U.S. patent application number 12/874167 was filed with the patent office on 2010-12-30 for methods of administering anti-inflammatory cyclooxygenase-2 selective inhibitors.
This patent application is currently assigned to Bioactives, Inc.. Invention is credited to Eric Kuhrts.
Application Number | 20100330215 12/874167 |
Document ID | / |
Family ID | 32711264 |
Filed Date | 2010-12-30 |
United States Patent
Application |
20100330215 |
Kind Code |
A1 |
Kuhrts; Eric |
December 30, 2010 |
Methods of Administering Anti-Inflammatory Cyclooxygenase-2
Selective Inhibitors
Abstract
Disclosed are novel anti-inflammatory pharmaceutical
compositions and related methods that exhibit potent and selective
inhibition of the cycloooxygenase-2 (COX-2) enzyme. The formulation
can comprise a hops extract that exhibits COX-2 selectivity as
defined by dividing the IC50 COX-2/IC50COX-1 concentrations that
are determined by testing with the William Harvey Whole Blood Assay
(WHMA), and can fall within the range of 0.011 to 0.2. Such
compositions may also optionally contain high levels of alpha acids
and low levels of beta acids, some flavonoid compounds, and
virtually no essential oils. Such compositions are useful for
treating conditions that manifest as inflammatory pain, or are
impacted by the COX-2 enzyme. The recited compositions are
particularly beneficial for treating osteoarthritis and rheumatoid
arthritis, and can be used for chronic pain with reduced gastric
side-effects.
Inventors: |
Kuhrts; Eric; (Bodega,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Bioactives, Inc.
Bodega
CA
|
Family ID: |
32711264 |
Appl. No.: |
12/874167 |
Filed: |
September 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11736551 |
Apr 17, 2007 |
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12874167 |
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10340183 |
Jan 9, 2003 |
7144590 |
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11736551 |
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Current U.S.
Class: |
424/778 ;
424/725 |
Current CPC
Class: |
A61K 31/12 20130101;
A61P 29/00 20180101; A61K 2300/00 20130101; A61K 31/12 20130101;
A61K 36/185 20130101 |
Class at
Publication: |
424/778 ;
424/725 |
International
Class: |
A61K 36/00 20060101
A61K036/00 |
Claims
1. A method of reducing inflammation in a warm-blooded animal,
comprising: formulating a hops extract comprising greater than 60
wt % of an alpha acid and from 0.5 wt % to 10 wt % of a beta acid;
and administering the hops extract to a warm-blooded animal.
2. The method of claim 1, wherein the step of administering is by
oral delivery.
3. The method of claim 1, wherein the step of administering is by
transmucosal delivery.
4. The method of claim 1, wherein the step of administering is by
parenteral delivery.
5. The method of claim 1, wherein the hops extract has an WHMA IC50
COX-2/IC50COX-1 ratio from about 0.011 to about 0.20.
6. The method of claim 5, wherein the hops extract has an WHMA IC50
COX-2/IC50COX-1 ratio from about 0.013 to 0.05.
7. The method of claim 5, wherein the hops extract has an WHMA IC50
COX-2/IC50COX-1 ratio from about 0.02 to 0.033.
8. The method of claim 1, wherein the alpha acid is present at from
75 Wt % to 99.5 wt %.
9. The method of claim 1, wherein the beta acid is present at from
1 wt % to 10 wt %.
10. The method of claim 1, wherein the alpha acid and beta acid are
derived from hops cone flowers.
11. The method of claim 1, wherein the alpha acid is selected from
the group consisting of humulone, cohumulone, adhumulone,
dihydrohumulone, dihydroadhumulone, and mixtures thereof.
12. The method of claim 1, further comprising an iso-alpha acid
selected from the group consisting of iso-humulone, iso-cohumulone,
iso-adhumulone, dihydro-iso-humulone, dihydro-iso-adhumulone, and
combinations thereof.
13. The method of claim 11, wherein the iso-alpha acid comprises a
member selected from the group consisting of trans-iso-humulone,
cis-iso-humulone, trans-iso-cohumulone, cis-iso-cohumulone,
cis-iso-adhumulone, trans-iso-adhumulone, and combinations
thereof.
14. The method of claim 1, wherein the hops extract is
substantially void of essential oils.
15. The method of claim 1, wherein the hops extract is formulated
to treat inflammatory pain by oral delivery with reduced gastric
side effects.
16. The method of claim 1, further comprising a flavonoid or
polyphenolic compound.
17. The method of claim 16, wherein the flavonoid or polyphenolic
compound is xanthohumol.
18. The method of claim 16, wherein the flavonoid or polyphenolic
compound is selected from the group consisting of isoxanthohumol,
8-prenylnaringenin, 6-prenylnaringenin, and combinations
thereof.
19. The method of claim 1, wherein the hops extract comprises
greater than 80 wt % alpha acid and not more than 10 wt % iso-alpha
acid.
20. The method of claim 1, said hops extract being administered in
a composition being devoid of myrcene, beta-caryophyleen,
undecane-2-on, and 2-methyl-but-3-en-ol.
21. The method of claim 1, wherein the step of administering
includes coadministering the hops extract with a pharmaceutical
carrier.
22. The method of claim 1, wherein the step of administering is by
a tablet, capsule, or suppository.
23. The method of claim 2, wherein the tablet, capsule, or
suppository is in a sustained release form.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/340,183, filed on Jan. 9, 2003, the
entirety of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Research into the mechanism of inflammatory pain led to the
discovery of the biochemical pathway associated with inflammation.
One such example of chronic inflammatory pain is osteoarthritis.
The elucidation of this pathway led to the association of the
pro-inflammatory prostaglandins, produced by the cyclooxegenase
enzyme, with pain and inflammation. The first generation of
anti-inflammatory pain relievers, were classified as non-steroidal
anti-inflammatory drugs or NSAIDs. Common NSAIDs such as aspirin,
ibuprofen, naproxen, and indomethacin inhibit the cyclooxygenase
enzyme, and thereby reduce inflammation by lowering the production
of prostaglandin E-2. Many new NSAIDs were developed over the last
30 years, most of which are available by prescription only.
[0003] Until the emergence of the discovery of a second form of the
cyclooxygenase enzyme, now called COX-2, there had been no
distinction made between the various NSAIDs, in terms of the
mechanism of action, and their effect. Only the magnitude of pain
relief, or the potency for inhibiting the COX enzyme was considered
important. However, side effects from the use of NSAIDs by patients
who suffer from chronic inflammatory pain began to emerge. The
principle side-effect was gastrointestinal toxicity, and it
manifested in the form of gastric erosion, or erosion of the
mucosal protective lining of the stomach. By the early 90s, as the
incidence of osteoarthritis and rheumatoid arthritis increased,
this side-effect became significant, leading to over 16,500 deaths
per year in the United States alone. A review article by Wolf, M et
al., Gastrointestinal Toxicity of Nonsteroidal Antiinfiammatory
Drugs, The New England Journal of Medicine, Vol. 340, No. 24,
1888-1899 (1999), is hereby incorporated by reference in its
entirety. According to this article, 13 of every 1000 patients with
rheumatoid arthritis who take NSAIDs for one year have a serious
gastrointestinal complication. According to data from the National
Center for Health Statistics and the Arthritis, Rheumatism, and
Aging Medical Information System, yearly deaths from NSAID toxicity
(1997) in patients suffering from rheumatoid arthritis or
osteoarthritis constitute the 15th leading cause of death in
America. This figure is similar to mortality from AIDS (16,685) and
only slightly less than deaths from Leukemia (20,197), but
considerably greater than the number of deaths from multiple
myeloma, asthma, cervical cancer, or Hodgkin's disease.
[0004] While most NSAIDs are more selective for the COX-1 form of
the enzyme, is they also inhibit the COX-2 form to varying degrees.
Some NSAIDs, such as indomethacin, reduce both COX-1 and COX-2 to
the same degree. Surprisingly, NSAIDs can also induce or
up-regulate COX-2.
[0005] The potency of NSAIDs to cause gastric erosion and rapidly
induce COX-2 can be illustrated by observing data from animal
studies in which COX-2 was induced in the rat stomach within 1 hour
of administration of aspirin or indomethacin. Both short term and
long term administration of NSAIDs have produced gastric erosion as
verified by endoscopy studies. Long term studies are defined as
NSAID ingestion for at least 3 months, but usually are done over
3-6 months.
[0006] In the late 90s, a new class of prescription drugs emerged
termed the COX-2 inhibitors. The first two compounds in this class
approved by the U.S. FDA were celecoxib and rofecoxib. These drugs
inhibited COX-2 with little or no effect on COX-1, and were
sufficiently potent to produce equivalent pain relief to other
NSIADs. While these compounds were no more effective than the NSAID
pain relievers, chronic use resulted in virtually little
gastrointestinal toxicity.
[0007] COX-2, or cyclooxygenase-2 inhibitors inhibit cyclooxygenase
and reduce prostaglandins without producing the degree of gastric
erosion associated with NSAID drugs such as aspirin. A COX-2
inhibitor selectively inhibits the COX-2 form of the enzyme more
than the COX-1 form. To be classified as a good COX-2 inhibitor, a
compound should inhibit COX-2 at least five times more than COX-1,
or should have at least a 5:1 ratio of COX-2 to COX-1. Preferably,
a COX-2 inhibitor should have an even greater selectivity than 5:1
for inhibiting COX-2, or from 5:1 to 100:1. A good COX-2 inhibitor
would be capable of producing a concentration level in the blood
that would reduce pain by 80 to 90% by inhibiting COX-2, with
little or no effect on the COX-1 form of the enzyme. The
terminology for quantifying the potency of a cyclooxygenase-2
inhibitor is the Inhibitory Concentration that produces a reduction
of prostaglandin E-2 by 50%, termed the IC50. An even better index
is the Inhibitory Concentration that produces an 80% reduction.
This is called the IC80. For purposes of this application, the term
IC80 shall refer to the concentration of the compound that produced
an 80% reduction in the principle pro-inflammatory cytokine or
prostaglandin, PGE-2. Conversely, the concentration of the compound
capable of producing an 80% inhibition of the COX-2 enzyme could
also be referred to as the IC80. Likewise, the 1050 shall mean the
concentration of the compound that produces a 50% reduction in
PGE-2, or a 50% reduction in the activity of the COX-2 enzyme.
[0008] In-vitro testing or screening of COX-2 inhibitors can be
conducted by measuring the inhibition of prostaglandin E-2, a
pro-inflammatory prostaglandin, in human whole blood. This results
in the calculation of the IC50 values, or the amount or
concentration of the compound needed to inhibit COX-2 by 50%, or
the IC80 value, the concentration of the compound necessary to
reduce prostaglandin E-2 by 80%. This testing model measures the
production of prostaglandin E2 (PGE2) by the COX-2 enzyme related
pathways, when stimulated by LPS or some other inducer of the COX-2
enzyme. COX-1 activity is also measured by measuring the production
of thromboxane (TxB2). Such assays are now considered to represent
a more complete in-vitro picture of COX-2/COX-1 selectivity and
potency.
[0009] An international group of scientists published a consensus
review related to COX-2 screening assays in: Brooks et al;
Interpreting the clinical significance of the differential
inhibition of cyclooxygenase-1 and cyclooxygenase-2, Rheumatology
1999; 38: 779-788. In this consensus paper, the committee stated
that the Human Whole Blood Assay developed by Patrignani et al (J
Pharmacol Exp Ther 1994; 271: 1705-12) was the best assay available
for assessing inhibition of COX-1 and COX-2, or evaluating new COX
inhibitors. More recently, the William Harvey Modified Human Whole
Blood Assay was developed as an extension of the original whole
blood assay, and most of the NSAID drugs, as well as the newer
COX-2 inhibitors have been screened using this method.
[0010] To determine the COX-2/COX-1 inhibitory activity according
to the invention the William Harvey Modified Human Whole Blood/Cell
Assay (WHMA) is used, as set forth in T. D. Warner et al.;
Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than
cyclo-oxygenase-2 are associated with human gastrointestinal
toxicity: A full in vitro analysis, Proc. Natl. Sci. USA 96:7563-68
(1999), hereby incorporated by reference in its entirety. The
results from this assay are used to calculate the IC50 and
IC80-WHMA COX-2/COX-1 ratio, which is simply the numerical ratio of
the COX-2 IC50 concentration divided by the COX-1 IC50
concentration, obtained using the WHMA. In addition, the potency of
the compound for reducing or inhibiting COX-2 is thereby
determined. This is done by measuring the inhibition of the two
isoforms of the enzyme at different concentrations of the
inhibitor, starting at very low concentrations, and increasing in a
log fashion until at least an 80% inhibition is produced. This
results in a log graph of the concentration versus inhibition
curve, or a dose response curve.
[0011] Numerous studies have shown that the relative incidence of
GI side effects from NSAIDs can be correlated to the relative COX-2
specificity of these anti-inflammatory agents. The higher the
specificity for COX-2 over COX-1, the lower the incidence of GI
upsets. Accordingly, cyclooxygenase inhibiting agents with
increased COX-2 specificity may provide improved anti-inflammatory
compositions having less incidences of gastrointestinal distress or
side effects. It is becoming increasingly apparent that the gastric
damage that can be caused by NSAIDs is not just related to their
effect on COX-1. Dual suppression of COX-1 and COX-2 seems to be
necessary for damage to occur (Wallace, J L et al., NSAID-Induced
Gastric Damage in the rat: Requirement for Inhibition of Both
Cyclooxygenase-1 and Cyclooxygenase-2. Gastroenterology, 2000;
119:706-14). Furthermore, selective inhibition of COX-1, which
greatly reduced prostaglandin synthesis, did not produce gastric
damage in the same study. On the other hand, selective inhibition
of COX-2 did not appear to have any effect on gastric prostaglandin
synthesis, and did not produce gastric damage.
[0012] Interestingly, when both COX-1 and COX-2 were inhibited,
gastric damage was consistently observed. This, and other research,
is providing a clearer picture of the relationship between COX-1,
COX-2, and gastric erosion. It now appears that combined inhibition
of COX-1 and COX-2 contribute to the side-effects, but more highly
selective inhibition of either COX-1 or COX-2 alone, is not
responsible.
[0013] However, too much selectivity for COX-2 over COX-1 may not
be desirable for other reasons. Certain side-effects may result
from COX inhibitors that are extremely selective for COX-2. For
example, the cardiovascular benefit of aspirin, a predominantly
COX-1 non-steroidal anti-inflammatory drug (NSAID), is thought to
be due to its activity as an anti-platelet aggregating drug. COX-2
inhibition does not result in anti-platelet aggregation. Current
pharmaceutical COX-2 inhibitors, such as celecoxib or rofecoxib,
are highly specific COX-2 inhibitors, and would not be expected to
have any COX-1 inhibitory activity at the doses used to reduce pain
and inhibit COX-2 activity. Thus, the cardiac-related side effects
that have been noted with the use of some COX-2 specific inhibitors
may be related to the lack of any COX-1 inhibition while
significantly inhibiting COX-2.
[0014] Furthermore, an additional problem associated with highly
specific COX-2 inhibitors is the increase in gastric erosion
produced by concurrent administration with other non-steroidal
anti-inflammatory drugs (NSAIDS). For example, if a patient is
taking a highly selective COX-2 inhibitor and also takes aspirin
for cardiovascular benefit, the aspirin will cause even worse
damage to the gastric mucosa. The reason for this is that some of
the prostaglandins that are inhibited by cyclooxygenase inhibitors,
such as prostaglandin E-2 (PGE2), are protective of the gastric
mucosa, and actually contribute to healing of ulceration. Low dose
aspirin produces small erosions in the stomach, and at the site of
these ulcerations, the COX-2 enzyme becomes up-regulated. When
COX-2 is blocked by selective COX-2 inhibitors, the protection
afforded by the beneficial prostaglandins is eliminated. The result
is that the ulcerative damage is made even worse. Concomitant
administration of selective COX-2 inhibitors with aspirin is
therefore contraindicated. This phenomenon is an indication of the
problems associated with the dual inhibition of both COX-1 and
COX-2. Thus gastric erosion will be worse with a single compound
that exhibits significant inhibition of both COX-1 and COX-2, or by
combining a COX-2 selective compound with a non-selective COX
inhibitor that also inhibits COX-1 to a large degree. The key to
overall risk reward benefits would be to have just the right amount
of COX-1 inhibition along with predominantly COX-2 inhibition. The
ratio of IC50COX-2/IC50COX-1 would be from about 1:5 to 1:100 or
numerically from 0.20 to 0.010. Preferably, the ratio of
IC50COX-2/IC50COX-1 would be at least 20:1 or numerically 0.05. For
example, a compound that is tested using the WHMA protocol might
have an IC50 for COX-2 of 1 .mu.g/ml and an IC50 for COX-1 of 20
.mu.g/ml, therefor, the IC50COX-2/IC50COX-1 ratio would be 1:20 or
0.05.
[0015] In summary, highly selective single entity. COX-2 inhibitors
such as rofecoxib and celecoxib, while important new drugs for the
treatment of pain associated with osteoarthritis and other
maladies, have some serious potential side-effects. These side
effects can be divided into two major groups; 1) cardiovascular,
and 2) worsening of gastric erosion when taken with aspirin or
other NSAIDS. Both of these side effects may be related to an
unbalanced total inhibition of the COX enzyme, and therefor,
virtually complete blocking of prostaglandin production. Because
prostaglandins have both positive and negative functions in the
body, their total inhibition is a double-edged sword. Furthermore,
there is a significant overlap in the patient populations that take
both aspirin for cardiovascular benefit, and a selective COX-2
inhibitor for pain. Most of these subjects primarily consist of the
elderly population. There is a significant need for
anti-inflammatory pain relief without the negative side effects of
the NSAIDs or the selective COX-2 inhibitors. Such a composition
would provide pain relief while also inhibiting platelet
aggregation, and providing protection for the gastric mucosa
through some gastroprotective or cytoprotective mechanism. These
second generation COX-2 inhibitors would be selective enough to
inhibit COX-2 over COX-1, but not so selective that they would
result in the additional side effects mentioned above. These
compounds may exhibit protective activity by virtue of the
existence of some other beneficial properties.
[0016] In the search for new anti-inflammatory compounds, many
potential candidates have come from the plant kingdom. These
botanicals are usually extracted and tested in-vitro for COX
inhibition using various cell lines and methods. Usually these
methods involve screening the compounds for COX-2 and COX-1
inhibition by measuring the inhibition of prostaglandin E-2 for
COX-2 inhibition, and TxB2 for COX-1 inhibition. Selectivity can
then be determined by calculating the COX-2/COX-1 ratio, or
conversely, the COX-1/COX-2 ratio.
[0017] It would be desirable to find compounds that exhibit good
selective COX-2 inhibition, but with the least amount of
cardiovascular or gastrointestinal side-effects. Such compounds
would result in a broader spectrum of therapeutic benefit, and be
tunable over a wide range of COX-2/COX-1 ratios, providing
effective pain relief with less side-effects. Such compounds could
provide some minimal amount of COX-1 inhibition for cardiovascular
benefit, without significant gastric erosion, while providing
significant COX-2 inhibition for pain.
[0018] What are needed are compositions and methods that address
the problems noted above.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
[0019] The present invention relates to compositions and methods
for reducing inflammation. In one embodiment, the invention relates
to a pharmaceutical composition comprising a therapeutic quantity
of a COX-2 inhibitor having an IC50-WHMA COX-2/COX-1 ratio ranging
from about 0.011 to about 0.20. An aspect of the present invention
would be decreased gastrointestinal and cardiovascular side
effects. An additional aspect is the treatment of a disease
impacted by the COX-2 enzyme, especially inflammation, or a disease
that is manifest in the up-regulation or induction of COX-2. Some
examples of such diseases include, but are not limited to:
osteoarthritis, rheumatoid arthritis, dysmenorrea, and psoriasis.
The compositions described herein may be used to treat any type of
inflammation or pain associated with inflammation.
[0020] Compositions are also described that consist primarily of
the alpha acids in hops, with little or no beta acids, and little
or no essential oils. Furthermore, compositions that contain the
various iso-alpha acids such as iso-humulone, iso-cohumulone,
iso-adhumulone, trans-iso-humulone, cis-iso-humulone,
trans-iso-cohumulone, cis-iso-cohumulone, trans-iso-adhumulone,
cis-iso-adhumulone, dihydro-iso-humulone, and combinations thereof,
may also be included.
[0021] In one aspect of the invention, an anti-inflammatory
composition of hops extract is provided including a
pharmaceutically acceptable amount of alpha acid and from 0.5 wt %
to 10 wt % of beta acid.
[0022] In a more detailed aspect of the invention, an
anti-inflammatory composition of hops extract having a WHMA IC50
COX-2/IC50 COX-1 ratio range from about 0.011 to 0.20 is
provided.
[0023] Yet another aspect of the invention includes a method of
reducing inflammation in a warm-blooded animal, including the steps
of formulating a hops extract comprising a pharmaceutically
effective amount of an alpha acid and from 0.5 wt % to 10 wt % of a
beta acid, and optionally, greater than 60 wt % alpha acid; and
administering the hops extract to a warm-blooded animal. In still
another aspect of the invention, the method includes the step of
formulating a hops extract having an WHMA IC50 COX-2 to COX-1 ratio
from about 0.011 to 0.20.
[0024] The present invention provides a composition that exhibits a
selective inhibition of the COX-2 isoform of the cycloxygenase
enzyme while having at least a minimal effect on the COX-1 isoform.
Minimal effect, for purposes of definition, shall mean at least 1%
COX-1 inhibition activity. For example, 1% inhibition of COX-1
would correspond to a ratio of IC50COX-2/IC50COX-1 of 1:100 or
0.010, in other words, it would take 100 times more of the compound
to inhibit COX-1 by 50% than the amount to inhibit COX-2.
[0025] As used in this application, the term IC50 or IC80 shall
mean the concentration of the compound or formulation that produces
a 50% or 80% is inhibition of COX-1 or COX-2 in the William Harvey
Modified Human Whole Blood/Cell Assay (WHMA).
[0026] As used in this application, WHMA shall be the abbreviation
for the William Harvey Whole Blood Assay.
[0027] As used herein, the concentration of the COX inhibitor shalt
be designated as either micrograms per milliliter (abbreviated as
.mu./ml) or micro molar (abbreviated as .mu.M).
[0028] As used herein, the COX ratios are calculated as the
IC50COX-2/IC50COX-1, or IC80COX-2/IC80COX-1, but COX-2 will always
be divided by COX-1.
[0029] As used herein, an oral dosage form shall mean a
pharmaceutical formulation designed to be administered orally,
consisting of various pharmaceutical carriers and excipients, and
can be in tablet, capsule, buccal, sublingual, or suppository
forms. Said oral dosage form will be absorbed in the human or
animal oral cavity, gastrointestinal tract or via suppository.
[0030] The oral dosage form described herein may also be formulated
in a sustained-release form, employing various polymers, fibers,
resins, waxes, oils, or other pharmaceutical excipients used by
those skilled in the art of medicinal chemistry to produce a
prolonged release of the active constituents from the
gastrointestinal tract. Such sustained-release dosage formulations
are designed to provide for a longer residence time of the compound
in the blood stream, thereby increasing the length of pain
relief.
[0031] One such candidate is a special extract of Humulus lupulus
L., or the plant commonly known as hops. Hops is derived from the
cone flowers of the hops plant, and has been used in the production
of beer for hundreds of years. Hops may exhibit some metabolic and
endocrine effects. There are at least six flavonoids that can be
isolated from hops, and some of these flavonoids have
antiproliferative, estrogenic, and cytotoxic effects. The
phytoestrogens in hops have also been shown to inhibit growth of
human breast cancer cells. The unique flavonoid compounds isolated
from hops (prenylated flavanoids) therefore may have potential as
cancer chemopreventative agents by effecting the metabolism of
carcinogens. The flavones contained in hops include xanthohumol,
isoxanthohumol, desmethyixanthohumol, 8-prenylnaringenin,
6-prenylnaringenin, and various other flavonoids. Hops also
exhibits antimicrobial and anti-fungal properties.
[0032] The primary constituents in hops consist of alpha acids and
beta acids. The alpha acids have been identified as humulone,
cohumulone, adhumulone, and dihydrohumulone. These alpha acids also
exist as dihydro-alpha acids and as various isomers. The iso-alpha
acids are iso-humulone, iso-cohumulone, iso-adhumulone,
trans-iso-humulone, cis-iso-humulone, trans-iso-cohumulone,
cis-iso-cohumulone, cis-iso-adhumulone, trans-iso-adhumulone,
dihydro-iso-humulone, and dihydro-iso-adhumulone. The beta acids
are lupulone, colupulone, adlupulone, prelupulone, and
postlupulone. There are no isomers of the beta acids. Hops also
contains various essential oils such as myrcene, caryophyllene,
humulene, undecane-2-on, and 2-methyl-but-3-enol. These oils can be
classified primarily as terpenes and sesqueterpenes. At least 50%
of the essential oils consist of the terpene, myrcene.
[0033] Topical application of humulone, one of the alpha acids
isolated from hops, inhibited arachidonic acid-induced inflammatory
ear edema in mice (Yasukawa, K et al, Oncology 1995, March; 52 (2):
156-158), and also inhibited skin tumor formation following
initiation with a chemical challenge. Pure humulon, has also been
shown to suppress cyclooxygenase-2 induction at the level of gene
transcription (Yamamoto K, et al, FEBS Lett 2000 Jan. 14, 465(2-3:
103-106). In this same study, humulone inhibited the catalytic
activity of COX-2 in osteoblast (bone) MC3T3-E1 cells with an IC50
of 1.6 .mu.M. Furthermore, humulone suppressed the
TNF-alpha-dependent cyclooxygenase-2 induction in the same cell
line. The direct inhibition of the COX-2 enzyme by humulone
required a greater concentration than the concentration necessary
to inhibit the gene transcription, or the suppression of COX-2
expression. Humulone appeared to be more effective at a lower
concentration in preventing the transcription or activation of
COX-2 by suppressing the gene transcription, than by direct
inhibition of the COX-2 enzymes catalytic activity. The IC50 for
suppression of COX-2 transcription was 30 .eta.M (10-9) whereas the
IC50 for direct inhibition of catalytic activity was 1.6 .mu.M
(10-6), or two orders of magnitude lower. Only pure humulone was
used in this study.
[0034] Special extracts of hops cone flowers can preferably be
prepared employing supercritical carbon dioxide. Supercritical CO2
extraction can result in extracts of hops that contain a very high
percentage of alpha acids, very little beta acids, and essentially
no essential oils. This invention, however, is not limited to the
extraction technique. Preferred amounts of alpha acids in the
instant invention can be from 75 wt % to 99.5 wt %. The alpha acids
may be humulone, cohumulone, adhumulone, dihydrohumulone, or
tetra-hydro-alpha acids such as tetra-hydrohumulone. The beta acids
can be from 1 wt % to 10 wt %, preferably around 3-5%, or even
less. Optionally, the composition may be substantially void of
essential oils such as myrcene or other terpenes or sesqueterpenes.
The composition may also contain iso-alpha acids to varying
degrees. Usually the level of iso-alpha acids will be from 0.5 to
10%. The iso-alpha acids may be iso-humulone, iso-cohumulone,
iso-adhumulone, di-hydro-iso-humulone, di-hydro-iso-adhumulone or
combinations thereof. The iso-alpha acids may be useful for tuning
the selectivity for COX-2 in the formulation, by boosting the COX-1
component, and changing the COX-2/COX-1 ratio to be less selective
for COX-2. The reason for these type of formulas may be to address
cardiovascular issues by contributing some anti-platelet
aggregation activity from the COX-1 inhibition via thromboxane.
[0035] The IC50 ratio of COX-2 to COX-1 or the IC50COX-2/IC50COX-1
can be in the range of 0.011 to 0.20, or from 1:90 to 1:5. The
percentage of alpha acids can be from 60% to 99.5%, but is not
limited to this range if the IC50COX-2/IC50COX-1 ratios fall within
the specified ranges of 0.011 to 0.20. As mentioned, the iso-alpha
acids may be from 0.5 wt % to 10 wt %. The beta acids range can be
from 0.5 wt % to 10 wt %. Various polyphenols or flavonoids may be
present such as xanthohumol, isoxanthohumol, 8-prenylnaringenin,
6-prenylnaringenin in varying amounts.
[0036] The compositions in this invention are not limited to the
amount of alpha acids and beta acids, or the method of processing
or extraction. For example, a hops extract that exhibits a WHMA
IC50COX-2 over IC50COX-1 ratio of 0.013 (1:75) would be part of
this invention regardless of the method of processing or the amount
of alpha acids, beta acids, or essential oils. Powders of hops can
be made which exhibit IC50COX-2/COX-1 ratios of 1:20 or 0.05, which
provide good pain relief from chronic osteoarthritis or rheumatoid
arthritis. Such compositions can also be useful for treating
dysmenorrhea or menstrual pain, psoriasis, and other diseases
impacted by COX-2.
EXAMPLES
[0037] In-vitro testing or screening of the recited COX-2
inhibitors can be conducted by measuring the inhibition of
prostaglandin E-2, a pro-inflammatory prostaglandin. This results
in the calculation of the IC50 and/or IC80 values, or the amount or
concentration of the compound needed to inhibit COX-2 by 50% and/or
80%. This model measures the production of prostaglandin E2 (PGE2)
by the COX-2 enzyme related pathways, when stimulated by LPS in an
in-vitro cell line model. However, the human whole blood assay has
been deemed the method of choice by a panel of experts for
assessing and screening COX inhibitors (Brooks, P et al,
Interpreting the clinical significance of the differential
inhibition of cyclooxygenase-1 and cyclooxygenase-2, Rheumatology;
1999; 38: 779-788). Such assays are now considered to represent a
more complete in-vitro picture of COX-2/COX-1 selectivity and
potency. A modified version of the human whole blood assay called
the William Harvey Modified Human Whole Blood Assay has been
selected as one of the best models for testing the compositions
described herein. To determine the COX-2/COX-1 inhibitory activity
according to the invention, the William Harvey Modified Human Whole
Blood/Cell Assay (WHMA) is used, as set forth in T. D. Warner et
al., Nonsteroid drug selectivities for cyclo-oxygenase-1 rather
than cyclo-oxygenase-2 are associated with human gastrointestinal
toxicity: A full in vitro analysis, Proc. Natl. Sci. USA 96:7563-68
(1999). The results from this assay are used to calculate the
IC50-WHMA COX-2/COX-1 ratio, which is simply the numerical ratio of
the COX-2 IC50 divided by the COX-1 IC50 ratio, obtained using the
WHMA.
[0038] Human whole blood (8 concentrations, n=4) is collected by
venapuncture into heparin. For determining COX-1, incubation of
test compound(s) was carried out for 1 hour, with addition of
stimulus (A23187) for 30 minutes. For COX-2, incubation of test
compounds in A549 cells in human whole blood was carried out for 1
hour, addition of stimulus (A23187) for 30 minutes. Following this,
TxB2 is measured by RIA as an index of COX-1 activity, and PGE2 is
measured by RIA as an index of COX-2 activity. The results are
expressed as % control, and the COX-2/COX-1 ratio is
calculated.
Example 1
COX-2 Inhibition Activity of Hops
[0039] A supercritical carbon dioxide extract of hops was produced
that yielded 91% alpha acids, of which the principle alpha acid was
humulone as verified by HPLC. The amount of beta acids in this
extract was verified to be 3.2% and the amount of iso-alpha acids
was about 3%. This extract was virtually devoid of the essential
oils normally found in a typical hops powder or extract.
Hops extract constituents as identified by HPLC are as follows:
TABLE-US-00001 Alpha acids 88% Beta acids 3.2% Iso-alpha acids 3%
Total alpha acids 91% (alpha and iso-alpha)
[0040] This extract was dissolved in DMSO and tested according to
the protocol described above.
[0041] The effects of test agents on COX-1 and COX-2 activity are
detailed in Tables 1-3. Results in Tables 1-3 are expressed as %
control and shown as mean.+-.s.e.m. (n=4) from which IC.sub.50
values were calculated (Table 3).
TABLE-US-00002 TABLE 1 COX-1 activity in human whole blood
(TxB.sub.2, % control) Concentration (log M) -10 -9 -8 -7 -6 -5 -4
-3 Hops 100 .+-. 6 103 .+-. 6 100 .+-. 5 94 .+-. 4 91 .+-. 5 80
.+-. 10 60 .+-. 15 18 .+-. 2 extract
TABLE-US-00003 TABLE 2 COX-2 activity in A549 cells in human whole
blood (PGE.sub.2, % control) Concentration (log M) -10 -9 -8 -7 -6
-5 -4 -3 Hops 97 .+-. 6 95 .+-. 6 87 .+-. 8 61 .+-. 8 54 .+-. 10 42
.+-. 10 16 .+-. 3 6 .+-. 2 extract Ibuprofen 85 .+-. 16 82 .+-. 10
80 .+-. 9 80 .+-. 5 60 .+-. 4 20 .+-. 5 4
[0042] As can be seen from tables 1 and 2, a rather large amount of
the hops extract is necessary to reduce COX-1 by 50% (the IC50 is
10 -3 to 10 -4), whereas, for COX-2, the concentration needed for
50% reduction is 10 -6 (IC50 was 1.4.times.10 -6 .mu.M). It takes
about 100 times as much of this 91% alpha acid hops extract to
reduce COX-1 by 50% as the concentration needed to reduce COX-2
activity by 50%. Ibuprofen is included for comparison. The hops
extract to was more potent and selective than ibuprofen for
inhibition of COX-2.
TABLE-US-00004 TABLE 3 Potencies of test agents on human COX-1 and
COX-2 (WHMA) IC.sub.50 (.quadrature.M) IC.sub.50 ratio COX-1 COX-2
(COX-2/1) Aspirin 1.7 7.5 4.4 Ibuprofen 7.6 20 2.6 Naproxen 9.3 35
3.8 Hops extract 110 1.4 0.0127 Celexocib 2.2 0.34 0.3 Rofecoxib 63
0.31 0.0049 Indomethacin 0.0031 0.021 7
[0043] Table 3 is a comparison of the 91% alpha acid containing
hops extract that was derived by supercritical carbon dioxide, with
other known COX inhibitors, including the prescription COX-2
selective inhibitors rofecoxib and celexocib, as well as the non
selective COX inhibitors aspirin, ibuprofen, naproxen and
indomethacin. As can be seen from the above data, hops extract is a
selective and potent COX-2 inhibitor when tested according to the
WHMA protocol. The IC50 COX-2 concentration of this hops extract
was about 1.4 .mu.M, a concentration that would be within a range
of pharmacological action of most non-steriodal anti-inflammatory
drugs, whereas the IC50 COX-1 concentration was about 110 .mu.M.
The ratio was therefor 0.0127 or 1:90.
TABLE-US-00005 TABLE 4 Potencies of test agents on human COX-1 and
A549 COX-2 (WHMA) IC.sub.50 (.mu.M) IC.sub.80 (.mu.M) ratios
(COX-2/1) COX-1 COX-2 COX-1 COX-2 IC.sub.50 IC.sub.80 Hops extract
110 1.4 >1000 85 0.01 <0.09 indomethacin 0.0069 0.055 0.027
0.21 8 8
[0044] Table 4 includes the IC80 data for the special hops extract
compared to indomethacin, a potent non-selective NSAID that lowers
COX-1 and COX-2 at a very low concentration. As can be seen from
table 4 above, the IC80 for COX-2 was about 85 .mu.M, and the IC80
COX-2/1 ratio was about 0.09 .mu.M, which is a ratio of about 1:11.
While not as potent as indomethacin, the selectivity for COX-2 is
much greater.
[0045] While the resin used in the above experiment resulted in
significant inhibition of the COX-2 enzyme with very little effect
on the COX-1 form, such a resin is difficult to use in a
pharmaceutical dosage form without converting to a powder. When
converted to a powder, various excipients must be used as carriers
which tend to dilute the potency of the resin, thereby reducing the
IC50 or IC80 by about 50%, and also enabling various mixtures of
alpha acids and iso-alpha acids to be employed.
Example 2
COX-2 Inhibition of a Hops Resin Converted to Powder
[0046] A hops resin is converted to powder using maltodextrin and
calcium silicate in a jacketed high intensity mixer. The resulting
powder was analyzed by HPLC and found to yield the following
principle constituents:
TABLE-US-00006 Alpha acids 20% Iso-alpha acids 9.4% Beta acids
8%
[0047] This powder was tested for COX-2 and COX-1 inhibition in a
cell line in whole blood by inducing COX-2 with LPS
(lipopolysaccharide) and measuring PGE-2 for COX-2 activity. COX-1
activity was assessed by measuring TxB2 (thromboxane B2). IC50
results for COX-2 and COX-1 were;
TABLE-US-00007 IC50 COX-2 IC50 COX-1 IC50COX-2/IC50COX-1 1 .mu.g/ml
30 .mu.g/ml 0.033 (1:30)
Example 3
[0048] This example is to demonstrate the reduction in gastric
erosion of a hops formulation versus a traditional non-selective
COX inhibitor NSAID such as aspirin. An oral formulation of a
tablet containing 750 mg of a hops extract powder consisting of 30%
alpha acids (225 mg alpha acids) is administered to 40 subjects in
a single blind, parallel-group, multiple dose study. The patients
are randomly assigned to treatment with either the hops alone or
1,000 mg aspirin per day for 4 days. Assessments are made based on
endoscopic evaluations of gastroduodenal irritancy performed 4
hours after the first dose and 3 hours after the final
administration on the fourth study day. To assure that all study
subjects had normal healthy gastroduodenal mucosa at baseline, an
endoscopic evaluation is also performed before subjects are
randomized.
[0049] Endoscopy is used to assess the extent and severity of
gastric and duodenal damage. During an endoscopic examination of
the stomach and duodenum, the number and location of submucosal
hemorrhages, erosions, and ulcerations, are determined by the
endoscopist. Based on the findings, the hemorrhagic damage is
graded on a scale of 0-4 and the erosive damage is graded on a
separate 0-4 scale. The stomach and duodenum are graded separately.
Under the test conditions described above, endoscopic evaluation is
expected to reveal virtually no gastric erosion form the hops
formulation, while the aspirin formulation exhibited significant
gastric submucosal hemorrhages and overall gastric erosions. The
difference between the two groups is expected to be statistically
significant. The reduction in gastric erosion of the hops
formulation in comparison to aspirin is believed to be related to
its selectivity for COX-2 over COX-1.
[0050] While the present invention is described above in connection
with the preferred or illustrative embodiments, those embodiments
are not intended to be exhaustive or limiting of the invention, but
rather, the invention is intended to cover any alternatives,
modifications, or equivalents that may be included within its scope
as defined by the appended claims.
* * * * *