U.S. patent application number 14/011874 was filed with the patent office on 2014-03-06 for pharmacological preparation for topical use containing npalmitoyl-vanillamide.
This patent application is currently assigned to EPITECH GROUP S.R.L.. The applicant listed for this patent is EPITECH GROUP S.R.L.. Invention is credited to Luciano De Petrocellis, Francesco Della Valle, Maria Federica Della Valle, Vincenzo Di Marzo, Sabatino Maione.
Application Number | 20140066399 14/011874 |
Document ID | / |
Family ID | 43533342 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140066399 |
Kind Code |
A1 |
Della Valle; Francesco ; et
al. |
March 6, 2014 |
PHARMACOLOGICAL PREPARATION FOR TOPICAL USE CONTAINING
NPALMITOYL-VANILLAMIDE
Abstract
The present invention relates to topical preparations containing
N-palmitoyl-vanillamide having hyperalgesic activity. In
particular, the invention relates to N-palmitoyl-vanillamide for
use in the treatment of pathologies selected from: post-herpetic
neuralgia, neuralgia of trigeminus, occipital neuralgia, dental
neuralgia, glottopharyngeal neuralgia, uremic neuralgia, diabetic
neuralgia, headache of different origin, neuropathic itch,
neurogenic itch, uremic itch, vulvodinia, vulvar vestibulitis,
ano-rectal pain and itch, balano-preputial pain and itch, painful
urogenital disorders of dogs and cats, psoriasis-associated
pruritus and pain, itching skin diseases (e.g. atopic dermatitis)
in the human and veterinary field, muscular pain, pain of the
tendon, osteoarthritis associated pain in humans, dogs and cats;
painful eye diseases in the human and veterinary field,
inflammatory pathologies of the oral cavity in the human and
veterinary field.
Inventors: |
Della Valle; Francesco;
(Padova, IT) ; De Petrocellis; Luciano; (Napoli,
IT) ; Maione; Sabatino; (Napoli, IT) ; Di
Marzo; Vincenzo; (Napoli, IT) ; Della Valle; Maria
Federica; (Padova, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EPITECH GROUP S.R.L. |
Milano |
|
IT |
|
|
Assignee: |
EPITECH GROUP S.R.L.
Milano
IT
|
Family ID: |
43533342 |
Appl. No.: |
14/011874 |
Filed: |
August 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13291294 |
Nov 8, 2011 |
8546352 |
|
|
14011874 |
|
|
|
|
Current U.S.
Class: |
514/54 ; 514/560;
514/616; 514/625 |
Current CPC
Class: |
A61P 25/02 20180101;
A61P 17/04 20180101; A61K 9/0031 20130101; A61K 9/06 20130101; A61K
9/006 20130101; A61K 9/0095 20130101; A61K 31/07 20130101; A61K
45/06 20130101; A61K 31/165 20130101; A61K 31/728 20130101; A61P
29/00 20180101; A61K 9/0048 20130101; A61K 9/0034 20130101; A61P
25/04 20180101; A61P 15/02 20180101; A61K 9/0014 20130101; A61P
1/02 20180101; A61K 31/194 20130101; A61P 25/06 20180101; A61P
15/00 20180101; A61P 19/02 20180101; A61K 9/7038 20130101; A61P
27/02 20180101; A61K 31/165 20130101; A61K 2300/00 20130101; A61K
31/07 20130101; A61K 2300/00 20130101; A61K 31/728 20130101; A61K
2300/00 20130101; A61K 31/194 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/54 ; 514/625;
514/560; 514/616 |
International
Class: |
A61K 31/165 20060101
A61K031/165; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2010 |
EP |
10190375.5 |
Claims
1. A method for treating or ameliorating hyperalgesia caused by
inflammatory or neuropathic conditions in a mammal comprising
administering to the mammal a composition comprising from about
0.05% to about 5% w/w of N-palmitoyl-vanillamide.
2. The method of claim 1, wherein the mammal is human.
3. The method of claim 1, wherein the mammal is a cat or a dog.
4. The method of claim 1, wherein said inflammatory or neuropathic
conditions are selected from the group consisting of post-herpetic
neuralgia, neuralgia of trigeminus, occipital neuralgia, dental
neuralgia, glossopharyngeal neuralgia, headache, uremic neuralgia,
diabetic neuralgia, neuropathic itch, neurogenic itch, uremic itch,
vulvodynia, vulvar vestibulitis, ano-rectal pain and itch,
balano-preputial pain and itch, painful urogenital disorders,
psoriasis-associated pruritus and pain, atopic dermatitis, itching
skin diseases, muscular pain, pain of a tendon,
osteoarthritis-associated pain, painful eye diseases, and
inflammatory pathologies of the oral cavity.
5. The method of claim 4, wherein the inflammatory pathologies of
the oral cavity are selected from the group consisting of
gingivitis, periodontitis, stomatitis, post implantation pain, and
burning mouth syndrome.
6. The method of claim 4, wherein the inflammatory pathologies of
the oral cavity are selected from the group consisting of: canine
and feline gingivitis, periodontitis, gingivostomatitis, and feline
resorptive lesions (FORL).
7. The method of claim 1, wherein said composition is in the form
of a topical composition.
8. The method of claim 7, wherein said topical composition is a
cream, gel, lotion, drops for otological application, patch,
eyewash, mouthwash, or suppository.
9. The method of claim 7, wherein the composition comprises one or
more additional active principles selected from the group
consisting of: adelmidrol, trans-traumatic acid, menthol,
hyaluronic acid or its salts, Vitamin A, and any combination
thereof.
10. A method for treating or ameliorating hyperalgesia caused by
inflammatory or neuropathic conditions in a mammal comprising
administering to the mammal a composition comprising
N-palmitoyl-vanillamide and one or more additional active
principles selected from the group consisting of adelmidrol,
trans-traumatic acid, menthol, hyaluronic acid or its salts,
Vitamin A, and any combination thereof.
11. The method of claim 10, wherein the mammal is human.
12. The method of claim 10, wherein the mammal is a cat or a
dog.
13. The method of claim 10, wherein said inflammatory or
neuropathic conditions are selected from the group consisting of
post-herpetic neuralgia, neuralgia of trigeminus, occipital
neuralgia, dental neuralgia, glossopharyngeal neuralgia, headache,
uremic neuralgia, diabetic neuralgia, neuropathic itch, neurogenic
itch, uremic itch, vulvodynia, vulvar vestibulitis, ano-rectal pain
and itch, balano-preputial pain and itch, painful urogenital
disorders, psoriasis-associated pruritus and pain, atopic
dermatitis, itching skin diseases, muscular pain, pain of a tendon,
osteoarthritis-associated pain, painful eye diseases, and
inflammatory pathologies of the oral cavity.
14. The method of claim 13, wherein the inflammatory pathologies of
the oral cavity are selected from the group consisting of
gingivitis, periodontitis, stomatitis, post implantation pain, and
burning mouth syndrome.
15. The method of claim 13, wherein wherein the inflammatory
pathologies of the oral cavity are selected from the group
consisting of: canine and feline gingivitis, periodontitis,
gingivostomatitis, and feline resorptive lesions (FORL).
16. The method of claim 10, wherein said composition is in the form
of a topical composition.
17. The method of claim 16, wherein the composition comprises from
about 0.05% to about 5% w/w of N-palmitoyl-vanillamide.
18. The method of claim 16, wherein said topical composition is a
cream, gel, lotion, drops for otological application, patch,
eyewash, mouthwash, or suppository.
Description
PRIOR APPLICATION DATA
[0001] This application is a divisional application of U.S.
application Ser. No. 13/291,294, filed on Nov. 8, 2011, which
claims priority to and benefit of European Patent Application
10190375.5, filed on Nov. 8, 2010, the contents of each of which
are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a topical preparation
containing N-palmitoyl-vanillamide with anti-hyperalgesic
activity.
BACKGROUND
[0003] The transient receptor potential of vanilloid type-1 (TRPV1)
channel is a non-specific cation channel and plays a major role in
nociceptive heat thermosensation and inflammatory hyperalgesia. It
is abundantly expressed in unmyelinated sensory fibers of the
A.delta.- and C-type as well as in the spinal cord, and its
activation in the peripheral afferents of dorsal root ganglia
causes entry of calcium, depolarization and local release of
algogenic peptides, such as calcitonin gene-related peptide and
substance P, as well as activation of ascending pathways of pain
transmission. The TRPV1 channel was discovered thanks to studies on
the mechanism of action of the pro-nociceptive and thermal
pain-mimicking actions of capsaicin, the pungent principle of
Capsicum annum. Apart from being up-regulated during chronic pain
conditions, TRPV1 exists in several phosphorylated/active and
dephosphorylated/inactive forms, only the former of which are
capable of mediating the gating of extracellular calcium into
neurons through channel pores and subsequent depolarization. TRPV1
phosphorylation can be triggered by several algogenic mediators
through the intermediacy mostly of protein kinases C and A, and,
together with release of the channel from the tonic inhibitory
action of phosphatidyl-inositol-bis phosphate, obtained through PI3
kinase or phospholipase C activation, it is the major molecular
mechanism for its sensitization to the action of temperature, low
pH (protons), endogenous agonists (endovanilloids) and toxins like
capsaicin. Calcium entry also causes the desensitization of the
channel, through the action of Ca.sup.2+-sensitive protein
phosphatases such as calcineurin. Therefore, TRPV1 is made
refractory by its agonists to further stimulation by noxious heat
or endogenous algogenic mediators, thus leading to paradoxical
anti-hyperalgesic actions, which are at the basis of the use of
capsaicin-based creams against chronic pain.
SUMMARY
[0004] It has now been discovered that N-palmitoyl-vanillamide,
known by the name of Palvanil, which is an highly stable synthetic
TRPV1 agonist, is capable of desensitizing TRPV1 from activation
operated by exogenous and endogenous pro-nociceptive and
pro-inflammatory stimuli, without displaying pungent effects, and
showing anti-nociceptive activity in vivo tests of nociception.
[0005] It has been observed that, as compared to capsaicin,
Palvanil exhibits a slower kinetics of TRPV1 activation, as
assessed by monitoring TRPV1-mediated elevation of intracellular
calcium in HEK293-TRPV1 cells and is significantly more potent at
desensitizing capsaicin- and anandamide/pH-induced activation of
TRPV1 in HEK293-TRPV1 cells, its maximal desensitizing effects
being observed with more rapid onset. A strong desensitizing effect
by Palvanil on anandamide 0.25 microM plus pH 6.5, which somehow
mimics conditions that might occur in the "inflammatory pathway",
in which TRPV1 is up-regulated, anandamide is overproduced and pH
is lowered, was observed at concentrations of Palvanil as low as
0.5 nM, as compared to capsaicin, which exerted a half-maximal
inhibition at concentrations 18-fold higher; 3) unlike capsaicin,
Palvanil is absolutely not pungent in the eye wiping assay; and 4)
although non-pungent per se, Palvanil inhibits in a long-lasting
and dose-dependent way the capsaicin-induced response in this
assay, in a way antagonized by a TRPV1-antagonist. These are very
relevant and innovative results because they indicate that it is
possible, with Palvanil, to obtain anti-hyperalgesic pharmaceutical
preparations, superior to capsaicin both in efficacy and safety, in
diseases characterized by neuropathic inflammation and chronic
inflammatory or neuropathic pain, in human and in veterinary
field.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A and 1B. Kinetics of TRPV1-mediated elevation of
intracellular [Ca.sup.2+] by capsaicin and Palvanil. (A) Time
necessary to produce a maximal elevation of intracellular calcium
in HEK-293-TRPV1 cells with different concentrations of Palvanil
and capsaicin; (B) kinetics of intracellular calcium elevation by 1
.mu.M capsaicin (continuous line) and Palvanil (dotted line).
Arrows denote time of administration of the compounds to cells. In
(A) data are means.+-.SEM of n=4 different determinations. The
values for Palvanil were always significantly different from those
of capsaicin as assessed by ANOVA followed by the Bonferroni's test
(P<0.05);
[0007] FIG. 2. Dose response curves for the desensitization of
capsicin (10 nM) effect on intracellular calcium in HEK-293-TRPV1
cells by capsaicin and Palvanil. Data are means.+-.SEM of n=4
different determinations. The values for Palvanil were
significantly different from those of capsaicin in the range of
log] [agonist] from -9 to -8, as assessed by ANOVA followed by the
Bonferroni's test (P<0.05);
[0008] FIG. 3. Extent of the desensitization of capsicin (10 nM)
effect on intracellular calcium in HEK-293-TRPV1 cells by capsaicin
and Palvanil as measured after different pre-incubation times of
cells with either compound. Data are means of n=4 different
determinations. SEM bars are not shown and were always <5% of
the means. The values for Palvanil were significantly different
from those of capsaicin at all times, as assessed by ANOVA followed
by the Bonferroni's test (P<0.05);
[0009] FIGS. 4A and 4B. Dose-dependent effects of Palvanil or
capsaicin on the effect on intracellular calcium in HEK-293-TRPV1
cells by anandamide 0.5 .mu.M (A) or 0.25 .mu.M (B) at pH 7.4 and
6.5. Data are means of n=4 different determinations. SEM bars are
not shown and were always <5% of the means;
[0010] FIG. 5. Effect of Palvanil (10 e 30 .mu.g/ml) alone or after
5'-iodo-resiniferatoxin (I-RTX) pre-treatment (1 .mu.g/ml) on the
number of wiping movements evoked by instillation of a capsaicin
(CAP) solution (10 .mu.g/ml) into the left eye. Palvanil was
administered 1, 3 and 6 hours before capsaicin instillation, and
I-RTX 30 minutes before Palvanil. Data are represented as
means.+-.S.E.M. of n=6 mice per group. *indicates P<0.05 vs.
CAP-treated and .degree. P<0.05 vs. Palvanil-treated mice.
One-Way ANOVA, post-hoc Tukey's.
DETAILED DESCRIPTION
[0011] An object of the present invention is
N-palmitoyl-vanillamide for use in the treatment of inflammatory
and neuropathic hyperalgesia in mammals.
[0012] A further object of the invention is N-palmitoyl-vanillamide
for use in the treatment of a pathology selected from:
post-herpetic neuralgia, neuralgia of trigeminus, occipital
neuralgia, dental neuralgia, glottopharyngeal neuralgia, uremic
neuralgia, diabetic neuralgia, headache of different origin,
neuropathic itch, neurogenic itch, uremic itch, vulvodinia, vulvar
vestibulitis, ano-rectal pain and itch, balano-preputial pain and
itch, painful urogenital disorders of dogs and cats,
psoriasis-associated pruritus and pain, itching skin diseases (e.g.
atopic dermatitis) in the human and veterinary field, muscular
pain, pain of the tendon, osteoarthritis associated pain in humans,
dogs and cats; painful eye diseases in the human and veterinary
field, inflammatory pathologies of the oral cavity in the human and
veterinary field.
[0013] According to an embodiment, the said inflammatory
pathologies of the oral cavity in the human are gengivitis,
parodontitis, stomatitis, post implantation pain, burning mouth
syndrome.
[0014] According to an embodiment, the said inflammatory
pathologies of the oral cavity in the veterinary field are canine
and feline gengivitis, parodontitis, gengivostomatitis, and feline
resorptive lesions (FORL). Another object of the invention is a
topical formulation containing N-palmitoyl-vanillamide for use in
the treatment of inflammatory and neuropathic hyperalgesia in
mammals.
[0015] N-palimitoyl-vanillamide is a known drug and can be prepared
according to conventional methods, such as the one disclosed in the
experimental section of the present description.
[0016] The topical composition according to the invention can be in
the form of a cream, gel, lotion, drops for otological application,
patch, eyewash, mouthwash, suppository or any other suitable
pharmaceutical form and may contain conventional excipients. The
preparation of the topical composition of the invention can be
performed according to well known technologies, such as the ones
described in Remington's Pharmaceutical Sciences Handbook, Mack
Pub. Co., N.Y., USA, 17th edition, 1985.
[0017] The composition may contain additional active principles,
such as adelmidrol, trans-traumatic acid, menthol, hyaluronic acid
or its salts or vitamin A.
[0018] Palvanil can be administered to a subject of about 70 kg of
body weight in a dosage between 0.1 mg and 1 g and preferably
between 1 mg and 100 mg per dosage unit. The dosage unit may be
administered for example from 1 to 4 times a day. The dosage can be
adjusted according to the pathology to be treated and the
conditions of the patient.
[0019] The composition of the invention may contain from 0.05% to
5% w/w of N-palmitoyl-vanillamide.
[0020] Experimental Part
EXAMPLE 1
[0021] Preparation of N-palmitoyl-vanillamide (Palvanil)
[0022] 4.475 g of 4-hydroxy-3-methoxybenzylamine hydrochloride and
0.556 g of 4-methylmorpholine are dissolved in 10 ml of
dimethylformamide at 0.degree. C. A solution of 0.605 g of
palmitoyl chloride in 5 ml of chloroform is added dropwise slowly
over 30 min with continuous stirring. The resulting mixture is
stirred overnight at 0.degree. C. and 25 ml of water are then added
and this mixture is extracted 3 times with 10 ml of ethyl
acetate.
[0023] The organic phases are washed twice with 5 ml of 1N
hydrochloric acid and twice with 4 ml of water; the organic phases
are then combined, decolorized with animal charcoal, dried over
anhydrous sodium sulfate and evaporated under vacuum.
[0024] The residue is crystallized from 7 ml of tert-butyl methyl
ether; the product, separated out by filtration, is washed twice
with 3 ml of cold tert-butyl methyl ether and is finally dried
under high vacuum.
[0025] The reaction yield is about 91%.
[0026] The physicochemical properties of the product
N-(4-hydroxy-3-methoxybenzyl)palmitoylamide are as follows:
[0027] Physical state: white crystalline powder
[0028] Empirical formula: C24H41NO3
[0029] Molecular weight: 391.60
[0030] Elemental analysis: C=73.61%; H=10.55%; N=3.58%;
O=12.26%
[0031] Solubility in organic solvents: >10 mg/ml in DMSO; >10
mg/ml in ethanol
[0032] Solubility in water: sparingly soluble
[0033] TLC: 65/30/5 toluene/ethanol/acetic acid eluent;
Rf=0.65.
[0034] Assays of TRPV1-Mediated Elevation of Intracellular
[Ca2+]
[0035] HEK-293 cells stably over-expressing recombinant human TRPV1
were selected by G-418 (Geneticin; 600 .mu.g/ml), grown on 100-mm
diameter Petri dishes as monolayers in minimum essential medium
supplemented with non-essential amino acids, 10% fetal bovine
serum, and 2 mM glutamine, and maintained under 5% CO2 at
37.degree. C. On the day of the experiment, the cells were loaded
for 1 h at 25.degree. C. with the cytoplasmic calcium indicator
Fluo-4AM (Invitrogen) 4 .mu.M in dimethyl sulfoxide containing
0.02% Pluronic F-127 (Invitrogen). After loading, cells were washed
twice in Tyrode's buffer (145 mM NaCl, 2.5 mM KCl, 1.5 mM
CaCl.sub.2, 1.2 mM MgCl.sub.2, 10 mM D-glucose, and 10 mM HEPES, pH
7.4), resuspended in the same buffer, and transferred to a quartz
cuvette of the spectrofluorimeter (excitation .lamda.=488 nm;
emission .lamda.=516 nm) (Perkin-Elmer LS50B) under continuous
stirring. Experiments were carried by measuring cell fluorescence
before and after the addition of various concentrations of the
compounds. The potency of test compounds (EC50 values) were
determined as the concentration of test substances required to
produce half-maximal increases in [Ca.sup.2+]i. The efficacy of the
agonists was determined by comparing their effect with the
analogous effect observed with 4 .mu.M ionomycin. Antagonist
behaviour was evaluated against capsaicin (10 nM) or anandamide 0.5
and 0.25 .mu.M at different pH values by adding the compounds in
the quartz cuvette 5 min before the agonist (capsaicin or
anandamide). Data were expressed as the concentration exerting a
half-maximal inhibition of agonist effect (IC50). The effect on
[Ca.sup.2-]i exerted by the agonist alone was taken as 100%. All
determinations were at least performed in triplicate. Dose response
curves were fitted by a sigmoidal regression with variable slope.
Curve fitting and parameter estimation were performed with GraphPad
Prism (GraphPad Software Inc., San Diego, Calif.).
[0036] Eye-Wiping Assays in Mice
[0037] The pain-inducing potency of Palvanil was determined by the
eye-wiping assay in mice, using a protocol similar to that
previously described in rats (Szallasi A, Blumberg P M.
Neuroscience, 1989; 30: 515-520), and its effect on
capsaicin-induced wiping was also evaluated. Male CD1 mice (weight
25 g) were maintained in a controlled lighting environment and
groups of 6 animals were used for each treatment. The animals
received drugs instillations (10 .mu.l) in the left eye and were
used for one treatment only. The number of the eye-wiping movements
following drugs instillation into the eye was considered as index
of pungency. Moreover, the eye-wiping reflexes in response to
capsaicin (10 .mu.g/ml), alone or after Palvanil or the TRPV1
antagonist 5'-iodo-resiniferatoxin (I-RTX), dropped in solution
into the eye, was determined during 30 sec after the
instillation.
[0038] The animals were treated as follows:
[0039] capsaicin (10 .mu.g/ml)
[0040] capsaicin (10 .mu.g/ml)+Palvanil (10 and 30 .mu.g/ml)
[0041] I-RTX (1 .mu.g/ml)+Palvanil (10 and 30 .mu.g/ml)+capsaicin
(10 .mu.g/ml)
[0042] I-RTX (1 .mu.g/ml)
[0043] The dose of I-RTX was chosen based on pilot experiments and
was the highest dose of the antagonist that did not antagonize the
effect of 10 .mu.g/ml capsaicin per se.
[0044] Results
[0045] Kinetics of TRPV1-Mediated Elevation of Intracellular
[Ca.sup.2+] by Capsaicin and Palvanil
[0046] Capsaicin and Palvanil were added at different
concentrations and produced a dose-dependent increase in
intracellular calcium with a EC50=3.9.+-.0.4 nM and 0.65.+-.0.04 nM
respectively. When we analyzed the time required for the agonists
to achieve 90% of total TRPV1 mediated calcium influx at different
concentrations we noted that the activation of TRPV1 by capsaicin
was always significantly faster than that obtained by Palvanil,
with the greatest difference at 1 .mu.M (FIG. 1A e 1B).
[0047] Effect of Palvanil and Capsaicin on Capsaicin-Induced
TRPV1-Mediated Elevation of Intracellular [Ca.sup.2+]
[0048] We evaluated the desensitizing effect of Palvanil and
capsaicin on TRPV1 mediated intracellular Ca.sup.2+ elevation
induced by 10 nM capsaicin, the two compounds being administered to
cells 5 min before exposure to capsaicin. Palvanil desensitized
TRPV1 to the effect of capsaicin significantly more potently than
capsaicin (IC50 0.81.+-.0.07 and 3.8.+-.0.5 nM, respectively,
P<0.01) (FIG. 2).
[0049] We next evaluated the influence of the pre-incubation time
on the desensitizing effect of the two TRPV1 agonists on 10 nM
capsaicin stimulation of TRPV1-mediated elevation of intracellular
calcium. The effects of 2 nM Palvanil and 10 nM capsaicin
(concentrations which per se produce a similar elevation of
intracellular calcium) were compared. Whilst the desensitizing
effect of Palvanil reached a maximum about with 50 min
pre-incubation, capsaicin produced a significantly less pronounced
desensitizing effect, which tended to increase only with 5 hour
incubation (FIG. 3). Therefore, Palvanil and capsaicin show
different time-dependent desensitizing abilities.
[0050] Effect of Palvanil and Capsaicin on Anandamide/Low
pH-Induced TRPV1-Mediated Elevation of Intracellular
[Ca.sup.2+]
[0051] The effects of Palvanil and capsaicin were evaluated on the
TRPV1-mediated elevation of intracellular Ca.sup.2+ induced by the
endogenous agonist anandamiide at physiological pH (7.4) and at
acid pH (6.5). Dose-response curves show that preincubation of both
Palvanil and capsaicin have strong desensitizing effects on the
activity of 0.5 .mu.M AEA. This effect is more pronounced with
Palvanil as compared to capsaicin at both physiological (IC50
0.9.+-.0.1 nM, and 7.4.+-.0.3 nM for
[0052] Palvanil and capsaicin respectively) and acid (IC50
0.3.+-.0.01 nM, and 3.8.+-.0.06 nM for Palvanil and capsaicin
respectively) pH. At the lower pH both compounds have a greater
inhibitory activity, although Palvanil was 90-fold more potent at
this pH, and capsaicin was only about 2-fold more potent. It is
important to note that at both pH values, Palvanil has a remarkable
inhibitory effect (>50%) at concentrations as low as 1 nM (FIG.
4A). Likewise, when using anandamide at a lower concentration (0.25
mM) the desensitizing effect of Palvanil was again more marked than
that of capsaicin at acid pH (IC50 0.5.+-.0.02 and 8.9.+-.0.3 nM
for Palvanil and capsaicin, respectively) (FIG. 4B).
[0053] Effect of Topical Capsaicin and Palvanil on Eye Wiping in
the Mouse
[0054] The eye wiping test was employed as an in vivo pungency test
in order to assess the pain-producing effects of topical Palvanil
in the mouse. We found that instillations into the eye of Palvanil
solution (10 and 30 g/ml) did not induce any eye-wiping reflexes in
the mouse, indicating the complete lack of pungency for this
compound (FIG. 5). On the contrary, the administration of capsaicin
solution (10 .mu.g/ml) evoked 12.+-.0.9 wiping movements monitored
within 30 seconds. However, Palvanil treatment (10 and 30
.mu.g/ml), performed 1 and 3 hours before capsaicin in the same
eye, significantly reduced the pain behavior to 8.2.+-.0.9 and
6.7.+-.0.7 and to 7.0.+-.0.9 and 6.0.+-.0.8, respectively (FIG. 5).
This effect was completely reverted by pre-treatment with I-RTX (1
.mu.g/ml) which did not cause any reflexes per se.
Examples of Pharmaceutical Formulations
EXAMPLE 1
Cream for Application on Scarred Skin
[0055] 100 g contain:
TABLE-US-00001 Palvanil g 0.50 Vitamin E acetate g 2.00 Sodium
hyaluronate g 0.02 Bronopol g 0.005 Hydrogenated Castor oil g 1.50
Noveon AA1 g 1.60 Water to g 100.00
EXAMPLE 2
Cream for Application on Healthy Skin
[0056] 100 g contain:
TABLE-US-00002 Palvanil g 0.50 PEG-5 plant sterols g 4.50 Stearic
Acid g 3.00 Cetostearylic alcohol g 3.00 Adelmidrol g 0.50 Glyceryl
monostearate g 1.50 Carbopol 940 g 0.40 2,4-dichlorobenzylic
alcohol g 0.15 Bronopol g 0.05 Water to g 100.00
EXAMPLE 3
Fluid Cream for Application on Broad Skin Areas
[0057] 100 g contain:
TABLE-US-00003 Palvanil g 1.00 Glycerine g 5.00 White mineral oil g
3.00 Silicone oil g 1.00 Glyceryl monostearate g 1.40 Cetostearylic
alcohol g 2.80 Stearic acid g 2.40 PEG plant sterols g 5.00
Carbomer g 0.10 Bronopol g 0.005 Water to g 100.00
EXAMPLE 4
Gel for Oral Use
[0058] 100 g contain:
TABLE-US-00004 Palvanil g 1.20 Glycerine g 10.00 Echinacea purpurea
glyc. Extract g 10.00 Sodium alginate g 2.50 Sodium Hyaluronate g
0.02 Triclosan g 0.25 Bronopol g 0.005 Water to g 100.00
EXAMPLE 5
Lotion for Trichological Use
[0059] 100 g contain:
TABLE-US-00005 Palvanil g 0.50 Adelmidrol g 0.20 D-biotine g 0.04
Echinacea pupurea glyc. Extract g 10.00 Ethyl alcohol g 40.00 Water
to g 100.00
EXAMPLE 6
Vaginal Gel
[0060] 100 g contain:
TABLE-US-00006 Palvanil g 0.50 Glycerine g 10.00 Vitamin A
palmitate g 0.20 2-phenylethanol g 0.15 Hydrogenated Castor oil 40
(OE) g 1.00 Methyl-paraoxybenzoate g 0.05 Noveon AA1 g 1.00 Sodium
Hyaluronate g 0.04 Water to g 100.00
EXAMPLE 7
Gel for Balano-Preputial Use
[0061] 100 g contain:
TABLE-US-00007 Palvanil g 0.25 Glycerine g 10.00 Vitamin A
palmitate g 0.20 2-phenylethanol g 0.18 Bronopol g 0.05 Noveon AA1
g 0.80 Sodium Hyaluronate g 0.04 Water to g 100.00
EXAMPLE 8
Drops for Otological Use
[0062] 100 g contain:
TABLE-US-00008 Palvanil g 0.30 Transcutol P g 49.00 Propylene
glycol g 30.00 Deo-Usnate g 0.30 Triclosan g 0.20 Phytosfingosin g
0.15 Trans-traumatic acid g 0.06 Water to g 100.00
EXAMPLE 9
Gel for Rectal Microclysma
[0063] 100 g contain:
TABLE-US-00009 Palvanil g 0.25 Glycerine g 8.00 Trans-traumatic
acid g 0.50 2-phenylethanol g 0.10 Hydrogenated Castor oil 40 (OE)
g 1.00 Methyl-paraoxybenzoate g 0.05 Noveon AA1 g 0.50 Water to g
100.00
EXAMPLE 10
Patch for Prolonged Dermal Application
[0064] 100 cm.sup.2 contain:
TABLE-US-00010 Palvanil mg 20.00 Trans-traumatic acid mg 2.00
Adelmidrol mg 10.00 Gluing vehicle to mg 80.00
EXAMPLE 11
Gel for Periungual Use
[0065] 100 g contain:
TABLE-US-00011 Palvanil g 0.50 Trans-traumatic acid g 0.10 Sodium
alginate g 2.50 Eumulgin L g 1.00 Undecylenic acid g 0.25 Bronopol
g 0.10 Hyaluronic acid g 0.10 Water to g 100.00
EXAMPLE 12
Sterile Eyewash for Corneal Use
[0066] 100 g contain:
TABLE-US-00012 Palvanil g 0.10 Trans-traumatic acid g 0.05
Phosphate buffer 0.1M to g 2.50
EXAMPLE 13
Mouthwash for Oral Use
[0067] 100 g contain:
TABLE-US-00013 Palvanil g 1.00 Adelmidrol g 0.50 Trans-traumatic
acid g 0.05 Glycerine g 7.00 Sodium Pyroglutamate g 3.00
Hydrogenated Castor oil 40 (OE) g 2.00 Noveon AA1 g 0.50 Hyaluronic
acid sodium salt g 0.05 2,4-dichlorobenzylic alcohol g 0.15
Bronopol g 0.10 Water to g 100.00.
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