Source: https://patents.google.com/patent/US7901713B2/en
Timestamp: 2018-10-22 15:40:28
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Matched Legal Cases: ['§119', 'application No. 60', 'application No. 60', '§119', 'application No. 60', 'application No. 60', 'art 2003', 'Application No. 02737562', 'Application No. 02784313', 'Application No. 05851567', 'Application No. 05723895']

US7901713B2 - Inhibition of COX-2 and/or 5-LOX activity by fractions isolated or derived from hops - Google Patents
Inhibition of COX-2 and/or 5-LOX activity by fractions isolated or derived from hops Download PDF
US7901713B2
US7901713B2 US10866315 US86631504A US7901713B2 US 7901713 B2 US7901713 B2 US 7901713B2 US 10866315 US10866315 US 10866315 US 86631504 A US86631504 A US 86631504A US 7901713 B2 US7901713 B2 US 7901713B2
US10866315
US20090118373A1 (en )
Matthew L. Tripp
Gary K. Darland
John G. Babish
A natural formulation of compounds isolated or derived from hops which inhibit the activity of cyclooxygenase-2 (COX-2) and/or 5-lipoxygenase (5-LOX) is disclosed. The hops formulations may be administered to a mammal to treat or inhibit a pathological condition associated with the activity of COX-2 and/or 5-LOX in such a mammal.
This patent application is a continuation-in-part of U.S. application Ser. No. 10/689,856, filed Oct. 20, 2003, now U.S. Pat. No. 7,270,835, which is a continuation-in-part of U.S. application Ser. No. 10/464,410, filed Jun. 18, 2003, which is a continuation-in-part of U.S. application Ser. No. 10/400,293, filed Mar. 26, 2003 (abandoned), and a continuation-in-part of U.S. application Ser. No. 10/401,283, filed Mar. 26, 2003 (abandoned), both of which claim the benefit under 35 U.S.C. §119(e) to provisional application No. 60/450,237, filed on Feb. 25, 2003, and provisional application No. 60/420,383, filed on Oct. 21, 2002; and is a continuation-in-part of U.S. patent application Ser. No. 10/464,834, filed Jun. 18, 2003, which is a continuation-in-part of U.S. application Ser. No. 10/400,293, filed Mar. 26, 2003 (abandoned), and a continuation-in-part of U.S. application Ser. No. 10/401,283, filed Mar. 26, 2003 (abandoned), both of which claim the benefit under 35 U.S.C. §119(e) to provisional application No. 60/450,237, filed on Feb. 25, 2003, and provisional application No. 60/420,383, filed on Oct. 21, 2002. This application is also a continuation-in-part of U.S. application Ser. No. 09/885,721, filed Jun. 20, 2001, now U.S. Pat. No. 7,205,151. The contents of each of these earlier applications are hereby incorporated by reference as if recited herein in their entirety.
This invention primarily relates to the method and use of fractions isolated or derived from hops as inhibitors of COX-2 and/or 5-LOX activity, particularly reduced isoalpha acids (RIAA), isoalpha acids (IAA), tetrahydroisoalpha acids (THIAA), hexahydroisoalpha acids (HHIAA), alpha acids, beta acids, spent hops, and hop essential oils.
Thrombosis—the current most common cause of ischaemic cardiovascular disease (CVD) such as myocardial infarction and stroke—is the late complication of atherosclerosis, a progressive inflammatory disease characterized by lipid infiltration in the wall of large arteries (atherosclerotic plaques). Platelet and leukocyte recruitment on endothelial cells constitutes an early mechanism of vascular inflammatory damage and consequent vessel occlusion. The increasing appreciation of the role of inflammation in atherosclerosis and thrombosis has renewed interest in the possibility that anti-inflammatory compounds might be effective in the prevention of CVD. Such an intriguing possibility was first raised when acetylsalicylic acid (aspirin) was shown to reduce platelet aggregation induced by several physiological stimuli. Because platelet aggregation was known to play a crucial role in thrombosis, it was anticipated that the newly described anti-aggregating activity of aspirin (at that time, a 70-year-old anti-inflammatory drug) might translate to a clinical benefit in CVD. Aspirin was then tested in dozens of clinical trials and was shown to reduce, by approximately 25%, both primary and secondary incidence of myocardial infarction and other CVDs. However, the gastric side-effects (mainly haemorrhagic) associated with aspirin limited its widespread clinical use for the prevention of cardiovascular events.
Prostacyclin (PGI2), another metabolite of arachidonic acid produced by the action of COX-1, has been identified in endothelial cells. Because PGI2, in contrast to TxA2, inhibits platelet aggregation, doubts were raised about the clinical potential of aspirin as an anti-thrombotic drug. The assumption was made that to achieve full anti-thrombotic efficacy, the inhibitory effect of aspirin on platelet COX-1 should be retained while that on vascular COX-1 should be minimized (the so-called ‘aspirin dilemma’). Low-dose aspirin (75-100 mg, daily, p.o., in healthy volunteers), which is virtually devoid of a measurable anti-inflammatory effect, was shown to abolish platelet TxA2 generation while leaving vascular PGI2 formation almost intact. However, the epidemiological observation that any dose of aspirin tested (between 30 and 1500 mg, daily, including the highest doses that inhibit both TxA2 and PGI2 generation) was equally effective as an anti-thrombotic, led many researchers to believe that inhibition of platelet COX-1 was indeed the crucial target of aspirin, with concomitant vascular COX-1 suppression having minor, if any, clinical relevance.
The invention provides a method of treating or inhibiting a pathological condition in a mammal involving inhibiting inducibility or activity of cyclooxygenase-2 (COX-2) and/or 5-lipoxygenase (5-LOX), the method comprising administering to the mammal a composition comprising a fraction isolated or derived from hops. The fraction isolated or derived from hops may be selected from the group consisting of alpha acids, isoalpha acids, reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids, beta acids, hop essential oils, and spent hops.
FIG. 1 is a bar graph representing the percent inhibition exhibited by 1, 5, or 10 μg agent/mL for the seven test materials and the Trolox positive control. Each of the three doses is represented by a bar with increasing concentration from left to right. The dark bars indicate those concentrations significantly different from the negative control (p<0.5).
FIG. 2 depicts relative change in DCF fluorescence over time in the Jurkat LPS-H2O2 oxidative stress model.
The present invention provides compositions, methods, and uses of fractions isolated or derived from hops to inhibit COX-2 and/or 5-LOX activity.
As used herein, the term “spent hops” refers to the solid and hydrophilic residue from the extraction of hops.
As used herein, the term “reduced isoalpha acid” (also sometimes referred to as dihydroisoalpha acids or rho-isoalpha acids) refers to alpha acids isolated from hops plant product and which subsequently have been isomerized and reduced, including cis and trans forms. Examples of reduced isoalpha acids (RIAA) include, but are not limited to, dihydro-isohumulone, dihydro-isocohumulone, and dihydro-isoadhumulone.
Tetrahydroiso-alpha-acids (tetrahydroisohumulones) usually are prepared from the beta-acids (or lupulones) in hop extracts. The hop extracts also contain alpha-acids (or humulones) but they are not normally used to make tetrahydroiso-alpha-acids for economical reasons. Alpha-acids and beta-acids are often referred to as “soft resins”. The alpha-acids consist of three major analogs: cohumulone, humulone and adhumulone. Beta-acids consist of three major analogs: colupulone, lupulone and adlupulone. Tetrahydroiso-alpha-acids can be prepared from either alpha-acids or from beta-acids which results in three analogs and two diastereoisomers. They are cis and trans-isomers of tetrahydroiso-cohumulone (THICO), tetrahydroiso-humulone (THISO) and tetrahydroiso-isoadhumulone (THIAD).
Cowles, et al., U.S. Pat. No. 4,644,084, disclose a process for making tetrahydroiso-alpha-acids by treating beta-acids to form desoxytetrahydro-alpha-acids which are dissolved in an aqueous alcoholic caustic solution and then oxidized with an oxygen-containing gas to form the desired tetrahydroiso-alpha-acids. The Cowles, et al. process does not use undesirable organic solvents and is superior to other known processes using beta-acids.
Hydrogenation and hydrogenolysis are well-known processes which are commonly employed in many organic chemical synthesis schemes, including the manipulation of lupulones and humulones, and their derivatives. Usually, low molecular weight organic compounds are used as solvents (C.sub.1-C.sub.6). For example, Carson, 73 J. Am. Chem. Soc. 1850-1851 (1951), discusses the hydrogenation of lupulone and humulone using methanol as a solvent. Anteunis, et al., Bull. Soc. Chim. Belg. 476-483 (1959), disclose carrying out the hydrogenation of humulone in methanol or ethanol.
Examples of compounds of an ingredient isolated or derived from hops, include, but are not limited to, humulone, cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone, dihydro-isoadhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-isoadhumulone, hexahydro-isohumulone, hexahydro-isocohumulone, and hexahydro-isoadhumulone. The preferred compounds can bear substituents, as shown in the formula above.
EXAMPLE 1 Inhibition of 5-Lipoxygenase Activity by Derivatives of Alpha-Acids from Hops (Humulus lupulus)
Test Materials and Reagents—Standardized (see Table 2) aqueous solutions of fractions isolated or derived from hops (Humulus lupulus) were obtained from BetaTech (Washington, D.C.). The solutions were diluted into DMSO to contain 1 mg/ml of the reference compounds. If necessary, the sample was clarified by centrifugation at 12000×g for 5 minutes. For testing, serial dilutions were made in DMSO. The Lipoxygenase Inhibitor Screening Assay Kit (LISAK) from Cayman (#760700, Chicago, Ill.) was used to assess the effects of test material on lipoxygenase activity. Included with the kit were soybean 15-lipoxygenase (#60700), and linoleic acid. Potato 5-lipoxygenase (#60401) was purchased from Cayman separately. Positive control compounds included caffeic acid (Cayman #70602), Trolox (Sigma 238813) and Rev 5901 (Sigma R5523); these were of the highest purity commercially available. Boswellin (RM07781) was provided by Metagenics, Inc., Gig Harbor, Wash.).
Description of hop preparations† tested.
Hop Concen-
Preparation Variety HPLC Analysis (w/w) tration
Alpha Hop Galena 82.1% α-acids, 2.7% β-acids, 1% (w/v)
3.0% Isomerized α-acids. α-acids
Beta Acids Galena 9.5-10.5% β-acids, 1% (w/v)
<.2% α-acids β-acids
Aromahop OE Galena + 25-30% Oil, ~10% β-acids,
Nugget <.2% Isohop
Isohop Galena 25.3% Isomerized α-acids 1% (w/v)
(IAA) IAA
Redihop Galena 30% α-acids 1% (w/v)
Tetrahop Galena 8.9% THIAA 1% (w/v)
Hexahop Gold Galena 3.9% THIAA, 4.4% HHIAA 1% (w/v)
†Obtained from Betatech Hops Products, Washington, DC; IAA = isomerized alpha-acids; THIAA = tetrahydro-isoalpha-acids; HHIAA = hexahydro-isoalpha-acids.
Assay—The 5-lipoxygenase (5-LOX) assay and calculations were performed in accordance with the manufacturer's protocol. Briefly, assay buffer was prepared by diluting the contents of LISAK vial #1 with nine parts of HPLC grade water to yield a final concentration of 0.1M Tris-HCl (pH 7.4). 5-LOX was diluted into assay buffer so that the final reaction rate was approximately 10 nmol min−1 mL−1.
The 5-LOX reaction was initiated by adding 10 mL linoleic acid to a reaction mixture consisting of 90 mL of diluted enzyme (or assay buffer for the reaction blank), 10 mL assay buffer and 10 mL of test inhibitor or DMSO. After 5 minutes at room temperature, the reaction was terminated by the addition of 100 mL of the proprietary LISAK chromagen, prepared by mixing equal amounts of LISAK vials 2 and 3. The absorbance was measured with a 492 nm (8 nm bandwidth) filter in a Victor™ Multilabel Counter equipped with an absorbance package (Perkin Elmer #1420-042, #1420-115; Boston, Mass.). The reaction rate was determined as follows:
ΔA min−1=(Absrx−Absenzyme blank)/5 minutes
Calculations—To assess the probability that the 5-LOX activity of enzyme plus test material was different from the solvent control enzyme activity, the lower 95% confidence limit was computed for the mean of all solvent control values. This lower limit represented a difference of 7.2 percent from the solvent average. Thus, means of duplicate determinations of 5-LOX enzyme plus test material that demonstrated a reduction of enzyme activity greater than 7.2 percent were considered significantly (p<0.05) different from the solvent controls.
We have demonstrated that the chemically modified α-acids from hops inhibit the activity of potato 5-LOX. Unexpectedly, while neither of the native α- or β-acids affected the enzyme until the highest concentration tested, the derivatives of the alpha-acids exhibited significant inhibition at 5 μg/mL, in line with the potent positive control Trolox (Table 3 and FIG. 1). Aromahop OE, an oil fraction of hops, also inhibited 5-LOX at the 5 μg/mL concentration. Tetrahop and Hexahop tended to increase in effectiveness more rapidly than Isohop and Redihop. The hops derivatives were similar in inhibitory activity to Trolox at 5 and 10 μg/mL. However, the synthetic 5-LOX inhibitor was more than twice as active as the natural compounds at the non-physiological 50 μg/mL concentration (data not presented). This finding indicates a steeper dose-response curve for Trolox compared to the hops derivatives. However, at the more physiologically relevant, lower concentrations, the hops derivatives and Trolox expressed similar inhibitory activity of 5-LOX.
activity at physiologically relevant concentrations†
Test Material 1 μg/ml 5 μg/ml 10 μg/ml
Alpha Hop 6.7 6.5 4.7
Beta Stab 0.0 0.0 2.7
Aromahop OE 5.2 13‡  17
Isohop 3.3 9.8‡ 17
Redihop 2.9 8.2‡ 15
Tetrahop Gold 3.3 12‡  21
Hexahop Gold 3.7 11‡  21
Caffeic acid 0.0 0.0 0.0
Boswellin 2.8 2.2 2.4
Rev5901 2.2 0.8 0.0
Trolox 0.5 13‡  27
†Means of duplicate determination relative to control activity of 14 nmol/min/mL, respectively, over the three sets of experiments.
‡The lowest concentration at which inhibition was significantly (p < 0.05) greater than the solvent control; minimum inhibition required for statistical significance (p < 0.05) was 7.2%.
Test Material IC25 †
Alphahop >50
Beta Stab >50
Aromahop 20 (10-39)
Redihop 29 (14-61)
Isohop 24 (17-33)
Tetrahop 17 (12-22)
Hexahop 18 (15-23)
Caffeic acid >50
Boswellin >50
Trolox 4.2 (2.6-6.9)
†values presented as μg/mL; parenthetic values are 95% confidence intervals.
EXAMPLE 2 Antioxidant Activity of Hops Fractions
Chemicals and reagents—Bacterial lipopolysaccharide (LPS; B E. coli 055:B5) was from Sigma (St. Louis, Mo.). 6-Carboxy-2′,7′-dichlorofluorescin diacetate (DCFH-DA) was purchased from Molecular Probes Inc. (Eugene, Oreg.); DCFH-DA was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and kept frozen at −20° C. For loading the cells, DCFH-DA from the stock solution was mixed with loading medium (99% RPMI and 1% FBS (fetal bovine serum)) to a final concentration of 20 μM. Standardized aqueous solutions of fractions isolated or derived from hops (Humulus lupulus) were obtained from BetaTech (Washington, D.C.) and are as described in Example 1 Table 1. Positive control compounds included caffeic acid and Trolox obtained from Sigma (St. Louis, Mo.) and were of the highest purity commercially available. Test compounds were dissolved in DMSO to deliver at the maximal concentration in cell cultures of 0.1% (v/v). Unless otherwise noted, all standard reagents were obtained from Sigma and were the purest commercially available.
Cell culture—Jurkat cells (human T cells) were obtained from the American Type Culture Collection (ATCC Number TIB-152, Manassas, Va.) and sub-cultured according to the instructions of the supplier. The cells were routinely cultured at 37° C. with 5% CO2 in RPMI 1640 containing 10% FBS, with 50 units penicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5% L-glutamine.
Jurkat T cells were grown and maintained in RPMI-1640 supplemented with 10% fetal calf serum. 6-Carboxy-2′,7′-dichlorofluorescin diacetate (DCFH-DA) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and kept frozen at −20° C. For incorporating DCFH-DA into cells, exponentially growing cells were treated with DCFH-DA in loading medium (99% RPMI and 1% FBS) at a final concentration of 20 μM for 15 minutes. Excess DCFH-DA was removed by centrifuging the cells for 10 minutes and resuspending them in the original growth medium. Cells were plated in microtiter wells at a concentration of approximately 106 cells per well. Fifteen minutes after the addition of the test compounds, LPS was added in 20 μL to each test well to achieve a final concentration of 1 μg/mL. Twenty μL of LPS-free solution were added to background control wells containing DCFH2-loaded cells. H2O2 was then added to each well in a volume of 20 μL to achieve final concentrations of 125, 250, 500 or 1000 μM per well. Background cells contained only DCFH-DA and oxidized controls contained DCFH-DA plus LPS and H2O2. Microtiter plates were placed in a Packard FluoroCount microplate fluorometer equipped with a temperature-controlled plate holder maintained at 37° C. The excitation filter was set at 485 nm and the emission filter was set at 530 nm. Fluorescence for each well was captured, digitized and stored on a computer using Cytofluor (Version 4.0). Measurements of fluorescence were made every ten minutes for 60 minutes. The change in DCF fluorescence is presented relative to the LPS-stimulated Jurkat cells alone; LPS treatment exhibited no increase in fluorescence measurements over the 60-minute experimental period.
The murine macrophage cell line RAW 264.7 was obtained from ATCC (TIB-71) and sub-cultured according to instructions from the supplier. For experiments, cells were cultured in DMEM (Dulbecco's Modification of Eagle's Medium) containing 10% FBS-HI (fetal bovine serum, heat inactivated), with added 50 units penicillin/mL and 50 μg streptomycin/mL, maintained in log phase in T-75 flasks (Corning, Corning, N.Y.) prior to experimental setup. Cells were maintained in a 5% CO2 humidified incubator at 37° C.
For experiments, exponentially growing Jurkat cells were harvested by centrifuging at 6,000 rpm for 10 minutes in 50 mL conical tubes, washing with serum-free RPMI 1640 and centrifuging again at 6,000 rpm. The supernatant fraction from the second centrifugation step was discarded and the cells were resuspended to 1×106 cells/mL for loading with DCFH-DA at a concentration of 20 μM for 30 minutes at 37° C. in 5% CO2. DCFH-DA loading medium was removed by centrifugation; cells were washed with serum-free RPMI medium and aliquoted to microtiter wells at 106 cells per well. Non-DCFH2 control wells (i.e., microtiter wells that do not contain the DCFH2 fluorescent indicator) were made using serum-free RPMI 1640 and DMSO in place of loading medium.
Adherent RAW 264.7 cells were loaded with DCFH-DA at a concentration of 20 μM for 30 minutes at 37° C. in 5% CO2. in the T-75 flask after removing the growth medium. The DCFH-DA medium was discarded and the cells were washed with PBS (phosphate-buffered saline), scrapped and aliquoted to microtiter wells at 106 cells per well. Non-DCFH2 control wells were made using serum-free RPMI 1640 and DMSO in place of loading medium.
Antioxidant activity of the test materials—Oxidative stress was measured in cells using DCFH-DA in a microtiter plate assay as previously described [Wang, H. and Joseph, J. A. (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Biology & Medicine 27:612-616] incorporated herein by reference. Based upon the conversion of the non-fluorescent 2′,7′-dichlorofluorescin (DCFH2) to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) by various free radicals, this indiscriminate probe produces concentration dependent changes in cellular fluorescence. Thus, the assay can be used to quantify overall oxidative stress in cells.
All RFU (relative fluorescence unit) measurements were preformed in a 96-well polypropylene plate with stirring; temperature was maintained at 37° C. The excitation filter was set at 485 nm and the emission filter was set at 530 nm. Fluorescence for each well was captured, digitized and stored on a computer using Cytofluor (Version 4.0). Data points were taken every 10 minutes for 60 minutes. Complete assays were exported to an Excel (Microsoft, Seattle, Wash.) spreadsheet for analysis.
Data handling and calculation of the median inhibitory concentration (IC50)—The extent of oxidation of intracellular DCFH2 during the 60-minute duration of the experiment was expressed as AUC(0-60) (area under curve) and calculated using the trapezoidal method. The area for each 10-minute reading period generated by the increase in RFU over time is trapezoidal. Summation of the area of each trapezoid (A(a−b)=[0.5*(RFUb+RFUa)]*[Tb−T8], where Tb−Ta represents the ten minute interval between RFU readings from 0 to 60 minutes) was used to compute the AUC(0-60).
[AUC(Cells+DCFH2+H2O2+LPS+Test Sample Dose)−AUC(Cells+DCFH2+H2O2+LPS)][AUC(Cells+DCFH2)−AUC(Cells+DCFH2+H2O2+LPS)]
The median inhibitory concentration (IC50) and its 95% confidence limits for the inhibition of ROS (reactive oxygen species) were calculated using CalcuSyn (BIOSOFT, biosoft.com). This statistical package performs multiple drug dose-effect calculations using the median effect methods described by T-C Chou and P. Talaly (Trends Pharmacol. Sci. 4:450-454) hereby incorporated by reference. The program correlates the “concentration” and the “effect” in the simplest possible form: fa/fu=(C/Cm)m, where C is the concentration of the compound and Cm is the median-effective concentration signifying the potency. Cm is determined from the x-intercept of the median-effect plot. The fraction affected by the concentration of the test material is fa and the fraction unaffected by the concentration is fu (fu=1−fa). The exponent m is the parameter signifying the sigmoidicity or shape of the dose-effect curve. It is estimated by the slope of the median-effect plot.
Cell viability—The CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.) was used to assess cellular respiration, as a measure of cell viability, following exposure to the test materials and oxidative stressors. The tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTS] is reduced by NADPH or NADH in living cells into a colored formazan product that is soluble in tissue culture medium. The assay was performed according to the procedure recommended by the manufacturer. MTS reagent was added directly into the microtiter wells, incubated for 1 hour at 37° C. and the absorbance at 490 nm was recorded on a BioTex Instruments ELX-800 microtiter spectrophotometer (Winooski, Vt.). The formazan product as measured at 490 nm is directly proportional to the number of living cells. Cell viability was not affected by the test materials at concentrations used in the assay.
The production of ROI as measured by increased relative RFU over the 60-minute experimental period is shown in FIG. 2. Relative to the LPS-stimulation alone, H2O2 increased fluorescence 3.7-, 3.8-, 4.0-, and 4.9-fold at 125, 250, 500 and 1000 μM, respectively. Neither LPS nor DMSO produced a change in absolute RFU during the experimental period. Thus, the model selected provided a robust test of antioxidant potential.
a. Test Material Jurkat H2O2 + LPS RAW H2O2 + LPS
Alpha Hop no activity† no activity
Beta Acids no activity no activity
Aromahop OE no activity no activity
Isohop no activity no activity
Tetrahop Gold (THIAA) no activity no activity
Redihop no activity no activity
Hexahop Gold (HHIAA) no activity no activity
Caffeic acid 3.2 (2.5-4.1) 3.3 (2.1-5.4)
Trolox (Vitamin E activity) 5.4 (3.1-9.3) Not tested
†No antioxidant activity was detected at the highest concentration tested (50 μg/mL).
EXAMPLE 3 Inhibition of PGE2 Synthesis in Stimulated and Nonstimulated Murine Macrophages by Hops (Humulus lupulus) Compounds and Derivatives
Chemicals and reagents—Bacterial lipopolysaccharide (LPS; B E. coli 055:B5) was from Sigma (St. Louis, Mo.). Hops fractions (1) alpha hop (1% alpha acids; AA), (2) aromahop OE (10% beta acids and 2% isomerized alpha acids, (3) isohop (isomerized alpha acids; IAA), (4) beta acid solution (beta acids BA), (5) hexahop gold (hexahydro isomerized alpha acids; HHIAA), (6) redihop (reduced isomerized-alpha acids; RIAA), (7) tetrahop (tetrahydro-iso-alpha acids THIAA) and (8) spent hops were obtained from Betatech Hops Products (Washington, D.C., U.S.A.). The spent hops were extracted two times with equal volumes of absolute ethanol. The ethanol was removed by heating at 40° C. until a only thick brown residue remained. This residue was dissolved in DMSO for testing in RAW 264.7 cells. Unless otherwise noted, all standard reagents were obtained from Sigma (St. Louis, Mo.) and were the purest commercially available. All other chemicals and equipment were as described in Examples 1 and 2 of U.S. patent application publication number 2004/0086580.
On day two of the experiment, test materials were prepared as 1000× stock in DMSO. In 1.7 mL microfuge tubes, 1 mL DMEM without FBS was added for test concentrations of 0.05, 0.10, 0.5, and 1.0 μg/mL. Two μL of the 1000×DMSO stock of the test material was added to the 1 mL of medium without FBS. The tube contained the final concentration of the test material concentrated 2-fold and the tube placed in an incubator for 10 minutes to equilibrate to 37° C.
For COX-1 associated PGE2 synthesis, 100 μL of medium were removed from each well of the cell plates prepared on day one and replaced with 100 μL of equilibrated 2× final concentration of the test compounds. Cells were then incubated for 90 minutes. Next, instead of LPS stimulation, the cells were incubated with 100 μM arachidonic acid for 15 minutes. Twenty-five μL of supernatant medium from each well was transferred to a clean microfuge tube for the determination of PGE2 released into the medium. The appearance of the cells was observed and viability was determined as described in Example 2 of U.S. patent application publication number 2004/0086580. No toxicity was observed at the highest concentrations tested for any of the compounds. Twenty-five μL of supernatant medium from each well was transferred to a clean microfuge tube for the determination of PGE2 released into the medium. PGE2 was determined and reported as previously described in Example 1 of U.S. patent application publication number 2004/0086580. The median inhibitory concentrations (IC50) for PGE2 synthesis from both COX-2 and COX-1 were calculated as described in Example 2 of U.S. patent application publication number 2004/0086580.
COX-2 COX-1
As seen in Table 6, all hops fractions and derivative selectively inhibited COX-2 over COX-1 in this target macrophage model. This was a novel and unexpected finding. The extent of COX-2 selectivity for the hops derivatives IAA and RIAA, respectively, 144- and 87-fold, was unanticipated. Such high COX-2 selectivity combined with low median inhibitory concentrations, has not been previously reported for natural products from other sources.
EXAMPLE 4 Reduced Isomerized Alpha Acids Relieve Headache Pain of Allergies
Three adult subjects (one females and two males) with ages ranging from 40 to 57 years) were given a caplet formulation containing 225 mg of reduced isomerized alpha acids per caplet to be taken two times per day. During a following week-long observation period, all subjects reported significant relief from their seasonal allergies. A wash-out period during which no test material was taken caused a return to the headaches suffered from their seasonal allergies in all three subjects. When the test formulation was re-administered, all subjects again reported significant headache relief occurring within 30 minutes of consuming the caplet. This observation indicates the reduced isomerized alpha acid composition of the present invention facilities rapid relief from seasonal allergies.
EXAMPLE 5 IAA and RIAA are not COX-1 or COX-2 Enzyme Inhibitors at Physiologically Relevant Levels
Indomethacin IAA RIAA
/ml % Inhibition ug/ml % Inhibition ug/ml % Inhibition
COX 1 COX 1 COX 1
10 60.6 200 9.6 200 2.9
1 34.6 100 3 100 0.5
0.01 28.2 10 4.2 10 2.5
0.001 −2.8 1 2.1 1 1.9
COX 2 COX 2 COX 2
200 48.4 200 0.3 200 1.9
50 77.6 100 −3.6 100 −0.7
0.5 67.5 10 −2 10 2.7
0.05 3.4 1 0.9 1 2.8
Methodology for Example 5
The Cayman Chemical COX Inhibitor Screening Assay Kit (CISAK, cat#560131) was used to assess the effect of test materials directly on the activity of both COX-1 and COX-2 enzymes. One mL aliquots of reaction buffer supplied in the kit were placed on a dry bath at 37° C., and 10 μLs of heme and either the COX-1 or COX-2 enzyme were added to the reaction buffer. Twenty μL of test compound were added, followed by a ten-minute incubation. Arachidonic acid was added to initiate the reaction, which was allowed to proceed for 2 minutes. The reaction was stopped by the addition of 56 μL of 1 M HCL, and the resulting PGH2 was reduced to PGF2, by the addition of 100 μL of stannous chloride. The reduced reaction mixture was allowed to sit at room temperature for 5 minutes, and then was refrigerated until used in the EIA assay. Each reaction was performed in duplicate, and each duplicate was plated on the EIA plate twice.
EIA Assay: The EIA assay was conducted by first preparing a 1:2000 dilution of the reduced reaction products in EIA buffer. Fifty μL of this dilution was added to each sample well of the 96-well EIA plate, followed by the addition of 50 μL of prostaglandin screening tracer (acetylcholineterase conjugated PGF2α) and 50 μL of prostaglandin screening antibody. Controls consisted of (1) blanks (left empty until the plate was actually developed), (2) maximum binding controls (containing tracer and antibody, without sample), and (3) non-specific binding controls that contained tracer without antibody. A standard curve was prepared, starting with 1000 pg/mL PGF2α, followed by 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.2 pg/mL, 15.6 pg/mL, and 7.8 pg/mL concentrations. The plates were incubated at 4° C. for 18 hours. After incubation, the plates were washed in wash buffer five times, then 200 μL Ellmans reagent was added to each well. One well was left as a total activity control, and 5 μL of tracer were added to this well after the addition of the Ellmans reagent. The plates were read at 405 nm after 60-90 minutes of gentle agitation in the dark.
EXAMPLE 6 Mite Dust Allergens Activate PGE2 Biosynthesis in A549 Pulmonary Cells
PGE2 assay—Determination of PGE2 in the culture medium was performed as previously described in Example 1 of U.S. patent application publication number 2004/0086580.
Mite allergen treatment increased PGE2 biosynthesis 6-fold in A549 cells (1844 pg PGE2/well) relative to the solvent treated controls (304 pg PGE2/well).
EXAMPLE 7 Hops Derivatives Inhibit Mite Dust Allergen Activation of PGE2 Biosynthesis in A549 Pulmonary Cells
The cell line and testing procedures are as described in Example 6. In addition to mite dust allergen, test materials included Hops fractions (1) alpha hop (1% alpha acids; AA), (2) aromahop OE (10% beta acids and 2% isomerized alpha acids, (3) isohop (isomerized alpha acids; IAA), (4) beta acid solution (beta acids BA), (5) hexahop gold (hexahydro isomerized alpha acids; HHIAA), (6) redihop (reduced isomerized-alpha acids; RIAA), and (7) tetrahop (tetrahydro-iso-alpha acids THIAA). Test materials at a final concentration of 10 μg/mL were added 60 minutes prior to the addition of the mite dust allergen.
Test Material PGE2 Biosynthesis
1. A method of treating an inflammatory condition in a mammal in need thereof, the method comprising administering to the mammal in need thereof a composition comprising a therapeutically effective amount of a compound selected from the group consisting of dihydro-isohumulone, dihydro-isocohumulone, dihydro-isoadhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-isoadhumulone, hexahydro-isohumulone, hexahydro-isocohumulone and hexahydro-isoadhumulone.
3. The method of claim 1, wherein the inflammatory condition is cancer.
4. The method of claim 3, wherein the cancer is breast cancer.
5. The method of claim 3, wherein the cancer is prostate cancer.
6. The method of claim 3, wherein the cancer is colon cancer.
7. The method of claim 3, wherein the cancer is pancreatic cancer.
8. The method of claim 1, wherein the inflammatory condition is asthma.
9. The method of claim 1, wherein the inflammatory condition is Alzheimer's Disease.
10. The method of claim 1, wherein the inflammatory condition is arthritis.
11. The method of claim 1, wherein the inflammatory condition is Crohn's Disease.
12. The method of claim 1, wherein the inflammatory condition is eczema.
13. The method of claim 1, wherein the inflammatory condition is Inflammatory Bowel Disease.
14. The method of claim 1, wherein the inflammatory condition is osteoarthritis.
15. The method of claim 1, wherein the inflammatory condition is psoriasis.
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