability.
Example 6
RHE IRRITATION ASSESSMENT METHOD 1
.1 Introduction
The purpose of this experiment was to investigate the potential of alpha-hydroxylglycine ( -
H-G) and L-homoserine (L-h-S) to cause skin irritation. The method was based on the
validated SOP for the "The SkinEthic Skin Irritation Test-42 bis assay" in accordance to
OECD guideline Test No. 439: In Vitro Skin Irritation. The protocol is provided in Section
1.2. Further details on the method can be found in the SkinEthic™ RHE SOP, Version 2.1
(July 2009), SkinEthic skin irritation test-42 bis test method for the prediction of acute skin
irritation of chemicals: 42 minutes application + 42 hours post-incubation. Available at:
[http://ecvam.jrc.ec.euiOpa.eu]. In this protocol a tissue viability score of less than 50% of the
negative control suggests the test solution is an irritant.
In addition, a further experiment was performed where the contact time of the test solution
with the tissue was increased to 24 h. Following which point an MTT assay was used to
determine the tissue viability and the test solutions potential to cause irritation.
1.2 The SkinEthic skin Irritation Test - 42 bis assay protocol
1.2.1 RHE irritation testing protocol
1.2. 1.1.1 Pre-incubation step
The following steps were completed on receipt of the tissues:
(i) Wells of 6-well plates were filled with 1mL growth culture medium.
(ii) Tissue were removed from their packaging, cleaned to remove the transport
agarose and inspected for signs of damage; damaged tissues were discarded.
(iii) The tissue were transferred into the growth culture medium then incubated at 37
°C, 5 % C0 until application of the test solution.
.2. .1.2 Preparation of Phosphate Buffered Saline (PBS)
(i) PBS tablets (x 5) were added to a 500 mL volumetric flask.
(ii) The volumetric flask from Step (i) was made to volume with deionised water
(18.2 MW) and stirred using a PTFE magnetic stirrer until the PBS tablets are
observed to dissolve.
(iii) The PBS was used as required
1.2.1.1.3 Preparation of MTT solution
(i) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (100.0 ±
5.0 mg) was weighed into a 20 mL volumetric flask. The solution produced had
a nominal MTT concentration of 5 mg/mL.
(ii) The volumetric flask form Step (i) was made up to volume with PBS.
(iii) A PTFE magnetic stirrer was added to the volumetric flask from Step (ii) and
the solution was left to stir until fully dissolved.
(iv) The solution from Step (iii) was filter sterilised using a 0.2 mh filters directly
into sterile tubes.
(v) This stock was protected from light and stored at -20 °C until required.
(vi) When required, the MTT solution was thawed and diluted with pre-warmed
maintenance medium to achieve a concentration up to 1 mg/mL.
1.2. 1.1.4 Preparation of positive control (5% SDS) solution
(i) Sodium dodecyl sulphate (SDS, 100 ± 5.0 mg) was weighed into a 20 mL
volumetric flask and made to volume with deionised water ( 8.2 MW) .
(ii) The volumetric flask from Step (i) was made to volume with deionised water
(18.2 MW) and stirred using a PTFE magnetic stirrer until the SDS was fully
dissolved,
(iii) The solution was filter sterilised using sterile 0.2 m h filter PES filters.
2. . .5 Application of 5 and 1% w/v a-H-G and 0 and 1% w/v L-h-S and rinsing
.1.6 Applications of 5 and 1% w/v a-H-G and 10 and 1% w/v L-h-S and controls
(i) 16 ± 0.5 m of the test solutions (5% w/v ot-hydroxylglycine (a-H-G) in
water, 1% w/v a-hydroxylglycine (a-H-G) in water, 0% w/v L-homoserine (Lh-
S) in water, 1% w/v L-homoserine (L-h-S) in water) and negative (PBS) and
positive (5% SDS) controls were dispensed onto the top of the epidermal tissue,
using positive displacement pipette. The test solutions or controls were
distributed over the surface epidermis using the tip of the pipette.
A nylon mesh was placed over the surface of the tissue using forceps and the
plate lid was replaced.
The plates from Step (ii) were either retained in the laminar flow cabinet at
room temperature for 42 minutes or incubated at 37 °C, 5 % C0 for 24h.
Rinsing and drying steps
(i) Following treatment the nylon mesh was removed from the surface of the tissue.
(ii) The tissues were rinsed 25 times with PBS ( 1 mL) at a distance of 5-8 cm from
the insert to remove all residual Test Item from the epidermal surface.
(iii) The inserts from Step (ii) were emptied and dried on sterile absorbent paper.
(iv) The surface of the Stratum cornewn was dried with a cotton tip.
(v) The washed tissue from Step (iv) was transferred in a fresh 6 well culture plate
prefilled with 2 mL of warmed growth culture medium,
1.2.1.1.8 Post treatment incubation: 42 hours
(i) Post treatment the tissues were incubated at 37 °C, 5 % C0 2, 95 % humidified
atmosphere for 42 h .
(ii) At the end of the culture period the subnatant underneath the treated RHE
tissues was retained.
(iii) The maintenance culture media was homogenised, by agitation at 300 RPM for
2 minutes.
(iv) The incubation medium was placed in centrifuge tubes and stored at -20 °C
until required for analysis.
1.2.1 .1.9 MTT Assay
(i) Required wells in 24-well plates were filled with 300 pL of the MTT solution
and protected from light.
(ii) The tissues were removed from the post treatment incubation plates and excess
culture media was removed with absorbent paper.
(iii) The treated tissues were transferred into wells in 24-well plates pre-filled with
the MTT solution.
(iv) The plates were incubated for 3 h (+/- 5 min) at 37 °C, 5 % C0 95 %
humidified atmosphere.
(v) A new plate was filled with 800 pL of isopropanol and treated tissues were
transferred into the plates. An additional 700 pL of isopropanol was added to
the top of each tissue.
(vi) The plates were parafilmed to stop evaporation and protected from light using
foil before being refrigerated (2-8 °C) overnight to extract.
(vii) Following extraction the tissue was pierced using a pipette tip in order to add
the whole extraction solution into the well. The solution was made homogenous
by mixing with a pipette.
(viii) 3 x 200 pL of extraction solution per well was pipetted into a 96 well plate and
the optical density was measured at 570 nm using a pQuant spectrophotometer.
1.3 Results and discussion
The results of the tissue viability assessments with 42 min and 24 h exposure are illustrated in
Figure 122 and Figure respectively. Tissues viability after exposure for all test solutions was
observed to have remained over 50 % of the negative control suggesting that the solutions
(5% w/v a-H-G, 1% w/v a-H-G, 10% w/v L-h-S, 1% w/v L-h-S) are non-irritating.
Figure 2 shows viability of RHE tissues post 42 minute exposure and 42 post exposure
incubation. Each point represents the mean tissue viability with error bars representing the
range; n=4.
Figure 3 shows viability of RHE tissues post 24 h exposure. Each point represents the mean
tissue viability with error bars representing the range; n=3.
2 RHE IRRITATION ASSESSMENT METHOD 2
2.1 Introduction
Method 1 was used to investigate the potential for alpha-hydroxylglycine (a-H-G) and Lhomoserine
(L-h-S) to cause skin irritation. The protocol is primarily designed and validated
for single compounds rather than solutions. Therefore to provide further insight into the
potential of N-hydroxyserine and N-hydroxylglycine to cause skin irritation a second
protocol was investigated using Mattek's MTT effective time (ET-50) assay which is
designed to test mixtures of compounds. In addition two commercially available amino acid
related compound (L-serine and glycine) were selected and tested as comparators for Nhydroxyserme
and N-hydroxylglycine.
Mattek's MTT effective time (ET-50) assay used a minimum of 3 exposure times (at each
exposure time the tissue viability was determined by MTT assay) to construct a dose response
curve to determine exposure time required for a chemical to reduce viability to 50 % of the
control (i.e. the ET-50 value), As a general guideline, the following groupings can be used in
assigning expected in vivo irritancy responses based on the ET-50 results obtained:
ET-50 h s expected In vivo Irritancy Example
< 0.5 strong/severe, possible corrosive cone. Nitric acid
0.5-4 moderate 1% Sodium Dodecyl Sulfate
4-12 moderate to mild 1% Triton X-100
12-24 very mild Baby shampoo
24 non-irritating 10 % Tween 0
2.2 MTT ET-50 assay protocol
The irritation assessment of the solutions was performed using the protocol described below.
Each of the test solutions; N-hydroxyserine (10% w/v, N-H-S in water), N-hydroxylglycine
(1% w/v, N-H-G in water), L-Serine ( 0% w/v, in water) and Glycine (10% w/v, in water)
positive (1% v/v Triton X-100 in PBS) and negative (untreated) controls were applied to
tissues at different dosing periods (2, 6 and 24 h for solutions, 5 h for negative control and 3
and 7 h for positive control, n=3).
2.2.1 Pre-incubation step of RHE tissue
Tissues were stored at 2-8 °C on arrival, after which the following procedure was performed:
(i) Wells of 6-well plates were filled with pre-warmed (37 °C) assay medium (0.9
mL, supplied with the tissues) using an automatic pipette.
(ii) Tissue were removed from their packaging, cleaned to remove the transport
agarose using cotton swabs and inspected for signs of damage. Damaged tissues
were discarded.
(iii) The tissue were transferred into the assay medium and incubated at 37 °C, 5 %
C0 until the application of the test solutions; N-hydroxyserine (10% w/v, N-HS
in water), N-hydroxylglycine (1% w/v, N-H-G in water), L-Serine (10% w/v,
in water) and Glycine (10% w/v, in water) on the following day.
2.2.2 Preparation of the working MTT solution
The working MTT solution was prepared using the following procedure:
(i) MTT concentrate was removed from the freezer and allowed to thaw. The
concentrate was diluted with the MTT diluent solution ( 1 mL of concentrate to 4
mL of MTT diluent solution).
(ii) MTT solution from Step (i) was centrifuged at a g-force of 300 for 5 min to
remove any particulates.
(iii) The supernatant of the MTT solution from Step (ii) was stored in the dark at 4
°C for a maximum period of 24 h and used as required.
2.2 . Applications of solutions and controls
(i) Prior to application of solutions and controls the pre-incubation media culture
medium of the RHE tissues was removed from the 6 well plates replaced with
fresh pre-warmed (37 °C) assay medium (0.9 mL, supplied with the tissues)
using an automatic pipette.
Using a positive displacement pipette, the solutions N-hydroxyserine ( % w/v,
N-H-S in water), N-hydroxylglycine (1% w/v, N-H-G in water), L-Serine (10%
w/v, in water) and glycine (10% w/v in water) or controls (negative and positive
control) were dispensed (100 ± 0.5 m onto the top of the RHE tissue. The
solutions or controls were then distributed over the surface of the RHE tissues
using a sterile glass rod.
The lid was placed on the well plate containing the RHE tissue and the tissues
were returned to the incubator (37 °C, 5 % C0 2) for the required dosing period
(2, 6 and 24 h for the solutions; 3 and 7 for positive control; 5 h for negative
control).
2.2.4 MTT Assay
To determine the viability of the RHE tissue post application of the solutions and controls,
the following procedure was used:
(i) The required wells in 24-well plates were filled with 300 m MTT solution and
protected from light.
(ii) The tissues were removed from the post treatment incubation plates and excess
solutions and controls were removed with sterile cotton swabs.
(iii) The RHE tissue was subsequently rinsed with 1000 iL of PBS five times to
remove the residual solution from the epidermal surface.
(iv) The tissue from Step (iii) was blotted dry with cotton swabs.
(v) The treated tissues from Step (iv) were transferred into wells in 24-well plates
pre-filled with MT solution (300 The plates were incubated for 3 h (+/- 5
min) a 37 °C, 5 % C0 and 95 % humidified atmosphere.
(vi) The treated tissues were subsequently transferred into a new plate filled with 1
mL of extractant solution.
(vii) The plates were sealed using Parafilm® to stop evaporation and protected from
light using foil before extract at 2-8 °C to minimise evaporation.
(viii) Following extraction the RHE tissue were removed from the wells and an
additional 1mL of extractant solution was added. Using a pipette the extraction
solution was mixed and made visually homogenous.
(ix) The solution from Step (viii) was transferred in to n=3 wells (200 per well)
using an automatic pipette of a 96 well plate and the optical density was
measured at a wavelength of 570 n .
2,3 Results and discussion
The tissue viability assessments at 2, 6 and 24 h are illustrated in Figure 14. The dose
response curve demonstrates that, 1% w/v N-H-G, 10% w/v L-Serine and 10% w/v Glycine
had no significant impact on the viability of the RHE tissues during the three dosing periods
(t= 2, 6 and 24 h). As such and in accordance with the ET-50 guidance documentation values
1% w/v N-H-G, 0% w/v L-H-S and 1 % w/v Glycine would be considered as non-irritating.
An ET-50 at 22.8 h was calculated for 10% w/v N-H-S which would categorise 10% w/v NH-
S as a very mild irritant. Both of the commercially available amino acid related compounds
(L-serine and glycine) were shown to be non-irritants.
Figure 14 shows tissue viability after exposure to solutions of 10% w/v N-H-G, 1% w/v N-HG,
10% w/v L-Serine and 10% w/v glycine at t= 2, 6 and 24 h and positive control ( 1 % w/v
Triton™ X-100, t= 3 and 7 h). Each time point represents the mean tissue viability and error
bars represent the range, n=3.
3 IN VITRO ASSESSMENT OF PSORIATIC TISSUE. METHOD 3
3.1 Introduction
A human cell based in vitro psoriatic tissue model consisting of psoriatic fibroblasts and
normal keratinocytes was used in this study. When compared to normal RHE tissues the
psoriatic tissue model exhibits hyperproliferation of basal epithelial cells and increased
expression of psoriasis markers compared to that of the normal RHE tissues (e.g. GM-CSF).
GM-CSF is a key activator of the innate immune system involved in chronic inflammatory
and autoimmune diseases including psoriasis (Lontz et al 1995), where macrophages,
granulocytes, neutrophils, eosinophils and dendritic cells can contribute to tissue damage and
disease progression (Krinner 2007). GM-CSF stimulates stem cells to produce granulocytes
and other macrophages and subsequently activates these differentiated immune cells. GMCSF
has also been identified as an inflammatory mediator in autoimmune disorders with
elevated levels of GM-CSF mRNA or protein being measured in a variety of inflammatory
sites including in allergic and psoriatic patients, arthritic and asthmatic patients (Plater-
Zyberk et al 2008). Numerous in vivo studies over the past few years have shown that
blockade of GM-CSF via neutralising antibodies can prevent or even cure pro-inflammatory
diseases in various models of inflammation including arthritis experimental autoimmune
encephalitis, psoriasis (Schon et al 2000), and lung disease (Plater-Zyberk et al 2008). In
addition treatment with recombinant GM-CSF was found to aggravate chronic inflammatory
diseases, for example patients with chronic psoriasis receiving GM-CSF therapy have been
shown to have exacerbated psoriatic lesions (Kelly et al 2007). Due to the importance of GMCSF
in psoriasis it was selected for analysis in this study.
3.2 Methods
In this investigation two types of RHE tissues were used, which were healthy and psoriatic
tissue sourced from the MatTek Corporation (Ashland, MA). For simplicity the protocol
below refers to both tissue types as RHE tissues and all media as assay medium as both
tissues were treated in the same manner. The only exception was that two different types of
media were required for the different types of tissue (healthy and psoriatic tissues). The
healthy tissue controls were used in this experiment demonstrate that the levels of GM-CSF
in the psoriatic tissue were evaluate as previously demonstrated by the manufacture.
3.2.1 Pre-incubation step of RHE tissite
Tissues were stored at 2-8 °C on arrival, after which the following procedure was performed:
(i) Wells of 6-well plates were filled with pre-warmed (37 °C) assay medium (0.9
mL, supplied with the tissues) using an automatic pipette.
(ii) Tissue were removed from their packaging, cleaned to remove the transport
agarose using cotton swabs and inspected for signs of damage. Damaged tissues
were discarded.
(iii) The tissue were transferred into the growth culture medium and incubated at 37
°C, 5 % C0 until the application of the solutions on the following day.
.2 Preparation MTT solution
(i) 3-(4,5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) (100.0 ±
5.0 mg) was weighed into a grade A 20 mL volumetric flask. The solution
produced had a nominal MTT concentration of 5 mg/mL.
(ii) The volumetric flask form Step (i) was made to volume with PBS.
(iii) A PTFE magnetic stirrer was added to the volumetric flask from Step (ii) and
the solution was left to stir until fully dissolved.
(iv) The solution from Step (iii) was filter sterilised using a 0.2 mih filters directly
into sterile tubes.
(v) This stock was protected from light and stored at -20 °C until required.
(vi) When required, the MTT solution was thawed and diluted with pre-warmed
maintenance medium to achieve a concentration up to 1 mg/mL.
. Tissue setup and application of test solutions
(i) At the end of the equilibration period, the media for each well was removed
then the tissues were lifted and placed on culture stands and fresh pre-warmed
(37 °C) assay medium (5 mL) was added to the well.
(ii) Using a positive displacement pipette, the test solutions 10% w/v, N-H-S (Nhydroxyserine)
in water, 10 % w/v, N-H-G (N-hydroxyglycine) in water, 0%
w/v, L-h-S (L-homoserine) in water and 10% w/v, combination 1:1:1 (N-H-S:
N-H-G: L-h-S) in water were dispensed (50 ± 0.5 \ S onto the top of the RHE
tissue. The solutions or controls were then distributed over the surface of the
RHE tissues using a sterile glass rod.
(iii) The lid was placed on the well plate containing the RHE tissue and the tissues
were returned to the incubator (37 °C, 5 % C0 2) 48 h between applications.
.4 Media change and reapplication of solutions
(i) Every 48 h culture media for the RHE tissues was removed from the 6 well
plates and transferred into tubes stored (-20 °C) for cytokine analysis.
(ii) Fresh pre-warmed (37 °C) assay medium (5 L) was added to each tissue using
an automatic pipette.
(iii) From each treatment 1 tissue was removed for viability assessment.
(iv) The RHE tissues were subsequently rinsed with 100 of PBS to remove the
residual solution from the epidermal surface.
(v) Using a positive displacement pipette, the solutions 10% w/v, N-H-S in water,
1% w/v, N-H-G in water, 10% w/v, L-h-S in water and 10% w/v combination
1:1:1 (N-H-S: N-H-G: L-h-S) were dispensed (50 ± 0.5 m ) onto the top of the
RHE tissue. The solutions or controls were then distributed over the surface of
the RHE tissues using a sterile glass rod.
(vi) The lid was placed on the well plate containing the RHE tissue and the tissues
were returned to the incubator (37 °C, 5 % C0 ) 48 h between applications at
t=0, 48 and 96 h.
3.2.5 7 Assay
To determine the viability of the RHE tissue post application of the solutions and controls,
the following procedure was used:
(i) The required weils in 24-well plates were filled with 300 i MTT solution and
protected from light.
(ii) The tissues were removed from the post treatment incubation plates and excess
solutions and controls were removed with sterile cotton swabs.
(iii) The RHE tissue was subsequently rinsed with 1000 of PBS five times to
remove the residual solution from the epidermal surface.
(iv) The tissue from Step (iii) was blotted dry with cotton swabs.
(v) The treated tissues from Step (iv) were transferred into wells in 24-well plates
pre-filled with MTT solution (300 m ) . The plates were incubated for 3 h (+/- 5
min) at 37 °C 5 % C0 and 95 % humidified atmosphere.
(vi) The treated tissues were subsequently transferred into a new plate filled with 1
mL of extractant solution.
(vii) The plates were sealed using Parafilm® to stop evaporation and protected from
light using foil before extract at 2-8 °C to minimise evaporation.
(viii) Following extraction the E tissue were removed from the wells and an
additional 1 L of extractant solution was added. Using a pipette the extraction
solution was mixed and made visually homogenous.
(ix) The solution from Step (viii) was transferred in to n=3 wells (200 per well)
using an automatic pipette of a 96 well plate and the optical density was
measured at a wavelength of 570 n .
3.2.6 Analysis of cytokine release
The conditioned media was analysed to determine the concentration of GM-CSF released. An
Invitiogen Human GM-CSF kit (a solid phase sandwich Enzyme Linked-Immuno-Sorbent
Assay (ELISA)) was utilised to quantify the concentration of GM-CSF. An antibody specific
for human GM-CSF is coated onto the wells of a microtiter plate. Each sample, including
standards of human GM-CSF, was pipetted directly into the coated wells, followed by the
addition of a biotinylated second antibody. The plate was incubated under ambient conditions
for 0.5 h during which period the human GM-CSF antigen binds simultaneously to the
immobilised (capture) antibody on one site, and to the solution phase biotinylated antibody
on a second site. The excess second antibody was removed and streptavidin-peroxidase
(enzyme) was added. This enzyme binds to the biotinylated antibody to complete a fourmember
sandwich. The sandwich was incubated for a second time under ambient conditions
for 0.5 h and any unbound enzyme was removed by washing, following which a substrate
solution was added, which results in the formation of a coloured product that was quantified
by measuring the absorbance of the solution at 450 run using a q ί spectrophotometer.
The intensity of this coloured product is directly proportional to the concentration of human
GM-CSF present in the original sample, and is quantified from the provided GM-CSF
standards.
3.3 Results and discussion
Figures 5 and 16 illustrate the effect of treatment with 10% w/v, N-H-S in water, 1% w/v,
N-H-G in water, 10% w/v, L-h-S in water and a 10% w/v combination 1:1:1 (N-H-S: N-H-G:
L-h-S) solution over 6 days in both psoriasis and control reconstructed human skin models.
This demonstrates that release of GM-CSF from psoriasis tissues is reduced by treatment
with N-H-S, L-h-S, N-H-G and combination solutions compared to untreated psoriasis
tissues. This reduction in GM-CSF release suggested that N-hydroxyserine (N-H-S), Lhomoserine
(L-h-S), N-hydroxyglycine (N-H-G) and a 1:1:1 combination could be beneficial
in the treatment of psoriasis.
None of the test solutions 10% w/v, N-H-S in water, 1% w/v, N-H-G in water, 10% w/v, Lh-
S in water and a 10% w/v combination 1:1:1 (N-H-S: N-H-G: L-h-S) solution had a strong
negative impact on tissue viability over 6 day experimental period. This data supports the
conclusions from the irritation assessments that the solutions are non-irritating i.e. tissue
viability remained above 50% for the duration of the experiment (Table 6).
Table 6. Tissues viability after exposure to test items (n=l)
Figure 15 shows release of GM-CSF from psoriasis tissues. Each point represents the
average cumulative release of GM-CSF over 6 day with measurements made at 2, 4, and 6
days with error bars representing the range n=2-3.
Figure 6 shows total release of GM-CSF. Each bar represents the total release of GM-CSF
over 6 day from both psoriasis model and control RHE tissues. Error bars representing the
range n=2-3.
4 SUMMARY
4.1 RHE irritation assessment (method 1)
The skin irritation potential of L-homoserine (L-h-S) and alpha-hydroxyglycine (a-H-G) was
investigated using the 42 bis assay protocol with the addition of an increased dosing period of
24 h. Analysis of the tissue viability after both incubation periods (42 min and 24 h)
demonstrated the tissue viability remained over 50 % which suggested that L-homoserine (Lh-
S) and alpha-hydroxyglycine (a-H-G) are both non-irritants.
4.2 RHE irritation assessment (method 2)
To further investigate the potential for skin irritation in response to N-hydroxyglycine (N-HG)
and N-hydroxyserine (N-H-S) the ET-50 skin irritation protocol (MatTek) was used. This
was selected as it is suitable for formulations and mixtures. In this assay in addition to the
test item N-hydroxyglycine (N-H-G) and N-hydroxyserine (N-H-S) two commercially
available amino acid related compounds were selected for comparison (L-serine and glycine).
In this study alpha-hydroxyglycine (a-H-G) was found to be a non-irritant (i.e. had an ET-
50< 24 h) and 10% w/v N-hydroxyserine (N-H-S) was categorised as a very mild irritant (i.e.
had an ET-50 between 12 - 24 h). Both of the commercially available amino acid related
compounds (L-serine and glycine) were shown to be non-irritants.
4.3 In vitro assessment of psoriatic tissue
The effect of N-hydroxyserine (N-H-S), L-homoserine (L-h-S), N-hydroxyglycine (N-H-G)
on GM-CSF release in an RHE model for psoriasis was investigated, It was observed that Nhydroxyserine
(N-H-S), L-homoserine (L-h-S), N-hydroxyglycine (N-H-G) and the
combination treatment reduced the production of GM-CSF which suggests that Nhydroxyserine
(N-H-S), L-homoserine (L-h-S), N-hydroxyglycine (N-H-G) maybe beneficial
in the treatment of psoriasis. Thus, there is provided the use of amino acids of the present
invention as anti-inflammatory agents, especially where a contributory factor in said
inflammation is GM-CSF. Particularly preferred amino acids for such use are Nhydroxyserine,
L-homoserine, N-hydroxyglycine, and combinations containing one or more
thereof.
The tissue viability of the RHE model for psoriasis during dosing with N-hydroxyserine (NH-
S), L-homoserine (L-h-S), N-hydroxyglycine (N-H-G) was in agreement with the previous
studies for both L-homoserine (L-h-S), N-hydroxyglycine (N-H-G) further confirming them
as non-irritants, as RHE viability remained above 50 % of the untreated controls over a 6 day
dosing period. This study also suggested that N-hydroxyserine (N-H-S) was a non-irritant.
Thus, there is provided the use of amino acids of the present invention as anti-irritants.
Particularly preferred amino acids for such use are N-hydroxyserine, L-homoserine, Nhydroxyglycine,
and combinations containing one or more thereof.
References
Clar EJ, Fourtanier A (1981). Pyrrolidone carboxylic acid and the skin [in French]. Int J
CosmetScl 3: 101-13.
Harding CR, Watkinson A, Rawlings AV, et al (2000). Dry skin, moisturisation and
corneodesmolysis. Int J Cosmet Sci, 22: 21-52.
Horii I, Nakayama Y, Obata M, et al (1989). Stratum corneum hydration and amino acid
content in xerotic skin. Brit J Dermatol. 121 : 587-592.
Jokura Y, Ishikawa S, Tokuda H, Imokawa G (1995). Molecular analysis of elastic
properties of the stratum corneum by solid state C-nuclear magnetic resonance
spectroscopy. J Invest Dermatol 104: 806-812.
Kezic S, Kemperman P M, Koster E S, De Jongh C M, Thio H B (2008). Loss-of-function
mutations in the filaggrin gene lead to reduced level of natural moisturizing factor in the
stratum corneum. J Invest Dermatol 8: 2 117-21 19.
Kezic S, O'Regan GM, Yau N, et al (201 1). Levels of filaggrin degradation products are
influenced by both filaggrin genotype and atopic dermatitis severity. Allergy 66: 934-40.
Nakagawa N, Sakai S, Matsumoto M, Yamada K (2004). Relationship between NMF (lactate
and potassium) content and the physical properties of the stratum corneum in healthy
subjects, J Invest Dermatol 122: 755-763.
Palmer CN, Irvine atopic dermatitis, Terron-Kwiatkowski A, et a (2006). Common loss offunction
variants of the epidermal barrier protein filaggrin are a major predisposing factor for
atopic dermatitis. Nat Genet 38: 441-446.
Rawlings AV, Scott IR, Harding CR, et al (1994). Stratum corneum moisturisation at the
molecular level. J Invest Dermatol. 103: 731-741.
Sasaki O, Kanai I, Yazawa Y (2007). Relationship between the chemical structure of humic
substances and their hygroscopic properties. Science 1: 17-22.
Scott IR, Harding CR, Barrett JG (1982). Histidine-rich protein of the keratohyalin granules.
Source of the free amino acids, urocanic acid, and pyrrolidoiie carboxylic acid in the stratum
corneum. Biochim Biophys Acta. 719: 10-1 17.
Weber, M. T., et al (20 ) ., J Clin Aesthet Dermatol. 5: 29-39.
White-Chu EF, Reddy M. Dry skin in the elderly: complexities of a common problem. Clin
Dermatol. 201 1; 29(l):37-42.

Claims:
1. An unnatural, hygroscopic amino acid for use in enhancing hydration and/or the
moisture retention and/or uptake properties of an external keratinaceous structure of an
animal.
2. An unnatural, hygroscopic amino acid for use in accordance with claim 1, wherein
said structure is the skin.
3. An unnatural, hygroscopic amino acid for use according to claim 1 or 2, wherein the
amino acid is capable of absorbing and retaining moisture from the atmosphere at a relative
humidity (RH) of <50%, at 32°C.
4. An unnatural, hygroscopic amino acid for use according to claim 3, wherein the
amino acid is capable of absorbing and retaining moisture from the atmosphere at a relative
humidity (RH) of <40%, at 32°C
5. An unnatural, hygroscopic amino acid for use according to any preceding claim,
wherein said host is a mammal.
6. An unnatural, hygroscopic amino acid for use according to claim 5, wherein said host
is a human.
7. A topical, including ocular, formulation comprising at least one amino acid as defined
in any of claims 1 to 4.
8. A formulation according to claim 7, further comprising one or more additional
moisturising ingredients.
9. A formulation according to claim 7 or 8, further comprising one or more additional
further amino acids.
10. A formulation according to any of claims 7 to 9, wherein said formulation mimics
NMF, by using one or more amino acid ingredients of NMF.
11. A formulation according to claim 10, wherein said one or more amino acid
ingredients ofNMF are present in amounts and/or ratios approximating those found in NMF.
12. A formulation according to any of claims 7 to 11, wherein said formulation mimics
NMF, by using one or more non-amino acid ingredients of NMF.
13. A formulation according to claim 12, wherein said one or more non-amino acid
ingredients of NMF are present in amounts and/or ratios approximating those found in NMF.
14. A formulation according to claims 12 or 13, wherein said one or more non-amino acid
ingredients of NMF include one or more salts.
15. A formulation according to claim 4, wherein said one or more salts are selected from
the sodium, potassium, HC1, and diethylamine salts.
16. An amino acid for use, or a composition according to, any preceding claim, wherein
the amino acid is selected from N-hydroxyserine, N-hydroxyglycine, L-homoserine,
a-hydroxyglycine, 2-(aminooxy)-2-hydroxyacetic acid, 2-hydiOxy-2-(hydroxyamino) acetic
acid, 2-(aminooxy)acetic acid, and combinations thereof.
17. An amino acid according to claim 16, which is selected from a-hydroxyglycine, or Lhonioserine,
or both.
18. An amino acid, or a composition comprising said amino acid, as defined in any
preceding claim, for use in the cosmetic treatment of an external keratinaceous structure of an
animal.
1 . An amino acid according to claim 8, wherein said structure is the skin.
20. An amino acid according to claim 8 or , for a use selected from the group
consisting of:
a) use in emollient formulations;
b) use in ocular formulation for the treatment of dry eye;
c) use to moisturise thickened skin;
d) use in nail and/or hoof softening;
e) use in conjunction with hair removal techniques;
f ) use in face packs;
g) use to hydrate the skin in the treatment of skin conditions; and
h) and use in hydrating the skin before application of a barrier preparation.
i) use in wound healing
21. An amino acid as defined in any of claims 1 to 17, for use in facilitating transdermal
absorption of drugs.
22. An amino acid, or a composition comprising said amino acid, as defined in any of
claims 1 to 17, for use as an ant -inflammatory agent, optionally in combination with a
further anti-inflammatory agent.
23. An amino acid, or a composition, according to claim 22, wherein a contributory factor
in said inflammation is GM-CSF.
24. An amino acid, or a composition, according to claim 22 or 23, wherein said amino
acid is selected from N-hydroxy serine, L-honioserine, N-hydroxyglycine, and combinations
containing one or more thereof.
25. An amino acid, or a composition comprising said amino acid, as defined in any of
claims 1 to 17, for use as an anti-irritant.
26. An amino acid, or a composition, according to claim 25, wherein said amino acid is
selected from N-hydroxyserine, L-homoserine, N-hydroxyglycine, and combinations
containing one or more thereof.
27. A composition for transdermal administration of a drug, comprising an amino acid as
defined in any of claims 1 to 17, to assist in dermal penetration of said drug
28. A composition for transdermal administration of a drug, comprising an amino acid as
defined in any of claims 1to 7, to reduce the irritation of said drug.
29. An amino acid, or a composition comprising said amino acid, as defined in any
preceding claim, wherein said amino acid lias a deliquescence relative humidity (DRH) of no
greater than 80% at 32°C.
30. An amino acid, or a composition comprising said amino acid, as defined in any
preceding claim, wherein said amino acid has an O/C ratio of at least 0.7.