Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
METHOD FOR AMELIORATING PRURITUS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention relates to a method for ameliorating pruritus,
and in
particular relates to a method of using a phenylbutyric acid or short-chain
fatty acid
derivative for preventing, treating, or ameliorating pruritus associated with
localized or
systemic diseases or disorders.
Description of the Related Art
[0002] As it is known, the cutaneous sensation referred to as pruritus, is
characterized
by an unpleasant, itchy sensation of the skin which provokes scratching. The
scratching is
sometimes severe enough to irritate and inflame the skin of afflicted
subjects. Pruritus may
also be characterized as a uniformed response to a wide variety of physical,
chemical,
and/or biological stimuli, which may be of an endogenous or exogenous nature
that may be
associated with specific dermatologic conditions such as allergic reactions to
drugs, insect
bites and to environmental allergens, or a systemic disease such as
thyrotoxicosis, diabetes
mellitus, uremia, iron deficiency anemia, delusions of parasitosis,
polycythemia rubra vera,
cholestasis and Hodgkin's disease. Although usually occurring in the skin,
pruritus can also
occur in non-cutaneous areas such as mucous membranes. Thus, the cause of
pruritus can
be multifactorial or due to a single underlying disorder. The pathophysiology
of pruritus
involves central and peripheral nervous systems as well as multiple cytokine
release and
molecular mediators.
[0003] When the origin of pruritus is in the skin, sensory nerve endings in
the
dermoepidermal junction are stimulated. The sensation of pruritus is
transmitted along
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dedicated unmyelinated C fibers which are distinct from fibers that transmit
pain and touch.
The irritated skin will transmit the sensation of pruritus by stimulating
local nerves in the
spinal cord. From there, the stimulus travels via the lateral spinothalamic
tract to the
thalamus, and then on to the cerebral cortex, where it causes the sensation of
pruritus
(Weldon D. Allergy Asthma Proc 28: 153-62, 2007). Gastrin-releasing peptide
receptor
(GRPR), histamine, substance P, and tumor necrosis factor a(TNF-(X) seem to
play
significant roles in the perception of pruritus (Sun YG, et al. Nature 448:700-
703, 2007).
Moreover, for the central neural mechanism where itching is detected, the
opioid peptides
and the receptor have been implicated in provoking the pruritus of
cholestasis, which
responds to intravenous naloxone (Jones EA, et al. JAMA 268:3359-62, 1992).
Meanwhile,
serotonin reuptake inhibitors can improve systemic pruritus induced by
cholestasis,
suggesting that serotonergic pathways are also important in the perception of
itching (Mayo
MJ, et al. Hepatology 45:666-74, 2007).
[0004] On the other hand, locally-released substances including histamine,
tachykinins,
serotonin (5-hydroxytryptamine (5-HT)), interferon (IFN)-gamma, interleukin
2(IL-2) and
IL-4 released from activated macrophage, mast or T cells at the site of
pruritoceptive origin
have been implicated to cause the symptoms and signs of itching sensation,
scratching,
swelling, rash, urticaria, and/or scaling (Greaves MW, et al. Lancet 348:938-
40, 1996;
Inagaki N, et al. Eur J Pharmacol 546:189-96, 2006). Subjects suffering from
pruritus
induced by a dermatological disorder or systemic disease can possibly worsen
the pruritus
by excessively scratching the affected area so extensively that the excessive
scratching will
lead to irritation, inflammation, wound formation and possibly infection. For
the peripheral
mechanism of pruritus, the role of multiple cytokine release and molecular
mediators in the
generation of signs and symptoms of pruritus in diseased skin or mucosa has
been defined.
Histamine-induced pruritus involves H1 receptors (Davies MG, et al. Br J Clin
Pharmacol
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9:461-65, 1980). Tachykinins including the neuropeptides substance P,
calcitonin gene-
related peptide, and vasoactive intestinal peptide are found in the cutaneous
free-nerve
endings of unmyelinated nociceptor neurons which initiate the sensations of
pruritis.
Intradermal 5-HT can evoke itching and scratching by acting on 5-HT2 and 5-HT3
receptors (Nojima H, et al. J Pharmacol Exp Ther 306:245-52, 2003). These
observations
have led to the use of a 5-HT2 or 5-HT3 receptor antagonist for treating
pruritus (Schworer
H, et al. Lancet 341:1277, 1993). IL-2 when given subcutaneously causes
intense localized
itching in both atopic and normal subjects (Wahlgren CF, et al. Arch Dermatol
Res
287:572-80, 1995). Inhibition of IL-2 biosynthesis by immunosuppressive agents
such as
cyclosporine A relieves the pruritus of atopic dermatitis (Wahlgren CF, et al.
Acta Derm
Venereol (Stockh) 70:323-29, 1990).
[0005] Although antihistamines are widely used for suppression of pruritis,
the extent
to which suppression is attributable to the side-effect of central sedation
rather than local
histamine antagonism in the skin is unclear (Krause L, et al. BMJ 287:1199-
200, 1983).
Many patients report persistent pruritus even with current antihistamines
therapies, 5-HT
receptor antagonists, and/or immunosuppressive agents, as most are ineffective
for chronic
pruritus, and only provide short-term relief with side-effects. Pruritus may
be quite
debilitating for some patients. Thus, there is a continuing need for
development of new and
improved methods and compositions for preventing, treating, or ameliorating
pruritus
resulting from a wide variety of causes.
[0006] Phenylbutyrate, a short-chain fatty acid, has been approved by the FDA
as an
orphan drug for inborn error with urea cycle disorder to treat hyperammonemia
(Brusilow
SW, et al. N Engl J Med 310: 1630-4, 1984). In the human body, phenylbutyrate
is
metabolized to phenylacetate via (3-oxidation. Phenylacetate subsequently
undergoes
conjugation with glutamine to form phenylacetylglutamine, which serves as a
vehicle for
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waste nitrogen excretion. Recently, phenylbutyrate has also been found to have
the ability
to inhibit deacetylase, to increase acetylation on histones and non-histone
proteins, to
remodel chromatin structures and to alter activities of multiple
transcriptional factors,
resulting in simultaneously, epigenetically modulating many genes and thus,
controlling
diseases (Marks PA, et al. J Natl Cancer Inst 92: 1210-6, 2000). In
preclinical and clinical
studies, the gene modulatory effects of phenylbutyrate have exhibited
therapeutic potential
in many hematologic and solid tumors, inherited genetic disorders such as
cystic fibrosis,
sickle cell anemia, [3-thalassemia, X-linked adrenoleukodystrophy, spinal
muscular atrophy,
and neurodegenerative disorders, aging, and inflammatory diseases such as
autoimmune
diseases (Kemp S, et al. Nat Med 4: 1261-8, 1998; et al. Proc Natl Acad Sci
USA 102:
11023-8, 2005; Kang HL, et al. Proc Natl Acad Sci USA 99: 838-43, 2002;
Blanchard F, et
al. Drug Discov Today 10: 197-204, 2005). Moreover, phenylbutyrate can also
act as a
chemical chaperone to protect normal cells from oxidative stress injury and
prevent
neurotoxicity (I'am GH, et al. Invest Ophthalmol Vis Sci 48:1683-90, 2007).
BRIEF SUMMARY OF INVENTION
[0007] The invention provides a pharmaceutical composition and method for
preventing, treating, or ameliorating pruritus associated with a cutaneous,
mucosal or
systemic disease or disorder. The method comprises administering to a subject
with
pruritus or topically applying to an affected area with pruritus an effective
amount of a
pharmaceutical composition comprising a phenylbutyric acid or short-chain
fatty acid
derivative and a pharmaceutically acceptable carrier, salt or solvate thereof.
[0008] The invention further provides a pharmaceutical composition and method
for
preventing, treating, or ameliorating pruritus associated with a cutaneous,
mucosal or
systemic disease or disorder. The method comprises administering to a subject
with
pruritus or topically applying to an affected area with pruritus an effective
amount of a
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pharmaceutical composition comprising a phenylbutyric acid or short-chain
fatty acid
derivative of 2 to 6 carbons in length in combination with other anti-pruritic
agents and a
pharmaceutically acceptable carrier, salt or solvate thereof.
[0009] A detailed description is given in the following embodiments with
reference to
the accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
The present invention can be more fully understood by reading the subsequent
detailed
description and examples with references made to the accompanying drawings,
wherein:
[0010] FIG. 1 shows a topical 2.5% phenylbutyric acid gel rapidly relieving
pruritus
associated with skin disorders caused by radiation dermatitis, sun burn,
surgical wound
healing, psoriasis, and atopic dermatitis;
[0011] FIGs. 2A-2C are human Thl-Th2-Th3 gene expression profiling
demonstrating
phenylbutyrate simultaneously suppressing the induction of multiple cytokine
expression in
activated T cells stimulated with PMA and ionomycin. Jurkat T cells were pre-
incubated
with phenylbutyrate (1 mM) for 24 hrs, and then stimulated with ionomycin (1
M) and
PMA (10 ng/ml) for 6 hrs. Using real-time PCR, the expression of a panel of
genes related
to helper T cells with or without T-cell stimulation and phenylbutyrate
treatment was
analyzed. The array includes cytokine genes representative of Thl, Th2 and Th3
cells, gene
encoding transcriptional factors regulating the expression of cytokines as
well as other
markers of CD4+ T lymphocytes, genes involved in immune cell activation in the
Thl and
Th2 type immune responses, and genes involved in the antimicrobial humoral
response.
Results are the mean SE of three determinations, expressed as the fold
induction
(observed experimental relative unit/basal control relative unit in the
absence of any stimuli
or treatment);
[0012] FIGs. 2131-2132 disclose the various human Thl-Th2-Th3 genes.
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[0013] FIG. 3 is a chromatin immunoprecipitation (ChIP) assay demonstrating
the
modulatory effects of phenylbutyrate on the status of histones that remodel
chromatin
structure and the binding of transcription-factors that regulate gene
expression. Results
show that 4-phenylbutyrate sodium was effective in not only modifying histones
but also
decreasing binding of transcriptional factors of NF-KB, NFAT, and AP-1 to the
IL-2
promoter in activated Jurkat T cells stimulated by ionomycin and PMA. Anti-Sp
1 antibody
was used as a negative control because Sp 1 does not bind to IL-2 promoter.
DETAILED DESCRIPTION OF INVENTION
[0014] The following description is of the best-contemplated mode of carrying
out the
invention. This description is made for the purpose of illustrating the
general principles of
the invention and should not be taken in a limiting sense. The scope of the
invention is best
determined by reference to the appended claims.
[0015] The invention is broadly intended for use of phenylbutyric acid or a
short-chain
fatry acid and its pharmaceutically acceptable derivatives to prevent, treat
or ameliorate all
types of pruritus from various diseases or disorders including, but not
limited to, allergic
dermatoses, pruritic dermatoses, vascular dermatoses, sebaceous gland
disorders,
autoimmune disorders, rheumatoid arthritis, systemic lupus erythematosus,
progressive
systemic sclerosis, sjogren's syndrome, dermatomyositis, mixed connective
tissue disease,
papulosquamous dermatoses, bacterial dermatoses, viral dermatoses, mycolic
skin
infections, granulomatous dermatoses, parasitic skin dermatoses, exfoliative
dermatitis,
bullous dermatoses, pigmented dermatoses, photosensitive dermatoses,
dermatoses caused
by collagen diseases, dermatoses due to internal diseases, xerosis, urticaria,
atopic
dermatitis, eczyma, vasculitis, lichen simplex chronicus, psoriasis, scabies,
pediculosis
corporis and pubis, multiple sclerosis, thyrotoxicosis, diabetes, renal
insufficiency, uremia,
iron deficiency anemia, delusions of parasitosis, polycythemia rubra vera,
cholestasis,
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wound, sun burn, cold sores, acne, insect bite, radiotherapy or chemotherapy-
induced
dermatitis or mucositis, paraneoplastic syndrome, malignancy, primary skin
cancer, and
metastatic skin cancer.
[0016] In the invention, the compounds of phenylbutyric acid derivatives
include, but
are not limited to, phenylbutyric acid, phenylproprionic acid, phenylacetic
acid,
phenylbutyrate, phenylproprionate, phenylacetate, phenylacetylglutamine,
phenoxybutyric
acid, phenoxyproprionic acid, phenoxyacetic acid, phenoxybutyrate,
phenoxyproprionate,
phenoxyacetate, bromophenylbutyric acid, bromophenylproprionic acid,
bromophenylacetic acid, bromophenylbutyrate, bromophenylproprionate,
bromophenylacetate, chlorophenylbutyric acid, chlorophenylproprionic acid,
chlorophenylacetic acid, chlorophenylbutyrate, chlorophenylproprionate,
chlorophenylacetate, fluorophenylbutyric acid, fluorophenylproprionic acid,
fluorophenylacetic acid, fluorophenylbutyrate, fluorophenylproprionate,
fluorophenylacetate, iodophenylbutyric acid, iodophenylproprionic acid,
iodophenylacetic
acid, iodophenylbutyrate, iodophenylproprionate, iodophenylacetate,
hydroxyphenylbutyric
acid, hydroxyphenylproprionic acid, hydroxyphenylacetic acid,
hydroxyphenylbutyrate,
hydroxyphenylproprionate, hydroxyphenylacetate, methylphenylbutyric acid,
methylphenylproprionic acid, methylphenylacetic acid, methylphenylbutyrate,
methylphenylproprionate, methylphenylacetate, ethylphenylbutyric acid,
ethylphenylproprionic acid, ethylphenylacetic acid, ethylphenylbutyrate,
ethylphenylproprionate, ethylphenylacetate, naphthylbutyric acid,
naphthylproprionic acid,
naphthylacetic acid, naphthylbutyrate, naphthylproprionate, naphthylacetate,
and tributyrin.
Also other short-chain fatty acids of 2 to 6 carbons in length include, but
are not limited to,
butyric acid, butyrate, 2,2 dimethyl butyric acid, a-methylhydrocinnamic acid,
3,5
dimethoxy-4-hydrocinnamic acid, cinnamic acid, butyryl hydroxamate,
propionate,
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-
bromopropionate, E-3-3 pyridyl-2-propenoic acid, levulinic acid, Kemp triacid,
isovalerate,
valerate, butrymide, isobutyramide, valproic acid, and valproate.
[0017] The second compounds for combination with the phenylbutyric acid or
short-
chain fatty acid derivative include, but are not limited to, an anti-
histamine, an
anticholinergics, a non-steroid anti-inflammation drug, a steroid, an anti-
oxidant agent, a
vitamin, a leukotriene modifier, an interleukin antagonist, a mast cell
inhibitor, an anti-IgE
antibody, a selective serotonin reuptake inhibitor (SSRI), a 5-
hydroxytryptamine (5-HT)
receptor antagonist, an antibiotics, a calcineurin inhibitor, a histone
deacetylase inhibitor, a
gastrin-releasing peptide receptor antagonist, gabapentin, and naloxone.
[0018] The compounds of the invention can be formulated as pharmaceutical
compositions. Such compositions can be administered orally, parenterally, by
inhalation
spray, rectally, vaginally, intradermally, transdermally, or topically in
dosage unit
formulations containing conventional nontoxic pharmaceutically acceptable
carriers,
adjuvants, and vehicles as desired. Topical administration may also involve
the use of
transdermal administration such as transdermal patches or iontophoresis
devices. The term
parenteral as used herein includes subcutaneous, intravenous, intramuscular,
or intrasternal
injection, or infusion techniques. Formulation of drugs is discussed in, for
example,
Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton,
Penn. (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage
Forms,
Marcel Decker, New York, N.Y. (1980).
[0019] In one embodiment, the preparations for treatment of skin pruritis are
generally
aimed at providing a condition for increasing skin manageability. There are
recognized
categories of formulations for skin care compositions, including creams,
ointments, gels,
sprays, lotions, skin tonics, shampoos or mousses as referred to above. Skin
sprays are
generally composed of aerosolized copolymers, such as polyvinylpyrrolidone,
vinyl acetate
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and the like, and may also function as a setting lotion. Skin gel preparations
are similar to
sprays in composition, but are in gel and alcohol free form, and can coat the
skin. Skin
mousse is foam released under pressure from an aerosolized can. The
phenylbutyric acid
derivative or short-chain fatty acid active ingredient in a topical skin care
composition
according to the present invention is preferably present at a concentration of
0.00001 to
100.00% by weight relative to the total weight of the composition, or in a
dosage of 1 to
1000 mg. A skin care composition for treating pruritus according to the
present invention
may be formulated as a hydrophobic or hydrophilic cream, ointment, gel,
emollient, spray,
lotion, skin tonic, shampoo or mousse as referred to above, suitably with
additional
ingredients suitable for use in skin care compositions of types known in the
art, and such
further ingredients can include petrolatum, waxes, lanolin, silicone,
liposomes, vegetable,
mineral oils, plasticizers, fragrances, preservatives, a penetration enhancing
agent, a pH
adjusting agent or other suitable ingredients for topical skin compositions.
Such ingredients
can moisturize skin, stabilize the active compound, increase drug-skin contact
and local
concentration, control drug slow release, and/or aid in decreasing skin
breakage, preventing
skin atrophy, fibrosis and infection, and promoting skin wound healing.
[0020] The invention also provides a method for treatment of skin pruritus as
described
herein, which method comprises a composition providing at least a
phenylbutyric acid
derivative or short-chain fatty acid thereof, together with at least one or
more other agents,
including an anti-histamine, an anticholinergics, a non-steroid anti-
inflammation drug, a
steroid, an anti-oxidant agent, a vitamin, a leukotriene modifier, an
interleukin antagonist, a
mast cell inhibitor, an anti-IgE antibody, a selective serotonin reuptake
inhibitor, a 5-
hydroxytryptamine receptor antagonist, an antibiotics, a calcineurin
inhibitor, a histone
deacetylase inhibitor, gabapentin, and naloxone, in which active ingredients
are present in
free form or in the form of a pharmaceutically acceptable salt and optionally
at least one
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pharmaceutically acceptable carrier, for systemically or topically
simultaneous, separate or
sequential use.
[0021] Suitable salts for the components to be employed according to the
present
subject matter are also those with inorganic cations, for example alkali metal
salts, in
particular sodium, potassium, or ammonium salts, alkaline earth metal salts
such as, in
particular, the magnesium or calcium salts, as well as salts with bi- or
tetravalent cations,
for example the zinc, aluminum, or zirconium salts. Also contemplated are
salts with
organic bases, such as dicyclohexylamine salts; methyl-D-glucamine; and salts
with amino
acids, such as arginine, lysine, histidine, glutamine and so forth. Also, the
basic nitrogen-
containing groups can be quaternized with such agents as: lower alkyl halides,
such as
methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl
sulfates, such as
dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as
decyl, lauryl,
myristyl, and stearyl chlorides, bromides, and iodides; asthma halides, such
as benzyl and
phenethyl bromides; and others. Salt-forming agents, for example, low
molecular weight
alkylamines such as methylamine, ethylamine, or triethylamine can also be
employed.
Water or oil-soluble or dispersible products are thereby obtained.
[0022] The amount of active ingredient that can be combined with the carrier
materials
to produce a single dosage form will vary depending upon the subject and the
particular
mode of administration. The dosage required will vary according to a number of
factors
known to those skilled in the art, including, but not limited to, the compound
or compounds
used, the species of subject, the size of the subject, and the severity of the
associated
disease condition that causes pruritus. The compounds can be administered in a
single dose,
in multiple doses throughout a 24-hour period, or by continuous infusion. When
administered by continuous infusion, the compounds can be supplied by methods
well
known in the art, such as, but not limited to, intravenous gravity drip,
intravenous infusion
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pump, implantable infusion pump, or any topical routes. Length of treatment
will vary
depending on many factors, for example, the duration and severity of the skin,
mucosa or
systemic diseases or disorders that cause localized or generalized pruritus.
Treatment of the
subject with the phenylbutyric acid derivative or short-chain fatry acid
derrivative alone or
in combination with other agents of the invention may last until pruritus
disappears, or
treatment will continue for the life of the subject.
[0023] EXAMPLE
[0024] Example 1: Various topical compositions-oleag;inous ointment, cream,
and g_el
[0025] A. Preparation of an Oleaginous Ointment of Phenylbutyrate:
[0026] 65 g of white petrolatum (Riedel-de Haen), 15 g of cetyl alcohol
(Riedel-de
Haen), 260 g of soft paraffin (Merck), 155 g of liquid paraffin (Merck), and 5
g of 4-
phenylbutyrate (Merck) were mixed in a beaker and heated at 70 C to form a
paste. The
paste was stirred at 400 rpm for 1 hour, and then cooled at room temperature.
[0027] B. Preparation of Cream of Phenylbutyrate:
[0028] Part I: 70 g of Tefose 63®, 20 g of Superpolystate®, 10 g of
Coster
5000®, 15 g of Myriyol 318®, 15 g of Coster 5088®, and 15 g of GMS
SE® (all commercially available from a local supplier) were mixed in a
beaker and
heated at 70 C.
[0029] Part Il: 5.739 g of sodium 4-phenylbutyrate (Triple Crown America,
Inc.),
0.125 g of inethylparaben (Merck), 0.075 g of propylparaben (Merck), and
149.061 g of
deionized water were mixed in a beaker and heated at 70 C.
[0030] Part 11 was slowly added into part I and continually stirred at 400 rpm
for 5
minutes to form a mixture. 2% Stabileze QM® (prepared by dissolving 2 g of
Stabileze QM® in 98 g of deionized water, heating and stirring at 70 C to
form a paste,
and cooling at room temperature) was added into the mixture and stirred for 5
minutes. The
.
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pH of the mixture was adjusted to 5.34 with 0.85% phosphoric acid (Merck), and
stirred at
600 rpm for 20 minutes. The mixture was cooled at room temperature.
[0031] C. Preparation of Gel of Phenylbutyrate:
[0032] Part I: 10 g of Stabileze QM® and 232.035 g of deionized water were
mixed in a beaker and heated at 70 C.
[0033] Part II: 5.739 g of sodium 4-phenylbutyrate (Triple Crown America,
Inc.),
0.125 g of inethylparaben (Merck), 0.075 g of propylparaben (Merck), 232.035 g
of
deionized water, and 20 g of 10% NaOH were mixed in a beaker and heated at 70
C.
[0034] Part II was slowly added into part I and continually stirred at 400 rpm
for 20
minutes to form a mixture. The mixture was cooled at room temperature.
[0035] D: Preparation of Liposomal Formulation of Phenylbutyrate:
[0036] In this liposomal formulation, egg phosphatidylcholine (EPC) and
cholesterol
were used in equi- or different-molar concentrations as primary lipid
components. Various
liposomes located with 4-phenylbutyrate were obtained by varying the
lipid:phenylbutyrate
ratio. Liposomes were prepared by thin film hydration, sized by membrane
extrusion, and
physically evaluated.
[0037] Example 2: Topical phenylbutyric acid to the affected skin of different
disorders to treat pruritus
[0038] A 2.5% phenylbutyric acid gel was applied to the affected skin six
times per
day for 1 week. There were four patients in each group, who completed a daily
itch diary
in which they graded the severity of their pruritus on a continuous scale from
0( no pruritus)
to 10 (the worse pruritus imaginable) using a visual analog scale (VAS) with
points
anchored with facial expressions to guide their selection (Mayo MJ, et al.
Hepatology
45:666-74, 2007).
[0039] Referring to FIG. l, the 2.5% phenylbutyric acid gel rapidly relieved
the itchy
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sensation within 2-10 minutes, and improved the mean VASs from 7.25, 7, 6.75,
8, and
7.75 to 2, 1, 1.5, 2.5 and 2.25 in 1-2 days in patients with radiation-induced
dermatitis, sun
burn, surgical wound healing, psoriasis, or atopic dermatitis, respectively.
The pruritus-
related symptoms of erythema, urticaria, swelling, and desqumation also
subsided
simultaneously.
[0040] Example 3: suppression of multiple itching-associated molecular
mediators bv_
4-12henylbuWate sodium
[0041] Itching is the most important problem in many allergic and inflammatory
skin
diseases. The skin barrier (stratum corneum) is a major factor for determining
the nature of
immune responses to allergens presented at the skin surface.
[0042] Abnormalities in skin barrier function may result in Thl, Th2 and Th3
responses to infectious agents, chemicals, or protein antigens, which induce
several
cytokines and molecular mediators in the skin lesion to cause symptoms and
signs of
pruritus. The cytokine profile subsequently produced depends upon the type of
allergen
stimulation. Distinct subsets of helper T cell activation have been identified
by virtue of
cytokines that they produce. Activated Thl cells produce IFN-gamma and IL-2.
Thl cells
regulate delayed type hypersensitivity reactions. Thl responses are promoted
by Iocal
release of the IL-12 superfamily of cytokines. These responses are further
enhanced by IL-
15 and IL-18 production. Activated Th2 cells produce IL-4 and IL-10. Th2 cells
mediate
allergic and antibody responses. Th2 responses are favored by local production
of IL-4, IL-
33, and IL-18 in synergy. Some cytokines, such as IL-3, GM-CSF (CSF2), and TNF-
alpha,
are produced by both Thl and Th2 subsets. On the other hand, IL-6 is a pro-
inflammatory
cytokine secreted by activated T cells to stimulate immune response to trauma,
especially
burns or other tissue damage leading to inflammation. Th3 cells are related to
negative
regulation of immune response.
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[0043] In order to demonstrate whether phenylbutyrate can suppress multiple
itching-
associated cytokines, molecular mediators or markers at one time, a panel of
gene
expression profiling was analyzed by using real-time PCR (RT2 ProfilerTM PCR
Array
Human Thl-Th2-Th3: APHS-034, SuperArray Bioscience Corporation).
[0044] Jurkat T cells were treated in the presence of increasing
concentrations of 4-
phenylbutyrate, ionomycin, and/or phorbol 12-myristate 13-acetate (PMA) for 24-
72 hr at
370C. At the doses of 1 mM of 4-phenylbutyrate for 48 hrs, and 1 M of
ionomycin plus 10
ng/ml of PMA incubated for 24 hrs, no significant differences were found by
flow
cytometry in cell proliferation, cytotoxicity, and apoptosis between control
and treated cells.
However, 48 hrs after stimulation with ionomycin (1 M) plus PMA (10 ng/ml), T
cells
were full cycling and progressed through the S, G2, and M phases of the cell
cycle due to
the induction of T cell growth and survival factors (interleukins), whereas
pre-treatment
with phenylbutyrate (1 mM) for 24 hrs almost completely prevented entry of the
cells into
the S phase of the cell cycle.
[0045] Jurkat T-cells were nonstimulated or stimulated with ionomycin (1 M)
and
PMA (10 ng/ml) for 6 hrs in the absence or presence of pre-incubation of 4-
phenylbutyrate
sodium (1 mM) for 24 hrs, RNA was extracted and then RT-PCR was performed for
profiling the expression of 84 genes related to Thl-Th2-Th3 responses shown in
the gene
table (FIG. 2).
[0046] Referring to FIGs. 2A-21) and Table 1, phenylbutyrate completely
suppresses
or significantly decreases the induction of Thl cytokines and related genes
(CCR5, CSF2,
IFN-gamma, IL1213, IL12RB2, IL18, IL18R1, IL2, IL2RA, IRF1, STAT4, TLR4,
TLR6),
Th2 cytokines and related genes (CCL11, CCL7, CCR2, CCR4, IL13, IL13RA1,
IL1R1,
IL1R2, IL4R, IL9, IRF4, MAF), T-cell activation markers (BCL3, CD69, IL6,
IL6R, JAK2,
LAT, TNFRSF9), T-helper 1 type imniune response (I1,1213, IL18, IRF4, SFTPD,
TLR4,
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TLR6), T-helper 2 type i.mmune response (IL18, IL4R, IRF4), and antimicrobial
humoral
response (CCL7, CCR2, IL12B, IL13, SFTPD) in PMA/ionomycin-induced Jurkat T-
cell
activation. On the other hand, phenylbutyrate upregulates more expression of
SOCS1, a
suppressor of cytokine signaling as a Th3 response, which is involved in
negative
regulation of cytokines.
[0047] IL-1 and IL-6 are pro-inflammatory cytokines. Antigen binding to the T
cell
receptor stimulates the secretion of IL-2, and the expression of IL-2
receptors. The IL-2/IL-
2R interaction then stimulates the growth, differentiation and survival of
antigen-selected
cytotoxic T cells. IL-4 stimulates activated B-cell and T-cell proliferation,
and the
differentiation of CD4+T-cells into Th2 cells, and induces B-cell class
switching to IgE.
IL-9 elicits many functions on lymphoid cells and mast cell lineages, and has
been thought
to have a role in asthma. IL-12 is known as a T cell stimulating factor in
response to
antigenic stimulation, which can stimulate the growth and function of T cells.
IL-13
secreted by many cell types, but especially Th2 cells, is an important
mediator of allergic
inflammation. IL-18 works together with IL-12 to induce cell-mediated immunity
following infection with microbial products like lipopolysaccharide. Taken,
together, the
effects of phenylbutyrate on inhibiting the complicated interrelated signaling
network
pathways of IL-1, IL-2, IL-4, IL-6, IL-9, IL-12, IL-13, and IL-18, and on
upregulating
SOCS 1(a suppressor of cytokine signaling) are correlated with the novel
finding in the
present invention that phenylbutyrate has the ability to ameliorate pruritus
in some allergic
and inflammatory dermatitis-associated pruritus.
[0048] Table 1. At least 2-Fold difference in induction by T-cell stimulation
when
compared to control, and suppressive effects of phenylbutyrate
Thl cytokines and related genes Mitogen stimulation Phenylbutyrate + mitogen
stimulation
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CCR5 2.68 0.98
CSF2 229.13 88.77
IFN-gamma 126.24 99.18
IL12B 2.68 0.98
IL12RB2 2.68 0.98
IL18 2.68 0.98
IL18R1 19.97 0.98
IL2 831.75 304.86
IL2RA 151.17 64.98
IRF1 5.86 1.7
SOCS 1 4.82 8.59
STAT4 7.89 3.32
TLR4 2.68 0.98
TLR6 29.04 10.43
Th2 cytokines and related genes
CCL11 2.68 0.98
CCL7 2.68 1.04
CCR2 4.20 0.84
CCR4 7.41 1.93
IL13 2.68 0.98
IL13RA1 2.68 0.98
IL1R1 11.31 3.92
IL 1 R2 2.25 0.82
IL4R 42.81 9.53
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IL9 2:68 0.98
IRF4 26.17 16.50
MAF 2.68 0.98
Other T-cell activation markers
BCL3 58.08 19.73
CD69 87.43 48.57
IL6 2.68 0.98
IL6R 2.68 0.98
JAK2 4.72 2.27
LAT 3.12 1.57
SFTPD 2.68 0.98
TNFRSF9 89.26 13.01
[0049] The induction of multiple cytokine expression depends on the
coordinated
activation of transcription factors, mostly including NFKB, NF-AT, and AP-1
(Sancho R, et
al. J Immunol 172:2341-51, 2004; Li-Weber M, et al. Eur J Immunol 34 :1111-18,
2004).
Because induction of cytokines is regulated mainly at the transcriptional
level, chromatin
immunoprecipitation (ChIP) analysis in Jurkat T cells was performed to
determine the
binding of NFKB, NF-AT, and AP-1 to IL-2 promoter. Jurkat T cells were
nonstimulated or
stimulated with ionomycin (1 M) and PMA (10 ng/ml) for 6 hrs in the absence
or presence
of pre-incubation of 4-phenylbutyrate sodium (1 mM) for 24 hrs, then
formaldehyde
crosslinking between protein and DNA and ChIP using anti- NFKB, NF-AT, AP-1,
Spl or
acetyl H3 antibodies (Santa Cruz) were performed. PCR primers were designed to
amplify
the human IL-2 promoter: F 5'-GAGTTACTTTTGTATCCCCACCCCC (-317 to -292 in
the IL-2 promoter), R 5'-CCTGTACATTGTGGCAGGAGTTGAGG (+33 to 58). PCR
amplifications used a three-step protocol with 90 C (30 s) denaturing
temperature, 59 C (45
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s) primer annealing temperature, and 72 C (30 s) enzyme reaction temperature
for 35
cycles. Referring to FIG. 3, phenylbutyrate affects chromatin structure by
altering acetyl H3
status, and decreases DNA binding of NFKB, NF-AT, and AP-1 to IL-2 promoter,
suggesting the suppression of phenylbutyrate on cytokine expression during T-
cell
activation could be mediated by decreasing binding of transcriptional factors
to promoters.
[0050] While the invention has been described by way of example and in terms
of the
preferred embodiments, it is to be understood that the invention is not
limited to the
disclosed embodiments. To the contrary, it is intended to cover various
modifications and
similar arrangements (as would be apparent to those skilled in the art).
Therefore, the scope
of the appended claims should be accorded the broadest interpretation so as to
encompass
all such modifications and similar arrangements.
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