USOO9084726B2

(12) United States Patent (10) Patent No.: US 9,084,726 B2 Egberg et al. (45) Date of Patent: Jul. 21, 2015

(54) SUSPENSION COMPOSITIONS OF 6,855.359 B2 2/2005 Khare PHYSIOLOGICALLY ACTIVE PHENOLC 3: R: 658. Hy st al COMPOUNDS AND METHODS OF MAKING 8,900,635- - - B2 12/2014 Elderer, etJr. al. et al. AND USING THE SAME 2004/0164194 A1 8/2004 Reed et al. 2004/022O11.6 A1* 11/2004 Behnam ...... 514/27 (71) Applicant: Humanetics Corporation, Eden Prairie, 2006/01 1651.0 A1 6/2006 Behnam MN (US) 2006/0292214 A1* 12/2006 Jenkins et al...... 424/451 2007/002O197 A1 1/2007 Galli et al. 2007.0043.121 A1 2/2007 Brown et al. (72) Inventors: David C. Egberg, Bonita Springs, FL 2007/0087104 A1* 4/2007 Chanamai ...... 426,602 (US); Michael D. Kaytor, Maplewood, 2007,0269.526 A1 11/2007 BOS et al. MN (US); John C. Dykstra, Eden 2008/0311209 A1 12/2008 Beumer et al. Prairie, MN (US) 2009.0035336 A1* 2, 2009 Volhardt et al...... 424/401 2009/0035366 A1* 2/2009 Liversidge et al...... 424/451 (73) Assignee: Humanetics Corporation, Minneapolis, 2012fO1641902012/O121654 A1 6,5, 2012 Ei.Elder, Jr. et al.al MN (US) (*) Notice: Subject to any disclaimer,- the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 WO WO 2012/068140 5, 2012 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 14/090,864 Atkinson, et al., “The effects of on bone (22) Filed: Nov. 26, 2013 density in women: a double-blind, randomized, placebo-controlled trial'', Am. J. Clin. Nutr., vol. 79, pp. 326-333, (2004). (65) Prior Publication Data (Continued) US 2015/O147359 A1 May 28, 2015 Primary Examiner — Robert A Wax (51) Int. Cl. Assistant Examiner — Melissa Mercier 3. Ig52 R (74) Attorney, Agent, or Firm — Stoel Rives, LLP: (2006.01) Zhi-Xiang (Alex) Oh (52) U.S. Cl. CPC ...... A61 K9/10 (2013.01); A61 K3I/352 (2013.01) (57) ABSTRACT (58) Field of Classification Search The present invention is directed to compositions comprising None physiologically active phenolic compounds and methods for See application file for complete search history. making and using the same. In particular embodiments, the compositions described herein include Suspension formula (56) References Cited tions including a physiologically active phenolic compound provided as a nanoparticulate material and dispersed within U.S. PATENT DOCUMENTS an edible lipid. 4,390,559 A * 6/1983 Zilliken ...... 426,545 5,302.401 A 4, 1994 Liversidge et al. 30 Claims, 4 Drawing Sheets

Genivida (before milling) 13 Meann SizeSi :35.7961.5cm) OO 12 Median Size : 29.54557(Lm) 90 Mode Size 27.8086{um) S.P. Area :2199.7(cnicm) 80 10 SDev, : 22.1223(um) Span : OFF 70 c 8 Diameter on Cumulative%: (1)10.00(%)- 17.1811 (um) s : (2)50.00(%)-29.5456(um) 60 S. q(%) 6 ; (5)99.00(%)-SE.00%). 92,25:E) 40so Sass: : (6)99.90%)- 181,0281(lam) SS 4. 30 20 2 O O O O. 0.100 1.000 1000 1000 1000 3OOO Diameterium) US 9,084.726 B2 Page 2

(56) References Cited Si, et al., “Improving the Anti-Tumor Effect of with a Biocompatible Superparamagnetic Drug Delivery System.” Journal OTHER PUBLICATIONS of Nanoscience and Nanotechnology, vol. 10, No. 4, pp. 2325-2331, (2010). Bhathena, et al., “Beneficial role of dietary in obesity Sun, et al., “Pilot Study of a Specific in Tumor and diabetes'’.” Am. J. Clin. Nutr., vol. 76, pp. 1191-1201, (2002). Bearing Mice and in Stage IIIB and IV Non-Small Cell Lung Cancer Chait, A., “Low-Density Lipoprotein Oxidation and the Pathogenesis Patients.” Nutrition and Cancer, vol.39, No. 1, pp. 85-95, (2001). of Atherosclerosis.” WJM, vol. 160, No. 2, pp. 183-184, Feb. 1994. Takimoto, et al., “Phase I Pharmacokinetic and Pharmacodynamic Clifton-Bligh, et al., “The effect of isoflavones extracted from red Analysis of Unconjugated Soy Isoflavones Administered to Individu clover (Rimostil) on lipid and bone metabolism.” : The als with Cancer.” Cancer Epidemiology, Biomarkers & Prevention, Journal of The North American Menopause Society, vol. 8, No. 4, pp. vol. 12, pp. 1213-1221, (Nov. 2003). 259-265, (2001). Cotter, et al., “Genistein Appears to Prevent Early Postmenopausal Urban, et al., “The Effect of Isolated Soy Protein on Plasma Biomark Bone Loss as Effectively as Hormone Replacement Therapy.” Nutr ers in Elderly Men with Elevated Serum Prostate Specific Antigen.” tion Reviews, vol. 61, No. 10, pp. 346-351, (2003). The Journal Urology, vol. 165, pp. 294-300, (Jan. 2001). Fitzpatrick, L.A., “Soy isoflavones: hope or hype?,” Maturitas, vol. Wei, et al., “ Genistein: Photoprotection and Clinical 44. Suppl. 1, pp. S21-S29, (2003). Implications in Dermatology'.” The Journal of Nutrition, vol. 133, Goodman-Gruen, et al., “Usual dietary isoflavone intake and body pp. 3811S-3819S. (2003). composition in postmenopausal women. Menopause: The Journal of Zhou, et al., “Genistein Stimulates Hematopoiesis and Increases the North American Menopause Society, vol. 10, No. 5, pp. 427-432, Survival in Irradiated Mice.” J. Radiat. Res., vol. 46, pp. 425-433, (2003). (2005). Landauer, et al., “Radioprotective and Locomotor Responses of Mice Office Action issued Mar. 21, 2012 in U.S. Appl. No. 12/946,711. Treated with Nimodipine Alone and in Combination with Response to the Mar. 21, 2012 Office Action filed Jun. 21, 2012, in WR-151327.” Journal of Applied Toxicology, vol. 21, pp. 25-31, U.S. Appl. No. 12/946,711. (2001). Office Action issued Aug. 31, 2012 in U.S. Appl. No. 12,946,711. Landauer, et al., “Genistein Treatment Protects Mice from Ionizing Response to the Aug. 31, 2012 Office Action filed Feb. 14, 2013, in Radiation Injury,” Journal of Applied Toxicology, vol. 23, pp. 379 U.S. Appl. No. 12/946,711. 385, (2003). Final Rejection issued Oct. 8, 2013, in U.S. Appl. No. 12,946,711. Landauer, et al., “Prevention of Gamma Radiation-Induced Mortality Office Action issued Mar. 28, 2012 in U.S. Appl. No. 13/411,405. in Mice by the Isoflavone Genistein.” NATO RTG-099, pp. 17-1-17 Response to the Mar. 28, 2012 Office Action filed Jun. 27, 2012, in 11, (2005). Leonarduzzi, et al., “Design and Development of Nanovehicle-Based U.S. Appl. No. 13/411,405. Delivery Systems for Preventive or Therapeutic Supplementation Office Action issued Sep. 11, 2012 in U.S. Appl. No. 13/411,405. with .” Current Medicinal Chemistry, vol. 17, pp. 74-95, Response to the Sep. 11, 2012 Office Action filed Dec. 11, 2012, in (2010). U.S. Appl. No. 13/411,405. Magee, et al., “Phyto-Oestrogens, their mechanism of action: current Third Office Action issued Feb. 22, 2013, in U.S. Appl. No. evidence for a role in breast and prostate cancer.” British Journal of 13/411,405. Nutrition, vol. 91, pp. 513-531, (2004). International Search Report and Written Opinion issued Mar. 23. Messina, M.J., “Emerging Evidence on the Role of Soy in Reducing 2012, in International Application No. PCT/US2011/060829. Prostate Cancer Risk.” Nutrition Reviews, vol. 61, No. 4, pp. 117 International Preliminary Report on Patentability issued May 21, 131. (Apr. 2003). 2013, in International Application No. PCT/US2011/060829. Mohammad, et al., “Genistein sensitizes diffuse large cell lymphoma Young, et al., “Why are Low-Density Lipoproteins Atherogenic.” to CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) WJM, vol. 160, No. 2, pp. 153-164, Feb. 1994. chemotherapy.” Molecular Cancer Therapeutics, vol. 2, No. 12, pp. Applicant Initiated Interview Summary issued Dec. 14, 2012 in U.S. 1361-1368, (2003). Appl. No. 12/946,711. Morabito, et al., “Effects of Genistein and Hormone-Replacement Office Action issued Oct. 8, 2013 in U.S. Appl. No. 12/946,711. Therapy on Bone Loss in Early Postmenopausal Women: A Random Response to the Oct. 8, 2013 Office Action filed Mar. 7, 2014 in U.S. ized Double-Blind Placebo-Controlled Study,” Journal of Bone and Appl. No. 12/946,711. Mineral Research, vol. 17, No. 10, pp. 1904-1912, (2002). Applicant Initiated Interview Summary issued Dec. 14, 2012 in U.S. Nicosia, et al., “Oncogenic pathways implicated in ovarian epithelial Appl. No. 13/411,405. cancer.” Hematology/Oncology Clinics of North America, vol. 17. Response to the Feb. 22, 2013 Office Action filed May 22, 2013 in pp. 927-943, (2003). U.S. Appl. No. 13/411,405. Park, et al., "Chemopreventive potential of Applicant Initiated Interview Summary issued May 29, 2013 in U.S. and genistein: evidence from epidemiological and laboratory stud Appl. No. 13/411,405. ies.” Toxicology Letters, vol. 150, pp. 43-56. (2004). Notice of Allowance issued Jun. 24, 2013 in U.S. Appl. No. Roomans, G.M., “Pharmacological Approaches to Correcting the Ion 13/411,405. Transport Defect in Cystic Fibrosis.” Am. J. of Respir. Med., vol. 2, Notice of Allowance issued Jul. 30, 2014 in U.S. Appl. No. No. 5, pp. 413-431, (2003). 12/946,711. Sarkar, et al., "Soy Isoflavones and Cancer Prevention.” Cancer Notice of Allowance issued Sep. 9, 2014 in U.S. Appl. No. Investigation, vol. 21. No. 5, pp. 744-757. (2003). 12/946,711. Sarkar, et al., “Lesson Learned from Nature for the Development of Notice of Allowance issued Sep. 18, 2014 in U.S. Appl. No. Novel Anti-Cancer Agents: Implication of Isoflavone, Curcumin, and 12/946,711. their Synthetic Analogs.” Curr. Pharm. Des., vol. 16, No. 16, pp. International Search Report and Written Opinion issued Feb. 6, 2015 1801-1812, (Jun. 2010). in International Application No. PCT/US14/67141. Setchell, et al., “Dietary phytoestrogens and their effect on bone: Preliminary Amendment filed Oct. 27, 2014 in U.S. Appl. No. evidence from in vitro and in vivo, human observational, and dietary 14/524,936. intervention studies'.” Am. J. Clin. Nutr., vol. 78(suppl.), pp. 593S 609S. (2003). * cited by examiner U.S. Patent Jul. 21, 2015 Sheet 1 of 4 US 9,084,726 B2 U.S. Patent Jul. 21, 2015 Sheet 2 of 4 US 9,084,726 B2 U.S. Patent Jul. 21, 2015 Sheet 3 of 4 US 9,084,726 B2

2 5 Bonistein dE20 t -o- initial measurement 515 -- 3 month measurement

O E 10 S. 5 A. O Bilgit O. 1. 10 1OO Channel Diameter (micron) FIG. 3A

25 Genivida pre-grind 20 -o- initial measurement 515 -- 3 month measurement

O E 10 S. 5 a. O Sr.R igur O.O1 0.1 1. 10 100 Channel Diameter (micron) FIG. 3B

25 Genivida NO pre-grind

20 -o- initial measurement 315 -- 3 month measurement g E 10 S. 5 A. O O.O1 1OO Channel Diameter (micron) FIG. 3C

US 9,084,726 B2 1. 2 SUSPENSION COMPOSITIONS OF materials after storage for three months (3-Month Measure PHYSIOLOGICALLY ACTIVE PHENOLC ment’) at ambient temperature. COMPOUNDS AND METHODS OF MAKING AND USING THE SAME DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS TECHNICAL FIELD Compositions of physiologically active phenolic com The present disclosure relates to compositions including pounds are described herein. In certain embodiments, the physiologically active phenolic compounds and methods for compositions described herein are suitable for administration producing and utilizing Such compositions. In certain 10 to or consumption by a Subject as a pharmaceutical formula embodiments, the compositions described herein comprise a tion, a medical food, or a dietary Supplement. In particular nanoparticulate phenolic compound dispersed in an edible embodiments, the compositions described herein include a lipid, and in further embodiments, methods of making and phenolic compound provided as a nanoparticulate material using Such compositions are provided. suspended within an edible lipid. Providing the phenolic 15 compound as nanoparticulate material serves to increase the BACKGROUND bioavailability of the phenolic compound and may addition ally facilitate administration or consumption of the phenolic There are a variety of physiologically active phenolic Sub compound at amounts Sufficient to achieve a desired nutri stances, including naturally-occurring phenolic compounds, tional or therapeutic benefit. that have been shown to exhibit favorable medicinal or nutri Compositions prepared according to the present descrip tional properties. However, for a variety of reasons, it is often tion have been shown to exhibit desirable physical stability difficult to provide these phenolic compounds in a composi characteristics. Often, where active compounds are provided tion that is readily ingested and Suited for delivering the as a nanoparticulate material, particle agglomeration over compounds to Subjects in Sufficient amounts to achieve a 25 time causes a significant shift in the particle size distribution desired nutritional or therapeutic effect. (PSD) of the active material, resulting in a loss of the advan Many phenolic substances are not readily bioavailable. As tages sought by providing the active as a nano-sized material a result, in order to achieve any benefit from consumption or in the first place. Embodiments of the compositions described administration of the phenolic Substance, a subject must con herein serve to preserve the PSD of the phenolic compound Sume or be administered large quantities of the phenolic 30 included therein over time so that, even after months of stor compound. In addition, oral delivery of many desirable phe age, the PSD of the phenolic compound is maintained as a nolic compounds has proven difficult or unpalatable to Sub nanoparticulate material. jects, which can lead to a perceived requirement for parenteral Methods for preparing the compositions described herein routes of administration and/or the formulation of relatively are also provided. In general, a physiologically active phe complex, multi-component, pharmaceutical grade composi 35 nolic compound is provided as a nanoparticulate material and tions designed to increase the targeted compound's bioavail then dispersed within an edible lipid, resulting in a Suspension ability. Suitable for administration to or consumption by a Subject. Any suitable size reduction process may be utilized to reduce DESCRIPTION OF THE FIGURES 40 the phenolic compound to an active material exhibiting a desired, nano-sized PSD. In certain embodiments, the nano FIGS. 1 and 2 show the particle size distribution of two particulate phenolic compound material may be provided by different grades of an exemplary physiologically active phe a nano-milling procedure. The term “active material as it is nolic compound prior to preparation according to the meth used in the present disclosure, refers to an amount of one or ods described herein or inclusion in a composition according 45 more physiologically active phenolic compound(s). to the present description. FIG. 1 provides the particle size Nano-mills typically include a milling media (e.g., balls, distribution of GenividaTM (food grade genistein) upon beads, pellets, satellites, crystalline media, etc.) into which receipt from a commercial supplier. FIG. 2 provides the par the active material to be size-reduced is introduced. Once the ticle size distribution of Bonistein(R) (pharmaceutical grade active material is introduced into the milling media, the mill genistein) upon receipt from a commercial Supplier. 50 ing media is agitated and the active material is Subjected to FIGS. 3A-3C show the initial and 3-month particle size grinding and shearing forces that reduce the materials PSD. distributions for two different grades of an exemplary nano In order to facilitate milling, the active material to be size particulate, physiologically active, phenolic compound mate reduced can be provided in a liquid vehicle or a liquid vehicle rial (nanoparticulate genistein) prepared according to the may be introduced into the milling media. In specific embodi methods described herein and included within compositions 55 according to the present description. ments of the methods described herein, the vehicle used in FIG. 4 provides particle size distribution data for an exem nano-milling the active material may be an edible lipid suit plary physiologically active phenolic compound (genistein) able for use in a composition according to the present descrip prepared according to the present description and included tion. within a composition as described herein. The genistein was 60 The compositions described herein are suitable for con Sourced in two different grades, a pharmaceutical grade mate Sumption by or administration to human and/or animal Sub rial (Bonistein(R) and a food grade material (GenividaTM). jects. A composition according to the present disclosure may FIG. 4 provides initial particle size distribution data for the be prepared as a food-grade composition. Alternatively, a genistein materials prepared according to the methods composition as described herein may be prepared as pharma described herein and included in a composition according to 65 ceutical-grade composition. In certain embodiments, the the present description (“Initial Measurement'). FIG. 4 also compositions described herein may be prepared for direct provides particle size distribution data for the same genistein administration or consumption. In other embodiments, the US 9,084,726 B2 3 4 compositions described herein may be prepared for combi the incident light, the Mie equation reduces to the Fraunhofer nation with one or more other constituents prior to adminis equation. An array of detectors, including high-angle and tration or consumption. back-scatter detectors, and multiple light sources of different wavelengths are typically employed to allow measurement of I. Definitions the full size range in one analysis. Equipment Suited for use in analyzing particle size by laser light diffraction is commer As used herein and in the appended claims, the singular cially available and manufactured, for example, by Horiba forms “a,” “an and “the include plural reference unless the Instruments, Irvine, Calif. context clearly dictates otherwise. In the context of the present description, the particle size Ranges can be expressed herein as from “about one par 10 distribution of a given material is provided in Volume diam ticular value, and/or to “about another particular value. eter as measured in accordance with USP 429 using a laser When such a range is expressed, another embodiment diffraction particle size analyzer operating in the Mie Scat includes from the one particular value and/or to the other tering Theory diffraction mode and equipped with a Suspen particular value. Similarly, when values are expressed as sion dispersion sample chamber (e.g., as available from approximations, by use of the antecedent “about, it will be 15 Horiba Instruments, Irvine, Calif., USA). For purposes of the understood that the particular value forms another embodi present description, Volume diameter is given as a particle ment. It will be further understood that the endpoints of each size distribution defined by one or more of D (0.10), D (0.50) of the ranges are significant both in relation to the other and D (0.90). When referred to herein, the term D (0.10) endpoint, and independently of the other endpoint. It is also indicates the volume frequency distribution of particles for understood that there area number of values disclosed herein, which 10% of the sample is below the referenced size, the and that each value is also herein disclosed as “about that term D (0.50) indicates the volume frequency distribution of particular value in addition to the value itself. For example, if particles for which 50% of the sample is below the referenced the value "10' is disclosed, then “about 10” is also disclosed. size, and the term D (0.90) indicates the volume frequency It is also understood that each unit between two particular distribution of particles for which 90% of the sample is below units are also disclosed. For example, if 10 and 15 are dis 25 the referenced size. In addition to measurement of the particle closed, then 11, 12, 13, and 14 are also disclosed. size distribution via laser diffraction, the results from such As used herein, "nanoparticulate” refers to material exhib particle size analysis and/or the morphology of the particles iting a Volume diameter, as measured using laser light diffrac may be confirmed using known electron microscopy tech tion, wherein the D (0.50) of the material is 0.5um or less. In niques. certain embodiments, the term “nanoparticulate” refers to 30 A “subject” for purposes of this disclosure is an animal to material exhibiting a Volume diameter, as measured using which a composition as described hereincan be administered laser light diffraction, wherein the D (0.50) of the material is in order to achieve a targeted benefit. In certain embodiments, 0.5um or less and the D (0.90) is 2 um or less. Particle size the Subject is a human being. analysis using laser light diffraction is a technique based on light being scattered through various angles which are 35 II. Compositions of Physiologically Active Phenolic directly related to the size of the particles. By measuring the Compounds angles of light scattered by the particles being analyzed and the intensity of this scattered light, a particle size distribution The compositions described herein include one or more can be calculated. Techniques for use in analyzing particle physiologically active phenolic compound. The phenolic size in the context of the present disclosure can be referred to 40 compound may be a non-naturally occurring or a naturally as static light scattering, Rayleigh light scattering, low angle occurring compound. In addition, the phenolic compound light scattering (LALS), multiple angle light scattering may be chemically synthesized (even if also produced by (MALS) Fraunhofer diffraction, or Mie Scattering. Measure natural sources) or isolated, purified, or derived from natural ment of particle size distributions using Mie Scattering allows Sources. In particular embodiments, the phenolic compound for the determination of particle size distributions through the 45 is a naturally occurring compound found in or derived from direction measurement of mass. one or more species or varieties of plant. Examples of Such Two theoretical applications to the analysis of particle size compounds suitable for use in the compositions described by laser light diffraction are based on assumptions about the herein include, but are not limited to, compounds selected properties of the particles. Fraunhofer theory considers the from isoflavones, curcuminoids, , and . following: particles are spherical, non-porous and opaque; 50 Curcuminoids are natural phenols found, for example, in particle diameters are greater than the wavelength of the laser turmeric. Curcuminoids include curcumin, desmethoxycur light used in the analysis; and particles are distant enough cumin, and bis-desmethoxycurcumin. Curcumin can exist in from each other not to interfere in the diffraction of light, several tautomeric forms, including a 1,3-diketo form and exhibit random motion, and diffract light with the same effi two equivalent enol forms. Curcuminoids generally exhibit ciency regardless of size and shape. Mie theory considers the 55 poor water solubility and poor bioavailability. The chemical differences in refractive index between the particles and the name for curcumin is (1E,6E)-1.7-bis(4-hydroxy-3-methox Suspending medium, which allows the measurement tech yphenyl)-1,6-heptadiene-3,5-dione (IUPAC). The chemical nique to account for particles in the size range below the name for desmethoxycurcuminis (1E,6E)-1,6-Heptadiene-3, wavelength of the laser light used in the analysis. The relative 5-dione, 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphe amounts of different size particles are determined by measur 60 nyl) (IUPAC). The chemical name for bis-desmethoxycur ing the intensity of light scattered at different angles. As the cuminis (1E,6E)-1.7-bis(4-hydroxyphenyl) hepta-1,6-diene particles get close to or Smaller than the wavelength of light, 3,5-dione (IUPAC). Curcuminoids are commercially more of the light intensity is scattered to higher angles and available and can be isolated from plant material or manufac back-scattered. Mie Scattering Theory accounts for this dif tured in purified form using known chemical syntheses. ferent behavior. In order to make particle size measurements, 65 Isoflavones include a variety of naturally occurring com the light intensity pattern is measured over the full angular pounds and belong to a class of organic compounds related to range. When the particle size is larger than the wavelength of isoflavonoids. Many isoflavones act as phytoestrogens in US 9,084,726 B2 5 6 mammals, and Some are termed antioxidants because of their characterized by a D (0.50) as detailed herein, the nanopar ability to trap singlet oxygen. Isoflavones are produced ticulate active material may exhibit a D (0.90) of 2.0 um or almost exclusively by the members of the Fabaceae family less. In particular embodiments, the nanoparticulate active (i.e., the Leguminosae or bean family). Soybeans are the most material exhibits a D (0.50) as detailed herein and a D (0.90) common source of isoflavones in human food, and the major 5 selected from a D (0.90) of 1.5um or less, a D (0.90) of 1.0 um isoflavones in Soybean are genistein and . In general, or less, and a D (0.90) of 0.5um or less. isoflavones also exhibit poor water solubility and bioavail Nanoparticulate active material suitable for use in the com ability. Genistein is commercially available and may be positions disclosed herein may be prepared according to obtained in Synthetic, purified form. Synthetic genistein is known methods for producing materials exhibiting a Sub available, for example, as BONISTEIN from DSM Nutri 10 micron PSD. In one embodiment, naturally derived or syn tional Products (DSM Nutritional Products, Inc. Parsippany, thetically manufactured phenolic compound material may be N.J.). Genistein's chemical name is 5,7-dihydroxy-3-(4-hy nanomilled according to milling techniques known in the art. droxyphenyl)-chromen-4-one (IUPAC). Daidzein's chemical Nanomilling may include wet bead milling utilizing an agi name is 7-hydroxy-3-(4-hydroxyphenyl) chromen-4-one tator bead mill in a grinding container for continuous disper (IUPAC). Like genistein, daidzein is commercially available 15 sion and fine wet grinding. Alternatively, the necessary and can be extracted or isolated from plant material or manu energy for dispersion and grinding of a material within the factured in purified form using known chemical syntheses. milling media of a nanomill may be transmitted to the grind Flavanols that may be utilized in compositions according ing media through agitator discs mounted on an agitator shaft. to the present description include . As used herein, Several different nanomills and milling techniques are com the term “' refers to a family of compounds as well as mercially accessible and offered from, for example, CB the compound itself. Catechin has the chemical name (2R, Mills, of Gurnee, Ill., and NETZSCH Premier Technologies, 3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3, LLC of Exton, Pa. 5,7-triol (IUPAC). The family of catechin compounds Though nanomilling is generally referenced herein as a includes, for example, epicatechin, and epigallocatechingal means for producing nanoparticulate active material Suitable late (EGCG), which may also be used in compositions, 25 for use in the compositions described herein, the nanoparticu described herein. As used herein, "epicatechin’ refers to one late active material can be produced by other suitable tech or both of two isomers of catechin in the cis configuration, and niques as well. For example, the desired phenolic compound when used herein as a compound name, the term “catechin' may be provided as a nanoparticulate material through one or refers to one or both of two isomers of catechin in the trans more known wet milling techniques, Super-critical or com configuration. The chemical name for EGCG is (2R,3R)-5, 30 pressed fluid techniques, hot or high-pressure homogeniza 7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl 3,4, tion, emulsification techniques, evaporative precipitation, 5-trihydroxybenzoate (IUPAC). Catechins are commercially antisolvent precipitation, microprecipitation, cryogenic tech available and can be found in and isolated from, for example, niques, complexation techniques, ultrasonication techniques, green tea, cocoa beans, the kola nut, raw apples, apricots, Solid dispersion techniques, or spray drying and lyophiliza nectarines, pears and plums, blackberries, red raspberries, 35 tion techniques. cranberries, cherries, broad beans. Catechins can also be The active material included in the compositions described manufactured in purified form using known chemical synthe herein is dispersed within an edible lipid to form a nanopar SCS. ticulate Suspension of the phenolic compound. The term Stilbenoids are naturally occurring phenols produced by “edible lipid refers to naturally occurring and synthetic lip several different plants. A of particular interest in 40 ids, fats, and oils suitable for consumption by or administra the context of the compositions described herein is resvera tion to a subject. In certain embodiments, the edible lipid may trol. The chemical name for is 5-(E)-2-(4-hy be an edible glyceride selected from glycerides having a chain droxyphenyl)ethenylbenzene-1,3-diol (IUPAC). Like other length ranging from one carbonacetate to 22 carbons. Where stilbenoids, resveratrol produced naturally by several differ an edible glyceride is used as the edible lipid, the fatty acid ent species of plants, and it is often extracted for commercial 45 chain of the edible glyceride may be saturated or exhibit purposes from the roots of the Japanese Knotweed. Like the varying degrees of unsaturation. In particular embodiments other phenol compounds specifically described herein, res where the edible glyceride includes an unsaturated fatty acid Veratrol is commercially available and can be isolated from chain the degree of unsaturation may range from one double naturally occurring plant material or manufactured in purified bond (e.g., oleic acid) up to six double bonds (e.g., docosa form using known chemical syntheses. 50 hexaenoic acid). Further, the fatty acid residues of edible The composition described herein may include one or glycerides suitable for use in the compositions described more phenolic compounds as described herein. For example, herein may form an ester linkage on one (mono-glyceride), in particular embodiments, the compositions include a phe two (di-glyceride), or all three glycerol hydroxyl groups (trig nolic compound selected from curcumin, desmethoxycur lyceride). Any hydroxyl groups not esterified may be free cumin, bis-desmethoxycurcumin, genistein, daidzein, cat 55 hydroxyl groups or chemically linked to, for example, phos echin, epicatechin, EGCG, resveratrol, and combinations of phate, inositols, choline, serine, or ethanol amine, such as two of more of such compounds. In each embodiment, one or found in lecithin. more phenolic compound included in the composition is pro The edible lipid used in the compositions described herein vided as a nanoparticulate material. In specific embodiments, may also be selected from edible lipids, oils, and fats from the compositions disclosed herein may include one or more 60 plant and animal sources. Plant oils that may be suitable for phenolic compounds provided as nanoparticulate material use in the compositions described herein include, for exhibiting a D (0.50) of 0.4 um or less. In certain such example, olive, corn, Soy, marine, coconut, palm, palm ker embodiments, the compositions disclosed herein may include nel, cotton seed, peanut, safflower, Sesame, Sunflower, one or more phenolic compounds provided as nanoparticulate almond, cashew, macadamia, pecan, pine nut, walnut, lemon, material exhibiting a D (0.50) selected from a D (0.50) of 0.35 65 orange, flaxseed, and borage oils. Where the edible lipid is a um or less, a D (0.50) of 0.30 Lum or less, a D (0.50) of 0.25um plant oil or is derived from one of the plant oils detailed or less, and a D (0.50) of 0.20 um or less. In addition to being herein, the edible lipid may also be selected from cocoa butter US 9,084,726 B2 7 8 or an inter-esterified plant oil. Such as medium-chain triglyc a composition as described herein include providing a phe erides formed from one or more of the plant oils detailed nolic compound as a nanoparticulate material, providing an herein. Animal oils and fats that may be suitable for use in the edible lipid as described herein, and mixing the nanoparticu compositions described herein include fish oils and dairy late phenolic compound material with the edible lipid to derived fats, such as butter. disperse the nanoparticulate phenolic compound material and The nanoparticulate active material can be suspended form a Suspension composition. In such embodiments, one or within the edible lipid at various concentrations. The compo more additional materials may also be provided for inclusion sitions described herein can be prepared with high concen in the composition. For example, one or more dispersants trations of the nanoparticulate material, and in certain embodiments, the nanoparticulate material is suspended in may be provided and combined with the nanoparticulate phe the edible lipid at a concentration of 200 mg/ml or higher. For 10 nolic compound material and the edible lipid. Where one or example, compositions according to the present description more dispersants are included, the dispersant may be com may include the nanoparticulate material Suspended within bined with the edible lipid prior to the introduction of the the edible lipid at a concentration selected from 200 mg/ml or nanoparticulate phenolic compound. Alternatively, one or higher, 250 mg/ml or higher, 300 mg/ml or higher, 350 mg/ml more dispersants may be combined with the nanoparticulate or higher, 400 mg/ml or higher, 450 mg/ml or higher, and 500 15 phenolic compound material prior to combination of the dis mg/ml or higher. persant and phenolic compound with the edible lipid. Even Compositions according to the present description may further, one or more dispersants may be combined with the additionally include edible colorants, flavorants, and dispers edible lipid and nanoparticulate phenolic compound after the ants. A wide variety of colorants and flavorants suitable for nanoparticulate phenolic compound has been dispersed human and animal consumption are well known and readily within the edible lipid. commercially available. Where included in the compositions In yet further embodiments, the compositions described described, a dispersant, which may be an edible emulsifier, herein can be prepared by forming the nanoparticulate active may serve to help maintain or improve particle size stability material in the presence of the edible lipid, where the edible within the suspension and/or bioavailability of the phenolic lipid forms part of the medium within which the size commi compound(s) dispersed therein. The edible dispersant may be 25 nution of the phenolic compound takes place. In certain Such selected from, for example, lecithin, and Sorbitan fatty acid esters, such as polysorbate 80, Steroyl-2-lactate, polyoxyeth embodiments, the active material is processed to a desired ylene esters, Sucrose esters offatty acids, polyglycerol esters, nanoparticulate PSD using a nanomill, wherein the active fatty acid esters of propyleneglycol, and glycerol fatty acid material is combined with the edible lipid prior to introduc esters. Where included, a dispersant may be provided in a tion into the nanomill. In other such embodiments, the active relative amount selected from about 0.01% to about 5% 30 material and edible lipid are introduced into the nanomill (w/v). separately (e.g., the phenolic compound material and edible The compositions described herein can be prepared for use lipid may be introduced to the milling media in separate in different contexts. For example, the compositions may be process steps, the edible lipid may be combined with the prepared for oral consumption as a dietary Supplement, a milling media prior to introduction of the active material, or medical food, or even as a pharmaceutical formulation. 35 the active material may be combined with the milling media Where prepared as a dietary Supplement or a medical food, prior to introduction of the edible lipid). the phenolic compound(s), edible lipid, and, where included, In specific embodiments where the active material and any colorant, flavorant, or dispersant included in the compo edible lipid are combined in a nanomill, the active material sition can be prepared and provided as a food-grade material and edible lipid are fed into the nanomill and milled in a Such that the resulting composition is a food-grade composi 40 manner that results in a composition characterized by nano tion Suitable for use as a medical food or dietary Supplement. particulate active material suspended within the edible lipid. If desired, in other embodiments, the compositions described In one such embodiment, a suspension of active material and herein can be prepared for use as a pharmaceutical composi edible lipid may be fed continuously through the nanomill tion. In Such embodiments, each of the components must be until a suspension composition containing nanoparticulate prepared as pharmaceutical grade materials such that a phar maceutical grade composition is produced. 45 active material of a desired PSD is reached. For example, the The compositions described herein may be prepared in any active material may be nanomilled by recirculating a Volume Suitable manner and using any suitable devices. Moreover, of the active material suspended within the edible lipid, fol compositions according to the present description can be lowed by one or more single passes through the nanomill to prepared for oral consumption or oral or parenteral adminis reach a composition that includes a phenolic compound mate tration via a variety of delivery devices or mechanisms. For 50 rial exhibiting the desired PSD. The particle size of the active example, the Suspension compositions described herein can material Suspended within a composition as described herein be prepared for delivery from any desired metering device, can be controlled by adjusting the parameters of the nanomill including a syringe, measuring spoon, cup, or vial, and where and the grinding conditions. For example, the particle size desired to ease administration or delivery, the compositions produced by nanomilling the active material or a combination according to the present description can be metered in pre 55 of active material and edible lipid may be controlled by the measured amounts into Syringes, Sachets, or capsules, such as size of the milling media, load/suspension weight ratio of the gelatin or Soft capsules, Suited for delivery of Suspension or milling media, Suspension composition (e.g., the amount of dispersion compositions. Particularly when used as a medical active material relative to the amount of edible lipid), agita food or dietary Supplement, the compositions described tion rate, and milling time. herein may also be prepared for distribution over or within 60 Depending on the nature of the active material, one or more other foods, liquids, or dry goods for consumption by the pre-milling steps may also be utilized prior to a final nanom intended subject. illing process. Pre-milling of the active material may be car ried out using any suitable method and system for size com III. Methods minution of the active material. Pre-milling may be 65 particularly helpful where the active material, as Supplied, Methods for preparing the compositions described herein exhibits a relatively coarse PSD. By subjecting the active are provided. In certain embodiments, methods for preparing material to a pre-milling step, the PSD of the active material US 9,084,726 B2 10 can be reduced to better approximate the targeted nanopar D(0.50) particle size 10.7 um. FIG. 1 and FIG. 2 illustrate the ticulate PSD and, thereby, reduce the process time required in PSD of the GenividaTM and Bonistein(R) materials as supplied the final milling process. Such an approach may be particu from DSM. larly advantageous where the active material is to be milled in A food grade, 100% olive oil was obtained for use as an an edible lipid, as the edible lipid and/or active material may 5 edible lipid. The olive oil was purchased from a supermarket be adversely affected by prolonged exposure to the heat, and was a generic Mediterranean olive oil blend containing shear, and grinding forces that could be required to reduce the olive oils from Italy, Spain, Tunisia, Turkey, and Morocco. PSD of a coarse active material to a suitable nanoparticulate Methods: range. A premilling step may also allow sourcing and use of The GenividaTM and Bonistein(R) materials were dispersed active material that exhibits relatively coarse initial particle 10 within the olive oil to provide a suspension composition that size characteristics, which, in turn, may lead to cost savings in included 25% (w/v) genistein. Several samples (s200 ml per the materials used for preparation of compositions according sample) of the 25% w/v genistein/olive oil suspension were to the present description. divided and milled using a, MiniCer(R) nanomill from Methods for administering the physiologically active phe Netzsch. The samples containing Bonistein(R) were milled for nolic compounds included in the compositions described 15 60 minutes in a 0.3 mm YTZ media (yttria stabilized zirco herein are also provided. The active materials described nium oxide (ZrO.) beads). Samples containing GenividaTM herein have several potential nutritional and therapeutic ben were milled for 60 minutes in a 0.5 mm YTZ media. efits, with many being recognized as powerful antioxidants A selection of the samples containing GenividaTM were and considered to confer nutritional and therapeutic benefits Subjected to a pre-milling process (referred to or labeled as to Subjects Suffering from or at risk of conditions ranging “pre-grind samples). The pre-milling process also utilized a from various forms of inflammation to certain forms of can MiniCer(R) nanomill from Netzsch with YTZ milling media. cer. Additionally, physiologically active phenolic compounds However, the milling media utilized in the pre-milling step disclosed herein have been associated with potential cardio was 1.25 mm YTZ media. Pre-milling was carried out for 20 protective and neuroprotective effects, particularly when con minutes, with the purpose of reducing the D(0.50) particle Sumed over time. Even further, physiologically active phe 25 size of the GenividaTM to below 10 um. After pre-milling, nolic compounds specified herein have been used to those samples Subjected to the pre-milling procedure were ameliorate symptoms associated with changes in hormone milled in a MiniCerR nanomill for 60 minutes in a 0.3 mm levels or production in Subjects. YTZ media. The compositions described herein will typically be con After the milling steps, particle size analysis was com Sumed or administered orally, and in Such embodiments, the 30 pleted for each sample. Milling of the genistein compositions amount and frequency with which the composition is con resulted in a size comminution of the Bonistein(R) and Sumed by a subject may depend on the desired nutritional or GenividaTM materials, with each sample exhibiting a nano therapeutic benefit to be achieved, the nature of the phenolic particulate PSD (see, FIG. 4). An amount of each nanosus compound(s) included in the composition, and the physical pension composition was aliquoted into 3.7 cc glass vials, characteristics of the Subject (e.g., age, weight, gender). The 35 which were then stored at ambient temperature. compositions described herein may be administered or con At approximately two months, samples were taken from Sumed, for example, as a single dose, a regular daily dose, a each vial and Subjected to laser diffraction particle size analy two-times daily dose, a three-times daily dose, or according to sis and TEM analysis. Particle size analysis and distribution another desired schedule. Moreover, in specific embodi obtained by TEM analysis was consistent with that obtained ments, a composition as described herein may be adminis 40 by laser diffraction analysis, and showed that the genistein tered to or consumed by a Subject in an amount Sufficient to material Suspended within the samples maintained a nanopar deliver a dose of a phenolic compound contained therein that ticulate PSD. ranges from about 50 mg/day to about 10,000 mg/day. In At the 3-month time point, a sample of each nanosuspen certain Such embodiments, the composition is delivered to or sion was taken from each vial and again subjected to laser consumed by the Subject in an amount Sufficient to deliver a 45 diffraction particle size analysis and TEM analysis. Even dose of phenolic compound selected from about 50 mg/day to after three months, the genistein material Suspended in the about 9,000 mg/day, about 50 mg/day to about 8,000 mg/day, olive oil remained nano-sized, with the D(0.50) at 3 months in about 50 mg/day to about 2,000 mg/day, about 100 mg/day to all cases being <300 nm (see, FIGS. 3A-3C). about 9,000 mg/day, about 100 mg/day to about 5,000 For each of the samples at each time point, particle size mg/day, about 100 mg/day to about 4,000 mg/day, about 100 50 analysis conducted by laser diffraction was carried out using mg/day to about 2,000 mg/day, and about 100 mg/day to a LA-950 particle size analyzer from Horiba Scientific, Edi about 1,000 mg/day. son, N.J. EXAMPLES The invention claimed is: 55 1. A composition Suitable for consumption by a subject, the Example 1 composition comprising: an edible lipid; and Preparation of Nano Particulate Compositions of an active material suspended within the edible lipid form Genistein ing a nanoparticulate Suspension, wherein the active 60 material comprises a physiologically active phenolic Materials: compound and exhibits a particle size distribution char GenividaTM, a food grade genistein, and Bonistein(R), a acterized by a D (0.50) of 0.5 um or less. pharmaceutical grade genistein, were obtained from a com 2. The composition according to claim 1, wherein the mercial supplier (DSM, http://www.dsm.com/corporate/ active material exhibits a particle size distribution character home.html) and Subjected to particle size analysis. As pro 65 ized by a D (0.50) selected from a D (0.50) of 0.4 um or less, vided, the GenividaTM material exhibited a D(0.50) particle a D (0.50) of 0.35um or less, a D (0.50) of 0.30 um or less, a size 29.5 um, and the Bonistein(R) material exhibited a D (0.50) of 0.25um or less, and a D (0.50) of 0.20 um or less. US 9,084,726 B2 11 12 3. The composition according to claim 2, wherein the particle size distribution characterized by a D (0.50) selected active material exhibits a particle size distribution character from a D (0.50) of 0.4 um or less, a D (0.50) of 0.35um or less, ized by a D(0.50) of 0.3 um or less. aD (0.50) of 0.30pum or less, a D (0.50) of 0.25um or less, and 4. The composition according to claim 2, wherein the a D (0.50) of 0.20 um or less. active material exhibits a particle size distribution character 16. The method according to claim 15, wherein milling the ized by a D (0.90) of 2.0 um or less. active material in the presence of the edible lipid comprises 5. The composition according to claim 4, wherein the milling the active material until the active material exhibits a active material exhibits a particle size distribution character particle size distribution characterized by a D(0.50) of 0.3 um ized by a D (0.90) selected from a D (0.90) of 1.5um or less, or less. a D (0.90) of 1.0 um or less, and a D (0.90) of 0.5um or less. 10 17. The method according to claim 15, wherein milling the 6. The composition according to claim 2, wherein the active material in the presence of the edible lipid comprises physiologically active phenolic compound is selected from milling the active material until the active material exhibits a the group consisting of isoflavones, curcuminoids, flavonols, particle size distribution characterized by a D (0.90) of 2.0 um stilbenoids, and combinations thereof. or less. 7. The composition according to claim 6, wherein the 15 18. The method according to claim 17, wherein milling the physiologically active phenolic compound is selected from active material in the presence of the edible lipid comprises the group consisting of curcumin, desmethoxycurcumin, bis milling the active material until the active material exhibits a desmethoxycurcumin, genistein, daidzein, catechin, epicat particle size distribution characterized by a D (0.90) selected echin, EGCG, resveratrol, and combinations of two of more from a D (0.90) of 1.5um or less, a D (0.90) of 1.0 um or less, of Such compounds. and a D (0.90) of 0.5um or less. 8. The composition according to claim 6, wherein the 19. The method according to claim 15, wherein the physi physiologically active phenolic compound is genistein. ologically active phenolic compound is selected from the 9. The composition according to claim 6, further compris group consisting of isoflavones, curcuminoids, flavonols, stil ing a dispersant selected from the group consisting of lecithin, benoids, and combinations thereof. polysorbate 80, steroyl-2-lactate, polyoxyethylene esters, 25 20. The method according to claim 19, wherein the physi Sucrose esters of fatty acids, polyglycerol esters, fatty acid ologically active phenolic compound is selected from the esters of propylene glycol, and glycerol fatty acid esters. group consisting of curcumin, desmethoxycurcumin, bis 10. The composition according to claim 6, wherein the desmethoxycurcumin, genistein, daidzein, catechin, epicat edible lipid is an edible glyceride having a chain length rang echin, EGCG, resveratrol, and combinations of two of more ing from one carbon acetate to 22 carbons, and wherein the 30 of such compounds. edible glyceride is selected from the group consisting of 21. The method according to claim 20, wherein the physi mono-glycerides, di-glycerides, and triglycerides. ologically active phenolic compound is genistein. 11. The composition according to claim 6, wherein the 22. The method according to claim 19, further comprising edible lipid is an edible plant oil or animal oil selected from providing a dispersant, wherein the dispersant is blended with the group consisting of olive, corn, Soy, marine, coconut, 35 the edible lipid and the active material, and wherein the dis palm, palm kernel, cotton seed, peanut, safflower, Sesame, persant is selected from the group consisting of lecithin, Sunflower, almond, cashew, macadamia, pecan, pine nut, wal polysorbate 80, steroyl-2-lactate, polyoxyethylene esters, nut, lemon, orange, flaxseed, borage oils, fish oils, and dairy Sucrose esters of fatty acids, polyglycerol esters, fatty acid derived fats. esters of propyleneglycol, and glycerol fatty acid esters. 12. The composition according to claim 8, wherein the 40 23. The method according to claim 19, wherein the edible edible lipid comprises olive oil. lipid is an edible glyceridehaving a chain length ranging from 13. A method for preparing an edible Suspension of a one carbon acetate to 22 carbons, and wherein the edible physiologically active phenolic compound, the method com glyceride is selected from the group consisting of mono prising: glycerides, di-glycerides, and triglycerides. providing an edible lipid; 45 24. The method according to claim 19, wherein the edible providing an active material comprising a physiologically lipid is an edible plant oil or animal oil selected from the active phenolic compound; and group consisting of olive, corn, Soy, marine, coconut, palm, milling the active material in the presence of the edible palm kernel, cotton seed, peanut, safflower, Sesame, Sun lipid to form a nanoparticulate Suspension of the active flower, almond, cashew, macadamia, pecan, pine nut, walnut, material suspended within the edible lipid, wherein the 50 lemon, orange, flax seed, borage oils, fish oils, and dairy active material in the nanoparticulate Suspension exhib derived fats. its a particle size distribution characterized by a D(0.50) 25. The composition according to claim 1, wherein the of 0.5um or less, and wherein milling the active material concentration of the active material in the nanoparticulate in the presence of the edible lipid comprises nanomilling Suspension is 200 mg/ml or higher. the active material in a milling media that comprises the 55 26. The composition according to claim 25, wherein the edible lipid. concentration of the active material in the nanoparticulate 14. The method according to claim 13, wherein, prior to Suspension is selected from a concentration of 250 mg/ml or milling the active material in the presence of the edible lipid, higher, a concentration of 300 mg/ml or higher, a concentra the active material is Suspended within the lipid and milling tion of 350 mg/ml or higher, a concentration of 400 mg/ml or the active material in the presence of the edible lipid com 60 higher, a concentration of 450 mg/ml or higher, and a con prises introducing the combined active material and edible centration of 500 mg/ml or higher. lipid into a nanomill and processing the combined active 27. The method according to claim 13, wherein the con material and lipid within the nanomill until the active material centration of the active material in the nanoparticulate Sus exhibits a desired particle size distribution. pension is 200 mg/ml or higher. 15. The method according to claim 13, wherein milling the 65 28. The method according to claim 27, wherein the con active material in the presence of the edible lipid comprises centration of the active material in the nanoparticulate Sus milling the active material until the active material exhibits a pension is selected from a concentration of 250 mg/ml or US 9,084,726 B2 13 14 higher, a concentration of 300 mg/ml or higher, a concentra tion of 350 mg/ml or higher, a concentration of 400 mg/ml or higher, a concentration of 450 mg/ml or higher, and a con centration of 500 mg/ml or higher. 29. The method according to claim 19, wherein the con centration of the active material in the nanoparticulate Sus pension is 200 mg/ml or higher. 30. The method according to claim 29, wherein the con centration of the active material in the nanoparticulate Sus pension is selected from a concentration of 250 mg/ml or 10 higher, a concentration of 300 mg/ml or higher, a concentra tion of 350 mg/ml or higher, a concentration of 400 mg/ml or higher, a concentration of 450 mg/ml or higher, and a con centration of 500 mg/ml or higher. k k k k k 15