EP2063898A1 - Utilisation d'équol pour améliorer ou prévenir des maladies ou des troubles neuropsychiatriques et neurodégénératifs - Google Patents

Utilisation d'équol pour améliorer ou prévenir des maladies ou des troubles neuropsychiatriques et neurodégénératifs

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Publication number
EP2063898A1
EP2063898A1 EP06813546A EP06813546A EP2063898A1 EP 2063898 A1 EP2063898 A1 EP 2063898A1 EP 06813546 A EP06813546 A EP 06813546A EP 06813546 A EP06813546 A EP 06813546A EP 2063898 A1 EP2063898 A1 EP 2063898A1
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EP
European Patent Office
Prior art keywords
equol
phyto
fed
dht
rats
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP06813546A
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German (de)
English (en)
Other versions
EP2063898A4 (fr
Inventor
Edwin Douglas Lephart
Trent D. Lund
Kenneth David Reginald Setchell
Robert J. Handa
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Cincinnati Childrens Hospital Medical Center
Brigham Young University
Colorado State University Research Foundation
Colorado State University
Original Assignee
Cincinnati Childrens Hospital Medical Center
Brigham Young University
Colorado State University Research Foundation
Colorado State University
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Application filed by Cincinnati Childrens Hospital Medical Center, Brigham Young University, Colorado State University Research Foundation, Colorado State University filed Critical Cincinnati Childrens Hospital Medical Center
Publication of EP2063898A1 publication Critical patent/EP2063898A1/fr
Publication of EP2063898A4 publication Critical patent/EP2063898A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to equol and its use as a therapeutic compound for treating and preventing physiological and pathophysiological conditions mediated by androgens. Specifically, the present invention relates to the use of equol for preventing or treating neuropsychiatric or neurodegenerative diseases or disorders.
  • phytoestrogens have received increased investigative attention due to their potential protective effects against age-related diseases (e.g. cardiovascular disease and osteoporosis) and hormone-dependent cancers (i.e., breast and prostate cancer).
  • age-related diseases e.g. cardiovascular disease and osteoporosis
  • hormone-dependent cancers i.e., breast and prostate cancer.
  • isoflavones derived principally from soybeans
  • lignans found in flaxseed in large quantities
  • coumestans derived from sprouting plants like alfalfa
  • human consumption of isoflavones has the largest impact due to its availability and variety in food products containing soy.
  • genistin the precursor of gen- istein
  • malonylglucoside and acetylglucoside forms also are found.
  • These conjugates are metabolized in the GI tract by intestinal bacteria, which hydrolyze the carbohydrate moiety to the biologically active phytoestrogen, genistein.
  • the same metabolic step occurs for the aglycone daidzein, which is converted from the glycosidic form daidzein.
  • Daidzein is then further metabolized to equol in an "equol-producing" mammal. Thereafter, equol circulates in the blood stream at very high concentrations.
  • Equol is not normally present in the urine of most healthy adults unless soy is consumed.
  • the formation of equol in vivo is exclusively dependent on intestinal microflora as evidenced from the finding that germ- Phyto-Free animals do not excrete equol, and that equol is not found in the plasma and urine of newborn or 4-month old infants fed exclusively soy foods from birth due to the fat that the intestinal flora has not yet developed in neonates. See Setchell K. D. R. et at, The Lancet 1997; 350:23-27.
  • the phenolic ring structures of isoflavones enable these compounds to bind estrogen receptors (ER) and mimic estrogen. Although genistein and daidzein bind to ER, it is with a lower affinity when compared to estradiol, and with a greater affinity for ER ⁇ than to ERa. Additionally, phytoestrogens have been reported to act like natural selective estrogen receptor modulators (SERMs) at various tissue sites throughout the body. In some tissues, there is evidence that phytoestrogens act as estrogen agonists, whereas in others, they display antagonistic characteristics comparable to that of tamoxifen or raloxifene where SERM activity appears to be sex-hormone and gender dependent. [0006] While the bulk of the scientific literature has focused on the natural isoflavones in soy or clover, little has been reported on the actions or effects of their intestinally derived metabolites.
  • SERMs selective estrogen receptor modulators
  • Equol (7-hydroxy-3(4'hydroxyphenyl)-chroman) represents the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abun- dantly in soybeans and soy-foods. Equol, however, is not a phytoestrogen, because it is not a natural constituent of plants. Equol does not occur naturally in any plant-based products. Rather, it is a non-steroidal isoflavone that is exclusively a product of intestinal bacterial metabolism (relatively few individuals, ⁇ 30- 400%, have the micro flora necessary to convert soy isoflavones to equol).
  • equol possess some weak estrogenic properties, binds sex hormone binding globulin, binds ⁇ -fetoprotein, and has antioxidant activity.
  • equol is unique among the plant-derived isoflavones in that it possesses a chiral center and as such exists as two distinct enantiomeric forms, the R- and S-enantiomers.
  • the S-enantiomer of equol is the exclusive equol form found in the urine and plasma of "equol-producing" mammals consuming soy, and is the only equol enantiomer made by human intestinal bacteria. All previous studies on equol appear to have been conducted with the racemic form of equol.
  • the prostate gland depends on androgen hormone action for its development and growth, and the development of human benign prostatic hyperplasia (BPH) clearly requires a combination of testicular androgens during the aging process.
  • testosterone is not the major androgen responsible for growth of the prostate.
  • the principal prostatic androgen is 5 ⁇ -dihydrotestosterone (5 ⁇ - DHT), as evidenced by current treatments of prostatic cancer are directed toward reducing 5 ⁇ -DHT with 5 ⁇ -reductase inhibitors.
  • 5 ⁇ -DHT levels in the prostate remain at a normal level with aging, despite a decrease in the plasma testosterone.
  • Testosterone is converted to 5 ⁇ -DHT by 5 ⁇ - reductase in prostatic stromal and basal cells.
  • 5 ⁇ -DHT is primarily responsible for prostate development and the pathogenesis of BPH.
  • Inhibitors of 5 ⁇ -reductase reduce prostate size by 20% to 30%. This reduction in glandular tissue is achieved by the induction of apoptosis, which is histologically manifested by ductal atrophy.
  • 5 ⁇ -reductase occurs as 2 isoforms, type 1 and type 2, with the prostate expressing predominantly the type-2 isoform, and the liver and skin expressing primarily the type-1 isoform. Patients have been identified with deficiencies in the type-2 5 ⁇ -reductase, but not type 1.
  • Knockout mice with the type-2 5 ⁇ -reductase null-mutation demonstrate a phenotype similar to that seen in men with 5 ⁇ - reductase deficiency.
  • Type-1 5 ⁇ -reductase knockout male mice are phenotypically normal with respect to reproductive function. Enzymatic activity for 5 ⁇ -reductase or immunohistochemical detection has been noted in other genitourinary tissues, such as the epididymis, testes, gubemaculum, and corporal cavemosal tissue. [0009] Quantitatively, women secrete greater amounts of androgen than of estrogen.
  • the major circulating steroids generally classified as androgens include dehydroepiandrosterone sulphate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T), and 5 ⁇ -DHT in descending order of serum concentration, though only the latter two bind the androgen receptor to a significant degree.
  • DHEAS dehydroepiandrosterone sulphate
  • DHEA dehydroepiandrosterone
  • A testosterone
  • 5 ⁇ -DHT is primarily a peripheral product of testosterone metabolism.
  • Testosterone circulates both in its free form, and bound to protein including albumin and sex steroid hormone-binding globulin (SHBG), the levels of which are an important determinant of free testosterone concentration.
  • SHBG albumin and sex steroid hormone-binding globulin
  • the postmenopausal ovary is an androgen-secreting organ and the levels of testosterone are not directly influenced by the menopausal transition or the occurrence of menopause.
  • the work of some research has focused on the development of steroidal compounds for the treatment of androgen dependent diseases such as: hirsutism, androgenic alopecia, benign prostatic hyperplasia (BPH) and prostate cancer.
  • 5 ⁇ -DHT has been implicated as a causative factor in the progression of these diseases, largely through the clinical evaluation of males who are genetically deficient of steroid 5 ⁇ -reductase enzyme.
  • the present invention is a method of preventing or ameliorating a neu- ropsychiatric or neurodegenerative disease or disorder in a subject.
  • the method includes administering a composition comprising equol in an amount sufficient to prevent or ameliorate the neuropsychiatric or neurodegenerative disease or disorder.
  • the equol may be a racemic mixture of R-equol and S-equol.
  • the equol is enantiomerically enriched with R-equol.
  • the equol may be enantiomerically enriched with S-equol.
  • the neuropsychiatric or neurodegenerative disease or disorder may be depression, anxiety, bipolar disorder, obsessive-compulsive disorder, hyperactive disorder, weight gain or obesity.
  • the neuropsychiatric or neurodegenerative disease or disorder may be Alzheimer's disease or Parkinson's disease.
  • the neuropsychiatric or neurodegenerative diseases or disorder may be a peri- or postmenopausal symptom.
  • the composition may further comprise an excipient.
  • the composition is in an oral formulation selected from the group consisting of a tablet, capsule, powder, trouche, buccal tablet, and sublingual tablet.
  • the composition may be an oral formulation comprising at least about 0.25 mg of equol.
  • the composition may be a food product and a beverage.
  • the composition may be a delayed or a sustained release formulation.
  • the equol is in an amount sufficient to bind free 5 ⁇ - dihydrotestosterone and inhibit its binding with androgen receptors.
  • the equol is in an amount sufficient to bind free 5 ⁇ -dihydrotestosterone and inhibit its binding with androgen receptors, and sufficient to bind estrogen receptor subtypes.
  • the present invention is a method of reducing obesity in a subject.
  • the method includes administering a composition comprising a therapeutically effective amount of equol.
  • the present invention is a method of reducing depression in a subject.
  • the method includes administering a composition comprising a therapeutically effective amount of equol.
  • the present invention is a method of reducing anxiety in a subject.
  • the method includes administering a composition comprising a therapeutically effective amount of equol.
  • the present invention is a method of providing a personalized treatment of a neuropsychiatric or neurodegenerative disease or disorder in a subject.
  • the method includes assessing a condition of the emotional, mental or endocrine health of the patient; assessing an equol-producer status of a patient; and determining an optimally beneficial course of treatment, selected from the group consisting of a) mode of administration, b) a dose amount, and c) a dose interval.
  • FIG. 1 shows the chemical structures of S-equol and R-equol enanti- omers.
  • FIG. 2A shows an appearance/disappearance plot of R-equol in plasma after oral administration of R-equol to a healthy adult.
  • FIG. 2B shows an appearance/disappearance plot of S-equol and R- Equol in plasma (mean levels) after oral administration of S-equol or R-equol in healthy human adults (from: K.D. Setchell et al, Am J. Clin. Nutr., 81: 1072-1091, 2005).
  • FIG. 3 shows a mass chromatogram of the elution of the equol enanti- omers from a sample of urine from an adult consuming soy food, compared against pure enantiomeric standards that had been characterized by optical dichro- ism.
  • FIG. 4 shows the GC-MS analysis of the trimethylsilyl ether derivative of synthesized product.
  • FIG. 5 shows a mass chromatogram of a chiral separation of S-equol and R-equol from a racemic mixture.
  • FIG. 6A shows prostate weight for in intact male rats subcutaneously injected with DMSO or equol.
  • FIG. 6B shows lutenizing hormone (LH) for in intact male rats subcutaneously injected with DMSO or equol.
  • FIG. 7 shows a distinct peak in [3H] DHT+equol but not [3H] DHT alone.
  • FIG. 8A shows two distinct peaks in [3 H] -DHT+equol incubated with prostate (A), while,
  • FIG. 8B shows only a single peak is present in [3H]-DHT incubated with prostate (B).
  • FIG. 9 shows the specific binding of equol to [3H]-DHT.
  • FIG. 1OA shows prostate weight in gonadectomized (GDX) male rats sc injected with DMSO, 5 ⁇ -DHT, equol, or both 5 ⁇ -DHT and equol.
  • FIG. 1OB shows plasma LH in gonadectomized (GDX) male rats sc injected with DMSO, 5 ⁇ -DHT, equol, or both 5 ⁇ -DHT and equol.
  • FIG. 11 shows plasma 5 ⁇ -DHT levels in rats treated with DMSO 5 DHTP, equol, or both DHTP and equol.
  • FIG. 12 shows the histological effects of equol in the prostate gland of GDX (A-D) and intact (E & F) rats treated with either Trent: DMSO (A & E), equol (B & F), DHT (C), or DHT plus equol (D).
  • FIG. 13 shows the histological effects of equol on the epididymis of intact rats treated with DMSO (A) or equol (B).
  • FIG. 14 shows body weight in male rats fed either an isoflavone-rich (Phyto-600) or a phytoestrogen-free (Phyto-Free) diet.
  • FIG. 15 shows the white adipose tissue mass in male rats fed either a Phyto-600 or Phyto-Free diet.
  • FIGS. 16A and 16B show food and water intake in male rats fed a Phyto-600 or a Phyto-Free diet, respectively.
  • FIGS. 17 A and 17B show plasma leptin and insulin levels from male rats fed a Phyto-600 or a Phyto-Free diet, respectively.
  • FIG. 18 shows serum glucose levels from male rats (non-fasting) fed either a Phyto-600 or Phyto-Free diet.
  • FIG. 19 shows thyroid (T3) serum levels in male rats fed either a Phyto- 600 or Phyto-Free diet.
  • FIG. 20 shows body weights of female rats fed either a Phyto-600 or Phyto-Free diet.
  • FIG. 21 shows the white adipose tissue mass from female rats fed either a Phyto-600 or a Phyto-Free diet.
  • FIG. 22 shows serum glucose levels from female rats fed either a Phyto-600 or Phyto-Free diet.
  • FIG. 23 shows serum T3 levels from female rats fed either a Phyto-600 or Phyto-Free diet.
  • FIG. 24 shows the body weights of female rats fed either a Phyto-600 or a Phyto-Free diet after 50 days of age.
  • FIG. 25 shows the white adipose tissue mass of female rats fed either a Phyto-600 or al Phyto-Free diet after 50 days of age.
  • FIG. 26 shows the serum leptin levels of female rats fed either a Phyto- 600 or a Phyto-Free diet after 50 days of age.
  • FIG. 27 shows the serum insulin levels of female rats fed either a Phyto-600 or a Phyto-tree diet after 50 days of age.
  • FIG. 28 shows body weights of OVX rats fed either a Phyto-600 (black bars) or a Phyto-Free (white bars) diet after and placed on a behavioral estrus induction regimem.
  • FIG. 29 shows the white adipose tissue mass of OVX rats fed either a Phyto-600 or 4 Phyto-Free diet.
  • FIG. 30 shows the serum leptin levels of OVX rats fed either a Phyto- 600 or a Phyto-Free diet.
  • FIG. 31 shows the body weights of 112-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 32 shows the body weights of 279-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 33 shows the body weights of 350-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 34 shows the adipose tissue mass from 350-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 35 shows serum insulin levels in 350-day-old male rats fed AIN- 76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 36 shows serum leptin levels in 350-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 37 shows body weights of 112-day-old female rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 38 shows body weights of 279-day-old female rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diets.
  • FIG. 39 shows body weights of 145-day-old male Noble rats fed Phyto- Free or Phyto-600 diets.
  • FIG. 40 shows white adipose tissue mass from 145-day-old male Noble rats fed Phyto-Free or Phyto-600 diets.
  • FIG. 41 shows body weights of 145-day-old female Noble rats fed Phyto-Free or Phyto-600 diets.
  • FIG. 42 shows white adipose tissue mass from 145-day-old female Noble rats fed Phyto-Free or Phyto-600 diets.
  • FIG. 43 shows baseline body weights of three groups of rats on a Phyto-Free diet prior to receiving equol injections.
  • FIG. 44 shows body weights of three groups of rats after 21 days on a Phyto-Free diet prior to receiving equol or vehicle injections.
  • FIG. 45 shows body weights of three groups of rats on a Phyto-Free diet 7 days after receiving equol or vehicle injections.
  • FIG. 46 shows body weights of three groups of rats on a Phyto-Free diet 15 days after receiving equol or vehicle injections.
  • FIG. 47 shows body weights of three groups of rats on a Phyto-Free diet 22 days after receiving equol or vehicle injections.
  • FIG. 48 shows body weights of three groups of rats on a Phyto-Free diet 28 days after receiving equol or vehicle injections.
  • FIG. 49 shows adipose tissue mass from three groups of rats on a Phyto-Free diet 28 days after receiving equol or vehicle injections.
  • FIG. 49 shows adipose tissue mass from three groups of rats on a Phyto-Free diet 28 days after receiving equol or vehicle injections.
  • FIG. 50 shows testes weight from three groups of rats on a Phyto-Free diet 28 days after receiving equol or vehicle injections.
  • FIG. 51 shows number elevated-plus maze anxiety-related behavior (entries into open arms) of 300-day-old male rats fed 4 different diets.
  • FIG. 52 shows elevated-plus maze anxiety-related behavior (time in open arms) of 300-day-old male rats fed 4 different diets.
  • FIG. 53 shows elevated-plus maze anxiety-related behavior (entries into open arms) of 330-day-old female rats fed 4 different diets.
  • FIG. 54 shows elevated-plus maze anxiety-related behavior (time in open arms) of 330-old female rats fed 4 different diets.
  • FIG. 55 shows serum isoflavone levels in 300-day-old male rats fed 4 different diets.
  • FIG. 56 shows serum isoflavone levels in 330-day-old female rats fed 4 different diets.
  • FIG. 57 shows the observed change in BMI for individuals after 5 weeks of strict adherence to a diet containing isoflavones.
  • FIG 58 is a graph of the total distance traveled in meters before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 56 shows serum isoflavone levels in 330-day-old female rats fed 4 different diets.
  • FIG. 57 shows the observed change in BMI for individuals after 5 weeks of strict adherence to a diet containing isoflavones.
  • FIG 58 is a graph of the total distance traveled in meters before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a
  • 59 is a graph of the overall average speed (in meters per second) before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 60 is a graph of the total time mobile in seconds before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 61 is a graph of the total number of dives before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 60 is a graph of the total time mobile in seconds before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 62 is a graph of the total time immobile in seconds before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 63 is a graph of the total number of boli excreted before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 63 is a graph of the total number of boli excreted before the equol injections (A) and after the equol injections (B) in Phyto-free fed mid-aged female rats compared to animals fed a Phyto-600 diet in the Porsolt swim test.
  • FIG. 63 is a graph of the total number of boli excreted before the equol injections (A) and after
  • FIG. 65 is a graph of the number of entries into the open arms of the elevated plus maze (EPM).
  • FIG. 66 is a graph of the time spent in the open arms of the EPM.
  • FIG. 67 is an illustration of the hormone sensitive hypothalamic regions of the brain in adult male rats after being treated with equol for 25 consecutive days.
  • FIG. 68 is a graph of the number of entries into the open arms of the EPM.
  • FIG. 69 is another graph of the time spent in the open arms of the EPM of male rats treated during prenatal and early postnatal development with equol compared to genistein or control animals.
  • the invention is based on the unexpected discovery by the inventor that equol may be used to prevent and/or treat neuropsychiatric disorders, such as depression, anxiety; and neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease or minor dementia.
  • neuropsychiatric disorders such as depression, anxiety
  • neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease or minor dementia.
  • the inventor further noted that equol may influence motivation for and regulation of feeding and weight control.
  • These findings have important ramifications in health and disease and indicate potential broad and important usage for equol in the treatment of androgen mediated pathologies. Without intending to be bound by a particular mechanism it is believed that equol can act as an anti-androgen.
  • the anti-androgenic properties of equol are unique in that equol does not bind the androgen receptor (AR) but specifically binds 5 ⁇ -dihydrotestosterone (5 ⁇ -DHT) with high affinity and thereby prevents 5 ⁇ -DHT from binding the AR. Furthermore, both the R- and S-enantiomers of equol may specifically bind 5 ⁇ -DHT, sequester 5 ⁇ -DHT from the AR and block 5 ⁇ -DHT's actions in physiological processes in vivo. Racemic equol, which constitutes R-equol and S-equol and R-equol or S-equol alone, selectively bind 5 ⁇ -DHT.
  • 5 ⁇ -DHT is recognized as the most potent androgen in the mammalian body.
  • the AR which is encoded by a single-copy gene located on the human X-chromosome specifically mediates the actions of androgens.
  • testosterone and 5 ⁇ -DHT bind the AR, certain tissues (i.e. prostate gland, hair follicles, etc.) that are only slightly influenced by testosterone are greatly influenced by 5 ⁇ -DHT.
  • 5 ⁇ -DHT has been implicated in a number of diseases and disorders.
  • Equol has a structure similar to the steroidal estrogen estradiol. Equol is unique among the isoflavones in that it possesses a chiral center and as such exists as two distinct enantiomeric forms, the R- and S-enantiomers. All previous studies on equol appear to have been conducted with the racemic form of equol.
  • R- and S-equol specifically bind 5 ⁇ -DHT. Equol racemic, R-equol or S-equol does not bind the androgen receptor. R-equol does not bind the estrogen receptor system. S-equol only binds ER ⁇ (with an affinity approximately 5-times less than 17 ⁇ -estradiol). Thus, R- and S-equol have SERM-like properties along with having the capability to selectively bind the most potent circulating androgen, 5 ⁇ -DHT.
  • the present invention addresses the effects of the enantiomeric forms of equol, and specifically, the ability of both S-equol, the natu- ral metabolite of daidzein, and R-equol antagonize the actions of the potent androgen dihydrotestosterone, 5 ⁇ -DHT. Also, without wishing to be bound by a particular theory, it is currently believed that equol plays a positive role acting as a estrogen-like molecule via the estrogen receptor subtypes. Such effects open up novel possibilities for dietary, nutraceutical, and pharmacological approaches to prevention and treatment of disease where 5 ⁇ -DHT plays a detrimental role.
  • estrogenic actions of S-equol may also be of benefit in treating or preventing prostate cancer because the combined actions of equol acting at the estrogen receptor level and as an antiandrogen.
  • Equol (7-hydroxy-3(4'hydroxyphenyl)-chroman) represents the major metabolite of phytoestrogens daidzin and daidzein, isoflavones found abundantly in soybeans and soy-foods, and is an important biologically active molecule.
  • the major circulating isofiavone is equol, which accounts for 70-90% of the total circulating isofiavone levels.
  • the present invention discloses a novel model of equol' s biological properties. In binding studies, equol enantiomers specifically bind 5 ⁇ -DHT, but not testosterone, DHEA or estrogen.
  • one embodiment of the present invention is a method of preventing 5 ⁇ -DHT binding to the AR by contacting 5 ⁇ -DHT with equol prior to DHT-AR binding occurs.
  • the enantiomeric equol may be brought into contact with the 5 ⁇ -DHT in vitro or in vivo.
  • the equol may be administered by any route that allows absorption of equol to the blood stream.
  • Biologically available 5 ⁇ -DHT is free and unbound by any native ligand prior to binding with equol.
  • Reproductive organs such as the prostate and epididymis are known to be under androgenic control.
  • rats fed a diet containing high levels of soy-derived isoflavones have prostate weights that are not altered by the consumption of this diet.
  • prostate weights are significantly decreased in phytoestrogen-rich-diet fed rats compared to animals fed a phytoestrogen-free diet.
  • equol-treated intact rats display significant decreases in prostate and epididymis weights (without alterations in testes or pituitary weights during short-term studies).
  • the ratios are still significantly different between equol-treated and control values. Equol also blocked 5 ⁇ -DHT's androgenic trophic influence on the prostate and epididymis, without significantly altering testosterone levels.
  • 5 ⁇ -DHT has negative feedback effects on circulating plasma levels of luteinizing hormone (LH). It is believed that equol significantly increases LH levels by binding 5 ⁇ -DHT and preventing this feedback effect. For example, equol completely reverses the inhibitory action of 5 ⁇ -DHT on LH levels in go- nadectomized (GDX) males, whereas 5 ⁇ -DHT plus equol-treated male rats display LH levels similar to that of control values. These data further suggest that equol may have a specific ability to bind 5 ⁇ -DHT, presumably in the blood circulation system, and block the hormonal action of 5 ⁇ -DHT in suppressing LH production or secretion.
  • LH luteinizing hormone
  • an embodiment of the present invention is a method of modulating LH levels in an individual by contacting the 5 ⁇ -DHT of the individual with enantiomeric equol.
  • the equol can be administered by any route that allows absorption of equol to the blood stream with the amount administered in accordance with the nature of the ailment to be treated and size of the individual.
  • Equol is distinct from most isoflavones in having a chiral center due to the lack of a double bond in the heterocyclic ring.
  • the phytoestrogen isoflavones from soy (daidzein, glycitein and genistein), clover (formononetin and bio- chanin A), and kudzu, (peurarin), do not have a chiral center.
  • FIG. 1 shows the chemical structures of R-equol and S-equol.
  • the present invention includes a composition having an at least physiological acceptable quantity of equol that is able to bind and sequester free 5 ⁇ -DHT (but not testosterone or DHEA) thereby preventing it binding to the androgen receptor following administration to an individual thereby having important ramifications in health and disease and a broad and important use in the treatment of androgen-mediated pathologies.
  • the compositions comprising equol may be used to prevent or treating neuropsychiatric or neurodegenerative diseases or disorders.
  • compositions containing equol are made for oral consumption.
  • equol is in an amount sufficient to prevent or treat diseases or disorder according to the methods of this invention.
  • a therapeutically effective amount of equol is used in the methods of this invention.
  • a therapeutically effective amount refers to the amount or quantity of equol (S-equol, R-equol, a racemic equol mixture, or a non-racemic equol mixture), which is sufficient to elicit the required or desired prophylactic or therapeutic response, or in other words, the amount which is sufficient to elicit an appreciable biological response when administered to a subject.
  • a therapeutically effective amount of equol may reduce anxiety or depression in a subject.
  • the composition or a product containing the composition may be a marketed or institutional food product, a pharmaceutical (i.e., drug), and an over- the-counter (OTC) medicament (i.e., nutraceutical).
  • a food composition comprises at least about 0.25 mg, more preferably, the food composition comprises about 1 mg, and preferably up to about 200 mg, enantiomeric equol or equol mixtures, per serving. Other amounts of the equol in a food composition are also contemplated.
  • an orally-administered medicament comprises at least about 0.25 mg, more preferably about 1 mg, and preferably up to about 200 mg, enantiomeric equol or equol mixture, per dose. Other amounts of the equol in a medicament composition are also contemplated.
  • a product for topical application can comprise at least about 0.1%, and up to about 10%, by weight S-equol, or R-equol, or enantiomeric mixtures. Other concentrations of equol are also contemplated for topical products containing equol.
  • a composition of the present invention may also include other cosmetic and pharmaceutical actives and excipients.
  • suitable cosmetic and pharmaceutical agents include, but are not limited to, antifungals, vitamins, antiinflammatory agents, antimicrobials, analgesics, nitric oxide synthase inhibitors, insect repellents, self-tanning agents, surfactants, moisturizers, stabilizes, preservatives, antiseptics, thickeners, lubricants, humectants, chelating agents, skin penetration enhancers, emollients, fragrances and colorants.
  • An enantiomeric equol can also be an enantiomeric equol conjugate, conjugated at the C-4' or the C-7 position with a conjugate selected from the group consisting of glucuronide, sulfate, acetate, propionate, glucoside, acetyl-glucoside, malonyl-glucoside, and mixtures thereof.
  • a composition or preparation comprising enantiomeric or mixture of equol, for administering to subjects for the treatment and/or prevention of, or for reducing the predisposition to, androgen-related diseases and conditions related thereto may also comprise one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.
  • Pharmaceutically acceptable adjuvants, carriers and/or excipients are well known in the art, for example as described in the Handbook of Pharmaceutical Excipients, second edition, American Pharmaceutical Association, 1994 (incorporated herein by reference).
  • the composition of the invention comprises a non-racemic mixture of S-equol and R-equol, having an EE for S-equol of more than 0% and less than 90%.
  • a composition that has an EE of 0% is a 50:50 race- mic mixture of the two enantiomers.
  • the composition can be made directly from a racemic mixture, by an incomplete separation and removal of either the R-equol or S-equol enantiomer from the racemic mixture.
  • the composition can also be made by combining a first equol component comprising a mixture (either a non- racemic or racemic mixture) of equol, with a second component comprising a composition consisting essentially of S-equol or R-equol. This produces a non- racemic composition that has an excess of S-equol or R-equol.
  • a composition can be prepared comprising S-equol and R-equol at a ratio of S-equol to R-equol preferably from greater than about 50:50 to about 99.5: 1, more preferably about 51:49 to about 99:1, and preferably from less than about 50:50 to about 1 :99.5, more preferably about 49:51 to about 1 :99.
  • the composition may be administered in the form of tablets, capsules, powders for reconstitution, syrups, food (such as food bars, biscuits, snack foods other standard food forms well known in the art), or in drink formulations. Drinks may contain flavoring, buffers and the like.
  • compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, trouche, tablets, buccal tablets, sublingual tablet, each containing a predetermined amount of the extract; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water or water-in-oil emulsion.
  • composition typically does not comprise a significant amount of any other androgen-receptor binding compound.
  • Equol is formed following the hydrolysis of the glycoside conjugates of daidzein from soy, and the methoxylated isoflavone formononetin, or its glycosidic conjugates found in clever. Once formed, equol appears to be metab- olically inert, undergoing no further biotransformation, save phase II metabolism or a minor degree of additional hydroxylation in the liver. As with daidzein and genistein, the predominant phase II reactions are glucuronidation and, to a minor extent, sulfation.
  • Cut-off values have been empirically derived permitting assignment of individuals to either of these categories. People who have plasma equol concentrations of less than 10 ng/mL (40 nmol/L) can be classified as 'non-equol producers' and where levels are above 10 ng/mL (40 nmol/L) this defines 'equol producers'. This distinction can also be derived from the levels in urine, an equol producer being someone excreting greater than 1000 nmol/L. Although the excretion of equol is highly variable among individuals there is a large demarcation be- tween those that can produce equol and those that cannot, consistent with a precursor-product relationship in enzyme kinetics catalyzing the reaction. There is consequently an inverse relationship between urinary daidzein and equol levels, and thus far no significant gender differences have been defined.
  • Enantiomeric equol can be prepared per se or as the racemic mixture.
  • Chemical synthesis routes can be used to produce the racemic mixture in good yields. In a typical synthesis process, standard chemical treatments are used to hydrogenate the double-bond of the heterocyclic ring and to remove the car- bonyl at position C-3.
  • Typical starting materials are isoflavones such as daidzein, genistein, glycitein, horrarin, formononetin and biochanin A and their glucoside conjugates. Any conjugated form would be reduced to its aglycon by hydrolysis.
  • Suitable solvents for the reaction include organic acids such as glacial acetic acid, lower alcohols such as isopropanol, and mixtures thereof.
  • Reduction catalysts typically employed include Palladium, such as 10% Pd on charcoal. Reactions can run at temperatures from ambient to 60° C, with pressures ranging from slightly above ambient, up to 200 psig (14 atm. gauge), and with reaction times of up to 30 hours or more.
  • the catalyst is removed and any filtrate evaporated.
  • the crude residue is purified, typically by chromatography employing a silica gel column, with an eluent comprising C2-C4 alcohols, C3-C7 alkanes, and mixtures thereof.
  • the purified residue can be crystallized from n-hexane to produce ( ⁇ )equol as a pure product, typically of at least 99%, with a yield typically of at least 75%.
  • the equol crystallized product is colorless, not hygroscopic, and stable in air, and does not decompose during the final filtration procedure.
  • a racemic mixture of equol can be separated into its two distinct enantiomers using a chiral-phase column with a mobile phase comprising a C4-C8 alkyl and a C2-C4 alcohol.
  • a typical example of a chiral-phase column is a Chiralcel OD column or OJ column, supplied by Daicel Chemical Industries Ltd.
  • a preferred example of a mobile phase comprises 70% hexane and 30% ethanol.
  • the first eluent is typically S-equol. After a second period of time from passing the racemic mixture into the inlet, the second eluent R-equol is obtained.
  • the elution of an equol enantiomer from the column can be detected by UV absorbance at 260-280 nm or by a more specific detection system such as a mass spectrometer and monitoring of ions specific to equol.
  • S-equol can be produced biologically in bulk using conventional food technology.
  • a base solution media, food product or plant extract can be provided that comprises daidzein or another related isoflavone from which daidzein can be derived.
  • the daidzein or other isoflavone can be converted to S-equol by a standard bacterial or enzyme fermentation process, to provide a bulk solution, food product or plant extract that comprises S-equol.
  • Conversion of daidzein to equol involves three major steps: 1) hydrolysis of any glucoside conjugate group, 2) conversion of the isoflavone agly- cons to a dihydro-intermediate, and 3) conversion of the dihydro-intermediate to equol.
  • the metabolic pathway and enzymes for each of the three steps required may not necessarily be present in one bacterium.
  • Anecdotal evidence from human studies suggests that there may be one or more bacteria that act in conjunction to perform these reactions, as evidenced from the fact that often dihydrodaidzein can be present in significant amounts in plasma and urine yet equol may be low or barely detectable.
  • equol may be produced from daidzein by a single organism it is believed that better or more efficient conversion can be achieved when using a mixture of bacterial species, each with its own metabolic profile.
  • Important conditions for effective conversion to S-equol include the selection of the bacterial organism or mixture of organisms, the temperature of incubation, and the amount of oxygen available to the organisms. These conditions can be optimized by techniques well known to persons skilled in this art. The organisms used to effect this change can be inactivated by standard techniques used in the food industry or, alternately, allowed to remain in an active state in the product.
  • one or more bacterial strains are required to convert the daidzein (or other related isoflavone) through intermediate products to S-equol, which generally involves one or more of the three major reactions: the conversion of isoflavone glycone to aglycon isoflavone; the conversion of aglycon isoflavone to dihydro isoflavone; and the conversion of dihydro isoflavone to the product, equol.
  • a mixed culture of organisms isolated from equine feces or a mixed culture of organisms derived from the gastrointestinal tract of a person known to an 'equol producer' can convert, as they do in vivo, the glycone daidzein to the final product S-equol.
  • Enterococcus faecalis include Enterococcus faecalis, a Lactobacillus plan- tarum, Listeria welshimeri, a mixed culture of organisms isolated from the intestinal tract of an 'equol producing' mammal, Bacteriodes fragilis, Bifidobacterium lactis, Eubactria limosum, Lactobacillus casei, Lactobacillus acidophilous, Lactobacillus delbrueckii, Lactobacillus paracasei, Listeria monocytogenes, Micrococcus luteus, Proprionobacterium freudenreichii and Sacharomyces boulardii, and mixtures thereof.
  • Typical bacterial strains that can convert an aglycon to equol include Proprionobacteria freundenreichii, a mixed culture containing: Bifidobacterium lactis, Lactobacillus acidophilus, Lactococcus lactis, Enterococcus faecium, Lactobacillus casei and Lactobacillus salivarius; and a mixed culture of organisms isolated from the intestinal tract of an 'equol producing' mammal.
  • the pH range for bacterial conversion of the isoflavone glucosides to aglycon isoflavones is from about 3 to about 9. The optimum pH depends primarily upon the type of bacteria used, and should be selected accordingly.
  • the time required for enzymatic conversion of the glucosides to aglycons, and aglycons to the equol product depends upon enzyme-related factors, particularly concentration, and the temperature and pH of the system. In most instances it is possible to achieve substantially complete conversion within 24 hours, more preferably within about 2 hours, and most preferably within about 1 hour.
  • S-equol produced in bulk can be separated from the resulting bulk solution of a bacterial production of S-equol, by methods well known in the art, including crystallization, solvent extraction, distillation, and precipitation/filtration.
  • the resulting bulk solution can contain unreacted daidzein or other related isoflavone used, by-products, and any reactants.
  • Such methods can include the use of a reverse-phase or straight-phase liquid chromatography column and these can be combined with chiral-phase chromatography.
  • a typical method of removing S-equol from a bulk solution or solid phase is by extraction. An extractant solution is added to the solution or solid phase containing the S-equol.
  • the extractant is a low molecular weight alcohol such as methanol, ethanol, isopropyl alcohol, or propyl alcohol, or an aqueous solution having a pH in the range from 3.5 to 5.5.
  • aqueous alcohol method is being used, sufficient alcohol is added to bring the alcohol/water ratio to between a minimum of 40:60 and a maximum of 95:5. More typically, the ratio is at least 60:40, and even more typically a ratio between 65:35 and 90:10.
  • an aqueous acid solution is prepared with the pH adjusted to about 3.5 to about 5.5, and more preferably within the pH range of about4.0 to about 5.0. Sufficient water is added to make a dilute liquid with a sufficiently low viscosity to permit separation of solids from liquids by centrifugation or filtration.
  • the liquid, from which insoluble solid matter has been removed, is concentrated by conventional methods for removing liquids. Methods used typically include, but are not limited to, removal of solvent by evaporation, preferably under reduced pressure.
  • the residual liquid is concentrated to at least about 15% solids, and up to about 55% solids, more typically to between 30% and 50% solids.
  • the concentrate is then diluted with water to reduce the solids content and increase the water to alcohol ratio.
  • the amount of water added can be varied over a wide range, though a final solid content between 6% and 15%, and more typically about 13%, is preferred.
  • the pH of the mixture is adjusted between about pH 3.0 and about pH 6.5, with a preferred value between about pH 4.0 and about pH 5.0.
  • the temperature is between about 20° C. to about 10° C, and more typically about 5° C. to 7° C.
  • the solid material is then separated from the liquid by standard separation techniques (centrifugation or filtration) and yields an equol-rich solid material.
  • the equol-rich material can optionally be purified, typically by chromatography employing a silica gel column, with an eluent comprising C2-C4 alcohols, C3-C7 alkanes, and mixtures thereof.
  • the purified residue can be crystallized from n-hexane to produce S-equol as a pure product, typically of at least 99%, with a yield typically of at least 75%.
  • the equol crystallized product is colorless, not hygroscopic, and stable in air, and does not decompose during the final filtration procedure.
  • the S-equol product can be authenticated by GC-MS analysis of the trimethylsilyl ether or tert-butyldimethylsilyl ether derivative, or some other appropriate volatile derivative of synthesized product as a single pure peak and a mass spectrum that is consistent with the published electron ionization spectrum of the trimethylsilyl (TMS) ether derivative of authentic equol. Confirmation of the product can also be established by direct mass spectrometry using electrospray ionization after introducing the sample into the instrument via an HPLC chiral- phase column.
  • This present invention provides a means for an individual subject to overcome the problem of not being able to produce equol in vivo, or to supply R- equol in particular, by providing delivery of equol enantiomers, the S-equol or R- equol, or non-racemic mixtures of S-equol and R-equol directly, circumventing the need for intestinal bacteria for its production or for the need to consume soy foods with equol's precursor isoflavones.
  • the delivery of S-equol can also supplement the in vivo production of S-equol in 'equol-producers', as well as in 'non-equol producers'.
  • Supplementing the diet of an equol producer with an equol enanti- omer or mixture may provide benefits when the ordinary level of S-equol produced by the equol producer is inadequate because of 1) insufficient consumption of isoflavones to produce equol, 2) antibiotic use that ablates the activity of intestinal bacteria to make equol from precursor isoflavones, or 3) other health factors that impact the level of equol production, e.g. short bowel syndrome or surgical construction of an intestinal stoma such as ileostomy.
  • a supplemental level of equol is believed to provide enhanced effect on the health and well-being of the person.
  • This invention provides a method for delivering S-equol, R-equol, racemic equol, or non-racemic mixtures of equol, in sufficient amounts to have health benefits toward androgen-related diseases and conditions associated therewith.
  • the anti-androgenic activity of equol can affect a number of tissues throughout the body.
  • the blocking of androgenic activity of 5 ⁇ -DHT may be beneficial for the treatment and prevention of (A) growth of the prostate gland with aging, benign prostatic hyperplasia (BPH) and prostate cancer, (B) female- and male-pattern baldness, (C) facial and body hair growth (hirsutism), skin health (acne, anti-aging and anti-photo aging), skin integrity (collagen and elastin robustness); (D) body weight gain (and loss), reduction in adipose tissue deposi- tion and metabolism of lipids, as well as general regulatory behaviors and effects, such as food and water intake, blood pressure changes, thyroid, glucose, leptin, insulin and the influence on the immune system; and (E) Alzheimer's disease and emotional, mental health issues, such as, mood, depression, anxiety and learning and memory by reducing the 5 ⁇ -steroid metabolites (covering androgens and progesterone) that are potent modulators of the GAB A A receptor in the brain that influences all of the
  • the amount of composition comprising the equol is administered in an amount sufficient to produce a transient level of enantiomeric equol in the blood plasma of the mammal of at least 5 nanograms per milliliter (ng/mL), more typically at least 10 ng/mL or greater, or transient levels of enantiomeric equol in urine of greater than 1000 nmol/L.
  • the composition is administered orally in a dose amount of at least about 0.25 mg, more preferably about 1 mg, more preferably of at least about 5 mg, and of up to about 200 mg, more preferably, up to about 50 mg, of enantiomeric equol.
  • a typical level of bioavailability of R-Equol in plasma after oral administration of 20 mg of R-equol enantiomer to a healthy adult is shown in the appearance/disappearance plots of R- equol in FIGS. 2 A and B.
  • R- and/or S-equol in sufficient amounts is believed to provide several advantages over delivery of a racemic mixture of equol.
  • the potency of R-equol or S-equol alone would typically be at least twice that of the racemic mixture.
  • the human body only produces the S-equol, and therefore, a composition comprising only S-equol represents a "natural" product with an ingredient, S-equol, with which the body is familiar.
  • a treatment composition comprising only, or substantially only, the R-enantiomer can produce beneficial and/or therapeutic effects.
  • administration of R-equol would supplement any endogenous S-equol present and allow for both estrogenic and anti-androgenic actions to occur in the body.
  • the invention includes the use of enantiomeric equol to treat and prevent diseases and conditions related to male- and female-pattern baldness.
  • 5 ⁇ - DHT is a known cause of scalp hair loss.
  • An androgen, specifically the principal circulating androgen, testosterone, is converted to the more potent androgen, 5 ⁇ - DHT (in the hair follicle), and the hormonal action of 5 ⁇ -DHT on scalp hair follicles cause hair loss.
  • the hormonal action of 5 ⁇ -DHT can be blocked, such as by the use in the present invention of equol to bind 5 ⁇ -DHT in the circulation (within blood vessels) and within the hair follicle, then scalp hair loss can be decreased or prevented.
  • the invention includes the use of enantiomeric equol to treat and prevent diseases and conditions related facial and body hair. Facial and body hair are regulated by androgens, but oppositely to that of the regulation of scalp hair. Specifically, the more potent androgen, 5 ⁇ -DHT, increases facial and body hair. 5 ⁇ -DHT also increases the production of sebum (oil) from the sebaceous gland, which can contribute to an increase in acne. Thus, the binding of 5 ⁇ -DHT by equol can cause a decrease in facial and body hair and in secretion of sebum (oil), and a reduction or prevention of acne.
  • the invention includes the use of enantiomeric equol to treat and prevent diseases and conditions related to skin effects, skin quality and integrity, skin aging, skin photoaging, and skin pigmentation and lightening.
  • Estrogens before but especially after menopause, improve skin health by increasing elastin and collagen content to improve skin characteristics or robustness.
  • the repair mechanism is faster and the skin heals better if estrogen or estrogen-like compounds, such as equol, are present. It is believed that an equol enantiomer mixture, and particularly S-equol, is a good stimulator of elastin and collagen and also can protect against photo-aging.
  • Equol's blocking the hormone action of 5 ⁇ -DHT can decrease sebum oil production from the sebaceous gland, which can decrease or eliminate acne. Since S-equol (though not R-equol) binds estrogen receptor(s) (mainly ER ⁇ ), the protective effects of this estrogen-like molecule would stimulate elastin and collagen in the skin. Additionally, since equol is a strong antioxidant, it can protect the skin from aging, including photo- aging. [00148] The invention includes the use of enantiomeric equol to treat and prevent diseases and conditions related to improved prostate health. The conversion of testosterone to the more potent androgen 5 ⁇ -DHT is the result of the action of the enzyme 5 ⁇ -reductase within the prostate.
  • 5 ⁇ -DHT causes benign prostatic hyperplasia (BPH), increases in the prostate weight, and can result in the need for prostatectomies and radiotherapy to treat these conditions. Finally, consumption of soy foods has received increased attention due to their 'health benefits' of decreasing hormone dependent cancers such as prostate and breast cancer. Thus, blockage of 5 ⁇ -DHT by equol decreases prostate weight in animal models and presumably will block BPH to prevent prostate cancer.
  • the invention includes a method of preventing or ameliorating neu- ropsychiatric and neurodegenerative diseases or disorders by administering equol in an amount sufficient to prevent or ameliorate the neuropsychiatric or neurodegenerative disease or disorder.
  • Neurodegenerative disorders which are often associated with aging, such as Alzheimer's disease and minor depression, as well as Parkinson's disease, are prevalent in the general population and are expensive to treat. About 4 million people in the U.S. now are thought to have Alzheimer's disease resulting in just over 100 billion dollars in cost a year, according to the Lasker Foundation. The risk of developing Alzheimer's doubles every five years after age 65 and almost 40 percent of people over age 85 have the disease (J.M. Lyness et at, Ann. Inter. Med., 144: 496-504, 2006; and Lephart et al, Curr Neurovas. Res., 1: 455-464, 2004). Minor depression is more frequent than major depression, with almost 50% percent of older community residents (60 years or older) displaying symptoms that are associated with minor depression (J.M. Lyness et ah, Ann. Inter. Med., 144: 496-504, 2006).
  • the invention includes the use of equol, including enantiomeric equol to treat and prevent diseases and conditions related to brain function and mental health, including brain disorders, such as Parkinson's diseases, dementia of the Alzheimer type, as well as other reduced or impaired cognitive functions associated with advancing age and with short- and long-term mem- ory loss.
  • brain disorders such as Parkinson's diseases, dementia of the Alzheimer type, as well as other reduced or impaired cognitive functions associated with advancing age and with short- and long-term mem- ory loss.
  • Brain mechanisms are more complex, and attempting to define what molecules and factors regulate, influence, etc., mood, depression, anxiety and so on, can be difficult.
  • estrogens or estrogen-like molecules like isoflavones can assist cognitive function in conditions such as Alzheimer's disease, and may help to prevent the onset of such disorders, especially in postmenopausal women.
  • neuropsychiatric disorders represent the second largest cause of morbidity and premature mortality worldwide.
  • the World Health Organization estimates, collectively, that neuropsychiatric disorders comprise 13 % of all reported diseases. These disorders include depression, anxiety, schizophrenia, bipolar disorder, obsessive-compulsive disorder, panic disorder, bulimia, alcohol and substance abuse, overeating and obesity, and attention-deficit hyperactivity disorder (World Health Organization. The world health report 2000 - health systems: improving performance. Geneva: WHO 2000). Approximately one out of five Americans will experience an episode of a psychiatric illness such as depression or anxiety in any given year (Mental Health Report of the U.S. Surgeon General, 1999).
  • depression is an effective disorder, or a disorder of mood. Symptoms include feelings of sadness, worthlessness, despair and loss of interest in life's activities or all pleasures. Most individuals suffering from depression also experience mental slowing, confusion, loss of energy and an inability to make decisions or concentrate. The symptoms can range from mild or minor to severe and are often associated with anxiety and/or agitation (Diagnostic Criteria form DSM- IV, American Psychiatric Association, Washington, D.C., 1994).
  • Depression has been referred to as the "common cold" of mental illness. It is a prevalent disorder with approximately 18 million adults in the U.S. population afflicted on an annual basis (National Institutes of Health, Invisible Disease: Depression. Bethesda, M.D., NIH publication 01-4591, 2001). It is estimated to be the second leading cause of disability, surpassed only by heart dis- ease (C. J. Murray & A.D. Lopez (eds.), in The Global Burden of Disease, Harvard Univ. Press, Boston, 1996). Worldwide, its prevalence is 21 % in women and 13 % in men (CB. Nemeroff and MJ. Owens, Nature Neuroscl, 5: 1068- 1070, 2002).
  • Anxiety disorders are often characterized by excessive worry, fear and apprehension, occurring frequently and causing a significant impairment of functioning in a daily routine. Symptoms can include restlessness, fatigue, difficulty concentrating, irritability, muscle tension and sleep disturbances.
  • the lifetime prevalence of anxiety disorders in the general population is approximately 5 to 6% (DJ. Nutt et al., Int. J. Neuropsychopharm., 5: 315-325, 2002). Women are affected more frequently than men, with a prevalence of 10% in women over the age of 35, anxiety rates tend to increase in midlife and in older adults (H. W. Wit- tchen, Depress. Anxiety, 16: 162-171, 2002), that are similar to that seen for depression.
  • Benzodiazepines e.g., diazepam, Valium
  • Benzodiazepines possess anxiolytic, sedative-hypnotic, anticonvulsant and muscle relaxant properties.
  • benzodiazepines may cause impairments of mental and motor function, confusion and amnesia.
  • Benzodiazepines may also be subject to dose escalation and abuse by some patients. Withdrawal from long- term use can precipitate anxiety, insomnia, restlessness and agitation (R.M. Julien, Primer of Drug Action, 7 th ed., W.H. Freeman Co., New York, 1995; and M.E. Likey and B. Gordon, Medicine and Mental Illness, W.H. Freeman Co., Boston, 1991).
  • the second line of research is more complex and supports the view that 5 ⁇ -reduced steroids, especially progesterone, have the ability to bind GABA A receptors in the brain and cause sedation.
  • GABA A is the major inhibitory neurotransmitter in the brain and its receptors are abundant in brain areas that control mood/emotion.
  • individuals express less anxiety. For example, most women during pregnancy, report that they feel OK but they are usually tired or sleepy.
  • estrogen on the one hand decreases anxiety and hence increases activity. Conversely, blocking the action of 5 ⁇ -DHP also increases activity and thus in behavioral tests is interpreted as de- creasing anxiety. For example, when the conversion of progesterone to 5 ⁇ -DHP in pregnant rats has been blocked, this results in a significant increase in their locomotor activity levels.
  • equol has the ability to bind 5 ⁇ -DHP (mainly seen in women) and 5 ⁇ -DHT (mainly seen in men). This would decrease the potent 'neurosteroid' effects at the GABA A receptor and decrease sedation and thus increase activity or decrease anxiety. Moreover, the ability of S-equol to bind the estrogen receptor(s) beta would also increase activity. Finally, our studies using young- or mid-aged adult rats, in males or fe-
  • the elevated plus maze is a behavioral test used to quantify anxiety- related behavior and identify anxiolytic drags (Pellow S. et al., JNeurosci Methods, 14:149-147 (1985); Current Protocols In Neuroscience (1997) 8.3.1-8.3.15, John Wiley & Sons. NY, N. Y.).
  • the test relies on the inherent conflict between exploration of a novel environment and avoidance of its aversive features. Normally, animals spend little time and make few entries into the open arms of the maze compared to the closed arms of the maze (Imhof J. T. et ah, , Behav Brain Res, 56:177-180 (1993).
  • the invention also includes the use of equol (R- and S-enantiomeric mixtures, etc.) to treat or prevent peri- or postmenopausal symptoms. These symptoms include mood alterations, depression, anxiety, fatigue and emotional and mental health, weight gain and obesity, and polycystic ovarian disease among others [00164]
  • the invention is a method of use of equol to prevent or treat neuropsychiatric diseases, including depression, anxiety, and others mentioned above.
  • the invention includes the use of enantiomeric equol to treat and prevent diseases and conditions related to body weight and body fat formation.
  • Weigh gain and obesity represent a huge health concern in the U.S. and developed countries throughout the world. For example, in the U.S., about 55 % of adults are overweight by international standards, about 23 % of American adults are considered obese; and one in five American kids are considered overweight. Obesity in the U.S. cost the United State Government 12 % of the national health care budget in the late 1990s or $118 billion (World Watch Institute, March, 4, 2000).
  • Phytoestogens including equol have the ability to decrease the formation of white adipose (fat) tissue and increase white adipose tissue breakdown, thus decreasing body weight. Also, the estrogen-like nature of phytoestrogen molecules decreases LDL (so-called "bad” cholesterol), blood pressure, and prevents insulin resistance (or in other words, provides beneficial effects to the diabetic condition). Since equol is a more potent isoflavone molecule compared to the other phytoestrogens, it presumably provides the health benefits and protects against the conditions outlined above.
  • the invention includes the use of equol to prevent or treat increase in body weight and obesity.
  • the term "increase in body weight” refers to any increase in body weight above an optimal healthy level that would be dependent upon age, sex and statute of the subject. A person is considered to be obese if they are more than about 20 % over their ideal weight. That ideal weight must take into account the person's height, age, sex, and build.
  • Overweight or obesity may also be defined using the body mass index, for instance.
  • body mass index For adults, overweight and obesity ranges are determined by using weight and height to calculate a number called the "body mass index” (BMI).
  • BMI body mass index
  • the invention includes the use of equol, as enantiomeric equol or mixture thereof, to treat and prevent lipid disorders such as high cholesterol (hypercholesterolemia), lipidemia, lipemia and dyslipidemia (disturbances in lipids).
  • lipid disorders such as high cholesterol (hypercholesterolemia), lipidemia, lipemia and dyslipidemia (disturbances in lipids).
  • soy protein The failure of soy protein to have significant cholesterol- lowering effects in adults with normal blood cholesterol levels, is, with few exceptions, probably because of heterogeneity in the study populations with regard to the metabolism of soy isoflavones and the failure to recognize the relevance of equol formation (and specifically, non-formation in non-equol producers). These data suggest that enantiomeric equol influences lipids in a favorable manner, and that the effect is mediated by androgens.
  • the composition comprising equol is administered in an amount sufficient to reduce the level of lipids in the blood stream.
  • the invention further includes the use of R- and/or S-equol to improve diminished blood vessel quality, by increasing reactivity or flexibility in response to acute changes in blood pressure, improving blood flow, and reducing blood pressure.
  • the invention also includes the use of R- and/or S-equol to treat and prevent cancer, including benign prostate cancer, prostate cancer, and skin cancer.
  • Equol may also be used to prevent enlarged prostate or epididymis in individuals believed to be at risk for development of these pathologies, without alterations in testes, pituitary or body weights.
  • the equol may be administered by any route that allows absorption of equol to the blood stream.
  • equol as a diagnostic agent in androgen-related disorders as well as disorders arising from disturbances in estrogenic/androgenic balance.
  • equol is administered to an individual to bind 5 ⁇ -DHT and thereby prevent 5 ⁇ -DHT binding to androgen receptors.
  • the changes in estrogenic balance are then measured or the change in androgen-binding is assessed to diagnose or further elucidate androgen-related anomalies.
  • Equol can be administered to bind 5 ⁇ -DHT prior to or along with other therapeutic moieties in order to assess the binding capacity of 5 ⁇ -DHT with respect to the therapeutic moiety in question.
  • androgen-binding moieties can be administered following administration of equol to assess the efficacy of the androgen-binding moiety to restore androgen activity and balance estrogenic activity in the absence of 5 ⁇ -DHT binding.
  • equol can be administered in the presence of 5 ⁇ -DHT-binding moieties in order to displace these naturally- occurring or xenobiotic 5 ⁇ -DHT-binding moieties from 5 ⁇ -DHT.
  • the equol may be administered by supplying an oral dosage form of equol to either an "equol-producing" mammal or a "non-equol producing” mammal, or an oral dosage of daidzein, daidzin, isoflavone mixtures containing daidzein, or soy protein preparations to an "equol- producing" mammal, wherein the administration of the oral dosage form results in effective absorption of equol to the blood stream.
  • Administration of equol may be made by routes other than oral, if desired.
  • rectal or urethral administration may be used to administer equol for the treatment of enlarged prostate or to prevent prostate enlargement.
  • the active ligand binding site of the equol molecule may be isolated and synthesized for administration, which may provide 5 ⁇ -DHT binding without the full equol molecule.
  • the dose of the equol molecule or fragment thereof having 5 ⁇ -DHT-binding abilities is dependent upon the route of admiration and the condition to be treated.
  • equol antagonize much higher doses of 5 ⁇ -DHT, and this may be explained by the marked differences in the binding of equol to serum protein compared with 5 ⁇ -DHT. The latter circulates mostly bound to proteins, while equol is 50% free.
  • the dose may be increased dramatically without incurring significant dose-limiting side effects to greater than about 10 mg/kg.
  • Oral administration can be effected in microencapsulated forms that can provide delayed or sustained release of the medicament.
  • Equol can be administered topically, transdermally, and subder- mally in a variety of forms, including lotions, ointments, foams (including shaving creams), nasal sprays, skin patches (such as described in U.S. Pat. No. 5,613,958, incorporated herein by reference in its entirety), electromechanical devices, including micropumps systems (such as described in U.S. Pat. No. 5,693,018 and U.S. Pat No. 5,848,991, incorporated herein by reference in their entirety), and subder- mal implants (such as described in U.S. Pat. No. 5,468,501, incorporated herein by reference in its entirety).
  • Equol Enantiomer in 'Equol-Producing' Adults
  • the urine samples from adults consuming soy foods previously identified as being 'equol-producers' were analyzed. Equol was isolated from urine (25 mL) by passage of the sample through a solid-phase Bond Elut Cl 8 cartridge. After washing the cartridge with water, the isoflavones were recovered by elution with methanol (5 mL) and the methanolic phase was taken to dryness under a stream of nitrogen. The sample was subjected to enzymatic hydrolysis with Helix pomatia and then re-extracted on a Bond Elut Cl 8 cartridge.
  • Equol enantiomers were identified by HPLC using a Chiral- cel OJ chiral phase column as described herein above.
  • the detection of equol was achieved by selected ion monitoring electrospray ionization mass spectrometry (ESI-MS). Mass chromatograms of a pure standard of S-equol, and of urine from an adult consuming soy food are shown in FIG. 3. Similar studies have demonstrated that soy-derived isoflavones are converted to equol in rats, as well, thus validating rodent models of isoflavone metabolism.
  • Daidzein (200 mg, 0.8 mmol) is dissolved in a mixture of glacial acetic acid (20 mL) and isopropanol (20 mL), and is reduced with 10% Pd on charcoal (150 mg) at 55 p.s.i.g. (3.7 atm gauge).
  • Pd on charcoal 150 mg
  • the catalyst is filtered off, and the filtrate is evaporated.
  • a racemic mix of S-equol and R-equol were separated by chiral chromatography on a Chiralcel OJ Column using a flow-rate of 1.0 mL/min and with a gradient elution consisting of an initial mobile phase of 10% ethanol in hexane and increasing to 90% ethanol in hexane over a time period of 15 minutes according to the program shown in Table 1 :
  • FIG. 5 shows the mass chromatogram of the ions recording (m/z
  • Enanti- omer-2 material (1.7 mg exact weight) had first and second measurements of +0.023 and +0.023, resulting in an optical rotation of +13.5[+0.023xl000/1.7], which corresponds with the R-enantiomer of equol.
  • Hormone Receptor Proteins Full length rat ERa expression vector (pcDNA-ERa; RH Price UCSF) and ER ⁇ expression vector (pcDNA-ER ⁇ ; T A Brown, Pfizer, Groton, Colo.) were used to synthesize hormone receptors in vitro using the TnT-coupled reticulocyte lysate system (Promega, Madison, Wis.) with T7-RNA polymerase, during a 90 min reaction at 30° C. Translation reaction mixtures were stored at -80° C. until further use.
  • the study shows that only the S-equol enantiomer binds ER with sufficient affinity to have potential relevance to circulating equol levels reported in humans.
  • FIG. 2A shows an appearance/disappearance plot of R-equol.
  • FIG. 2B shows an appearance/disappearance plot of S-equol and R-Equol in plasma (mean levels) after oral administration of S-equol or R-equol in healthy human adults (from: K.D. Setchell et al., Am J. Clin. Nutr., 81: 1072-1091, 2005).
  • equol In addition to equol's effects on prostate racemic equol blocks the effects of DHT in other tissues, and decreases body weight.
  • DMSO control
  • equol 0.5 mg/kg
  • Rats injected with R-equol have a significant decrease in body weight gain compared to control rats, as shown in Table 3.
  • Testis and pituitary gland weights are not significantly altered by the treatments (data not shown).
  • the slight decreases in body weight (around 10%) from the equol experiments are very similar to those seen between animals fed a Phyto-Free diet (a diet containing very low levels of phytoestrogens) vs. a Phyto-600 diet (a phytoestrogen-rich diet containing 600 ppm of isoflavones).
  • the significant reduction in white adipose tissue deposition with the racemic equol injections (around 36%) is also comparable with that seen with the data sets derived from the dietary treatment studies.
  • LH-20 columns are used in order to identify elution peaks establishing the binding of [3H]DHT to equol.
  • a peak of [3H]DHT is apparent in the elution fractions between 5 and 9 niL when the [3H]DHT+equol column incubate is applied. This peak is not present when [3H]DHT alone is applied to the column.
  • 5 ⁇ -DHT or 5 ⁇ -DHT+equol are incubated with prostate supernatant and then passed through the 30 cm column (FIG. 8A) two distinct binding peaks are identifiable.
  • the first peak of [3H]DHT represents that bound to the AR in prostate.
  • This example demonstrates the effects of racemic equol on andro- gen-sensitive tissues.
  • animals are gonadectomized (GDX) under isoflurane anesthesia and allowed to recover for 7 days.
  • animals are assigned to the following groups 1) DMSO, 2) DHTP (2mg/l kg), 3) racemic equol (0.25 mg/kg), or 4) both DHTP and racemic equol.
  • Injections are given subcutaneously daily for 4 days. Animals are killed via decapitation and trunk blood and tissues are collected for analysis. Plasma 5 ⁇ -DHT is measured, shown in FIG. 11.
  • Plasma 5 ⁇ -DHT As expected there were significant elevations of plasma 5 ⁇ -DHT in animals treated with DHTP (GDX+DHTP, GDX+equol+DHTP groups). Plasma 5 ⁇ -DHT was further elevated, although, not significantly, by co-treatment with equol. Tissues, including prostate, testes and epididymis, are removed from the animal, dissected free of fat and connective tissues, weighed, fixed by immersion in 4% paraformaldehyde, and then sectioned at 15 ⁇ m on a cryostat.
  • Tissue sections are mounted on charged slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA) prewarmed to 23 °C, and stained with hematoxylin and eosin (H&E), dehydrated in ascending alcohol and cleared with xylene. Histological sections are shown in FIGS. 12 and 13. H&E stained prostates reflect a change due to both GDX and treatments.
  • the prostate glands of control, equol, and DHTP plus equol treated groups show similar histology (FIG. 12 A, B, D). In these animals prostates are characterized by very small atrophic glands with little volume in the gland lumen. In DHTP-treated animals (FIG. 12C), the glands show signs of cell proliferation.
  • Lumen size is increased compared to GDX animals; the epithelium is of a tall columnar type (FIG. 12C).
  • the epithelium is of a tall columnar type (FIG. 12C).
  • the prostate of equol treated males show involution and consist of more closely spaced, atrophic glands (FIG. 12F).
  • the epididymal histology of equol-treated intact males shows overall smaller ducts, as evidenced by shrunken lumen (FIG. 13).
  • T3 serum glucose and thyroid
  • This example demonstrates the effects of a Phyto-600 or Phyto-Free diet on adult ovariectomized (OVX) rats.
  • the OVX rat is a well-established animal model of postmenopausal human females.
  • OVX permits the subcutaneous injections of estrogen and progesterone to stimulate behavioral estrus in rats, to determine the effects of a Phyto-600 or Phyto-Free diet.
  • Adult ovariec- tomized rats are fed a phytoestrogen diet of approximately 200 ppm of isoflavones ("Phyto-200") until 50 days of age (all animals are ovariectomized at approximately 40 days of age).
  • the female rats are age and weight matched at 50 days of age and placed into one of two diet treatments: either the Phyto-600 (black bars) or Phyto-Free (white bars) until 94 days of age.
  • Baseline body weights are taken at 50 days of age before the animals are placed on the diet treatments, again at 58 days (8 days of diet treatment), at 92 days of age (before injection of estradiol), and at 94 days of age (before injection of progesterone, and 6 hours later at 94 days of age (after chemical induction of behavioral estrus), shown in FIG. 28.
  • the Phyto-600-fed rats display a slight but significant reduction in body weight (of about 7%) compared to Phyto- Free-fed. This reduction in body weight is maintained before and during the chemical induction of behavioral estrus by the estrogen and progesterone (steroid) injections.
  • White adipose tissue is measured at 94 days of age after the chemical induction of behavioral estrus, Phyto-600-fed OVX rats have approximately 50% less white adipose tissue mass compared to Phyto-Free-fed OVX rats, shown in FIG. 29, consistent with findings in Examples 9 and 10.
  • Serum leptin levels in Phyto-600-fed OVX rats are decreased by approximately 30% compared to Phyto-Free-fed rats, shown in FIG. 30, reflecting the decreased white adipose tissue mass.
  • AIN-76 diet containing approximately ⁇ 5 ppm isoflavones
  • Phyto-Free containing approximately ⁇ 5 ppm isoflavones
  • Phyto-600 diet containing approximately ⁇ 5 ppm isoflavones
  • the AIN-76 diet contains extremely low concentrations of isoflavones, its formulation is quite different compared to the other three diets.
  • the sucrose content is very high (almost approaching 50% of the total diet formulation) and has a dense white consistency that the rats may not enjoy consuming as much as the regular plant-based ingredient diets (i.e., the Phyto-Free diet uses corn and wheat in its formulation which contains very low levels of isoflavones); the Phyto-200 or Phyto-600 diets use varying amount of soy meal in their formulations.
  • the male rats are maintained on their assigned diets until 350 days of age (equivalent to middle-age in humans).
  • the female rats are maintained on the diets until 279 days of age (approaching middle-age in humans). Food and water intake is measured to determine the potential influence of these parameters on body weight changes. In each case these factors do not contribute to the reductions in body weight with consumption of the isoflavone-containing diets (i.e., Phyto-200 and Phyto-600 diets; data not shown).
  • the male rats have a similar profile to that observed at 112 days of age, shown in FIGS. 32 and 33, respectively.
  • the males fed the Phyto-Free diet display the heaviest body weights and the Phyto-600-fed males display the lowest body weights, while the males on the AN-76 and Phyto-200 diets fall in between these two group values.
  • Males fed the AIN-76 or the Phyto-Phyto-Free fed males display the highest white adipose tissue weights, measured at 350 days of age.
  • the Phyto- 200-fed males show a 19% non-significant reduction in white adipose tissue weight compared to AIN-76 or Phyto-Free-fed rats.
  • Male rats fed the Phyto-600 diet have significantly less adipose tissue mass, an approximate 40% reduction, compared to AIN-76 or Phyto-Free-fed rats, shown in FIG. 34.
  • Both serum insulin and leptin levels are significantly reduced as a function of increasing concentrations of isoflavones in the diet treatments, shown in FIGS. 35 and 36, respectively.
  • males fed the Phyto-200 or Phyto- 600 diets have significant reductions in insulin levels compared to AIN-76 fed males.
  • Phyto-600-fed males show an approximate 50% reduction in insulin levels compared to Phyto-Free-fed male Serum leptin profiles display a similar pattern to that of the insulin results, where Phyto-200- or Phyto-600-fed males have significant reductions in serum insulin levels compared to either AIN-76 or Phyto-Free-fed males. Insulin levels in the Phyto-600-fed males are 46% lower compared to the Phyto-200-fed males. However, the difference between these two diet groups do not reach significance (p ⁇ 0.065). [00227] Females-
  • the body weights are measured at 112 and 279 days of age.
  • females fed the Phyto-Free and the Phyto-200 diets have the heaviest body weights and the Phyto-600-fed females have the lowest, while the AIN-76 diet group fall in between the values of the other three groups, shown in FIG. 37.
  • Body weights of the Phyto-600-fed groups are significantly lower, by approximately 10%, compared to the Phyto-Free- and the Phyto-200-fed females.
  • Noble rats were used to determine whether an inbred strain of rat has body and adipose tissue changes similar to those of out-bred strains of rats such as the Long-Evans animals when placed on isoflavone-rich diets. Due to inbreeding, Noble rats are more fragile animals. For example, pregnant dams do not always carry their litters to term and frequently have smaller litters. Noble rats have been used for more than twenty years because they spontaneously generate tumors with aging, especially in hormonal-dependent organs of the reproductive tract. Thus, Noble rats have been extensively studied in the area of cancer research (e.g., R. L. Noble, "Prostate carcinoma of the Nb rat in relation to hormones," Int Rev Exp Pathol, 1982, 23: 113-159).
  • Male and female Noble rats are fed either the Phyto-Free or Phyto- 600 diets from conception until 145 days of age.
  • Male Noble rats fed the Phyto- 600 diet have significantly lower body weights at 145 days of age compared to age-matched males fed the Phyto-Free diet, shown in FIG. 39.
  • the significant reduction in body weight represents a modest but consistent decrease of approximately 8% compared to Phyto-Free-fed males.
  • white adipose tissue mass is significantly decreased in Phyto- 600-fed males compared to Phyto-Free-fed, shown in FIG. 40.
  • Female Noble rats fed the Phyto-600 diet have a 6% reduction in body weight compared to Phyto-Free-fed females, shown in FIG. 41.
  • White adipose tissue mass is markedly decreased female Noble rats fed the Phyto-600 diet, shown in FIG. 42.
  • the Phyto-600 diet group has a 61% reduction in adipose tissue compared to Phyto-Free- fed rats. The decrease in white adipose tissue is similar to that seen in Long-Evans rats.
  • Phyto-Free diet Prior to initiation of a Phyto-Free diet period Male Long-Evans rats are fed a Phyto-200 diet, as described in previous examples. The rats are placed on a diet containing the Phyto-Free diet at approximately 52 days of age and randomly assigned to three groups. Baseline body weights after 14 days and 21 days on the Phyto-Free diet for all rats are similar, shown in FIGS. 43 and 44, respectively. Beginning at 73 days of age, rats receive daily subcutaneous 0.
  • testis weights are quantified in these animals. There are no significant alterations in testes weight with the equol injections, with testicular weight essentially the same among the injection treatment groups, shown in FIG. 50.
  • the Phyto-600 fed males display the highest percentage of time spent in the open arms, while the lowest percentage of time spent in the open arms is display by animals fed the AIN-76 diet, with Phyto-free and Phyto- 200 values falling in between these maximal responses in a dose-dependent fashion, shown in FIG. 52.
  • Equol Decreases Depression and Body Weight in Mid-Aged Female
  • Isoflavones have been shown to possess beneficial effects on cardiovascular and hormone-dependent diseases, however, little is known about their influences on brain and behavior, especially behaviors such as depression and despair. In this study, mid-aged Long-Evans female rats were examined in this context.
  • Age-associated morphological and physiological changes in rodents are equivalent to humans using the following age ranges: Mid-aged rats (10 - 12 months of age) are roughly equivalent to humans 45 - 55 years of age, while aged rats (21-23 months of age) are roughly equivalent to humans 65 - 75 years of age (D. Mileusnic et al., Neurobiol. Aging, 20: 19-35, 1999). Additionally, mid-aged rats (at 12 months old) are equivalent in age to postmenopausal women at approximately 55 years of age. The average age of menopause in the United States is approximately 51 years of age (National Institutes of Health, USA). [00249] All female rats in this study were examined for hormonal patterns of estrus cycling characteristics. AU animals in this study were not cycling which is consist with scientific reports using this aging animal model (CR. Anzalone et al, Biol. Reprod, 64: 1056-1062, 2001).
  • the test is designed to force rats to swim vigorously. Over time, some of the animals may become immobile which is considered a condition of behavioral despair. The total time an animal is immobile is an index of despair or depression. Other parameters include: a) total distance traveled, b) total time mobile (the opposite of the immobile index), c) number of dives the animal attempts demonstrating escape behaviors and d) the number of defecations or boli excreted during the test which indexes the level of emotionally (CS. Hall, J. Comp. Psych, 18: 385-403, 1934), increased boli numbers during the test would indicate heighten emotional behavior. All of these parameters were recorded in this study and quantified by AnyMaze® computer software and the total time of the Porsolt forced swim test was 480 seconds or 8 minutes.
  • Test 1 compared diet exposure alone, shown as A in FIGS. 58-69 and labeled as "before equol injections.”
  • DMSO dimethylsulfoxide
  • Test 2 assessed depressive behaviors following the equol treatments, shown as B in FIGS. 58-69 and labeled as "after equol injections.”
  • Test 2 assessed depressive behaviors following the equol treatments, shown as B in FIGS. 58-69 and labeled as "after equol injections.”
  • Phyto-600 fed females displayed significantly greater a) distance traveled (meters), see FIG. 58 A; b) overall average speed (meters/second), see FIG. 59 A; c) total time mobile (seconds), see FIG. 60 A; and d) number of dives compared to Phyto-free females, see FIG. 61 A.
  • the Phyto-free females displayed significantly greater a) total time immobile (seconds), see FIG. 62 A and b) boli excreted, see FIG. 63 A, compared to Phyto-600 females.
  • the Phyto-free females displayed values that were similar to those exhibited by the Phyto-600 females for all of the parameters tested above. For example, a) total distance traveled was increased and similar to control values, see FIG. 58 B; b) increased average speed was also similar to controls, see FIG. 59 B; c) along with increased total time mobile, see FIG. 60 B (the total time mobile significantly increased in the Phyto-free rats after the equol injections compared to the before equol injections levels (see **)); d) and the number of dives was significantly reduced and comparable to control levels, see FIG. 61 B. Furthermore, the total time immobile was significantly reduced and similar to controls, see FIG. 62 B. Finally, as illustrated in FIG. 63 B, the number of boli was reduced after equol treatments and this was essentially the same to that of control values.
  • Equol Decreases Anxiety-Related Behaviors and Body Weight
  • SDN serotonin-derived neuropeptide
  • AVPV anterioventroperiventicular nucleus
  • SDN and AVPV structures are sensitive to isoflavone influences in adult rats and, in general, many reports have demonstrated that phytoestrogens are neuroprotective (E.D. Lephart et al., J. Steroid Biochem. MoI. Biol, 85: 299-309, 2003; E. D. Lephart et al, Curr. Neurovas. Res., 1 : 455- 464, 2004; and L. Bu & E.D. Lephart, Neurosci. Lett, 385: 153-157, 2005).
  • equol the major isoflavone metabolite in rodents (and many other animal models) has not been examined in brain studies.
  • the EPM test relies on the inherent conflict between exploration of a novel environment and avoidance of its aversive features. Typically, animals spend little time and make few entries into the open arms (new environment) compared to the closed arms of the maze (secure environment). For example, an anti-anxiety agent or drug would increase the number of entries into and total time spent in the open arms of the maze.
  • the rats (10 ppm of isofiavones) and remained on this diet until 215 days of age.
  • the rats were age and body weight matched and then divided into control or equol treatment groups.
  • the male rats received daily subcutaneous (0.1 cc) injections of control vehicle (DMSO) or equol (2.5 mg/Kg) for 25 consecutive days.
  • DMSO control vehicle
  • equol 2.5 mg/Kg
  • the brains were processed via standard staining and analyzed via
  • Bioquant® for morphometric SDN and AVPV parameters by treatments The serum equol levels in equol-treated animals were equivalent to consuming a phy- toestrogen-rich soy diet.
  • Equol Is More Potent Compared To Genistein In Decreasing Anxiety Levels Via Pre- and Early Postnatal Treatments In Male Rat Offspring Tested As Adults
  • Pregnant Long-Evans rats were treated via daily subcutaneous injections from gestational day 14 through 20 and during lactation from postnatal day 2 through 25 with: 1) control-DMSO vehicle or, 2) genistein @ 3 mg/day or 3) equol @ 3 mg/day according to the treatment groups. All animals were fed a phy- toestrogen-low diet throughout these studies (10 ppm isoflavones). There were no significant differences in maternal body weights just before birth among the treatment groups. At birth, body weights were slightly (but not significantly) higher in genistein- and equol-treated animals compared to controls. The maternal and male offspring genistein and equol levels (@ 25 days post birth) paralleled that of the treatments administered.

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Abstract

La présente invention concerne un procédé permettant de prévenir ou d'améliorer une maladie ou un trouble neuropsychiatrique ou neurodégénératif chez un sujet. Le procédé consiste à administrer une composition contenant de l'équol en une quantité suffisante pour prévenir ou améliorer la maladie ou le trouble neuropsychiatrique ou neurodégénératif. L'équol peut être un mélange racémique de R-équol et de S-équol. L'équol peut être énantiomériquement enrichi avec du R-équol ou énantiomériquement enrichi avec du S-équol.
EP06813546A 2006-08-18 2006-08-18 Utilisation d'équol pour améliorer ou prévenir des maladies ou des troubles neuropsychiatriques et neurodégénératifs Withdrawn EP2063898A4 (fr)

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JP2010501485A (ja) 2010-01-21
WO2008020853A1 (fr) 2008-02-21

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