United States Patent and Trademark Office
| United States Patent Application |
20080175935 |
| Kind Code |
A1 |
| Kern; Dale |
July 24, 2008 |
Method to Treat Skin Conditions with Narcissus Tazetta Bulb
Extract
Abstract
Methods for the prevention or treatment of disorders and complications of disorders resulting from cell damage caused by an aging-related isoform of NADH oxidase (arNOX) are described. The agent for such inhibition comprises processed various Narcissus tazetta extracts, preferably IBR-DORMIN.RTM. Narcissus tazetta bulb extract, both alone and in combination with other inhibition agents, including ubiquinones like coenzyme Q. These agents bind arNOX and inhibit the ability of arNOX to generate reactive oxygen species, thereby decreasing the ability of arNOX to generate reactive oxygen species. Such agents, and their methods of administration, as extremely effective as part of anti-aging treatments.
| Inventors: |
Kern; Dale; (Hyde Park, UT)
|
| Correspondence Name and Address: |
DORSEY & WHITNEY LLP;INTELLECTUAL PROPERTY DEPARTMENT
SUITE 1500, 50 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402-1498
US
|
| Assignee Name and Adress: |
NuSkin International, Inc.
|
| Serial No.: |
019163 |
| Series Code: |
12 |
| Filed: |
January 24, 2008 |
| U.S. Current Class: |
424/773 |
| U.S. Class at Publication: |
424/773 |
| Intern'l Class: |
A61K 36/896 20060101 A61K036/896;
A61P 43/00 20060101 A61P043/00 |
Claims
1-45. (canceled)
46. A method to inhibit the generation of
reactive oxygen species by aging-related isoform of NADH oxidase, comprising the
step of:administering a therapeutically effective amount of a composition
comprising at least one of Narcissus tazetta bulb extract, Schisandra chinensis,
Lonicera japonica, Fagopyrum cymosum, methylparaben, L-carnosine, propylparaben,
ethylparaben, L-ergothioneine, betulinic acid, or Solanum lycopersicum to a
patient in need thereof, such that generation of reactive oxygen species by
aging-related isoform of NADH oxidase, is inhibited.
47. A method to
inhibit the generation of reactive oxygen species by aging-related isoform of
NADH oxidase, comprising the step of:administering a therapeutically effective
amount of a composition comprising Narcissus tazetta bulb extract to a patient
in need thereof, such that generation of reactive oxygen species by
aging-related isoform of NADH oxidase, is inhibited.
48. The method of
claim 47, wherein the composition further comprises a preservative.
49.
The method of claim 47, wherein the total daily amount of Narcissus tazetta bulb
extract administered is between about 0.2% and about 2.0% in a composition.
50. The method of claim 47, wherein the composition further comprises a
ubiquinone.
51. The method of claim 50, wherein the ubiquinone is
coenzyme Q.sub.10.
52. The method of claim 51, wherein coenzyme Q.sub.10
is administered with a ubiquinone wherein the ubiquinone is at least one of
coenzyme Q.sub.5, coenzyme Q.sub.7, coenzyme Q.sub.8, or coenzyme Q.sub.9.
53. The method of claim 47, wherein the composition further comprises at
least one of Schisandra chinensis, Lonicera japonica, Fagopyrum cymosum,
methylparaben, L-carnosine, propylparaben, ethylparaben, L-ergothioneine,
betulinic acid, or Solanum lycopersicum.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application
is a divisional application of, and claims priority to U.S. patent application
Ser. No. 11/049,585, filed Feb. 2, 2005, which claims priority to U.S. Patent
Application Ser. No. 60/541,615, filed Feb. 4, 2004, entitled "Agents for
Inhibiting Serum Aging Factors and Uses Therefor," both hereby incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002]The
invention relates to agents for sequestering serum aging factors, and methods
for using the same. More particularly, the invention relates to agents and
methods for using the same, to prevent or treat disorders and complications of
disorders resulting from cell damage caused by an aging-related isoform of NADH
oxidase (arNOX).
BACKGROUND OF THE INVENTION
[0003]The plasma
membrane NADH oxidase (NOX) is a unique cell surface protein with hydroquinone
(NADH) oxidase and protein disulfide-thiol interchange activities that normally
responds to hormone and growth factors. NOX (or CLOX) are a family of growth
related proteins that are associated with aging cells. A hormone-insensitive and
drug-responsive form of the NOX designated tNOX has been described that is
specific for cancer cells. For example, see U.S. Pat. No. 5,605,810, which is
incorporated herein by reference.
[0004]The aging-related isoform of
NADH oxidase (arNOX) is a member of this family of proteins. The circulating
form of arNOX increases markedly in human sera and in lymphocytes of
individuals, especially after the age of 65. The arNOX is uniquely characterized
by an ability to generate superoxides, which may contribute significantly to
aging-related changes including atherogenesis and other action-at-a-distance
aging phenomena. Activity of arNOX in aging cells and in sera has been described
previously. See, for example, PCT Pub. App. No. WO 00/57871, which is
incorporated by reference in its entirety herein.
[0005]This model is
consistent with the Mitrochondrial Theory of Aging, which holds that during
aging, increased reactive oxygen species in mitochondria cause mutations in the
mitochondrial DNA and damage mitochondrial components, resulting in senescence.
The mitochondrial theory of aging proposes that accumulation of spontaneous
somatic mutations of mitochondrial DNA (mtDNA) leads to errors of mtDNA encoded
polypeptide chains. (Manczak M et al., J Neurochem. 2005 February;
92(3):494-504). These errors, occurring in mtDNA encoded polypeptide chains, are
stochastic and randomly transmitted during mitochondrial division and cell
division. The consequence of these alterations is defective oxidative
phosphorylation. Respiratory chain defects may become associated with increased
oxidative stress amplifying the original damage (Ozawa, 1995, Biochim. Biophys.
Acta 1271:177-189; and Lenaz, 1998, Biochim. Biophys. Acta 1366:53-67). In this
view, therefore, mutated mitochondrial DNA, despite being present only in very
small quantities in the body, may be the major generator of oxidative stress.
[0006]Where accumulation of somatic mutations of mitochondrial DNA
(mtDNA) leads to defective oxidative phosphorylation a plasma membrane
oxido-reductase (PMOR) system has been suggested to augment survival of
mitochondrially deficient cells through regeneration of oxidized pyridine
nucleotide. (de Grey, 1997, BioEssays 19:16 1-166; de Grey, 1998, Anti-Aging
Med. 1:53-66; Yoneda et al, 1995, Biochem. Biophys. Res. Comm, 209:723-729;
Schon et al., 1996, Cellular Aging and Cell Death, Wiley and Sons, New York, pp.
19-34; Ozawa, 1997, Physiol. Rev. 77:425-464; and Lenaz, 1998, BioFactors
8:195-204). A model to link accumulation of lesions in mtDNA to extracellular
responses, such as the oxidation of lipids in low density lipoprotein (LDLs) and
the attendant arterial changes, was first proposed with rho.degree. cells (Larm
et al., 1994, Biol. Chem. 269:30097-30100; Lawen et al., 1994, Mol. Aspects.
Med. 15:s13-s27; de Grey, 1997, BioEssays 19:161-166; and de Grey, 1998,
Anti-Aging Med. 1:53-66). Similar studies have been conducted with transformed
human cells in culture. (Vaillant et al., 1996, Bioenerg. Biomemb. 28:53 1-540).
[0007]Under conditions where plasma membrane oxidoreductase (PMOR) is
overexpressed electrons are transferred from NADH to external acceptors by a
defined electron transport chain, resulting in the generation of reactive oxygen
species (ROS) at the cell surface. Such cell surface-generated ROS may then
propagate an aging cascade originating in mitochondria to both adjacent cells as
well as to circulating blood components such as low density lipoproteins. See
PCT Pub. App. No. WO 00/57871.
[0008]Consequently, there is a need to
find agents that reduce the ability of arNOX to generate reactive oxygen species
(ROS) for the purposes of reducing or treating the resultant physiological
conditions, such as oxidation of lipids in low density lipoprotein (LDLs) and
attendant arterial changes. The arNOX activity of aging cells has been shown to
be inhibited by co-enzyme Q (ubiquinone). See PCT Published Application Numbers
WO 00/57871, WO 01/72318, and WO 01/72319, the disclosures of which are
incorporated herein by reference. However, the use of co-enzyme Q is not
completely satisfactory for several reasons: it is costly, it oxidizes easily
losing its efficacy, and preparations containing coenzyme Q must be specially
packaged to prevent loss of function. Thus, while some agents and methods
currently exist, which may inhibit arNOX activity, challenges still exist.
Accordingly, it would be an improvement in the art to augment or even replace
previously disclosed agents and techniques with the agents and techniques
disclosed in this invention.
[0009]The skin in particular is vulnerable
to damage by reactive oxygen species. The skin is made of several layers, or two
major layers. The stratum corneum, or epidermis, is the top layer and forms a
protective covering for the skin and controls the flow of water and substances
in and out of the skin. The dermis is the lower level of the skin and provides
the strength, elasticity and the thickness to the skin. The main cell type of
the dermis is fibroblasts, which is responsible for synthesis and secretion of
all the dermal matrix components such as collagen, elastin and
glycosaminoglycans. Collagen provides the strength, elastin the elasticity, and
glycosaminoglycans the moistness and plumpness of the skin.
[0010]In
addition to being damaged by reactive oxygen species the skin is subject to
various damaging stressors. The skin may be damaged abused by soaps,
emulsifier-based cosmetics, hot water, organic solvents, dermatological
disorders, environmental abuse (wind, air conditioning, central heating) or
through the normal aging process (chronoaging), which may be accelerated by
exposure of skin various external stressors (e.g. photoaging).
[0011]Anti-aging" cosmetic and medical products, which treat or delay
the visible signs of actual aging and weathered skin such as wrinkles, lines,
sagging, hyperpigmentation and age spots are desirable. Accordingly, there is a
demand for effective natural skin treatments and preventative compositions and
methods for using the same.
SUMMARY OF THE INVENTION
[0012]The
invention relates to agents for sequestering serum aging factors, and methods
for using the same. More particularly, the invention relates to agents and
methods for using the same, to prevent or treat disorders and complications of
disorders resulting from cell damage caused by an aging-related isoform of NADH
oxidase (arNOX). In a preferred embodiment the agents of the invention comprise
at least one processed Narcissus tazetta product.
[0013]The invention
described herein encompasses pharmaceutical compositions, pharmaceutical kits
and methods for the prevention or treatment of disorders and complications of
disorders resulting from cell damage caused by an aging-related isoform of NADH
oxidase (arNOX). The agent for such inhibition in some embodiments of the
invention comprise ingredients extracted from various plant species. One
embodiment comprises the use of a processed Narcissus tazetta product. A
preferred embodiment of the processed Narcissus tazetta product is
IBR-DORMIN.RTM. Narcissus tazetta bulb extract, which comprises Narcissus
tazetta extract. Another embodiment comprises the use of the processed Narcissus
tazetta product, both alone and in combination with other inhibition agents,
including ubiquinones like coenzyme Q, extracts of Schisandra chinensis,
extracts of Lonicera japonica, and or extract of Fagopyrum cymosum. Extracts
from each of the foregoing may be utilized individually or in combination with
other active and inactive ingredients.
[0014]The agents of this
invention may bind arNOX and inhibit, or otherwise decrease, the ability of
arNOX to generate reactive oxygen species. The inhibition of arNOX results in a
decrease in the generation of reactive oxygen species by arNOX. A decrease in
reactive oxygen species results in a decrease of oxidative damage resulting from
said reactive oxygen species. Such agents, and their methods of administration,
are an effective part of anti-aging treatments. Thus, one embodiment of the
invention described herein encompasses methods of preventing or treating
disorders caused by oxidative damage by an aging-specific isoform of NADH
oxidase (arNOX).
[0015]The invention described herein further
encompasses methods for detecting cell membrane associated arNOX and soluble
arNOX in sera. Further, the invention encompasses methods of assaying,
screening, and identifying agents that inhibit arNOX, as well as methods using
agents comprising processed Narcissus tazetta products, preferably
IBR-DORMIN.RTM. Narcissus tazetta bulb extract, in combination with ubiquinone
to inhibit the ability of arNOX to generate reactive oxygen species. These
agents may be formulated into pharmaceutical compositions in the prevention and
treatment of disorders caused by oxidative damage. The invention described
herein further encompasses properties of agents comprising at least one
processed Narcissus tazetta extract. The invention discloses the isolation and
characterization of arNOX using agents comprising at least one processed
Narcissus tazetta extract. Additional information about agents comprising at
least one processed Narcissus tazetta extract, including IBR-DORMIN.RTM.
Narcissus tazetta bulb extract can be found in U.S. Pat. Nos. 6,635,287 and
6,342,254, the disclosure of which is also incorporated herein by reference.
[0016]The pharmaceutical compositions of this invention may comprise
varying modes of administration. The modes of administration of compounds
comprise capsules, tablets, soft gels, solutions, suppositories, injections,
aerosols, or a kit.
[0017]The present invention provides compositions
comprising active agent(s), which prevent and/or ameliorates skin damage and
associated conditions. Further, the invention encompasses methods for utilizing
said compositions.
[0018]A preferred embodiment of the invention
provides active agents from processed plants for the treatment of skin. The
active agents prevent and/or ameliorate skin damage and associated conditions.
In one embodiment of the invention the processed plant products sequester arNOX
activity. In another embodiment of the invention, the processed plant products
inhibit radical oxygen species. In another embodiment agents and methods of the
invention prevent and/or improve the health of the skin. For example, the agents
may improve skin tone, and helps diminish the appearance of fine lines and
visible signs of aging. In another embodiment of the invention, the agents
positively affects the body's natural production of collagen and elastin. In
another embodiment, the agents of the invention minimize the effects of
environmental agitators such as pollution, sun, free radicals and stress.
[0019]These and other features and advantages of the present invention
will be set forth or will become more fully apparent in the description that
follows and in the appended claims. The features and advantages may be realized
and obtained by means of the instruments and combinations particularly pointed
out in the appended claims. Furthermore, the features and advantages of the
invention may be learned by the practice of the invention or will be obvious
from the description, as set forth hereinafter.
BRIEF DESCRIPTION OF THE
DRAWINGS
[0020]In order that the manner in which the above recited and
other features and advantages of the present invention are obtained, a more
particular description of the invention will be rendered by reference to
specific embodiments thereof, which are illustrated in the appended drawings.
Understanding that the drawings depict only typical embodiments of the present
invention and are not, therefore, to be considered as limiting the scope of the
invention, the present invention will be described and explained with additional
specificity and detail through the use of the accompanying drawings.
[0021]FIG. 1 illustrates periodic variation in the rate of
ferricytochrome c reduction. Ferricytochrome c reduction over 90 min showed four
sets of maxima (arrows indicate a 24.7 min period) for sera of a 100 y female.
The activity with the period length of 24.7 min is much reduced or absent from
sera of young individuals. Each maximum was resolved into a doublet pattern
indicated by double and single arrows. The doublet pattern was reproduced with
three additional serum samples, an 80 y male, an 83 y male and a 98 y female.
[0022]FIG. 2 illustrates rate of ferricytochrome c reduction by buffy
coats and sera of old and younger individuals. Buffy coat fractions (A-D) and
sera (E-H) pooled from 2040 y (A,B,E,F) and 70-100 y (C,D,G,J,H) individuals
were compared. Rates were monitored continuously at intervals of 1.5 min using a
SLM Aminco. DW-2000 spectrophotometer in the dual wavelength mode of operation
from the increase in absorbance at 550 nm with 540 nm as reference. Maxima
separated by ca 25 min are indicated by the single arrows (C,D,G,H). After 45
min of measurement, superoxide dismutase (SOD, 60 .mu.l/60 units) or coenzyme Q
in ethanol (30 .mu.l/450 .mu.g) were added at the curved arrows and the
measurements were continued for a total of 120 min. The oscillating activity was
unique to buffy coats and sera of the aged individuals and was reduced to basal
levels by the addition of either superoxide dismutase (SOD) or coenzyme Q.
[0023]FIG. 3 illustrates superoxide dismutase (SOD) inhibition of
age-related ferricytochrome c reduction. SOD was added to the reaction mixture
at the mid-point of the assay. The rates were determined before (solid symbols,
solid lines) and after (open symbols, dashed lines) the addition of SOD. Sera
were from old (80 to 100 y, circles) or young (20 to 40 y, triangles) subjects.
Results are means of 5 to 10 samples.+-.standard deviations. The lack of
complete inhibition is explained by the observation that the oscillating
age-related oxidase accounts for only about 30% of the total apparent activity
even with sera of these very old individuals. The oscillating activity was
completely inhibited by SOD (FIG. 2G).
[0024]FIG. 4 illustrates coenzyme
Q inhibition of aging-related ferricytochrome c reduction. As in FIG. 2 except
that the indicated amounts of coenzyme Q were added instead of SOD. Rates were
determined before solid symbols, solid lines and after open symbols dashed lines
coenzyme Q addition. Sera were from old (80 to 100 y, circles) or young (20 to
40 y, triangles) subjects. The oscillating activity is largely blocked by
coenzyme Q addition (FIG. 2H).
[0025]FIG. 5 illustrates rates of
NADH-cytochrome c reductase activity of pig liver microsomes. When determined
for 1 min every 1.5 min over a total of 90 min, the mean rate was 1.2.+-.0.6
.mu.moles/min/mg protein without any indications of repeating oscillatory
patterns.
[0026]FIG. 6 illustrates a Western blot of aging-related NOX
protein from sera. This Western blot comparing proteinase K digested pooled sera
from young individuals (Lane 1, .ltoreq.35 y females; Lane 2, .ltoreq.25 y
males; Lane 6, 36-45 y females; Lane 7, 36-45 y male) and aged individuals (Lane
3, .gtoreq.90 y females; Lane 4, 75-85 y males; Lane 5, 75-85 y females). A
protein band at .about.22 kD Lanes 3-5 arrow) was elevated in sera of aged
individuals. Detection was by polyclonal peptide antisera generated against the
C terminal adenine nucleotide binding region (H-KQEMTGVAGASLEKRWK-OH) of human
tNOX.
[0027]FIG. 7 illustrates the response of periodic superoxide
generation by arNOX of aged transfusion buffy coats to inhibition by
IBR-DORMIN.RTM. Narcissus tazetta bulb extract (upper figure) and lack of
inhibition by the product Pilinhib (lower figure). The solid arrows show
activity maxima with a period length of ca. 25 min. the preparation with
Pilinhib showed two sets of maxims neither of which was inhibited. The reaction
s were for 45 min without inhibitor. Inhibitor was added at the large open
arrows and the reaction continued for another 45 min in the presence of
inhibitor.
[0028]FIG. 8 illustrates 2-pyridyidithio substrates
generating two moles of pyridinethionine per mole of substrate will provide a
direct measure of protein disulfidethiol interchange activity.
[0029]FIG. 9 illustrates the total scoring parameter for each patient at
each follow-up visit related to the application of vehicle cream applied to the
right elbow of each patient.
[0030]FIG. 10 illustrates the total scoring
parameter for each patient at each follow up visit related to the application of
cream comprised of a processed Narcissus tazetta extract to the left elbow of
each patient.
[0031]FIG. 11 illustrates the average percent reduction of
scoring parameters for each elbow for each follow up visit.
[0032]FIGS.
12A-C depict graphically statistical data related to sensory analysis of several
concentrations of cosmetic cream, which comprise a processed Narcissus tazetta
extract. In particular 12A depicts perceived resistance against external
aggressions, 12B depicts skin sensitivity and 12C depicts skin protection when a
placebo, 1% by weight processed Narcissus tazetta extract in cosmetic cream and
3% by weight processed Narcissus tazetta extract in cosmetic cream were applied
to test subjects.
[0033]FIGS. 13A-C illustrate depict graphically
statistical data related to sensory analysis of several concentrations of
cosmetic cream, comprising a processed Narcissus tazetta extract. In particular
13A depicts skin irritability, 13B depicts skin fatigue and 13C depicts skin
tautness when a placebo, 1% by weight processed Narcissus tazetta extract in
cosmetic cream and 3% by weight processed Narcissus tazetta extract in cosmetic
cream were applied to test subjects.
[0034]FIGS. 14A-C illustrate depict
graphically statistical data related to sensory analysis of several
concentrations of cosmetic cream, which comprise a processed Narcissus tazetta
extract. In particular 14A depicts skin comfort, 14B depicts the appearance of
little lines on the skin and 14C depicts skin suppleness when a placebo, 1% by
weight processed Narcissus tazetta extract in cosmetic cream and 3% by weight
processed Narcissus tazetta extract in cosmetic cream were applied to test
subjects.
[0035]FIG. 15 illustrates the percent evolution of qualitative
sensory analysis for several categories of after applying a placebo, 1% by
weight processed Narcissus tazetta extract in cosmetic cream and 3% by weight
processed Narcissus tazetta extract in cosmetic cream four weeks related to a
group of patients.
DETAILED DESCRIPTION OF THE INVENTION
[0036]The invention relates to agents for sequestering serum aging
factors, and methods for using the same. More particularly, the invention
relates to agents and methods for using the same, to prevent or treat disorders
and complications of disorders resulting from cell damage caused by an
aging-related isoform of NADH oxidase (arNOX). In a preferred embodiment the
agents of the invention comprise at least one processed Narcissus tazetta
product. One embodiment of the invention comprises agents that bind arNOX and
inhibit the ability of arNOX to generate reactive oxygen species as well as
methods of using these agents to inhibit the ability of arNOX to generate
reactive oxygen species.
[0037]The invention provides pharmaceutical
compositions, methods of use, and pharmaceutical kits for the treatment of
disorders resulting from oxidative changes in cells that result in aging by
targeting an aging-related isoform of NADH oxidase (arNOX), shed into the sera
by aging cells. The compositions may contain agents extracted from plants. For
example the compositions of the invention may comprise at least one processed
Narcissus tazetta product, whether alone or with other inhibition agents and
inhibit the activity of an aging-related isoform of NADH oxidase shed into the
sera by aging cells, wherein the other inhibition agents may comprise
ubiquinones, extracts of Schisandra chinensis, or Lonicera japonica, or extracts
of Fagopyrum cymosum, In a preferred embodiment the processed Narcissus tazetta
extract is IBR-DORMIN.RTM. Narcissus tazetta bulb extract. IBR-DORMIN.RTM.
Narcissus tazetta bulb extract is available from Israeli Biotechnology Research
Ltd. Corporation, Tel Aviv, Israel.
[0038]As used herein, the term
"disorder" refers to any condition of a living animal or plant body or of one of
its parts that impairs normal functioning comprising any ailment, disease,
illness, clinical condition, pathological condition, weakened condition, unsound
condition, and any abnormal or undesirable physical condition.
[0039]As
used herein, the term "reactive oxygen species" refers to oxygen derivatives
from oxygen metabolism or the transfer of free electrons, resulting in the
formation of free radicals (e.g., superoxides or hydroxyl radicals).
[0040]As used herein, the term "antioxidant" refers to compounds that
neutralize the activity of reactive oxygen species or inhibit the cellular
damage done by said reactive species.
[0041]As used herein, the term
"pharmaceutically acceptable carrier" refers to a carrier medium that does not
interfere with the effectiveness of the biological activity of the active
ingredient, is chemically inert, and is not toxic to the patient to whom it is
administered.
[0042]As used herein, the term "pharmaceutically
acceptable derivative" refers to any homolog, analog, or fragment corresponding
to the formulations described in this application, which exhibit antioxidant
activity, and is relatively non-toxic to the subject.
[0043]The term
"therapeutic agent" refers to any molecule, compound, or treatment, preferably
an antioxidant, which assists in the prevention or treatment of the disorders,
or complications of disorders caused by reactive oxygen species.
[0044]The term "agent that sequesters arNOX" refers to any molecule,
compound, or treatment that interacts with arNOX, thus decreasing the reaction
of arNOX with other substrates and inhibits the ability of arNOX to generate
reactive oxygen species.
[0045]The antioxidants, cellular components,
and target proteins defined herein are abbreviated as follows:
TABLE-US-00001 mitochondrial DNA mtDNA nicotinamide adenine dinucleotide
NADH cell surface hydroquinone (NADH) oxidase with NOX protein disulfide-thiol
isomerase activity NOX specific to non-cancer cells CNOX NOX specific to aged
cells AR-NOX NOX specific to cancer cells tNOX low density lipoproteins LDLs
plasma membrane oxido-reductase chain PMOR ubiquinone or coenzyme Q CoQ coenzyme
Q.sub.10 CoQ.sub.10 reactive oxygen species ROS
[0046]The following
disclosure of the present invention is grouped into subheadings. The utilization
of the subheadings is for convenience of the reader only and is not to be
construed as limiting in any sense.
1. Plasma Membrane Hydroquinone
(NADH) Oxidase (NOX)
[0047]The plasma membrane NADH oxidase (NOX) is a
unique cell surface protein with hydroquinone (NADH) oxidase and protein
disulfide-thiol interchange activities that normally responds to hormone- and
growth factors. A hormone insensitive and drug-responsive form of the activity
designated tNOX also has been described, which is specific for cancer cells.
Evidence exists that NOX proteins, under certain conditions, are capable of the
production of ROS. For example, ultraviolet light as a source of oxidative
stress in cultured cells is used to initiate superoxide generation (Morre et
al., 1999, Biofactors 9:179-187) (See U.S. Pat. No. 5,605,810, which is
incorporated by reference in its entirety).
2. Plasma Membrane Levels of
Coenzyme Q
[0048]Plasma membrane ubiquinone or coenzyme Q (CoQ) plays a
major role in the PMOR system. Ubiquinone or coenzyme Q (CoQ) occurs
ubiquitously among tissues. The ubiquinone content of plasma membrane is two to
five times that of microsomes and only half that of mitochondria. Ubiquinone has
long been considered to have both pro and antioxidant roles over and above its
more conventional role in mediating electron transport between NADH and succinic
dehydrogenase and the cytochrome system of mitochondria (Emster and Daliner,
1995, Biochim. Biophys. Acta 127:195-204; and Crane and Barr, 1985, Coenzyme Q,
John Wiley & Sons, Chichester 1-37).
[0049]CoQ is normally a product
of cellular biosynthesis and provides a potentially important source of
one-electron pro-oxidant oxygen reduction (Anderson et al., 1994, Biochim.
Biophys. Acta 1214:79-87; Appelkvist et al., 1994, Molec. Aspects Med.
15S:37-46). In its reduced hydroquinone form (ubiquinol), it is a powerful
antioxidant acting directly upon either superoxide or indirectly on lipid
radicals alone or together with vitamin E (a-tocopherol) (Crane and Barr, 1985,
Coenzyme Q, Jolm Wiley & Sons, Chichester, pp. 1-37; Beyer and Emster, 1990,
Highlights of Ubiquinone Research, Taylor & Francis, London, pp. 191-213;
Beyer, 1994, J. Bioenerg. Biomemb. 26:349-358; Kagan et al., 1990, Biochem.
Biophys. Res. Comm. 169:851-857; and Emster et al., 1992, BioFactors 3:241-248).
[0050]The antioxidant action of ubiquinol normally yields the
ubisemiquinone radical. The latter is converted back to ubiquinol by
re-reduction through the electron transfer chain in mitochondria, or by various
quinone reductases in various cellular compartments including the plasma
membrane (Takahashi et al., 1995, Biochem. J. 309:883-890; Takahashi et al.,
1996, J. Biochem. (Tokyo) 119:256-263; Beyer et al., 1996, Proc. Natl. Acad.
Sci. U.S.A. 93:2528-2532; Beyer et al., 1997, Molec. Aspects Med. 18:slS-s23;
Navarro et al., 1995, Biochem. Biophys. Res. Comm. 2 12:138-143; Villalba et
al., 1995, Molec. Aspects Med. 18:s7-s13; and Arroyo et al., 1998, Protoplasma
205:107-113). Thus, ubiquinone may transform from a beneficial one-electron
carrier to a superoxide generator if the ubisemiquinone anion becomes protonated
(Nohl et al., 1996, Free Rad. Biol. Med. 20:207-15 213).
[0051]Exogenous
CoQ addition may prevent ROS production and concomitantly protect cells from
oxidative damage. For example, exogenous CoQ affects NOX-mediated ROS
production. (Valls et al., 1994, Biochem. Mol. Biol. Tnt. 33:633-642; Beyer et
al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2528-2532; and PCT Pub. App. No. WO
00/5787). The antioxidant effect at the plasma membrane may ameliorate LDL
oxidation by scavenging ROS by PMOR produced at the cell surface (Thomas et al.,
1997, Molec. Aspects Med. 8:s85-s 103).
[0052]Some studies have shown
that overall CoQ levels decrease with age (Beyer et al., 1985, Mech. Aging Dev.
32:267-281; Kalen et al., 1990, Lipids 25:93-99; and Genova et al., 1995,
Biochem. J. 311:105-109). However, this is not true for all tissues and
especially for the brain, where high CoQ levels are maintained throughout aging
(Soderberg et al., 1990, J. Neurochem. 54:415-423 and Battino et al., 1995,
Mech. Aging Dev. 78:173-187). Thus, the invention also encompasses particular
therapeutic levels of coenzyme Q for inhibiting or reducing the effects caused
by overactive or aberrant cell surface PMOR system and for sequestering NOX
isoforms.
3. Isolation and Characterization of arNOX
[0053]The
invention encompasses research related to arNOX, an aging isoform of the cell
surface NADH oxidase, which is capable of oxidizing reduced quinones. The NOX
protein is anchored in the outer leaflet of the plasma membrane (Morre, 1995,
Biochem. Biophys. Acta. 1240:201-208; and DeHahn et al., 1997, Biochem. Biophys.
Acta. 1328:99-108). NOX activity was shown to be shed in soluble form from the
cell surface (Morre et al., 1996, Biochim. Biophys. Acta 1280:197-206). The
presence of the shed form in the circulation provides an opportunity to use
patient sera as a source of the NOX protein for isolation and characterization
studies. A serum form of the CNOX activity specific to sera from elderly
subjects (arNOX) has been identified. (PCT Pub. App. No. WO 00/57871).
[0054]The invention is based on the identification of arNOX, which is a
constitutive cell surface NADH oxidase protein (CNOX) capable of oxidizing
reduced quinones. The NOX proteins have been postulated to link the accumulation
of lesions in mitochondrial DNA to cell surface accumulations of reactive oxygen
species as one consequence of its role as a terminal oxidase in a plasma
membrane electron transport chain (Morre, D. M. et al., 2000, J. Expl Biol
203:1513-1521). Cells with functionally deficient mitochondria become
characterized by an anaerobic metabolism. NADH accumulated from the glycolytic
production of ATP and an elevated plasma membrane electron transport activity
become necessary to maintain the NAD+/NADH homeostasis essential for survival.
Previous findings demonstrate that the hyperactivity of the plasma membrane
electron transport system results in an NADH oxidase activity capable of cell
surface generation of reactive oxygen species (Morre, D. J. et al., 1999
BioFactors 9:179-187). This would serve to propagate the aging cascade both to
adjacent cells and to oxidize circulating lipoproteins.
[0055]ArNOX has
a superoxide-generating and aging-related enzymatic activity, which is
substantially reduced by addition of coenzyme Q and processed Narcissus tazetta
products. A feature of the aging isoform of the NOX protein is that the
generation of superoxide by this protein associated with aging is inhibited both
by processed Narcissus tazetta products and by coenzyme Q. These findings
provide a rational basis for the antiaging activity of processed Narcissus
tazetta products with skin and by circulating coenzyme Q in the prevention of
atherosclerosis, and other oxidative changes in cell membranes and circulating
lipoproteins. Thus, one embodiment of the invention encompasses the findings
that arNOX provides a molecular target for processed Narcissus tazetta products
and ubiquinones (coenzyme Q) to offer protection to maintain skin vitality as
well as ablate cardiovascular changes associated with cellular aging. Another
embodiment of the invention prevents programmed cellular death, apoptosis, by
utilizing agents, which sequester, neutralize, bind, or otherwise block or
eliminate, the arNOX protein and inhibit its ability to generate reactive oxygen
species.
[0056]Generally, the characteristics of aged cells includes
those that express and/or shed arNOX, and include, but are not limited to, those
exhibiting one or more of the following characteristics: an age-related PMOR
system, the ability to generate reactive oxygen species, and have functionally
defective mitochondria. One embodiment of the invention is the utilization of
agents to reduce the negative effects of aging cells.
[0057]Another
embodiment of the invention is directed to utilizing agents, which switch the
NOX protein from oxygen reduction to protein disulfide reduction. For example
drugs or supplements may be utilized as agents. The advantage of such an
approach has already been observed with plant cells in response to auxins (Chueh
et al., 1997, Biol. Chem. 272:11221-1227).
[0058]NOX-specific polyclonal
antibody to the arNOX protein from lymphocytes have been produced. Once the
amino acid sequence of arNOX is deduced from the corresponding cDNA sequence,
the amino acid sequence may be used to strategically generate peptide sera with
therapeutic potential as probes specific to arNOX to investigate and ameliorate
NOX responses to aging. Using methods, which are well known to those skilled in
the art, recombinant cDNA libraries may be constructed using RNA prepared from
cells known to express arNOX. See, for example, the techniques described in
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, N.Y.; and Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Intersciences, N.Y. Alternatively, a human cDNA
library may be obtained from a commercial source, e.g., Stratagene. The
recombinant cDNA libraries may be screened using a number of different
techniques, which are well known to those skilled in the art.
[0059]In
yet another embodiment of the invention, a cDNA library may be engineered into a
mammalian expression vector and screened by transfection into the appropriate
mammalian cell line followed by assaying for arNOX activity in the tissue
culture supernatant.
[0060]In yet another embodiment of the invention, a
method for cloning arNOX by means of polymerase chain reaction may be used to
clone a cDNA coding for arNOX. Such a method may be utilized using RNA prepared
from lymphocytes of aged individuals.
[0061]Alternatively, arNOX may be
cloned by polymerase chain reaction (PCR) amplification of a human cDNA library
obtained from a commercial source (e.g., Stratagene). In addition, gene
expression assays using gene expression arrays or microarrays are now
practicable for identifying changes in gene expression patterns between
different cells or tissue types (see, e.g., Schena et al., 1995, Science
270:467-470: Lockhart et al., 1996, Nature Biotechnology 14:1674-1680; and
Blanchard et al., 1996, Nature Biotechnology 14:1649). Thus, in another
alternative embodiment of the invention, such gene expression arrays or
microarrays may be used to compare mRNA expression patterns in cells that
exhibit arNOX activity (e.g., as determined by one of the assays of the present
invention) to mRNA expression patterns in cells that do not exhibit arNOX
activity and thus, do not express arNOX.
4. Methods of Detecting arNOX
[0062]The invention encompasses methods for detecting cell-membrane
associated arNOX and soluble arNOX in sera. See, e.g., PCT Pub. App. No. WO
00/57871, which is incorporated by reference in its entirety. The invention
further contemplates using arNOX as a diagnostic tool when oxidative damage to
cells and/or tissue is suspected. As such, arNOX in tissue, cells, or
circulation may be detected. Embodiments include: detection by employing
antibodies specific to arNOX, which may be conjugated to a wide variety of
labels, wherein the label provides a detectable signal. For example
radioisotopes, enzymes, fluorescence and the like may be utilized as labels.
Examples of detection techniques comprise: detection based upon assays that
recognize that sera with arNOX exhibits a higher rate of cytochrome c reduction
than sera without arNOX; an assay which measures the disappearance of the
ascorbate radical spectrophotometrically by measuring the absorbance at about
265 nm since arNOX reduces an electron acceptor, e.g. ascorbate radical; by
measuring the reduction of NADH by arNOX using methods known in the art; assays
based on the unique oscillation property of arNOX; arNOX may be detected by
resistance to retinoic acid, since NOX from healthy cells is inhibited by
retinoic acid and arNOX is not inhibited by retinoic acid; a method using arNOX
to identify cells where mitochondrial functions are depressed and consequently,
PMOR is overexpressed; and cells may be identified in the presence of
overexpressed arNOX (Morre, 1998, Plasma Membrane Redox Systems and their Role
in Biological Stress and Disease 121-156; Morre et al., 1999, Mol. Cell.
Biochem. 200:7-13, wherein each of the referenced documents is incorporated by
reference in its entirety).
5. Methods of Identifying Agents that
Interact with arNOX
[0063]The present invention relates to in vitro and
in vivo methods for screening for agents which target arNOX. Within the broad
category of in vitro selection methods, several types of methods are likely to
be particularly convenient and/or useful for screening test agents comprising:
methods which measure a binding interaction between two or more components; and
methods which measure the activity of an enzyme which is one of the interacting
components, i.e., arNOX. See, for example, the description in Pub. App. No. WO
00/57871, the disclosure of which is incorporated herein by reference.
[0064]Binding interactions between two or more components can be
measured in a variety of ways known in the art. One approach is to label one of
the components with an easily detectable label, place it together with the other
component(s) in conditions under which they would normally interact (e.g.,
ubiquinone), perform a separation step which separates bound labeled component
from unbound labeled component, and then measure the amount of bound component.
The test agent may be labeled with a various detectable markers, and the he
separation step in this type of approach can be accomplished in various ways.
See, for example, Pub. App. No. WO 00/57871.
[0065]The invention also
comprises in vitro selection method which may be used is the screening of
combinatorial chemistry libraries using ubiquinone, ubiquinone derivatives,
plant extracts, dormin, IBR-DORMIN.RTM. Narcissus tazetta bulb extract, or
processed Narcissus tazetta products as a base molecule (U.S. Pat. No.
5,565,324, which is incorporated by reference in its entirety), in vivo
screening methods, gene therapy approaches (U.S. Pat. No. 5,093,246, which is
incorporated by reference in its entirety) and yeast two-hybrid assays to
identify test agents that interact with arNOX (Fields and Song, 1989, Nature
340:245-246, which is incorporated by reference in its entirety). The invention
further encompasses methods for monitoring patient response to the agents
described in this invention.
6. Target Disorders
[0066]Disorders
that can be treated by the methods of the present invention include any clinical
condition in which oxidative species have been implicated. Examples of clinical
conditions in which oxidative species have been implicated include, but are not
limited to ischemia-reperfusion injury (e.g., stroke/myocardial infarction and
organ transplantation), cancer, aging, arthritis associated with age, fatigue
associated with age, alcoholism, red blood cell defects (e.g., favism, malaria,
sickle cell anemia, Fanconi's anemia, and protoporphyrin photo-oxidation), iron
overload (e.g., nutritional deficiencies, Kwashiorkor, thalassemia, dietary iron
overload, idiopathic hemochromatosis), kidney (e.g. metal ion-mediated
nephrotoxicity, aminoglycoside nephrotoxicity, and autoimmune nephrotic
syndromes), gastrointestinal tract (e.g., oral iron poisoning, endotoxin liver
injury, free fatty acid-induced pancreatitis, nonsteroidal anti-inflammatory
drug induced gastrointestinal tract lesions, and diabetogenic actions of
alloxan), inflammatory-immune injury (e.g., rheumatoid arthritis,
glomerulonephritis, autoimmune diseases, vasculitis, and hepatitis B virus),
brain (e.g., Parkinson's disease, neurotoxins, allergic encephalomyclitis,
potentiation of traumatic injury, hypertensive cerebrovascular injury, and
vitamin E deficiency), heart and cardiovascular system (e.g, atherosclerosis,
adriamycin cardiotoxicity, Keshan disease (selenium deficiency) and alcohol
cardiomyopathy, eye (e.g, photic retinopathy, occular hemorrhage,
cataractogenesis, and degenerative retinal damage), amyotrophic lateral
sclerosis, and age-related macular degeneration (Slater, 1989, Free Rad. Res.
Comm. 7:119-390; Deng et al., 1993, Science 261:1047-1051; Seddon et al., 1994,
JAMA 272:1413-1420; Brown, 1995, Cell 80:687-692; and Jenner, 1991, Acta Neurol.
Scand. 84:6-15).
[0067]The invention is also directed to preventing or
alleviating complications of diabetes, atherogenesis, atherosclerosis, and
related diseases. Oxidative stress and LDL oxidation are common complicating
features in diabetics and circulating AR-NOX offers opportunities for redox
modulation of blood constituents important to aging, atherogenesis, and
atherosclerosis (Kennedy and Lyons, 1998, Metabolism 56;14-21).
[0068]In
one embodiment, the invention is directed towards a method of preventing a
complication of a primary disorder in patients wherein said complication results
from oxidative damage resulting from the generation of reactive oxygen species
by arNOX. The method comprises administering to a patient with a primary
disorder, in an amount effective to prevent said complication, an agent or
agents that sequesters arNOX, in a pharmaceutically acceptable carrier.
[0069]In another embodiment, the invention is directed towards a method
of preventing a secondary disorder in patients having a primary disorder that
causes oxidative damage resulting from the generation of reactive oxygen species
by arNOX. The method comprises administering to a patient having a primary
disorder, in an amount effective to prevent said secondary disorder, an agent or
agents that sequesters arNOX, in a pharmaceutically acceptable carrier.
[0070]One embodiment of the invention provides agents and method of
using said agents to ameliorate and prevent dermatological disorders comprising:
Acne Vulgaris, Adiposis Dolorosa, Albinism, Alopecia, alpha 1-Antitrypsin
Deficiency, Baldness, Behcet's Syndrome, Birthmarks, Birt-Hogg-Dube Syndrome
(not on MeSH), Blister, Cafe-au-Lait Spots, Cellulitis, Cholesteatoma,
Connective Tissue Diseases, Contractural Arachnodactyly, Congenital (Beal's
Syndrome) (not on MeSH), Cutis Laxa, Decubitus Ulcer, Dercum Disease,
Dermatitis, Dermatitis Exfoliative, Dermatitis Herpetiformis, Ectodermal
Dysplasia, Eczema, Ehlers-Danlos Syndrome, Epidermolysis Bullosa, Erysipelas,
Erythema Multiforme, Exanthema Subitum, Furunculosis, Granuloma Annulare,
Gustatory Sweating, Hailey-Hailey Disease, Hair Diseases, Hair Loss, Head Lice,
Hidradenitis Suppurativa, Hirsutism, Hives, Hypohidrosis, Ichthyosis, Immersion
Foot, Incontinentia Pigmenti, Keloid, Keratosis Actinic (not on MeSH), Keratosis
Follicularis, Keratosis Seborrheic, Leg Ulcer, Lentigo, Lichen Planus, Lichen
Sclerosus et Atrophicus, Lipodystrophy, Lupus, Lupus Erythematosus Cutaneous,
Lupus Erythematosus Systemic, Marfan Syndrome, Mastocytosis, Melanoma,
Melanosis, Mixed Connective Tissue Disease, Nail Patella Syndrome, Nail
Diseases, Nails Ingrown, Panniculitis, Parapsoriasis, Paronychia, Pemphigoid
Bullous, Pemphigus, Pemphigus Benign Familial, Photosensitivity Disorders,
Pigmentation Disorders, Pityriasis, Poison Ivy, Port-Wine Stain, Pruritus,
Pseudoxanthoma Elasticum, Psoriasis, Pyoderma Gangrenosum, Rosacea, Scabies,
Scleroderma, Scleroderma Systemic, Seborrheic Dermatitis, Skin Cancer, Skin and
Connective Tissue Diseases, Skin Diseases, Skin Diseases Infectious, Skin Ulcer,
Stevens-Johnson Syndrome, Stickler Syndrome (not on MeSH), Sweat Gland Diseases,
Sweet's Syndrome, Swimmer's Itch, Tinea Versicolor, Urticaria, Vitiligo, Warts,
Xanthomatosis, Xeroderma Pigmentosum
7. Processed Narcissus tazetta
Products
[0071]One embodiment of the invention comprises treating
patients with pharmaceutically active amount of processed Narcissus tazetta
products. A preferred embodiment of the processed product is IBR-DORMIN.RTM.
Narcissus tazetta bulb extract. IBR-DORMIN.RTM. Narcissus tazetta bulb extract
is comprised of a water extract of Narcissus tazetta bulbs, and therefore
soluble in water. The extraction process, such as extraction, precipitation and
filtration eliminates some of the bulb material as well as part of the water.
IBR-DORMIN.RTM. Narcissus tazetta bulb extract preferably is comprised of:
water, at least one Narcissus tazetta extract and at least one preservative.
Phenochem, a blend of paraben esters and phenoxyethanol, is an example of a
preferred preservative. Narcissus tazetta extracts may be present in various
amounts in agents used to treat mammals. For example processed Narcissus tazetta
products may be present in amounts measured by percentage of total volume:
between 25-49.9%, between 10-24.9%, between 5-9.9%, between 1-4.9%, between
0.1-0.99%, and less than 0.1%. Additional information about IBR-DORMIN.RTM.
Narcissus tazetta bulb extract can be found in U.S. Pat. Nos. 6,635,287 and
6,342,254 the disclosure of which is also incorporated herein by reference.
[0072]Narcissus tazetta bulb extract can be prepared by the procedure
described in U.S. Pat. No. 6,342,254. The bulbs are induced into dormancy if not
already in a dormant state by subjecting them to hot water having a temperature
of 45.degree. C. for 2-4 hours. The bulbs can be either used immediately for the
preparation of water soluble extracts or, alternatively, maintained in a dry
room at a temperature of 30.degree. C. The dormant bulbs are then disinfected in
soap water for a period of 1 hour. The bulbs are then cut and homogenized in
distilled water (30 sec..times.3) using a Homogenizer Ultra-Turbo-turax. The
homogenized preparation of the bulbs is then filtrated through a first 0.45 m.
sterile filter and then through a second 0.22 .mu.m filter and the preparation
which was not maintained on the filters is then collected.
[0073]A
feature of processed Narcissus tazetta products are their ability to slow cell
proliferation. Processed Narcissus tazetta products can induce reversible
dormancy in other plants. Processed Narcissus tazetta products have also shown
inhibitory effects on cell growth of human fibroblasts and keratinocytes primary
cultures as well as on cancerous strains. This effect is thought to take place
through a slowdown of the cell cycle in phase S, G2 and M, as FACS studies have
shown, resulting in a decrease of the cell pool in GI.
[0074]One
embodiment of the invention is the utilization of agents comprised of processed
Narcissus tazetta products to produce cutaneous antagonism between growth and
differentiation (e.g., psoriasis). For example an agent comprised of
IBR-DORMIN.RTM. Narcissus tazetta bulb extract in the form of a cream could be
used to treat psoriasis. Various concentrations of IBR-DORMIN.RTM. Narcissus
tazetta bulb extract may be utilized to affect desired efficacy of treatment.
[0075]In theory, processed Narcissus tazetta products may be used
wherever slowing cell proliferation is a benefit, such as: reduction the rate of
nail growth, prolonging sun tan, treatment of skin disorders including acne,
treatment of psoriasis, hair removal treatments, inhibition of alopecia and
hirsutism, decrease in pigmentation, treatment for people with high risk for
benign or malignant tumor.
[0076]One of the identified active fractions
in processed Narcissus tazetta products is at molecular size less than 5,000
Dalton. The extraction process therefore preferably includes an ultra-filtration
step through a 5,000 D cut-off membrane. This active fraction is heat stable. It
can be autoclaved at 120 C, 2 atmospheres for 30 min and retain 99% of initial
activity. The extract or agents containing the extract should preferably be kept
sterile, in closed containers at 4 to 24.degree. C. The inhibiting activity of
the extracts processed according to this invention is stable at room temperature
for two years with no loss of activity.
8. Inhibition of arNOX by
Narcissus tazetta Products
[0077]Processed Narcissus tazetta products
sequester arNOX activity. The inhibition of arNOX results in a decrease in the
generation of reactive oxygen species by arNOX. A decrease in reactive oxygen
species results in a decrease of oxidative damage resulting from said reactive
oxygen species. For example, IBR-DORMIN.RTM. Narcissus tazetta bulb extract is a
complex mixture from dormant Narcissus tazetta bulbs for which anti-aging
activity is claimed. The preparation specifically and completely inhibits the
arNOX activity of sera and of transfusion buffy coats (FIG. 7). The invention
encompasses the use of IBR-DORMIN.RTM. Narcissus tazetta bulb extract for
inhibition of arNOX. (FIG. 7). As such, the processed Narcissus tazetta product
preparations may be utilized as disclosed herein to ameliorate conditions
associated with a variety of aliments.
[0078]One embodiment of the
invention comprises the use of agents comprising 15 processed Narcissus tazetta
products, IBR-DORMIN.RTM. Narcissus tazetta bulb extract, and/or coenzyme Q,
alone or in combination with each other for inhibition of arNOX.
[0079]Another embodiment of the invention further comprises the use of
inhibition agents other than processed Narcissus tazetta products,
IBR-DORMIN.RTM. Narcissus tazetta bulb extract and coenzyme Q such as Schisandra
chinensis, Lonicera japonica, Fagopyrum cymosum and methylparaben.
[0080]The pharmaceutical compositions of this invention may comprise
varying modes of administration of compounds that sequester arNOX. The modes of
administration of compounds comprise capsules, tablets, soft gels, solutions,
suppositories, injections, aerosols, or a kit.
[0081]Embodiments of the
invention comprises the isolation and characterization of arNOX using processed
Narcissus tazetta products, preferably IBR-DORMIN.RTM. Narcissus tazetta bulb
extract as an inhibition agent.
[0082]The invention contemplates the
isolation and purification of arNOX, cloning of the arNOX cDNA and a complete
molecular characterization of the arNOX protein. Existing assays will be
employed to fractionate processed Narcissus tazetta products to identify the
active constituent(s) and to assay other natural product sources for anti-aging
activities. Based on the molecular information, a rapid and non-invasive RT-PCR
(reverse transcriptase-polymerase chain reaction) skin test for arNOX expression
will be developed along with models to test the necessity and/or sufficiency of
arNOX in the aging process.
[0083]The invention encompasses the use of
topical administration of processed Narcissus tazetta products to, maintain skin
vitality and for the oral administration of coenzyme Q as an approach to
ablation of age-related cell surface and lipoprotein oxidation. A preferred
embodiment of the invention comprises the topical administration of a cream,
which comprises IBR-DORMIN.RTM. Narcissus tazetta bulb extract, to the skin of
patients to maintain and improve skin vitality.
[0084]One embodiment of
the invention comprises therapeutic agents and the administration of a
therapeutically effective amount of a formulation comprised of at least one
therapeutic agent. One embodiment of the therapeutic agents of this invention
comprises at least one processed Narcissus tazetta product. The agent may
further comprise ubiquinones. The formulation may be administered to a patient
with a disorder or a complication of a disorder caused by oxidative damage
resulting from the generation of reactive oxygen species. For example the
formulation may be administered to a patient with a disorder or a complication
of a disorder caused by oxidative damage resulting from the generation of
reactive oxygen species by arNOX. In a preferred embodiment, the total daily
amount of the therapeutic agent administered is from about 1 to about 500 mg. of
a 1 g. composition. In a more preferred embodiment, the total daily amount
administered is from about 1 to 100 mg of a 1 g. composition of therapeutic
agent.
[0085]In one embodiment, the invention is used to identify
patients suffering from disorders associated with reactive oxygen species who
may be responsive to treatment with the therapeutic agents disclosed in this
invention. Such responsive patients may be identified by assay of serum or urine
for superoxide generation, which is responsive to treatment comprising the
therapeutic agents of the present invention. The generation of superoxide may be
followed by reduction of cytochrome c or any other suitable biological or
chemical method.
[0086]In one embodiment the invention further comprises
treating a patient with a pharmacologically effective amount of ubiquinones to
inhibit the generation of reactive oxygen species. In a preferred embodiment,
the ubiquinones are of the human derivative Q,.sub.10. In another embodiment,
the ubiquinones comprise the naturally occurring derivatives Q.sub.6, Q.sub.7,
Q.sub.8, and Q.sub.9. In another embodiment, the ubiquinones comprise other
derivatives Q.sub.1, Q.sub.2, Q.sub.3, Q.sub.4, Q.sub.5, Q.sub.11, and Q.sub.12.
In another embodiment, the invention comprises mixtures of the ubiquinone
derivatives described supra. The invention further comprises all
pharmaceutically acceptable derivatives of the compositions listed supra for
methods of treating a patient with an arNOX related disorder, with ubiquinone
administration in the range of 0.1 to 100 mg per kg body weight.
[0087]The invention also encompasses methods for monitoring patient
response to the agents of the present invention. Preferably the patients would
be monitored for responsiveness to treatments comprising the administration of
processed Narcissus tazetta products, and which may further comprise the
administration of ubiquinones. By monitoring circulating arNOX activity in
patient sera, it will be possible to determine therapeutic dosages and to
monitor therapeutic benefit from the therapeutic agents of the invention. The
response to the subject compositions may be monitored by assaying the blood or
urine of the patient for the arNOX activity that is responsive to the
compositions of this invention. By following the above monitoring procedures, an
effective dosage of the subject compositions may be administered in accordance
with the requirement of the individual patient.
9. Pharmaceutical
Formulations
[0088]Agents that interact with arNOX identified in this
invention may be formulated into pharmaceutical preparations for administration
to mammals for prevention or treatment of disorders in which oxidative species
have been implicated. In a preferred embodiment, the mammal is a human.
Compositions comprising a compound of the invention formulated in a compatible
pharmaceutical carrier may be prepared, packaged, and labeled for treatment. If
the complex is water-soluble, then it may be formulated in an appropriate
buffer, for example, phosphate buffered saline or other physiologically
compatible solutions.
[0089]Alternatively, if the resulting complex has
poor solubility in aqueous solvents, then it may be formulated with a non-ionic
surfactant such as Tween, or polyethylene glycol. Thus, the compounds and their
physiologically acceptable solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose) or oral,
buccal, parenteral, rectal administration or, in the case of tumors, directly
injected into a solid tumor.
[0090]For oral administration, the
pharmaceutical preparation may be in liquid form, for example, solutions, syrups
or suspensions, or may be presented as a drug product for reconstitution with
water or other suitable vehicle before use. Such liquid preparations may be
prepared by conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives or
hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia);
non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable
oils); and preservatives (e.g., methyl or propylhydroxybenzoates or sorbic
acid). The pharmaceutical compositions may take the form of for example, tablets
or capsules prepared by conventional means with pharmaceutically acceptable
excipients such as binding agents (e.g, pregelatinized maize starch, polyvinyl
pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g.,
magnesium stearate, tale or silica); disintegrants (e.g., potato starch or
sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The
tablets may be coated by methods well-known in the art.
[0091]Preparations for oral administration may be suitably formulated to
give controlled release of the active compound. For buccal administration, the
compositions may take the form of tablets or lozenges formulated in conventional
manner. For administration by inhalation, the compounds for use according to the
present invention are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of
a pressurized aerosol the dosage unit may be determined by providing a valve to
deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0092]The
compounds may be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multi-dose containers,
with an added preservative. The compositions may take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
[0093]The
compounds may also be formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases such as cocoa
butter or other glycerides. The compounds may also be formulated as a topical
application, such as a cream or lotion.
[0094]In addition to the
formulations described previously, the compounds may also be formulated as a
depot preparation. Such long acting formulations may be administered by
implantation (for example, subcutaneously or intramuscularly) or by
intramuscular injection.
[0095]Thus, for example, the compounds may be
formulated with suitable polymeric or hydrophobic materials (for example, as an
emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for hydrophilic drugs.
[0096]The composition may be formulated as compositions to be applied to
the skin of mammals. The composition may for example be comprised of active
agents and other carrier ingredients that facilitate the application of the
active agent to the surface of skin. For example, the composition may be
formulated as a cream or lotion for application to the skin.
[0097]The
compositions may, if desired, be presented in a pack or dispenser device, which
may contain one or more unit dosage forms containing the active ingredient. The
pack may for example comprise metal or plastic foil, such as a blister pack. The
pack or dispenser device may be accompanied by instructions for administration.
[0098]The invention also provides kits for carrying out the therapeutic
regimens of the invention. Such kits comprise in one or more containers
therapeutically or prophylactically effective amounts of the compositions in
pharmaceutically acceptable form. The composition in a vial of a kit of the
invention may be in the form of a pharmaceutically acceptable solution, e.g., in
combination with sterile saline, dextrose solution, or buffered solution, or
other pharmaceutically acceptable sterile fluid. Alternatively, the complex may
be lyophilized or desiccated; in this instance, the kit optionally further
comprises in a container a pharmaceutically acceptable solution (e.g., saline,
dextrose solution, etc.), preferably sterile, to reconstitute the complex to
form a solution for injection purposes.
[0099]In another embodiment, a
kit of the invention further comprises a needle or syringe, preferably packaged
in sterile form, for injecting the complex, and/or a packaged alcohol pad.
Instructions are optionally included for administration of compositions by a
clinician or by the patient.
10. Treatment of Skin
[0100]The
present invention provides compositions comprising active agent(s), which
prevent and/or ameliorates skin damage and associated conditions. Further, the
invention encompasses methods for utilizing said compositions. The stratum
corneum is the layer of the skin that forms the top surface layer and serves to
protect the skin while controlling moisture and the flow of substances in and
out of the skin. As this barrier function is broken down, the skin suffers
damaging effects, thus creating or contributing to premature aging. These
damaging effects causing premature aging of the skin are a concern for many
individuals wishing to maintain healthy, youthful looking and feeling skin.
Reactive oxygen species participate in a number of destructive reactions
potentially lethal to cells. Reactive oxygen species are responsible in part for
deleterious cellular interactions including impairing fibroblast cells ability
to produce healthy collagen and elastin. Furthermore, the skin is subject to
deterioration through dermatological disorders, environmental abuse (wind, air
conditioning, central heating) or through the normal aging process
(chronoaging), which may be accelerated by exposure of skin to sun (photoaging).
[0101]A preferred embodiment of the invention provides active agents
from processed plants for the treatment of skin. The active agents prevent
and/or ameliorate skin damage and associated conditions. In one embodiment of
the invention the processed plant products sequester arNOX activity. In another
embodiment of the invention, the processed plant products inhibit radical oxygen
species. In another embodiment agents and methods of the invention prevent
and/or improve the health of the skin. For example, the agents may improve skin
tone, and helps diminish the appearance of fine lines and visible signs of
aging. In another embodiment of the invention, the agents positively affects the
body's natural production of collagen and elastin. In another embodiment, the
agents of the invention minimize the effects of environmental agitators such as
pollution, sun, free radicals and stress.
[0102]One embodiment of the
invention provides compositions, and methods for using the same, for preventing
and/or ameliorating dermatological disorders and the effects thereof.
[0103]One embodiment of the invention provides composition for
preventing and reducing the effects of the production of reactive oxygen species
and methods for using the same. For example, the invention encompasses the use
of active agents derived from plants to sequester arNOX activity. Further, the
invention contemplates the use of other synthetic and natural compounds to
sequester arNOX activity.
[0104]The present invention discloses
compositions, which treat the skin and delays the visible signs of actual aging
and weathered skin such as wrinkles, lines, sagging, hyperpigmentation and age
spots. The present invention also decreases the appearance and condition of
sensitive, dry and/or flaky skin, serves to soothe red, and/or irritated skin,
and treats spots, pimples, blemishes, and other skin irregularities.
[0105]The present invention advances prior art compositions by providing
compositions and methods for using the same not previously disclosed. The
invention provides pharmaceutical compositions, methods of use, and
pharmaceutical kits for the treatment of disorders resulting from oxidative
changes in cells that result in aging by targeting an aging-related isoform of
NADH oxidase (arNOX), shed into the sera by aging cells. The compositions may
contain agents extracted from plants. For example, the compositions of the
invention may comprise at least one processed Narcissus tazetta product, whether
alone or with other inhibition agents and inhibit the activity of an
aging-related isoform of NADIR oxidase shed into the sera by aging cells. The
composition may comprise ubiquinones, extracts of Schisandra chinensis, Lonicera
japonica, Fagopyrum cymosum, methlyparaben, L-Carnosine, Propylparaben,
Ethylparaben, L-Ergothioneine, Betulinic acid, Solarium Lycopersicum,
Univestin.RTM. joint discomfort preparation, available from Unigen
Pharmaceuticals, Inc., Lacey, Wash., Soliprin.RTM. plant extract blend (water,
Scutellaria baicalensis root extract and Acacia catechu wood extract), available
from Unigen Pharmaceuticals, Inc., Lacey, Wash., coenzyme Q.sub.10, and/or
preservatives.
[0106]In a preferred embodiment the processed Narcissus
tazetta extract is IBR-DORMIN.RTM. Narcissus tazetta bulb extract. The active
agent(s) may be incorporated into various carriers suitable for application to
the skin. Additional elements such as colorants, fragrances, and other
ingredients, such as skin protectants, may also be present.
[0107]In one
embodiment a portion of, or all of these ingredients may be combined with other
ingredients commonly found in anti-aging and repair serum formulations.
Vehicles, other than, or in addition to water can include liquid or solid
emollients, solvents, humectants, thickeners and powders. The vehicle may be
from 0.1% to 99.9%, preferably from 25% to 80% by weight of the composition, and
can, in the absence of other cosmetic adjuncts, form the balance of the
composition. In one embodiment, the vehicle is at least 80% water, by weight of
the vehicle. In another embodiment water comprises at between about 50% to 85%
of the composition by weight. In yet another embodiment, water is present
between about 0.1% to 55%, by weight of the composition. In other embodiments
other vehicles are used in the above recited concentrations.
[0108]An
oil or oily material may be present, together with an emulsifier to provide
either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on
the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed.
[0109]The inventive compositions may also include sunscreens. Sunscreens
include those materials commonly employed to block ultraviolet light.
Illustrative compounds are the derivatives of PABA, cinnamate and salicylate.
For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy benzophenone (also
known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy
benzophenone are commercially available under the trademarks, Parsol MCX and
Benzophenone-3, respectively. The exact amount of sunscreen employed in the
emulsions can vary depending upon the degree of protection desired from the
sun's UV radiation.
[0110]Emollients may further be incorporated into
cosmetic compositions of the present invention. Levels of such emollients may
range from 0.5% to 50%, preferably between 5% and 30% by weight of the total
composition. Emollients may be classified under such general chemical categories
as esters, fatty acids and alcohols, polyols and hydrocarbons.
[0111]Esters may be mono- or di-esters. Acceptable examples of fatty
di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and
dioctyl succinate. Acceptable branched chain fatty esters include 2-ethyl-hexyl
myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid
esters include triisopropyl trilinoleate and trilauryl citrate. Acceptable
straight chain fatty esters include lauryl palmitate, myristyl lactate, and
stearyl oleate. Preferred esters include coco-caprylate/caprate (a blend of
coco-caprylate and coco-caprate), propylene glycol myristyl ether acetate,
diisopropyl adipate and cetyl octanoate.
[0112]Suitable fatty alcohols
and acids include those compounds having from 10 to 20 carbon atoms. Especially
preferred are such compounds such as cetyl, myristyl, palmitic and stearyl
alcohols and acids.
[0113]Among the polyols, which may serve as
emollients are linear and branched chain alkyl polyhydroxyl compounds. For
example, propylene glycol, sorbitol and glycerin are preferred. Also useful may
be polymeric polyols such as poly-propylene glycol and polyethylene glycol.
Butylene and propylene glycol are also especially preferred as penetration
enhancers.
[0114]Exemplary hydrocarbons which may serve as emollients
are those having hydrocarbon chains anywhere from 12 to 30 carton atoms.
Specific examples include mineral oil, petroleum jelly, squalene and
isoparaffins.
[0115]Other embodiments of the compositions of the present
invention comprise thickeners. A thickener will usually be present in amounts
anywhere from 0.1 to 20% by weight, preferably from about 0.5% to 10% by weight
of the composition. Exemplary thickeners are cross-linked polyacrylate materials
available under the trademark CARBOPOL.RTM. from the B.F. Goodrich Co. Gums may
be employed such as xanthan, carrageenan, gelatin, karaya pectin and locust
beans gum. Under certain circumstances the thickening function may be
accomplished by a material also serving as a silicone or emollient. For
instance; silicone gums in excess of 10 centistokes and esters such as glycerol
stearate have dual functionality.
[0116]Powders may be incorporated into
the cosmetic composition of the invention.
[0117]These powders include
chalk, talc, kaolin, starch, smectite clays, chemically modified magnesium
aluminum silicate, organically modified montmorillonite clay, hydrated aluminum
silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof.
[0118]Other adjunct minor components may also be incorporated into the
cosmetic compositions. These ingredients may include coloring agents, opacifiers
and perfumes.
[0119]Amounts of these other adjunct minor components may
range anywhere from 0.001% up to 20% by weight of the composition.
[0120]The composition of the invention may be used for topical
application to human skin, as an agent for conditioning, moisturizing and
smoothing the skin, increasing the flexibility and elasticity and preventing or
reducing the appearance of wrinkled, lined or aged skin. The unique formulation
of the present invention offers the complete response to the loss of skin tone
and promotes immediate and continuous benefits to effectively boost hydration
and firmness of the surface layer of the skin, all while working to repair the
underlying layers of the skin with antioxidants and other beneficial ingredients
to help diminish the appearance of fine lines and wrinkles and to restore
visible tone and elasticity.
[0121]In one embodiment a small quantity of
the composition comprised of from about 1 to 1000 ml of active agent, is applied
to the skin. In a preferred embodiment, a quantity of composition comprising
from about 1 to 100 ml of active agent is applied to the skin.
[0122]This process may be repeated several times daily for any period of
time. Preferably, the composition is applied to the skin once in the morning and
once in the evening.
[0123]The topical skin care composition of the
invention can be formulated as a lotion, a cream or a gel. The composition can
be packaged in a suitable container to suit its viscosity and intended use by
the consumer. For example, a lotion or a cream can be packaged in a bottle or a
roll-ball applicator, or a propellant-driven aerosol device or a container
fitted with a pump suitable for finger operation. When the composition is a
cream, it can simply be stored in a non-deformable bottle or squeeze container,
such as a tube or a lidded jar. The invention accordingly also provides a closed
container containing a cosmetically acceptable composition as herein defined.
EXAMPLE 1
Characterization of arNOX
1. Superoxide
Production by Buffy Coats
[0124]Reduction of ferric cytochrome c by
superoxide was employed as a standard measure of superoxide formation (Mayo, L.
A. and Cumutte, J. (1990) Meth. Enzyme. 186, 567-575. 7. Butler, J, Koppenol, W.
H. and Margollash, E. (1982) J. Biol. Chem. 257, 10747). If superoxide dismutase
was added to remove the superoxide as it was generated, the reduction of ferric
cytochrome c was prevented to confirm that ferric cytochrome c reduction in the
assay was due to superoxide.
[0125]Buffy coats were pooled from aged
individuals (70-100 y) and the reduction of ferric cytochrome c was observed
(FIG. 2) with an oscillating activity. The oscillations exhibited a period
length of ca. 25 min (arrows, FIGS. 2C and 2D). This oscillatory reduction of
cytochrome c was absent from buffy coat fractions from younger (20-40 y)
individuals (FIGS. 2A and 2B). The oscillating reduction of ferric cytochrome c
was inhibited completely by superoxide dismutase (SOD) (FIG. 2C) and by 100
.mu.M coenzyme Q (EC.sub.50 of 20 .mu.M) (FIG. 2D). The rate of coenzyme Q
inhibited ferric cytochrome c reduction was 7-fold greater in buffy coat
fractions of 90-94 y individuals as compared to 80-89 y individuals (Table 1).
Buffy coats of less than 65 y individuals lacked the activity.
2.
Superoxide Production and Inhibition by Coenzyme Q and Superoxide Dismutase
[0126]Assays of ferric cytochrome c reduction in sera compared 53
samples from young (20 to 40 y) and 65 samples from aged (80 to 100)
individuals. Activities were 0.2.+-.0.2 nmoles/min/100 .mu.l sera for young
compared to 1.4.+-.0.2 nanomoles/min/100 .mu.l sera for aged. With untreated
serum samples, addition of 30 units/ml of superoxide dismutase inhibited the
activity by 40.+-.10%. Addition of 300 .mu.g/ml coenzyme Q also inhibited the
activity by 40% although on average the results were more variable.
[0127]When SOD (30 units) and coenzyme Q (450 .mu.g) were added
sequentially to the same reaction mixture and assayed over 300 sec, no further
inhibition to the rate of ferric cytochrome c reduction in serum was observed
when coenzyme Q was added after maximal SOD inhibition and vice versa.
[0128]Inhibition of the rate of age-related cytochrome c reduction (ACR)
was in proportion to the SOD concentration between 8 and 35 units (FIG. 3). A
plateau was reached at 45 units. With coenzyme Q, inhibition was proportional to
amounts between 75 .mu.g to 450 .mu.g and reached a plateau at about 450 .mu.g
coenzyme Q (FIG. 4).
[0129]Additional reduction of cytochrome c by sera
was observed in the presence of NADH (NADH-cytochrome c reductase). However, the
NADH-stimulated activity was about 4 nmoles/min/ml of sera for sera of both
young and aged individuals. The aging-specific increment of ca. 2 nmoles/min/mg
protein was observed both in the presence or absence of NADH. Neither SOD nor
coenzyme Q inhibited the activity of NADH cytochrome c reductase in serum of
either young or aged individuals. Also, the addition of coenzyme Q did not
significantly inhibit the activity of authentic NADH cytochrome c reductase of
pig liver microsomes.
[0130]Thus the arNOX appears to be unrelated to
NADH-cytochrome c reductase. Composed of a large hydrophilic, catalytic domain
and a smaller hydrophobic membrane binding segment, proteases release the active
protein from, membranes. The NH.sub.2 terminal glycyl residue is linked to the
membrane via myristic aid. Solubilization can be achieved by enzymatic digestion
without loss of enzymatic activity. Lysosomal acid proteases, i.e., capsaicin D,
also release the activity. Also, as arNOX does not respond to capsaicin or
(-)-epigallocatechin gallate (EGCg), it is not one of the drug-responsive tNOX
isoforms.
[0131]An oscillating rate of enzymatic activity with a regular
period length of about 24 min is one of the defining characteristic of the CLOX
family of proteins. When the reduction of ferric cytochrome c of individual sera
of 90-100 y subjects was assayed over 1 min at intervals of 1.5 mm, the activity
was observed to oscillate with a regular period length but again with a period
length of 25 min rather than 24 min (FIG. 1). The oxidation of NADH measured in
parallel with the same sample showed two patterns of oscillations, one with a
period length of ca. 25 min corresponding to the age-related isoform and a
second pattern with a period length of 24 min corresponding to CNOX as reported
previously. Corresponding oscillations were not observed with the activity of
NADH cytochrome c reductase of pig liver microsomes as a positive control (FIG.
6).
TABLE-US-00002 TABLE 1 Reduction of Ferricytochrome c. Measure of
Superoxide Production by Buffy Coats from Blood of Aged Individuals and
Inhibition by Coenzyme Q. Rate of reduction of ferricytochrome c nmoles
min.sup.-1 10.sup.7 cells.sup.-1 Group N No addition +100 .mu.M Q.sub.10 35-65
years 5 ND ND 80-89 years 6 0.25 .+-. 0.02 -0.03 .+-. 0.02 90-94 years 6 0.36
.+-. 0.07 -0.07 .+-. 0.07 Values are means .+-. standard deviation; ND, not
detected Q.sub.10 ubiquinone-10 (CoQ.sub.10) In these experiments, the negative
rates reflect small negative slopes in the rate of NADH oxidation. Statistically
the rates were zero.
TABLE-US-00003 TABLE 2 Response of Rate of
Reduction of Ferricytochrome c of rum Samples to Proteinase K Digestion. Rate of
reduction of ferricytochrome c nmoles min.sup.-1 mi serum.sup.-1 Group N No
addition +Proteinase K <35 y females 12 0.24 .+-. 0.14 0.26 .+-. 0.17 35-45
males 9 0.21 .+-. 0.15 0.27 .+-. 0.16 75-85 males 10 0.7 .+-. 0.35 0.72 .+-.
0.14 75-85 females 8 1.1 .+-. 0.20 1.0 .+-. 0.18 >90 females 10 0.8 .+-. 0.11
0.94 .+-. 0.24 N = Number of subjects represented in each pooled sample.
TABLE-US-00004 TABLE 3 Superoxide Production (Reduction of
Ferricytochrome c) in Sera of Aged (80-98 y) individuals Following Treatment
with Proteinase K and Response to Superoxide Dismutase (n = 10).
Nmoles/min/mi/sera Group N No addition No proteinase K 0.7 .+-. 0.2 0.42 .+-.
0.1 After proteinase K 0.6 .+-. 0.2 0.22 .+-. 0.1
[0132]Source of
electrons for cytochrome c reduction with sera of aged individuals. The regular
pattern of oscillations with a period length of 25 min that correlates with a
corresponding pattern of oscillations for NADH oxidation dictates that the
source of electrons for the oscillating generation of superoxide reduction of
tonic cytochrome c for buffy coats and in the sera of aged patients is the
age-related NOX protein. The regular period length of ca 25 min distinguished
the activity from that of other proteins including the constitutive CNOX protein
of sera which has a period length of 24 min and does not generate superoxide
(i.e., reduce ferric cytochrome C). Within the age-related NOX protein, active
site cysteines and bound copper were considered as electron sources. The serum
activity was unaffected by the copper chelators bathocuproene or
bathocuproenedisulforiate. A protein thiol source was considered more likely
since the activity was inhibited by thiol reagents such as
p-chloromercuribenzoate.
[0133]The serum source to regenerate the NOX
protein thiols oxidized during the reduction of cytochrome c also appears to be
protein thiols. NOX proteins exhibit protein disulfide-thiol interchange
activity and are capable of undergoing protein thiol oxidation and protein
disulfide reduction at the expense of external protein sources. Copper as a
serum source of electrons is less likely since added copper did not enhance the
activity nor did the copper chelators inhibit.
[0134]Serum levels of
protein thiols are certainly adequate to fuel the reaction. By direct assay
using Ellman's reagent, the sera analyzed were calculated to contain sufficient
thiols to sustain the average rate of cytochrome c reduction for more than 10
days at room temperature and for several months in the cold assuming that all,
thiols were available for reaction. Serum samples where protein SH was oxidized
with dilute (0.01%) hydrogen peroxide followed by catalase to remove residual
hydrogen peroxide were inactive. Catalase by itself was without effect. Also,
oxidation of proteins by equilibration with low concentrations of GSSG
inactivates serum activity but not that of buffy coats where the source of
electrons is assumed to be from the electron transport pathway. Addition of GSH
neither stimulates nor inhibits but may eventually prolong the cytochrome c
reduction capacity of the sera by maintaining levels of protein thiols. The lack
of complete inhibition by SOD or coenzyme Q of FIGS. 3 and 4 results in large
measure from a basal activity that is insensitive to inhibition. As shown by
data of FIG. 2, the oscillating activity component in sera of aged patients is
eliminated by both SOD (FIG. 2G) and coenzyme Q (FIG. 2H). The source of the
basal activity appears to involve neither a specific enzyme nor cytochrome c
reduction. It is encountered in other serum NOX assays and appears to result
from light scattering changes due to aggregation of serum proteins.
3.
Proteinase K Digestion.
[0135]Resistance to proteinase K digestion is a
very important characteristic of the CLOX proteins. There was no significant
decrease in the rate of arNOX following proteinase K digestion (Table 2).
Inhibition by superoxide dismutase increased from 40% to 60% following
proteinase K digestion of sera from aged individuals (Table 3), due to a marked
reduction in the basal absorbance changes attributed to protein aggregation. The
protein thiol content of the sera was not affected by proteinase digestion.
4. Western Blotting for arNOX
[0136]To further verify that the
arNOX is the result of a NOX protein of the CLOX protein family, polyclonal
anti-sera to the C terminus of a previously-cloned and tumor-specific NADH
oxidase (tNOX) were utilized to identify an immunoreactive band on Western
blots. Serum samples, after proteinase K digestion to reduce background
proteins, were separated by 10% SDS-PAGE and transferred by electroblotting onto
nitrocellulose membranes. A reactive band at a molecular weight of ca. 22 kD was
observed on lanes from samples of sera of elderly subjects (FIG. 6). There was a
strong correlation between band intensity determined by densitometry and arNOX.
A similar correlation was seen between band density and the age of the subject.
To verify that the 22 kD region of the gel contained a CLOX protein, an SDS-PAGE
gel was cut into segments and the proteins were eluted. Activity was restored by
reduction of the protein with 100 .mu.M NADH, pH 7.0, followed by addition of
0.03.degree. h hydrogen peroxide to reoxidize the refolded protein. The region
of the gel corresponding to a molecular weight of 22 kD exhibited an oscillating
pattern of ferric cytochrome c reduction (FIG. 9).
[0137]The remainder
of the gel slices lacked a reproducible pattern of ferric cytochrome c
reduction.
5. Purification of Protein Catalyzing Aging-Related
Cytochrome a Reduction
[0138]The protein catalyzing arNOX activity was
separated from serum through immunoprecipitation with (NOX antibody. The
precipitated proteins were separated by SDS-PAGE and the proteins in the gel
were transferred to PVDF membranes. The protein on PVDF membrane was identified
by Coomassie blue staining. The target band on the PVDF membrane was excised and
submitted for N-terminal amino acid sequencing.
EXAMPLE 2
arNOX
Inhibition
[0139]Various compounds were analyzed to assess arNOX
inhibition according to the methods disclosed. The compounds, product codes and
names, etc. as provided in the table below (Table 4):
TABLE-US-00005
TABLE 4 arNOX inhibition assays No. Product Code Product Name Lot Number
Comments 1. UP566 Soliprin .RTM. plant E0404 Free B-ring flavanoids and extract
blend Flavans (water, Scutellaria baicalensis root extract and Acacia catechu
wood extract), 2. R44390 Univestin .RTM. joint G1702-COX-2 discomfort
preparation 3. 0301 IBR-DORMIN .RTM. BA0303161 Narcissus tazetta Bulb Narcissus
tazetta Japanese Name: bulb extract Fusazakisuisen Extract 4. 0601 IBR-TOM .RTM.
BA t4006L Solanum lycopersicum aqueous serum of Japanese Name: tomato Tomato
Ekisu (Tomato Extract) 5. 855057 Betulinic acid CAS No. 472-15-1 FW 456.71 6.
26547 L-Ergothioneine 12723P MW 229.3 L-ERGO .RTM. CAS No. 497-30-3
synthetically derived 1- ergothioneine 7. Ethylparaben 8. Propylparaben 9.
Methylparaben 10. C-9625-5G L-Carnosine CAS No. 305-84-0 FW 226.24 (Sigma)
[0140]The compounds listed above in Table 4 were tested initially in the
standard arNOX assay at a dilution of 1:50. Solids were prepared in water at an
initial concentration of approximately 100 mM and then also tested at a dilution
of 1:50, i.e., 2 mM. All compounds were evaluated using human buffy coats
prepared from an 86 y female. Several compounds were evaluated with sera from
aged patients as follow: IBR-DORMIN.RTM. Narcissus tazetta bulb extract (88 y
female), IBR-TOM.RTM. aqueous serum of tomato, available from Israeli
Biotechnology Research Ltd. Corporation, Tel Aviv, Israel, (88 y female),
L-carnosine (88 y female, 84 y female), L-ERGO.RTM. synthetically derived
1-ergothioneine, available from OXIS Health Products, Inc., Portland, Oreg. (89
y male). Serum and buffy coats gave consistent results. Compounds active at 1:50
dilutions were reassayed with buffy coats at a dilution of 1:500 and compounds
active at 1:500 were reassayed with buffy coats at a dilution of 1:5000.
[0141]Details of the assay protocol are as follow. Buffy coats, a
mixture of lymphocytes and platelets, were obtained from a commercial supplier.
The blood samples were maintained at 4.degree. C. prior to collection and assay.
Ca. 10.sup.7 cells were added to each assay. Cell numbers were determined using
a hemocytometer.
[0142]Measurement of arNOX activity based on
ferricytochrome c reduction as a measure of superoxide production were taken.
The rate of reduction of ferricytochrome c was determined from the increase in
absorbance at 550 nm with 540 nm as reference. This is a widely accepted method
when coupled to superoxide dismutase inhibition for the measurement of
superoxide generation. The assay consists of 150 .mu.l (2 mg/ml) of oxidized
ferricytochrome c solution and 150 .mu.l serum or 40 .mu.l buffy coats in PBSG
buffer (8.06 g NaCl, 0.2 g KCl, 0.18 g Na.sub.2HPO.sub.4, 0.26 g
KH.sub.2PO.sub.4, 0.13 g CaCl.sub.2, 0.1 g MgCl.sub.2, 1.35 g glucose dissolved
in 1000 ml deionized water, adjusted to pH 7.4, filtered and stored at 4.degree.
C.). Rates were determined using a SLM Aminco DW-2000 spectrophotometer (Milton
Roy, Rochester, N.Y., USA) in the dual wave length mode of operation with
continuous measurements over 1 min every 1.5 min. After 45 min, test compound
was added and the reaction was continued for 45 min. A millimolar extinction
coefficient of 19.1 cm.sup.-1 was used for reduced ferricytochrome c. (D. M.
Morre, F. Guo and D. J. Morre, 2003, Mol. Cell. Biochem. 254: 1010-109).
[0143]The following compounds were active at a dilution of 1:50 but were
inactive at a dilution of 1:500: Soliprin.RTM. plant extract blend (water,
Scutellaria baicalensis root extract and Acacia catechu wood extract),
propylparaben and methylparaben. The buffy coats used contained two distinct
arNOX activities. Methylparaben inhibited one and had no effect on the other. A
similar result was seen with Soliprin.RTM. plant extract blend (water,
Scutellaria baicalensis root extract and Acacia catechu wood extract). In one
experiment with a serum sample containing 3 arNOX activities, L-carnosine,
inhibited one arNOX, stimulated a second arNOX and was without effect on a
third. The following compounds gave mixed results at a dilution of 1:50 but were
inactive at a dilution of 1:500: IBR-TOM.RTM. aqueous serum of tomato, and
L-ERGO.RTM. synthetically derived 1-ergothioneine. Only the following compound
was active at a dilution of 1:500 IBR-DORMIN.RTM. Narcissus tazetta bulb
extract.
EXAMPLE 3
IBR-DORMIN.RTM. Narcissus tazetta Bulb
Extract Heat Resistance
[0144]A batch of IBR-DORMIN.RTM. Narcissus
tazetta bulb extract was produced the pH was measured as 5.84. Its color was
light yellow (607c by Pantone). The batch was kept in high-density polyethylene
container, at room temperature. As detailed in the table below, samples were
taken to determine color, pH and activity by seeds test. Color was defined by
Pantone color formula guide. pH was measured by pH meter (Radiometer,
Copenhagen, Denmark). Product pH range was 4.5-6.5.
[0145]Seed test were
performed as follows. Cucumber seeds were germinated over night on water-wetted
filter paper in closed tray at 28.degree. C. Seeds with 1-2 mm roots were taken
for the assay. IBR-DORMIN.RTM. Narcissus tazetta bulb extract (.times.2
concentrated) was applied in the following dilutions: 50%, 25%, 12.5%, 5% and
2.5%. Tap water served as a control. 2-ml of each dilution were applied on a
filter paper in a big Petri dish. Ten seeds were put in each Petri dish.
[0146]Root length was measured after 48 h. The average length of 10
seeds was calculated. A semi-logarithmic graph of root length vs. % extract was
drawn. ID.sub.50 (the percentage of extract required to reach 50% inhibition of
root growth) was calculated from the equation of the best-fit curve. Product
ID.sub.50 range is 9.5-13.5%.
TABLE-US-00006 TABLE 5 ID.sub.50 range PH
Color ID.sub.50 by seed test (%) 5.86 Light yellow (607c) 9.81 5.96 Light yellow
(607c) 10.99 5.84 Light yellow (607c) 10.60 5.55 Light yellow (607c) 11.34 5.59
Light yellow (607c) 10.30
[0147]By all the examined parameter,
IBR-DORMIN.RTM. Narcissus tazetta bulb extract pH was found to be stable for up
to 18 months. The pH and activity (ID.sub.50) are within the specified range,
and the color did not change.
[0148]IBR-DORMIN.RTM. Narcissus tazetta
bulb extract was examined for its stability to heat by examining the influence
of autoclaving on the liquid appearance and activity. A sample of
IBR-DORMIN.RTM. Narcissus tazetta bulb extract was autoclaved in the lab for 30
min at 120.degree. C. and 2 atmospheres. Three subsequent cycles of autoclave
were performed. After each cycle, a portion was taken to examine maximum
inhibition activity by seed test: Cucumber seeds were germinated over night on
wet filter paper in closed tray at 28.degree. C. Seeds with 1-2 mm roots were
taken for the assay. IBR-DORMIN.RTM. Narcissus tazetta bulb extract was applied
at 50%. Tap water served as a control. 2 mil of this dilution were applied on a
filter paper in a Petri dish with 10 seeds. Root length was measured after 48 h.
The average length of the 10 seeds was calculated, and percentages of inhibition
is given by the equation: (1-(Average of dormin treated root length/Average of
root length in water)).
[0149]Results of the heat stability assay were
as follows. Liquid appearance: after autoclaving, small precipitates could be
observed. Only a slight change in color was observed comparing non-autoclaved to
autoclaved sample. Activity: The table below summarizes the inhibition activity
of 50% IBR-DORMIN.RTM. Narcissus tazetta bulb extract on cucumber seed growth.
As shown, full activity was retained after three subsequent cycles of autoclave.
TABLE-US-00007 TABLE 6 Heat Stability Root inhibition (%) No autoclave
91.0 First autoclave 89.5 Second autoclave 90.0 Third autoclave 90.5
[0150]IBR-DORMIN.RTM. Narcissus tazetta bulb extract is heat stable. The
small precipitates and the slight change in color observed after the autoclaving
process, does not influence its activity.
EXAMPLE 4
Treatment
with IBR-DORMIN.RTM. Narcissus tazetta Bulb Extract
[0151]Plaque
psoriasis of mild to moderate severity is routinely treated with topical
steroids and coal tar, along with emollients. A safe and convenient new
treatment modality would be of value to most patients suffering from psoriasis.
A study was undertaken in order to assess the efficacy of 5% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract in cream in the treatment of mild
to moderate, persistent psoriasis. The results of the treatment of psoriasis in
this study show that the left elbows of the patients (those treated with
IBR-DORMIN.RTM. Narcissus tazetta bulb extract) exhibited a better overall
improvement compared to their right elbows (treated with vehicle cream only).
Additionally, no side effects were experienced on the IBR-DORMIN.RTM. Narcissus
tazetta bulb extract treated elbow.
1. Methods
[0152]The
application, twice daily, of 5% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb
extract in cream was compared with the application of its vehicle cream for up
to 10 weeks in a double blind, controlled study of 15 patients, with no
randomization. In the study, all of the patients applied 5% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract in cream to one elbow and the
vehicle cream to the other elbow.
[0153]Inclusion criteria for this
study were as follows: all of the patients were between the ages of 16 and 70,
and had mild to moderate stable psoriasis vulgaris. Exclusion criteria for this
study included the presence of acute pruritus, acute urticaria, scabies, other
systemic diseases that involve pruritus, steroidal treatment during the last
month, pregnancy, treatment of systemic retinoids, and the use of any
investigational drug within the last 30 days prior to study entry.
2.
Results
[0154]The psoriatic plaques were judged by the clinical
characteristics of thickness, dryness, desquamation, erythema, and pruritic
lesions. These criteria were graded on a scale of 0 to 4, where 0=cure or
absence and 4=severe. The clinical parameters were evaluated at baseline, 3, 6,
and 10 weeks after start of the treatment regimen.
[0155]Two of the
fifteen patients did not complete the study and follow-up of their conditions
was lost. One patient did not come to the last follow-up visit (Visit #4), but
was included in the study results. Total scoring parameter results for each
patient at follow-up visits are summarized in Table 7.
[0156]On the left
elbow (IBR-DORMIN.RTM. Narcissus tazetta bulb extract): 3 patients had a
complete cure; 2 patients had a cure rate of 75-99%; 4 patients had a cure rate
of 50-74%; 3 patients had a cure rate of less than 50%, and in 1 patient, there
was no change (FIGS. 10 and 11). On the right elbow (control): 3 patients had a
cure rate of 50-74%; 7 patients had a cure rate of less than 50%, and in 3
patients, there was no change (FIGS. 9 and 11).
[0157]When the study was
completed, it was revealed that the left elbows were treated with 5% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract in cream and the right elbows
were treated with the vehicle cream only.
[0158]The results of the
treatment of psoriasis in this study show that the left elbows of the patients
(those treated with IBR-DORMIN.RTM. Narcissus tazetta bulb extract) exhibited a
better overall improvement compared to their right elbows (treated with vehicle
cream only). No side effects.
TABLE-US-00008 TABLE 7 Total Scoring
Parameter Results for Each Patient at Follow-up Visits Total Score of Clinical
Parameters Patient Number Visit Number Right Left 1 1 8 10 2 7 6 3 4 5 4 4 6 3 1
9 8 2 7 2 3 6 0 4 6 0 4 1 9 6 2 6 2 3 5 0 4 4 0 5 1 12 11 2 12 4 3 12 2 4 11 2 6
1 10 11 2 10 11 3 10 11 4 10 11 7 1 16 16 2 14 6 3 16 6 4 16 6 8 1 8 8 2 8 8 3 4
2 4 Patient did not show up 10 1 15 15 2 11 15 3 11 15 4 5 5 11 1 20 20 2 20 20
3 20 15 4 20 12 12 1 15 15 2 10 5 3 I1 0 4 11 4 13 1 13 13 2 13 13 3 12 6 4 12 4
4 1 15 20 2 15 12 3 5 10 4 10 10 Legend: Visit 1 = baseline. Visit 2 = 3 weeks.
Visit 3 = 6 weeks. Visit 4 = 10 weeks.
EXAMPLE 5
Treatment with
IBR-DORMIN.RTM. Narcissus tazetta Bulb Extract
1. Materials and Methods
[0159]150 women (mean age: 37.77.+-.0.96) took part in the study. Three
creams containing respectively 0% (placebo), 1% by weight and 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract were produced for the study.
IBR-DORMIN.RTM. Narcissus tazetta bulb extract was used to replace some of the
water used in the formula for the excipient. The study lasted four weeks. The
creams were applied daily by the women users themselves. 135 subjects completed
the study; 48 in the placebo group (mean age: 36.71.+-.1.53), 44 in the group
testing the 1% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb extract in cream
(mean age: 37.93.+-.1.7) and 43 in the 3% by weight IBR-DORMIN.RTM. Narcissus
tazetta bulb extract in cream group (mean age: 38.77.+-.1.8).
[0160]Rating scales were administered and handed in before the first
application of the cream. Similar rating scales were administered at the end of
the four weeks test. The parameters or items used in the test were the
following: Resistance against external aggressions, Skin Sensitivity,
Protection, Skin Irritability, Skin Fatigue, Skin Tautness, Comfort, Little
Lines and Suppleness.
2. Data Analysis
[0161]Centimetric
measures were drawn on unstructured scales 10 cm in length. The mean values
before and after the cream application were obtained for each of the three
groups. The before/after comparison was obtained statistically by a t-test, when
applicable, using the SigmaStat 2.0 program. A Rank Sum Test (RST) was used each
time the normality test failed using the same SigmaStat 2.0 program. The
percentage of before/after change was calculated on the basis of means for each
item.
3. Results: Resistance Against External Aggressions
[0162]Results of the study for resistance against external aggressions
are depicted in Table 8 and FIG. 12A. No statistical difference was found for
the placebo and 1% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb extract
cream. Significant statistical difference was found for the 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream.
TABLE-US-00009
TABLE 8 Resistance Against External Aggressions CRITERIA PLACEBO 1% CREAM 3%
CREAM Number of women 48 43 44 Missing data 0 2 1 Mean before (B) 4.690 .+-.
2.553 4.198 .+-. 2.627 3.414 .+-. 2.145 Mean after (A) 5.181 .+-. 2.445 5.112
.+-. 2.049 5.288 .+-. 2.165 Difference (A - B) 0.491 0.915 1.874 Statistical
test RST t-test RST Probability of 0.395 0.083 <0.001 improvement (p)
Significance NS NS S(++)
4. Results: Skin Sensitivity
[0163]Results for skin sensitivity are depicted in Table 9 and FIG. 12B.
No statistical difference was found for the placebo and 1% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream. Significant statistical
difference was found for the 3% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb
extract cream.
TABLE-US-00010 TABLE 9 Skin Sensitivity CRITERIA PLACEBO
1% CREAM 3% CREAM Number of women 48 43 44 Missing data 1 1 0 Mean before (B)
4.294 .+-. 2.574 4.233 .+-. 2.396 3.745 .+-. 2.433 Mean after (A) 5.034 .+-.
2.477 5.124 .+-. 2.047 5.373 .+-. 2.180 Difference (A - B) 0.740 0.890 1.627
Statistical test t-test t-test t-test Probability of 0.159 0.07 0.003
improvement (p) Significance NS NS S(++)
5. Results: Protection
[0164]Statistical results for protection assays are depicted in Table 10
and FIG. 12C. Statistical difference was found for the placebo and 1% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream. Significant statistical
difference was found for the 3% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb
extract cream.
TABLE-US-00011 TABLE 10 Protection Statistics CRITERIA
PLACEBO 1% CREAM 3% CREAM Number of women 48 43 44 Missing data 0 1 3 Mean
before (B) 4.385 .+-. 2.203 4.112 .+-. 2.496 3.976 .+-. 2.425 Mean after (A)
5.496 .+-. 2.284 5.131 .+-. 2.087 5.507 .+-. 2.327 Difference (A - B) 1.11 1.019
1.532 Statistical test t-test t-test t-test Probability of 0.017 0.046 0.003
improvement (p) Significance S S S(++)
6. Results: Skin Irritability
[0165]Statistical results for skin irritability are depicted in Table 11
and FIG. 13A. No statistical difference was found for the placebo and 1% by
weight IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream. Significant
statistical difference was found for the 3% by weight IBR-DORMIN.RTM. Narcissus
tazetta bulb extract cream.
TABLE-US-00012 TABLE 11 Skin Irritability
Statistics CRITERIA PLACEBO 1% CREAM 3% CREAM Number of women 48 43 44 Missing
data 0 1 0 Mean before (B) 4794 .+-. 2.606 4.769 .+-. 2.336 4.405 .+-. 2.479
Mean after (A) 5.617 .+-. 2.372 5.690 .+-. 2.189 6.125 .+-. 2.128 Difference (A
- B) 0.823 0.921 1.720 Statistical test t-test t-test t-test Probability of
0.109 0.066 0.001 improvement (p) Significance NS NS S(++)
7. Results:
Skin Fatigue
[0166]Statistical results for skin fatigue survey are
depicted in Table 12 and FIG. 13B. No statistical difference was found for the
placebo and 1% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream.
Significant statistical difference was found for the 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream.
TABLE-US-00013
TABLE 12 Skin Fatigue Statistics CRITERIA PLACEBO 1% CREAM 3% CREAM Number of
women 48 43 44 Missing data 0 1 0 Mean before (B) 4.635 .+-. 2.522 4.684 .+-.
2.374 4.345 .+-. 2.640 Mean after (A) 5.602 .+-. 2.265 5.600 .+-. 2.323 5.784
.+-. 2.017 Difference (A - B) 0.967 0.916 1.439 Statistical test t-test t-test
t-test Probability of 0.051 0.074 0.005 improvement (p) Significance NS NS S(++)
8. Results: Skin Tautness
[0167]Statistical results for skin
tautness are depicted in Table 13 and FIG. 13C. No statistical difference was
found for the placebo and 1% by weight IBR-DORMIN.RTM. Narcissus tazetta bulb
extract cream. Strong statistical difference was found for the 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream.
TABLE-US-00014
TABLE 13 Skin Tautness Statistics CRITERIA PLACEBO 1% CREAM 3% CREAM Number of
women 48 43 44 Missing data 0 0 0 Mean before (B) 5.158 .+-. 2.890 5.858 .+-.
2.497 5.173 .+-. 2.864 Mean after (A) 5.933 .+-. 2.713 6.695 .+-. 2.561 6.457
.+-. 2.636 Difference (A - B) 0.775 0.837 1.284 Statistical test t-test RST RST
Probability of 0.179 0.092 0.032 improvement (p) Significance NS NS S
9.
Results: Skin Comfort
[0168]Statistical results for skin comfort survey
are depicted in Table 14 and FIG. 14A. No statistical difference was found for
the placebo. Significant statistical difference was found for the 1% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream. Significant statistical
difference, at a higher probability, was found for the 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream.
TABLE-US-00015
TABLE 14 Skin Comfort Statistics CRITERIA PLACEBO 1% CREAM 3% CREAM Number of
women 48 43 44 Missing data 3 2 3 Mean before (B) 5.678 .+-. 2.059 5.410 .+-.
2.298 5.700 .+-. 1.934 Mean after (A) 6.349 .+-. 2.156 6.502 .+-. 1.940 7.259
.+-. 1.781 Difference (A - B) 0.671 1.093 1.559 Statistical test t-test t-test
t-test Probability of 0.139 0.023 <0.001 improvement (p) Significance NS S S
10. Results: Little Lines
[0169]Statistical survey information
related to little lines is depicted in Table 15 and FIG. 14B. No statistical
difference was found for the placebo, 1% by weight and 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream. However, note the relative
young age of the women participated in the study (mean age: 37.77) and the
values obtained that indicated few wrinkles. Another study with 3% by weight
IBR-DORMIN.RTM. Narcissus tazetta bulb extract cream used by women who put
values indicating real lines showed an improvement of this item.
TABLE-US-00016 TABLE 15 Little Lines Statistics CRITERIA PLACEBO 1%
CREAM 3% CREAM Number of women 48 43 44 Missing data 0 0 0 Mean before (B) 6.583
.+-. 2.268 5.990 .+-. 2.582 5.939 .+-. 2.771 Mean after (A) 6.946 .+-. 2.062
6.867 .+-. 2.571 6.877 .+-. 2.361 Difference (A - B) 0.491 0.53 0.938
Statistical test RST RST RST Probability of 0.395 0.167 0.126 improvement (p)
Significance NS NS NS
11. Results: Suppleness
[0170]Statistical
survey results related to skin suppleness are depicted in Table 16 and FIG. 14C.
Statistical difference was found for the placebo. Significant statistical
difference was found for the 1% by weight and the 3% by weight IBR-DORMIN.RTM.
Narcissus tazetta bulb extract creams.
TABLE-US-00017 TABLE 16
Suppleness Statistics CRITERIA PLACEBO 1% CREAM 3% CREAM Number of women 48 43
44 Missing data 1 1 1 Mean before (B) 6.430 .+-. 1.978 6.174 .+-. 1.920 5.860
.+-. 2.045 Mean after (A) 7.330 .+-. 1.437 7.362 .+-. 1.520 7.170 .+-. 1.850
Difference (A - B) 0.9 1.188 1.309 Statistical test RST t-test t-test
Probability of 0.022 0.002 <0.001 improvement (p) Significance S S(++) S(++)
12. Results: Percentage of Change
[0171]Statistical survey data
related to percent change is depicted in Table 17 and FIG. 15.
TABLE-US-00018 TABLE 17 Evolution after 4 weeks [(after - before)/before
.times. 100] ITEMS PLACEBO 1% CREAM 3% CREAM External Aggressions 17 22 55 Skin
Sensitivity 17 21 43 Protection 25 25 39 Skin Irritability 17 19 39 Skin Fatigue
21 20 33 Skin Tautness 15 14 25 Comfort 12 20 27 Little Lines 6 15 16 Suppleness
14 19 22
13. General Conclusions and Discussion
[0172]After four
weeks of daily use, the findings suggest that the women users were able to
recognize the cream containing IBR-DORMIN.RTM.B Narcissus tazetta bulb extract
as superior each time (FIG. 15). The effect was stronger for the group using the
3% by weight preparation (Table 17). The most striking effects are related to
skin resistance and protection, sensitivity and skin irritability. This could
lend weight to the hypothesis that better maturation enables the skin to better
fulfill its protective functions. Comfort, fatigue, tautness and suppleness also
improved. There was no significant change as regards to wrinkles in this
experiment. However, as regards to these parameters, note the relative youth of
the women (mean age: 37.77) in the sample and the values indicating few wrinkles
at the start of the study. According to the results obtained in this study
IBR-DORMIN.RTM. Narcissus tazetta bulb extract at a concentration of 3% by
weight is best for cosmetic use.
[0173]The present invention is not to
be limited in scope by the specific embodiments described herein. Indeed,
various modifications of the invention in addition to those described will
become apparent to those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall within the scope
of the appended claims. Various publications are cited herein, the disclosure of
which are incorporated by reference in their entireties.
* * * * *