JP2010501485A - Use of equol to prevent or treat neuropsychiatric or neurodegenerative diseases or disorders - Google Patents

Abstract

The present invention relates to a method for preventing or ameliorating a neuropsychiatric or neurodegenerative disease or disorder in a subject. The method includes administering a composition comprising equol in an amount sufficient to prevent or ameliorate a neuropsychiatric or neurodegenerative disease or disorder. The equol may be a racemic mixture of R-equol and S-equol. The equol may be enantiomerically enriched with R-equol or enantiomerically enriched with S-equol.

Description

(Government-supported research or development)
The present invention relates to U.S. Pat. S Dept. This was done with government support under grant number NRI 2002-00798 and grant number NRI 2004-01811 awarded by of Agriculture (USDA).

  The government has certain rights in the invention.

(background)
(1. Technical field)
The present invention relates to equol and its use as therapeutic compounds to treat and prevent physiological and pathophysiological conditions mediated by androgens. In particular, the invention relates to the use of equol for preventing or treating neuropsychiatric or neurodegenerative diseases or disorders.

(2. Background information)
In recent years, phytoestrogens have been associated with research due to their potential protective effects against age-related diseases (eg, cardiovascular disease and osteoporosis) and hormone-dependent cancers (ie, breast and prostate cancer). Attention is increasing. There are three main classes of phytoestrogens: 1) isoflavones (mainly derived from soybeans), 2) lignans (found in flaxseed), and 3) cumestants (from germinating plants such as alfalfa) . Of these three main categories, human consumption of isoflavones has the greatest impact due to their availability and variety in foods containing soy. Among isoflavones, genistein and daidzein are considered to exert the most potent estrogen hormone activity, and thus most attention is directed to these molecules (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). However, these isoflavone molecules do not exist at high levels in their biologically active form in soy foods and are large in precursor form. For example, genistin, the precursor of genistein, is a glycoside type that contains the carbohydrate portion of the molecule. In addition, malonyl glucoside and acetyl glucoside forms are also found. These conjugates are metabolized in the GI tract by intestinal bacteria, which hydrolyze the carbohydrate component to genistein, a biologically active phytoestrogen. The same metabolic process occurs for aglycone daidzein converted from glycoside daidzein. Daidzein is then further metabolized to equol in mammals “equol-producing”. Thereafter, equol circulates in the bloodstream at very high concentrations. Equol is not normally present in most healthy adult urine unless soy is consumed. The formation of equol in vivo is due to the fact that germ-Phyto-Free animals do not excrete equol, as well as the fact that intestinal flora has not yet developed in newborns, so be fed a soy diet exclusively from birth. As evidenced by the finding that equol is not found in plasma and urine of newborn or 4 month old neonates, it relies exclusively on intestinal microflora. See Non-Patent Document 4.

  Due to the phenolic ring structure of isoflavones, these compounds can bind to the estrogen receptor (ER) and mimic estrogen. Genistein and daidzein bind to ER, which has a lower affinity compared to estradiol and has a greater affinity for ERβ than for ERα. Furthermore, 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, while on the other hand they exhibit antagonist-like characteristics comparable to those of tamoxifen or raloxifene (SERM activity is sex hormone and sex dependent) Conceivable).

  Most of the scientific papers are concentrated on the natural izoflavone of soybean or clover, but there are a few reports on the effects or effects of intestinal metabolites.

  Equol (7-hydroxy-3 (4'hydroxyphenyl) -chroman) is a major metabolite of phytoestrogen daidzein and one of the major isoflavones found abundantly in soybeans and soybean foods. But equol is not a phytoestrogen. Because it is not a natural component of plants. Equol does not occur naturally in any plant-based product. Rather, it is a non-steroidal isoflavone and exclusively the product of the intestinal bacterial metabolite (in relatively few individuals, about 30-400% convert soy isoflavone to equol. With the necessary microflora). Previous studies with equol have confirmed that equol has some weak estrogenic properties, binds to sex hormone binding globulin, binds to α-fetoprotein, and has antioxidant activity. However, equol is unique among plant-derived isoflavones in that it has an asymmetric center and therefore exists as two distinct enantiomeric forms, namely the R and S enantiomers. We have found that the S enantiomer of equol is a form of exclusive equol found in the urine and plasma of “equol-producing” mammals that consume soybeans, and human enteric bacteria It is the only equol enantiomer produced by. All previous work on equol is believed to have been conducted in the racemic form of equol. In general, there is a lack of recognition that there are two forms of equol, and previous studies on the specific effects or activities of individual enantiomers have been reported prior to our findings. Not. The R and S enantiomers differ in configuration and consequently affect their biological activity. For example, only the S enantiomer of equol binds to the estrogen receptor (ER) with sufficient affinity, thereby binding at the circulating equol level reported in humans. Compared to 17β estradiol, the relative binding affinity of the R and S equol enantiomers to ERα is 210.6 and 49.2 times, respectively. However, the S-equol enantiomer is ERβ selective and is considered to have a relatively high affinity for ERβ. The enantiomer S-equol binds to ERβ at about 20% of 17β estradiol [equol, Kd = 0.7 nM vs 17β-estradiol, Kd = 0.15 nM], while the R equol enantiomer is about 100 min. Join with 1 of R-equol is not naturally occurring, but is of considerable importance because of its ability to regulate androgen-mediated processes in the body.

  The prostate relies on androgen hormone action for its development and growth, and the development of human prostate hyperplasia (BPH) clearly requires a combination of testicular androgens during aging. However, testosterone is not the main androgen responsible for prostate growth. The major prostate androgen is 5α-dihydrotestosterone (5α-DHT), as evidenced by the current treatment of prostate cancer towards a 5α DHT reduction with 5α reductase inhibitors. Although not elevated with human BPH, prostate 5α-DHT levels remain at normal levels with aging, despite a decrease in plasma testosterone. Testosterone is converted to 5α-DHT by 5α reductase in the stroma and basal cells of the prostate. 5α-DHT is primarily responsible for prostate development and the pathogenesis of BPH. Inhibitors of 5α reductase reduce prostate size by 20-30%. This reduction in glandular tissue is achieved by induction of apoptosis, which is revealed histologically by vascular atrophy. 5α reductase exists as two isoforms, type 1 and type 2, the prostate preferentially expresses type 2 isoforms, and the liver and skin mainly express type 1 isoforms. Patients have been identified with differences in type 2 alpha reductase rather than type 1. Knockout mice with a type 2 5α reductase null mutation show a phenotype similar to that seen in humans with a deficiency of 5α reductase. Type 1 5α reductase knockout male mice are phenotypically normal with respect to reproductive function. Enzymatic activity for 5α reductase or immunohistochemical detection has been noted in other genitourinary tissues, such as epididymis, testis, cubemaculum and corporate cavemosal tissues.

  Quantitatively, women secrete more androgen than estrogen. The major circulating steroids are generally classified as androgens, which are, in descending order of serum concentration, dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T). And 5α-DHT, but only the last two bind to the androgen receptor to a significant extent. The other three steroids are considered even better as proandrogens. 5α-DHT is mainly a terminal product of testosterone metabolism. Both testosterone circulates in its free form and binds to proteins including albumin and sex steroid hormone binding globulin (SHBG) whose levels are important determinants of free testosterone concentration. The post-menopausal ovary is an androgen-secreting organ, and testosterone levels are not directly affected by the transition to menopause or the appearance of menopause.

Knight D.D. C. Obstet Gyneco, 187: 897-904, (1996). Setchell, K. et al. D. R. Am J Clin Nutr, 129: 1333S-1346S (1998) Kurzer, M.M. S. Et al., Annu Rev Nutr, 17: 353-381 (1997). Setchell K. D. R. The Lancet 1997; 350: 23-27.

  Several research efforts have focused on the development of steroidal compounds for the treatment of androgen-dependent diseases such as: (male pattern) hirsutism, androgen alopecia, benign prostatic hyperplasia ( BPH), and prostate cancer. 5α-DHT is regarded as a causative factor in the progression of these diseases, primarily through clinical evaluation of men who are genetically lacking the steroid 5α reductase enzyme. As a result of such studies, inhibition of this enzyme has become a pharmacological strategy for the design and synthesis of new antiandrogenic drugs. However, it is not clear whether inhibition of 5α reductase has a deleterious effect on this system, as evidenced by the contraindications resulting from the reported side effects of conventional treatments using 5α reductase inhibitors. The development of different strategies that target inhibition of 5α-DHT is a major advance in the treatment of androgen-mediated conditions.

(Summary)
The present invention is a method of preventing or ameliorating a neuropsychiatric or neurodegenerative disease or disorder in a subject. The invention includes administering a composition comprising equol in an amount sufficient to prevent or ameliorate this neuropsychiatric or neurodegenerative disease or disorder.

  The equol may be a racemic mixture of R-equol and S-equol. Preferably, the equol is enantiomerically enriched with R-equol. Alternatively, 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, hyperactivity 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 disease or disorder may be perimenopausal or postmenopausal symptoms.

  The composition may further comprise an excipient. Preferably, the composition is in an oral formulation selected from the group consisting of tablets, capsules, powders, troches, buccal tablets and sublingual tablets. The composition may be an oral formulation comprising at least about 0.25 mg equol. The composition may be food and beverages. The composition may be a delayed formulation or a sustained release formulation.

  Preferably, equol is in an amount sufficient to bind free 5α-dihydrotestosterone and inhibit its binding to the androgen receptor. Preferably, equol is in an amount sufficient to bind free 5α-dihydrotestosterone and inhibit its binding to the androgen receptor and to bind the estrogen receptor subtype.

  In another embodiment, 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.

  In another embodiment, 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.

  In yet another embodiment, the invention is a method of reducing anxiety in a subject. The method includes administering a composition comprising a therapeutically effective amount of equol.

  In a further embodiment, the present invention is a method for providing personal treatment of a neuropsychiatric or neurodegenerative disease or disorder in a subject. The method comprises the steps of assessing a patient's emotional health, mental health or endocrine health; assessing the patient's equol producer status; a) mode of administration, b) dosage, and c) dosing interval Determining an optimally beneficial course of treatment selected from the group consisting of:

FIG. 1 shows the chemical structure of the S-equol and R-equol enantiomers. FIG. 2A shows the appearance / disappearance of R-equol in plasma after oral administration of R-equol to healthy adults. FIG. 2B shows a plot (mean level) of the appearance / disappearance of S-equol or R-equol in plasma after oral administration of S-equol or R-equol in healthy human adults (KD Setchell et al., Am J. Clin. Nutr., 81: 1072-11091, 2005). FIG. 3 shows a mass chromatogram of the elution of equol enantiomers from a urine sample from a soy food consumed by adults compared to a pure enantiomeric standard characterized by optical dichroism. Show. FIG. 4 shows a GC-MS analysis of the trimethylsilyl ether derivative of the synthesized food. FIG. 5 shows the mass chromatogram of the chiral separation of S-equol and R-equol from the racemic mixture. FIG. 6A shows the prostate weight of intact (intact) male rats injected subcutaneously with DMSO or equol. FIG. 6B shows luteinizing hormone (LH) in intact male rats injected subcutaneously with DMSO or equol. FIG. 7 shows separate peaks in [3H] DHT + equol (but not [3H] DHT alone). FIG. 8A shows two separate peaks in [3H] -DHT + equol incubated with prostate (A), while FIG. 8B shows that there is only a single peak in [3H] -DHT incubated with prostate (B). FIG. 9 shows the specific binding of equol to [3H] -DHT. FIG. 10A shows prostate weight in gonadectomized (GDX) male rats injected sc with DMSO, 5α-DHT, equol or both 5α-DHT and equol. FIG. 10B shows plasma LH in gonadectomized (GDX) male rats injected sc with DMSO, 5α-DHT, equol or both 5α-DHT and equol. FIG. 11 shows plasma 5α-DHT values in rats treated with DMSO, DHTP, equol or both DHTP and equol. FIG. 12 shows GDX (AD) and intact (E & F) rats treated with either Trent: DMSO (A & E), equol (B & F), DHT (C), or DHT plus equol (D). Shows the histological effects of equol in the prostate. 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 phytoestrogen-free (Phyto-Free) diet. FIG. 15 shows white adipose tissue mass in male rats fed either Phyto-600 or Phyto-Free (no phytoestrogens) diet. Figures 16A and 16B show food and water intake in male rats fed a Phyto-600 or Phyto-Free diet, respectively. Figures 17A and 17B show plasma leptin and insulin values in male rats fed a Phyto-600 or Phyto-Free diet, respectively. FIG. 18 shows serum glucose values from male rats (not fasted) fed either Phyto-600 or Phyto-Free (no phytoestrogens) diet. FIG. 19 shows thyroid (T3) serum levels in male rats fed either Phyto-600 or Phyto-Free diet. FIG. 20 shows the weight of female rats fed either Phyto-600 or Phyto-Free diet. FIG. 21 shows the mass of white adipose tissue from female rats fed either Phyto-600 or Phyto-Free diet. FIG. 22 shows blood glucose values from female rats fed either Phyto-600 or Phyto-Free diet. FIG. 23 shows blood T3 values from female rats fed either Phyto-600 or Phyto-Free diet. FIG. 24 shows the weight of female rats fed either Phyto-600 or Phyto-Free diet after 50 days of age. FIG. 25 shows the mass of white adipose tissue of female rats fed either Phyto-600 or Phyto-Free diet after 50 days of age. FIG. 26 shows serum leptin levels in female rats fed either Phyto-600 or Phyto-Free diet after 50 days of age. FIG. 27 shows serum insulin levels of female rats fed either Phyto-600 or Phyto-Free diet after 50 days of age. FIG. 28 shows the body weight of OVX rats fed either a Phyto-600 (black bar) or Phyto-Free (white bar) diet placed after and in a behavioral estrous induction regimen. . FIG. 29 shows the mass of white adipose tissue of OVX rats fed either Phyto-600 or Phyto-Free diet. FIG. 30 shows serum leptin values in OVX rats fed either Phyto-600 or Phyto-Free diet. FIG. 31 shows the body weight of 112-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 32 shows the weight of 279 day old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 33 shows the body weight of 350 day old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 34 shows the adipose tissue mass of 350-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 35 shows blood insulin levels in 350-day-old male rats fed a diet of AIN-76, Phyto-Free, Phyto-200, or Phyto-600. FIG. 36 shows blood leptin levels of 350-day-old male rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 37 shows the body weight of 112-day-old female rats fed AIN-76, Phyto-Free, Phyto-200, or Phyto-600 diet. FIG. 38 shows the body weight of 279 day old female rats fed a diet of AIN-76, Phyto-Free, Phyto-200, or Phyto-600. FIG. 39 shows the body weight of a 145 day old male Noble rat fed a Phyto-Free or Phyto-600 diet. FIG. 40 shows white adipose tissue mass from 145 day old male Noble rats fed with Phyto-Free or Phyto-600 diet. FIG. 41 shows the body weight of 145 day old female Noble rats fed with Phyto-Free or Phyto-600 diet. FIG. 42 shows white adipose tissue mass from a 145 day old female Noble rat fed a Phyto-Free or Phyto-600 diet. FIG. 43 shows the baseline body weight of the three groups of rats on the Phyto-Free diet prior to receiving equol injection. FIG. 44 shows the body weights of the three groups of rats after 21 days on the Phyto-Free diet before receiving an equol or vehicle injection. FIG. 45 shows the weight of the three groups of rats on the Phyto-Free diet 7 days after receiving equol or vehicle injection. FIG. 46 shows the body weights of the three groups of rats on the Phyto-Free diet 15 days after receiving equol or vehicle injection. FIG. 47 shows the weights of the three groups of rats on the Phyto-Free diet 22 days after receiving equol or vehicle injection. FIG. 48 shows the body weights of the three groups of rats on the Phyto-Free diet 28 days after receiving equol or vehicle injection. FIG. 49 shows adipose tissue mass from three groups of rats to the Phyto-Free diet 28 days after receiving equol or vehicle injection. FIG. 50 shows the weight of testes from three groups of rats on the Phyto-Free diet 28 days after receiving equol or vehicle injection. FIG. 51 shows the number of elevated-plus maze anxiety-related behaviors (entry into open arms) of 300-day-old male rats fed four different diets. FIG. 52 shows elevated plus maze anxiety related behavior (time in open arms) of 300 day old male rats fed four different diets. FIG. 53 shows elevated plus maze anxiety related behavior (entry into open arms) of 330 day old female rats fed four different diets. FIG. 54 shows elevated plus maze anxiety related behavior (open arm time) of 330 day old female rats fed four different diets. FIG. 55 shows serum isoflavone values in 330 day old male rats fed four different diets. FIG. 56 shows serum isoflavone values in 330 day old female rats fed four different diets. FIG. 57 shows the changes observed in BMI for individuals after 5 weeks of being in close proximity to a diet containing isoflavones. FIG. 58 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsol swim test. It is a graph of total movement distance (meter). FIG. 59 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsol swimming test. It is a graph of the total average speed (meter per second). FIG. 60 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsolt swim test. It is a graph of total movement time (second). FIG. 61 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsol swimming test. It is a graph of the total number of dives. FIG. 62 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsol swim test. It is a graph of total movement time (second). FIG. 63 shows pre-equol injection (A) and post-equol injection (B) in middle-aged female rats fed Phyto-free compared to animals fed Phyto-600 diet in the Porsol swimming test. It is a graph of the total number of excreted boli. FIG. 64 is a graph of weight loss in equol-treated adult male rats compared to control animals fed Phyto- (Free) (n = 8 per treatment group). FIG. 65 is a graph of the number of times of entry into the open arm of the elevated plus maze (EPM). FIG. 66 is a graph of time elapsed in an EPM open arm. FIG. 67 is an illustration of brain hormone-sensitive hypothalamic regions in adult male rats after treatment with equol for 25 consecutive days. FIG. 68 is a graph of the number of times the EPM has entered the open arm. FIG. 69 is another graph of time elapsed in the open arm of male rats treated with equol compared to genistein or control animals during prenatal and early postnatal development.

(Drawings and Detailed Description of Preferred Embodiments of the Invention)
Despite recent advances in pharmacology understanding that equol is related to the effects of estrogens, the researchers have shown that equol's potent anti-androgen action is unique and novel, preventing orrogen-related conditions Publish a new approach to treatment. Binding or sequestering 5α-DHT provides a means to inhibit its effect on 5α-DHT sensitive tissues. Although there are no known ligands that are specific for 5α-DHT, such agents have distinct advantages over promiscuous compounds that directly target the androgen receptor or enzymes involved in androgen synthesis.

  The present invention provides that equol can 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 mild dementia. Based on unexpected discoveries by the people. The inventors further noted that equol can affect feeding regulation and weight management motivation.

  These findings are an important branch in health and disease, indicating a potential broad and important use of equol in the treatment of androgen-mediated pathologies. Without being limited to a particular mechanism, it is believed that equol can act as an antiandrogen. The antiandrogenic properties of equol show that equol does not bind to the androgen receptor (AR) but specifically binds to 5α dihydrotestosterone (5α-DHT) with high affinity, thereby preventing 5α-DHT from AR binding. This is unique. Furthermore, the R and S enantiomers of equol can specifically bind 5α-DHT, sequester 5α-DHT from AR, and block the action of 5α-DHT in physiological processes in vivo. Racemic equol constitutes R-equol and S-equol and R-equol or S-equol alone and selectively binds 5α-DHT.

  In mammals, there are two major androgens, testosterone and its 5α-reducing metabolite, 5α-DHT. 5α-DHT is recognized as the most powerful androgen in the mammalian body. AR is encoded by a single copy gene located on the human X chromosome and specifically mediates the action of androgens. Although both testosterone and 5α-DHT bind to AR, certain tissues that are only slightly affected by testosterone (ie, prostate, hair follicles, etc.) are greatly affected by 5α-DHT. Furthermore, 5α-DHT has been implicated in a number of diseases and disorders. This is because, as mentioned above, equol specifically binds to 5α-DHT and prevents its action, and therefore has the potential for a broad and important use of equol in the treatment of androgen-mediated pathology. Specifically, the inventors have discovered that equol can be used to prevent or treat neuropsychiatric or neurodegenerative diseases or disorders.

  Equol has a similar structure to the steroidal estrogen estradiol. Equol is unique among isoflavones in that it has an asymmetric center and exists as two distinct enantiomeric forms, the R enantiomer and the S enantiomer. All previous studies on equol are considered to be conducted in racemic form of equol. There is generally no recognition that there are two types of equol; previous studies on the specific action or activity that the individual enantiomers R-equol and S-equol specifically bind to 5α-DHT. Has not been reported prior to our findings. Racemic R-equol or S-equol does not bind to the androgen receptor. R-equol does not bind to the estrogen receptor system. Only S-equol binds to ERβ (approximately one-fifth the affinity of 17β-estradiol). Thus, R-equol and S-equol have SERM-like properties and the ability to selectively bind to 5α-DHT, the most potent circulating androgen.

  The most important genistein and to a lesser extent daidzein in soybean and its components isoflavones concentrate on its estrogenic effects, or on its non-hormonal effects, for example its effects on enzymes, growth factors or cytokines, or their antioxidant effects Was. There has never been a discussion of the potential antiandrogenic action of isoflavones before, and only the enantiomeric form of equol is mentioned. The present invention relates to the effect of the enantiomeric form of equol, in particular the ability of both S-equol and R-equol, natural metabolites of daidzein, to antagonize the action of the potent androgen dihydrotestosterone 5α-DHT. Work on. Also, without wishing to be bound by any particular theory, equol is now believed to play a positive role acting as an estrogen-like molecule through the estrogen receptor subtype. Such an effect opens up new possibilities for dietary, nutritional and pharmacological approaches to the prevention and treatment of diseases in which 5α-DHT plays a detrimental role. This includes, but is not limited to, prostate cancer, skin diseases, hair loss, neuropsychiatric diseases or disorders, and neurodegenerative diseases. Furthermore, the estrogenic action of S-equol may also be beneficial in treating or preventing prostate cancer. This is because the combined action of equol acts at the estrogen receptor level and as an antiandrogen.

Equol binds to 5α-DHT Equol (7-hydroxy-3 (4′hydroxyphenyl) -chroman) is an isoflavone found abundantly in soybean and soybean foods, phytoestrogens daidzin and daidzein Is an important biologically active molecule. In animals fed a diet rich in phytoestrogens, the primary circulating isoflavone is equol, which accounts for 70-90% of total circulating isoflavone levels. The present invention discloses a novel level of equol biological properties. In binding studies, the equol enantiomer specifically binds to 5α-DHT but not to testosterone, DHEA, or estrogen. By so binding, equol sequesters 5α-DHT from the androgen receptor without directly binding to the androgen receptor itself. In vivo studies have shown that treatment of equol in intact male rats significantly reduced prostate and epididymal weight but not testis weight. In castrated male rats treated with 5α-DHT after equol administration, equol blocks 5α-DHT trophic effects on the prostate, and its negative feedback affects plasma luteinizing hormone (LA) levels.

  Although not wishing to be bound by the mechanism of action of a particular theory, equol specifically binds 5α-DHT and does not bind itself to the androgen receptor (AR). It is currently believed that it can act as an antiandrogen by preventing its binding to AR. It has also been shown that 5α-DHT already bound to AR does not competitively bind by the enantiomer equol. Thus, one embodiment of the invention is a method of preventing 5α-DHT from binding to AR by contacting 5α-DHT with equol before DHT-AR binding occurs. This enantiomer of equol can be contacted with 5α-DHT in vitro or in vivo. When 5α-DHT is contacted in vivo, equol may be administered by any route that allows absorption of equol into the bloodstream. Biologically available 5α-DHT is free and is not bound to any natural ligand prior to binding to equol.

  Reproductive organs such as the prostate and epididymis are known to be under androgen control. For example, before puberty, if circulating androgen levels are very low, the prostate weight of rats fed a diet containing high levels of soy-derived isoflavones is not altered by consumption of this diet. However, after puberty, when androgen levels increase, prostate weight is significantly reduced in rats fed a diet rich in phytoestrogens compared to animals fed a diet without phytoestrogens. As shown herein, equol-treated intact rats show a significant decrease in prostate and epididymal weight (no change in testis or pituitary weight during short-term studies). Significantly, when prostate and epididymal values are normalized to body weight (per 100 grams), the ratio is also significantly different between equol treatment and control values. Equol also blocked the androgenic nutritional effects of 5α-DHT on the prostate and epididymis without significantly changing testosterone levels.

  5α-DHT has a negative feedback effect on circulating plasma levels of luteinizing hormone (LH). Equol is thought to significantly increase LH levels by binding to 5α-DHT and preventing this feedback effect. For example, equol completely reverses the inhibitory effect of 5α-DHT on LH levels in gonadectomized (GDX) males, whereas male rats treated with equol in addition to 5α-DHT have LH values similar to control values. Indicates. These data further indicate that equol has the specific ability to bind 5α-DHT, possibly in the blood circulation, and can block the hormonal action of 5α-DHT in suppressing LH production or secretion. It is suggested. Accordingly, an embodiment of the present invention is a method of adjusting an individual's LH value by contacting the individual's 5α-DHT with enantiomeric equol. Equol may be administered by any route that allows absorption of equol into the bloodstream and in an amount that is administered according to the nature of the disease being treated and the size of the individual.

Equol Structure Equol differs from most isoflavones in that it has an asymmetric center due to the loss of a double bond in the heterocycle. Plant estrogens isoflavones derived from soybean (daidzein, glycitein and genistein), clover (formononetin and biochanin A), and waste (purarin) do not have asymmetric centers. FIG. 1 shows the chemical structures of R-equol and S-equol.

  The R and S enantiomers differ in configuration and this is how the equol enantiomer fits the binding site in the dimerized ER complex cavity and it is 5α-DHT It is expected to affect how they combine.

  About 50% of equol circulates free or unbound in humans, which is significantly greater than the proportion of free daidzein (18.7%) or estradiol (4.6%) in plasma. Since this is the unbound fraction available for receptor occupancy, presumably for 5α-DHT binding, this effectively contributes to enhancing the overall titer of equol.

Compositions containing equol The present invention has at least a physiologically acceptable amount of equol capable of binding to and sequestering free 5α-DHT (but not testosterone or DHEA), thereby providing an individual. It includes compositions having a broad and important application in the treatment of androgen-mediated pathology, thereby preventing it from binding to androgen receptors after administration to, thereby important branching of health and disease. In particular, compositions comprising equol can be used to prevent or treat neuropsychiatric or neurodegenerative diseases or disorders.

  Preferably, compositions containing equol (which may be S-equol, R-equol, racemic equol mixture, or non-racemic equol mixture) are made for oral consumption.

  Preferably, equol is in an amount sufficient to prevent or treat a disease or disorder according to the methods of the invention. Preferably, a therapeutically effective amount of equol is used in the method of the invention. The term “therapeutically effective amount” is an amount of equol (S-equol, R-equol, racemic equol mixture, or non-racemic equol mixture) that elicits a necessary or desired prophylactic or therapeutic response. An amount sufficient to do so, or in other words, an amount sufficient to elicit a substantial biological response when administered to a subject. For example, a therapeutically effective amount of equol can reduce anxiety or depression in a subject.

  The composition or product containing the composition may be a commercial or institutional food, a pharmaceutical (ie, drug) and an over-the-counter (OTC) pharmaceutical (over-the-counter) (ie, a nutraceutical). .

  Preferably, the food composition comprises at least about 0.25 mg per dose, more preferably the food composition comprises about 1 mg, preferably up to about 200 mg of the enantiomeric equol or equol mixture. Other amounts of equol in the food composition are also considered.

  Preferably, the orally administered medicament comprises at least about 0.25 mg, more preferably about 1 mg, preferably up to about 200 mg enantiomers of equol or equol mixtures per dose. Other amounts of equol in the pharmaceutical composition are also contemplated.

  Preferably, the product for topical application may comprise at least about 0.1 wt%, up to about 10 wt% S-equol or R-equol, or enantiomeric mixtures. Other concentrations of equol are also considered for topical products containing equol.

  The compositions of the present invention may also contain other cosmetic and pharmaceutical active ingredients and excipients. Such suitable cosmetics and pharmaceuticals include, but are not limited to, antifungal agents, vitamins, anti-inflammatory agents, antibacterial agents, analgesics, nitric oxide synthase inhibitors, insect repellents, self-tanning agents, surfactants , Humectants, stabilizers, preservatives, disinfectants, thickeners, lubricants, humectants, chelating agents, skin penetration enhancers, emollients, fragrances, and colorants.

  The enantiomeric equol is also a conjugate selected from the group consisting of glucuronide, sulfate, acetate, propionate, glucoside, acetyl-glucoside, malonyl-glucoside and mixtures thereof and C-4 'or C- It may also be an enantiomeric equol conjugate conjugated at the 7 position.

  A composition or preparation comprising an enantiomeric equol or a mixture of equol for administration to a subject for the treatment and / or prevention of androgen related diseases and conditions and / or prevention thereof is One or more pharmaceutically acceptable adjuvants, carriers and / or excipients may also be included. Pharmaceutically acceptable adjuvants, carriers and / or excipients are described in the art as described, for example, in Handbook of Pharmaceutical Excipients, Second Edition, American Pharmaceutical Association, 1994 (incorporated herein by reference). Is well known.

  In one embodiment, the composition of the invention comprises a non-racemic mixture of S-equol and R-equol having an EE for S-equol of greater than 0% and less than 90%. A composition with 0% EE is a 50:50 racemic mixture of the two enantiomers. This composition can be made directly from the racemic mixture by completely separating and removing either the R-equol or S-equol enantiomer from the racemic mixture. The composition also comprises a first equol component comprising a mixture of equol (either non-racemic or racemic mixture) and a second component comprising a composition consisting essentially of S-equol or R-equol. Can be created by tailoring. This produces a non-racemic composition with an excess of S-equol or R-equol. Depending on the specific advantages or instructions for the R-equol and S-equol components in the composition, S-equol and R-equol are preferably in the ratio of S-equol to R-equol, preferably about 50. : 50 to about 99.5: 1, more preferably about 51:49 to about 99: 1, preferably about 50:50 to less than about 1: 99.5, more preferably about 49:51 to about 1:99. A composition comprising can be prepared.

  The composition may be in the form of a tablet, capsule, powder for reconstitution, syrup, food (eg, food bar, biscuit, snack food, other standard food well known in the art), or a formulation for drinking May be administered. The beverage may contain a flavoring agent, a buffer solution and the like.

  Compositions suitable for oral administration include discrete units such as capsules, cachets, lozenges, troches, tablets, buccal tablets, sublingual tablets, each containing a predetermined amount of extract. It may exist as a powder or granules; as a solution or suspension in an aqueous or non-aqueous solution; or as an oil-in-water or water-in-oil emulsion.

  The composition typically does not contain a significant amount of any other androgen receptor binding compound.

Identify equol producers and non-equol producers. Equol is formed after hydrolysis of soybean-derived daidzein and glycoside conjugates of methoxylated isoflavone formononetin, or its glycoside conjugates found in clover. The Once formed, equol is considered to be metabolically inactive and undergoes no further biotransformation, omitting hepatic second-phase metabolism or a slight degree of further hydroxylation. Similar to daidzein and genistein, the predominant second phase reaction is glucuronidation and sulfation to a lesser extent. In accordance with the original discovery that the presence of equol in the urine is a function of soy food intake, approximately 50-70% of the adult population turns equol into urine even if given soy food daily for unexplained reasons. It was observed that she did not excrete. Furthermore, it has been shown that many do not convert daidzein to equol even when a pure isoflavone compound is administered, thereby eliminating the maternal effects of the food. This phenomenon has led to the terminology of “equol producers” or “non-equor producers” (or “poor equol producers”) to describe these two distinct populations.

  Cut-off values have been obtained empirically, which allows individuals to be assigned to any of these categories. A person with a plasma equol concentration of less than 10 ng / mL (40 nmol / L) may be classified as a “non-equol producer” if the level exceeds 10 ng / mL (40 mmol / L). Producer. This definition may also be derived from levels in the urine, an equol producer is a human that excretes more than 1000 nmol / L. The excretion of equol can vary widely from individual to individual, but in accordance with the precursor-product relationship in the kinetics of the enzyme that catalyzes this reaction, there is a large difference between those that can and cannot produce equol. There is a boundary. As a result, there is an inverse relationship between urinary daidzein and equol, and so far no significant gender differences have been clarified.

Preparation and isolation of the enantiomers of equol The enantiomeric equol may be prepared as is or as a racemic mixture. A chemical synthesis route may be used to produce a racemic mixture in good yield. In a typical synthesis process, standard chemical treatments are used to hydrogenate the heterocyclic double bond and remove the carbonyl at the C-3 position. Typical starting materials are isoflavones such as daidzein, genistein, glycitein, puerarin, formononetin and biochanin A and their glucoside conjugates. Any conjugate type is reduced to its aglycone by hydrolysis. Suitable solvents for the reaction include organic acids such as glacial acetic acid, lower alcohols such as isopropanol and mixtures thereof. Typically used reduction catalysts include palladium, for example 10% Pd on charcoal. The reaction can be carried out at temperatures from ambient to 60 ° C., where the pressure is slightly above atmospheric pressure, with a maximum of 200 psig (14 atm, gauge) and a reaction time of up to 30 hours or more.

  After the reaction is complete, the catalyst is removed and the filtrate is evaporated. The crude residue is purified by chromatography, typically using a silica gel column, and the eluent comprises C2-C4 alcohol, C3-C7 alkane and mixtures thereof. The purified residue may be crystallized from n-hexane to produce (±) equol as a pure product, typically in a yield of at least 99%, typically at least 75%. The equol crystallization product is colorless, not hygroscopic, stable in air and does not degrade during the final filtration procedure.

Method for the isolation of individual R-enantiomers and S-enantiomers from racemic equol The racemic mixture of equol is a chiral phase column with a mobile phase comprising C4-C8 alkyl and C2-C4 alcohol. Can be separated into two separate enantiomers. A typical example of a chiral phase column is a Chiral OD column or OJ column sold by Daicel Chemical Industries, Ltd. A preferred example of the mobile phase includes 70% hexane and 30% ethanol. After the first time that the racemic mixture passes to the inlet, the time interval depends on the column type, the eluent type, the eluent flow rate, the temperature, and the mass of the racemic mixture. , Recovered from the outlet of the HPLC column. The first eluent is typically S-equol. After a second time for the racemic mixture to pass to the inlet, a second eluent R-equol is obtained. The elution of the equol enantiomer from this column may be detected by UV absorption at 260-280 nm or by monitoring more specific detection systems such as mass spectrometers and ions specific for equol.

Biological production of S-equol S-equol may be produced biologically in bulk using conventional food technology. A base solution medium, food or plant extract may be provided that includes daidzein or another related isoflavone from which daidzein can be derived. Daidzein or other isoflavones may be converted to S-equol by standard bacterial or enzymatic fermentation processes to obtain bulk solutions, food or plant extracts containing S-equol.

  Conversion of daidzein to equol involves three main steps: 1) hydrolysis of any glucoside linking group, 2) conversion of isoflavone aglycone to dihydro intermediate, and 3) dihydro intermediate to equol. conversion. The metabolic pathways and enzymes for each of the three required steps need not be present in one bacterium. Anecdotal evidence from human studies suggests that there may be one or more bacteria acting in connection with carrying out these reactions, which often means that dihydrodaidzein is plasma and Although it can be present in significant amounts in the urine, equol is evidenced by the fact that it is low or hardly detectable. Although equol can 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 having its own metabolic profile. Important conditions for effective conversion to S-equol include the selection of the bacterial organism or mixture of organisms, the culture temperature, and the amount of oxygen available to the organism. These conditions can be optimized by techniques well known to those skilled in the art. The organisms used to accomplish this change may be inactivated by standard techniques used in the food industry or may remain active in the product.

  Typically, one or more bacterial strains are required to convert daidzein (or other related isoflavones) to S-equol through intermediate products, which is generally one of three main reactions. Involves in more than one: conversion of isoflavone glycone to aglycone isoflavone; conversion of aglycone isoflavone to dihydroisoflavone; and conversion of dihydroisoflavone to product equol. For example, mixed cultures of organisms isolated from equine feces, or mixed cultures of organisms derived from the gastrointestinal tract of a person known to be an “equol producer,” are converted in vivo. Thus, glycone daidzein can be converted to the final product S-equol.

  Representative bacterial strains that can convert glycone to aglycone (eg, daidzein to daidzein) include Enterococcus faecalis, Lactobacillus plantarum, Listeria welsheri, Equol production "mixed cultures of organisms isolated from the mammalian intestinal tract, Bacteroides fragilis, Bifidobacterium lactis, Eubacteria lisumsum, Lactobacillus casei Lactobacillus acidophilus Lactobacillus acidophilophile), Lactobacillus delbrueckii, Lactobacillus probium leucobacterium, Lactobacillus parabusei, L. fredenreichii) and Saccharomyces boulardi, and mixtures thereof.

  Representative bacterial strains that can convert aglycone to equol (eg, daidzein to S-equol) include Propionibacterium flodenreich, mixed cultures containing: Bifidobacterium lactis, Lactobacillus acidophilus (Lactobacillus acidophilus), Lactococcus lactis, Enterococcus faecium, Lactobacillus casei and Lactobacillus salivarius organisms; Lactobacillus salivarius; Examples include mixed cultures of the body.

  The time required for bacterial conversion of glycosides to aglycones, or bacterial conversion of aglycones to equol products, depends on bacterial-related factors, specifically concentration, oxygen availability, and the temperature and pH of the incubating system. . In most cases it is possible to achieve a substantially complete conversion within 24 hours.

  The pH range for bacterial conversion of isoflavone glycosides to aglycone isoflavones is about 3 to about 9. The optimum pH mainly depends on the type of bacteria used and needs to be selected accordingly.

  The time required for enzymatic conversion of glycosides to aglycones and from aglycones to equol products depends on enzyme-related factors, in particular the concentration of the system, and the temperature and pH. In most cases, it is possible to achieve substantially complete conversion within 24 hours, more preferably within about 2 hours, and most preferably within 1 hour.

  S-equol produced in bulk can be separated from the resulting bulk solution of bacterial production of S-equol by methods well known in the art including crystallization, solvent extraction, distillation and precipitation / filtration. The resulting bulk solution may contain unreacted daidzein or other related isoflavones used, by-products and any reactants. Such methods include the use of reverse phase or normal phase liquid chromatography columns, which may be combined with chiral phase chromatography.

  A typical method for removal of S-equol from a bulk solution or solid phase is by extraction. The extraction solution is added to a solution or solid phase containing S-equol. Typically, 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 of 3.5 to 5.5. Typically, if the hydroalcoholic process is used, sufficient alcohol is added to bring the alcohol / water ratio between a minimum of 40:60 and a maximum of 95: 5. More typically, this ratio is at least 60:40, and even more typically is a ratio between 65:35 and 90:10.

  When an aqueous acid extraction method is used, the aqueous acid solution is prepared within a pH adjusted to about 3.5 to about 5.5, more preferably within a pH range of about 4.0 to about 5.0. Sufficient water is added to allow the diluent to have a sufficiently low viscosity to separate the solid from the liquid by centrifugation or filtration.

  The liquid from which insoluble solids have been removed is concentrated by conventional methods for removing the liquid. The method typically used is not limited, but preferably includes removal of the solvent by evaporation under reduced pressure. The remaining liquid is concentrated to at least about 15% solids and up to about 55%, more typically 30% to 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 vary widely, but the final solids content is preferably 6% to 15%, more typically about 13%. The pH of the mixture is adjusted between about pH 3.0 and about pH 6.5, with a value of about pH 4.0 to about pH 5.0 being preferred. Typically, the temperature is 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) to obtain an equol-rich solid material.

  The equol-rich material may be purified, if necessary, typically by chromatography using a silica gel column, with eluents including C2-C4 alcohols, C3-C7 alkanes and mixtures thereof. The purified residue may be crystallized from n-hexane to produce S-equol as a pure product, typically in a yield of at least 99%, typically at least 75%. The equol crystallization product is colorless, non-hygroscopic, stable in air and does not degrade during the final filtration procedure.

  The S-equol product is a trimethylsilyl ether or tert-butyldimethylsilyl ether derivative, or a synthesis as a single pure peak and mass spectrum consistent with the published electron ionization spectrum of a standard equol trimethylsilyl (TMS) ether derivative Can be confirmed by GC-MS analysis of some other suitable volatile derivatives of the produced product. Product validation may also be achieved by direct mass spectrometry using electrospray ionization after sample introduction into the instrument via an HPLC chiral phase column.

Treatment of disease by administering S-equol, E-equol and mixtures The present invention is directed to overcoming the problem of inability to produce equol in vivo, or R-equol, particularly enantiomers of equol. , S-equol or R-equol, or a soy food product that directly supplies the delivery of a non-racemic mixture of S-equol and R-equol, with the need for enterobacteria for its production or the equol precursor isoflavone Methods are provided to individual subjects to avoid the need to consume. Delivery of S-equol may also complement in vivo production of S-equol in “equol producers” and in “non-equol producers”.

  Supplementing the equol producer's diet with an equol enantiomer or mixture may provide benefits if the normal levels of S-equol produced by the equol producer are inadequate for the following reasons: 1 ) Inadequate consumption of isoflavones to produce equol, 2) Use of antibiotics to remove enterobacterial activity to make equol from precursor isoflavones, or 3) Others that affect the level of equol production Surgical construction of intestinal stoma, such as short bowel syndrome or ileostomy, for health factors. Furthermore, supplemental levels of equol are thought to improve the impact on human health and well-being.

The present invention relates to a method for delivering S-equol, R-equol, racemic equol, or a non-racemic mixture of equol in an amount sufficient to have health benefits for androgen related diseases and conditions associated therewith. I will provide a. The antiandrogenic activity of equol can affect a number of tissues throughout the body. Specifically, the block of androgenic activity of 5α-DHT is: (A) prostate growth with age, benign prostatic hyperplasia (BPH), and prostate cancer, (B) female and male buns, (C) Facial and body hair growth (hypertrichosis), skin health (acne, anti-aging and photoaging prevention), skin integrity (collagen and elastin robustness); (D) weight gain ( Reduced), adipose tissue accumulation and lipid metabolism, and general regulatory behaviors and effects such as food and water intake, blood pressure changes, thyroid, glucose, leptin, insulin and the immune system; And (E) for the treatment and prevention of Alzheimer's disease and emotional, mental health problems such as mood, depression, anxiety and learning and memory By reducing a strong modulator is 5α- steroid metabolites GABA _A receptor (covering androgen and progesterone) in the brain which affect all the features of, may be beneficial.

  Typically, the amount of composition comprising equol is at a transient level of at least 5 nanograms per milliliter (ng / mL), more typically at least 10 ng / mL or more, or over 1000 nmol / L. A transient value of enantiomeric equol, the enantiomeric equol in urine, is administered in an amount sufficient to produce in mammalian plasma. Preferably, the composition is administered orally at a dosage of at least about 0.25 mg of enantiomeric equol, more preferably about 1 mg, more preferably at least about 5 mg, and up to about 200 mg, more preferably up to about 50 mg. The Representative values of R-equol bioavailability in plasma following oral administration of 20 mg R-equol enantiomer to healthy adults are shown in the R / equol appearance / disappearance plots of FIGS. 2A and 2B. .

  The ability to deliver a sufficient amount of R-equol and / or S-equol is believed to provide several advantages over delivery of a racemic mixture of equol. First, the single titer of R-equol or S-equol is typically at least twice that of the racemic mixture. Secondly, only the human body produces S-equol, and therefore a composition containing only S-equol represents a “natural” product with the component S-equol that the body is familiar with. Third, since the R-equol enantiomer has unique properties, a treatment composition containing only the R enantiomer, or substantially only the R enantiomer, produces a beneficial and / or therapeutic effect. obtain. Fourth, administration of R-equol helps estrogenic and anti-estrogenic effects occur in the body, assisting in the presence of any endogenous S-equol.

  The present invention encompasses the use of enantiomeric equol to treat and prevent diseases and conditions associated with male and female buns. 5α-DHT is known as a cause of hair loss. Androgen, specifically testosterone, the main circulating androgen, is converted (in the hair follicle) to the more potent androgen 5α-DHT, and the hormonal action of 5α-DHT on the hair follicle results in hair loss. Thus, if the hormonal action of 5α-DHT can be blocked, such as by use in the equol of the present invention that binds 5α-DHT in the circulation (intravascular) and hair follicles, hair loss is reduced or Can be prevented.

  The present invention encompasses the use of enantiomeric equol to treat and prevent diseases and conditions associated with facial and body hair. Facial and body hair is regulated by androgens, as opposed to hair regulation. Specifically, the more powerful androgen 5α-DHT increases facial and body hair. 5α-DHT also increases the production of sebum (oil) from sebaceous glands, which may contribute to increased acne. Thus, the binding of 5α-DHT by equol can result in a decrease in facial and body hair and sebum (fat) secretion, and a reduction or prevention of acne.

  The present invention relates to the use of enantiomeric equol to treat and prevent diseases and conditions associated with skin effects, skin quality and integrity, skin aging, skin photoaging, and skin pigmentation and lightening. Is included. Estrogens improve skin health and improve skin characteristics or structural stability by increasing the content of elastin and collagen before, especially after menopause. Furthermore, if the skin is damaged by acne or other skin destruction (such as scratches, pimple breaks or small cuts), the repair mechanism is faster if estrogen or an estrogen-like compound, such as equol, is present, Skin healing is good. The equol enantiomeric mixture, and in particular S-equol, is a good stimulator of elastin and collagen and is believed to be able to protect against photoaging. Equol blocking the hormonal action of 5α-DHT can reduce sebum production from sebaceous glands, which can reduce or eliminate acne. Since S-equol (but not R-equol) binds to the estrogen receptor (s) (primarily ERβ), the protective effect of this estrogen-like molecule stimulates elastin and collagen in the skin. Furthermore, since equol is a powerful antioxidant, it can protect the skin from aging, including photoaging.

  The present invention encompasses the use of enantiomeric equol to treat and prevent diseases and conditions related to improving prostate health. The conversion of testosterone to the more potent androgen 5α-DHT is a result of the action of the enzyme 5α-reductase in the prostate. 5α-DHT can cause benign prostatic hyperplasia (BPH), increase prostate weight, and may require the need for prostatectomy and radiation therapy to treat these conditions. Finally, consumption of soy foods has increased attention due to “health benefits” of reducing hormone-dependent cancers such as prostate and breast cancer. Thus, blocking 5α-DHT with equol reduces prostate weight in animal models and possibly blocks BPH to prevent prostate cancer.

  The present invention prevents or reduces this neuropsychiatric or neurodegenerative disease or disorder by administering equol in an amount sufficient to prevent or treat a neuropsychiatric or neurodegenerative disease or disorder. Includes methods of remission.

  Neurodegenerative disorders, such as Alzheimer's and minor depression, and Parkinson's disease, often associated with aging, are prevalent in the general population and are expensive to treat. According to Lasker Foundation, 4 million people in the United States currently have Alzheimer's disease, which is estimated to cost over 100 billion yen per year. The risk of developing Alzheimer's disease doubles every year above the age of 65, and nearly 40 percent of people have the disease above the age of 85 (JM Lynessra, et al., Ann. Inter. Med., 144: 496). ˜504, 2006; and Lepart et al., Curr neurovas. Res., 1: 455-464, 2004). Small depression is more frequent than major depression, with nearly 50% of the older group (over 60 years) showing symptoms associated with minor depression (JM Lynes et al., Ann. Inter. Med., 144: 496-504, 2006).

  In one embodiment, the invention encompasses the use of equol, including enantiomeric equol, for treating and preventing diseases and conditions related to brain function and mental health, which include brain disorders. For example, Parkinson's disease, Alzheimer-type dementia, and other reduced or impaired cognitive functions associated with aging and short- and long-term memory loss. The brain mechanisms are more complex, and attempts to define which molecules and factors regulate, influence, etc. mood, anxiety, etc. can be difficult. However, the notion that estrogens, or estrogen-like molecules such as isoflavones, can help cognitive function in conditions such as Alzheimer's disease and can help to prevent the development of such disorders, especially in postmenopausal women. There are some data to support.

  It is well established that neuropsychiatric disorders are the second leading cause of prevalence and setbacks worldwide. World Health Organization estimates that, as a population, neuropsychiatric disorders comprise 13% of all reported diseases. These disorders include depression, anxiety, schizophrenia (schizophrenia), bipolar disorder, obsessive compulsive disorder, panic disorder, binge eating, alcohol and drug abuse, binge eating and obesity, and attention deficit hyperactivity. Disability (World Health Organization. World health report 2000-health systems: improving performance. Geneva: WHO 2000). Nearly one in five Americans experience episodes of psychiatric illness, such as depression or anxiety, in a given year (Mental Health Report of the US Surgenon General, 1999). In particular, most common concomitant psychiatric diagnoses with alcoholism are often drug abuse-related disorders such as depression and anxiety disorders (R. Hitzemann, Alchol Res. & Health 24: 149-158, 2000).

  Depression is an impressive or mood disorder. Symptoms include unhappiness, helplessness, despair and loss of interest in life activity or all enjoyment. Most individuals suffering from depression also experience mental retardation, confusion, loss of energy, and inability to determine or concentrate. Symptoms can range from moderate to mild to severe, often accompanied by anxiety and / or restlessness (Diagnostic Criteria form DSM-IV, American Psychiatric Association, Washington, DC, 1994).

  Depression is called the “common cold” of mental illness. This is a pervasive disorder affecting approximately 18 million adults in the US population per year (National Institutes of Health, Invisible Disease: Depression, Bethesda, MD, NIH publication 01-4591, 2001). This is regarded as the second leading cause of disability, and only surpasses heart disease (CJ Murray & AD Lopez (edit) The Global Burden of Disease, Harvard Univ.). Press, Boston, 1996). Worldwide, the prevalence is 21% for women and 13% for men (CB Nemerov and MJ Owens, Nature Neurosci., 5: 1068-1070, 2002).

  Although research on the background of the cause of depression continues, it is generally believed that impairments result from a decrease in neurotransmitter synaptic concentrations or related mechanisms of neurotransmitter action. Antidepressants are the current method of treatment. Unfortunately, most antidepressants typically require weeks of administration before clinical effects appear, despite the fact that their biochemical effects occur almost immediately. After all, there can be concerns regarding the side effects, contraindications and safety of antidepressants.

  Anxiety disorders are often characterized by excessive anxiety, fear and anxiety and occur frequently, resulting in significant impairments in daily work. Symptoms can include restlessness, fatigue, lack of concentration, excitement, muscle tone, and sleep disorders. The lifetime prevalence of anxiety disorders in the general population is approximately 5-6% (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 35 years of age, and the rate of anxiety tends to increase in middle-aged and older adults (HW Witchen) Depress. Anxiety, 16: 162-171, 2002), which is similar to the tendency seen in depression.

  Current methods of treatment for anxiety include pharmacological treatment with compounds containing anxiolytics such as benzodiazepines (eg, diazepam, valium). Benzodiazepines possess anti-anxiety, analgesic hypnosis, anticonvulsant and muscle relaxant properties. However, benzodiazepines can cause disturbances in mental and motor function, confusion (confusing) and forgetfulness when circulating plasma levels exceed the range of anxiolytic. Benzodiazepines can also be susceptible to dose escalation and abuse in some patients. Withdrawal from long-term use can induce anxiety, insomnia, restlessness and excitement (RM Julien, Primer of Drug Action 7th edition, WH Freeman Co., New York, 1995; and ME Likey and B. Gordon, Medicine and Mental Ilness, WH Freeman Co., Boston, 1991).

  There are two basic perspectives on mood, anxiety, depression and other mental health conditions. One line of research supports the view that estrogens help regulate anxiety (especially in women) and reduce anxiety levels. Both estradiol and progesterone alter anxiety-related behaviors and testosterone, a testicular androgen (Imhof JT et al., Behav Brain Res, 56: 177-180 (1993)). Equol's antiandrogenic activity acts in the brain by enhancing neurotransmitters and restoring synaptic density. While not being bound by a particular theory, we believe that R-equol and / or S-equol are active as estrogens in the brain at the same site and exert an estrogen response.

The second line of studies is more complex and supports the view that 5α-reducing steroids, especially progesterone, have the ability to bind to GABA _A receptors and cause sedation in the brain. GABA _A is the major inhibitory neurotransmitter in the brain, and its receptors are abundant in brain regions that control mood / emotion. By producing calmness, the individual has less expression of anxiety. For example, most pregnant women have been reported to feel OK, but they are usually tired or sleepy. This is due to the conversion of progesterone to 5α-dihydroprogesterone (5α-DHP) by the 5α-reductase enzyme in the body, but in particular in the brain, and the further metabolism of this molecule is GABA _A It produces the most potent “neurosteroid” that can bind to the receptor and enhance the action of GABA _A. Further supportive evidence that this 5α-dihydroprogesterone molecule (and its metabolites) can reduce brain activity is found in epileptic women (who experience epilepsy and therefore epileptic seizures), and these individuals are progesterone Due to the high level of circulation, epileptic seizures are almost never experienced during pregnancy. It is noted that 5α-reduced androgen-like 5α-DHT also has a similar effect on GABA _A receptors, resulting in sedation (in males) but at a much lower level compared to 5α-DHP. There is a need.

  Taken together these two views estrogen on the one hand reduces anxiety and thereby increases activity. Conversely, blocking the action of 5α-DHP also increases activity and is therefore interpreted as reducing anxiety in behavioral testing. For example, if the conversion of progesterone to 5α-DHP in pregnant rats is blocked, this results in a significant increase in their spontaneous activity level.

Taking a similar perspective on equol, equol has the ability to bind to 5α-DHP (predominantly seen in women) and 5α-DHT (predominantly found in men). This diminishes the potent “neurosteroid” effect at GABA _A receptors and reduces sedation, thereby increasing activity or reducing anxiety. Furthermore, the ability of S-equol to bind to estrogen receptor (s) β also increases activity. Finally, by our study, studies using male or female young or middle-aged adult rats, dietary phytoestrogens consumption (Lund TD et al., Brain Res, 913: 180-184 (2001); Lefart, ED, et al., Neurotoxicology Tetralogy, 24: 1-12 (2002)), or injection with equol significantly reduces the level of anxiety represented in the elevated plus maze test It has been shown.

  The elevated plus maze is a behavioral test used to quantify anxiety-related behaviors and identify anti-anxiety drugs (Pellow S et al., J. Neurosci Methods, 14: 149-147 (1985); Current Protocols In Neuroscience (1997) 8.3.1-8.3.15, John Wiley & Sons. NY, NY). This test relies on an intrinsic discrepancy between exploring a new environment and avoiding its nasty features. Normally, animals spend less time and less enter the maze open arm than the maze closed arm (Imhof JT et al., Behav Brain Res, 56: 177-180 (1993). However, animals treated with anxiolytics such as benzodiazepines (valium) spend more time in the open arm, and the number of entries into the open arm reflects a decrease in anxiety-related behaviors (Pellow S. et al., J. Neurosci Methods, 14: 149-147 (1985); Current Protocols In Neuroscience (1997) 8.3.1-8.15, John Wiley & Sons, NY, NY); Chopin P. et al., Psy hopharm, 110: 409~414 (1993).

  The invention also encompasses the use of equol (such as a mixture of R-enantiomers and S-enantiomers) to treat or prevent perimenopausal or postmenopausal symptoms. These symptoms include mood swings, depression, anxiety, fatigue and emotional and mental hygiene, weight gain and obesity, and polycystic ovary, among others.

  In another embodiment, the invention is a method of using equol to prevent or treat neuropsychiatric disorders, including depression, anxiety and others described above.

  The present invention encompasses the use of enantiomeric equol to treat and prevent diseases and conditions associated with weight and body fat formation. Weight gain and obesity represent a huge health concern in developing countries in the United States and around the world. For example, in the United States, about 55% of adults are overweight by international standards, and about 23% of American adults are considered obese; one in five American children is considered overweight. Obesity in the US costs 12% of the US government budget, or 111.8 billion yen, in the late 1990s (World Watch Institute, March 4, 2000).

  Phytoestrogens, including equol, have the ability to reduce the formation of white adipose (fat) tissue and increase the destruction of white adipose tissue, thereby reducing body weight. Also, the estrogenic nature of the phytoestrogenic molecule reduces LDL (so-called “bad” cholesterol), lowers blood pressure and prevents insulin resistance (or, in other words, has a beneficial effect on the diabetic state). Since equol is a more potent isoflavone molecule compared to other phytoestrogens, this probably provides health benefits and protects the conditions outlined above.

  Since equol binds to 5α-DHT, equol can also block the action of 5α-DHT to promote weight gain. Thus, equol, and in particular S-equol, combined with antiandrogens, but simultaneously with the estrogen hormone action from the same molecule (equol), further improves the health benefits of weight loss (and weight management). It helps to reduce LDL cholesterol, reduce blood pressure, and prevent the destructive effects of diabetes.

  The present invention encompasses the use of equol to prevent or treat weight gain and obesity. The term “weight gain” refers to any increase in body weight above an optimal health value that depends on the age, sex and condition of the subject. A person is considered obese if he exceeds his ideal weight by about 20%. Its ideal weight needs to take into account a person's height, age, gender and body shape. Overweight or obesity may also be defined using, for example, a body mass index. For adults, the range of overweight and obesity is determined by calculating a numerical value called “body mass index” (BMI) using weight and height. Since BMI is used by most people, it correlates with the amount of body fat. Adults with a BMI between about 25-29.9 are considered overweight. Adults with a BMI of about 30 or greater are considered obese. Overweight or obesity increases the risk of many diseases and conditions, including: hypertension, dyslipidemia (eg, high total cholesterol, or high levels of triglycerides), Type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis, sleep apnea and respiratory problems and some cancers (endometrial cancer, breast cancer and colon cancer) (US Department of Health and Human Services, Center for Disease Control (CDC) and Prevention, 2006).

  The present invention relates to equol, enantiomeric equol, or the like, for treating and preventing lipid disorders such as hypercholesterol (hypercholesterolemia), lipemia, lipemia and dyslipidemia (lipid disorders) Includes use as a mixture. Plasma total cholesterol levels decreased 7.2% (p = 0.04) for equol producers and 3.0% (p = NS) for non-equol producers compared to baseline values Has been shown by research. The fact that soy protein has no significant cholesterol lowering effect in adults with normal blood cholesterol levels has a few experiences, probably because of the heterogeneity in the research population regarding the metabolism of soy isoflavones. The relationship between sex and equol formation (especially non-formation in non-equol producers) is unrecognizable. These data suggest that enantiomeric equol affects lipids in an advantageous manner and that this effect is mediated by androgens. A composition comprising equol is administered in an amount sufficient to reduce the level of lipids in the bloodstream.

  The present invention further provides R for improving reduced blood vessel quality by increasing responsiveness or plasticity in response to precise changes in blood pressure, improving blood flow, and reducing blood pressure. Includes the use of equol and / or S-equol. The invention also encompasses the use of R-equol and / or S-equol for treating and preventing cancer, including benign prostate cancer, prostate cancer and skin cancer.

  Another embodiment of the present invention is the use of equol to treat an enlarged prostate or epididymis. Equol can also be used to prevent enlarged prostate or epididymis in individuals considered to be at risk of developing these pathologies without changes in testis, pituitary gland or body weight. Equol can be administered by any route that allows absorption of equol into the bloodstream.

DHT-Androgen Receptor Other embodiments of the invention include the use of equol as a diagnostic agent in androgen related disorders and disorders resulting from disturbances in the estrogen / androgen balance. In these embodiments, equol is administered to an individual and binds to 5α-DHT, thereby preventing 5α-DHT binding to the androgen receptor. This change in estrogen balance is then measured, or this change in androgen binding is assessed to diagnose or further elucidate androgen-related abnormalities.

Binding to 5α-DHT In order to assess the binding capacity of 5α-DHT for the relevant therapeutic moiety, equol may be administered and bound to 5α-DHT prior to or with other therapeutic moieties. The androgen binding moiety may also be administered after the administration of equol in order to evaluate the effectiveness of the androgen binding moiety to restore androgenic activity and the balance of estrogen activity in the absence of 5α-DHT binding. In addition, equol may be administered in the presence of a 5α-DHT binding moiety to replace these naturally occurring or xenobiotic 5α-DHT binding moieties from 5α-DHT.

Administration of equol In each of the embodiments of the invention described herein, if administration of equol needs to be oral, the equol is an “equol-producing” mammal or “no equol production”. Supplying an oral equol dosage form to any of the “non-equal producing” mammals, or daidzein, daidiyne, an isoflavone mixture containing daidzein or soy protein preparation to the “equol producing” mammals Administration of this oral dosage form results in effective absorption of equol into the bloodstream. Administration of equol can be performed by routes other than oral, if desired. For example, rectal or urethral administration may be used to administer equol for the treatment of enlarged prostate or to prevent prostate enlargement. It is further contemplated that the active ligand binding site of the equol molecule may be isolated and synthesized for administration, thereby obtaining 5α-DHT binding without the complete equol molecule. The dose of the equol molecule or fragment thereof having 5α-DHT binding ability depends on the route of administration and the condition being treated. Based on our in vivo studies, it is clear that relatively low doses of equol antagonize fairly high doses of 5α-DHT, which in the binding of equol to serum proteins compared to 5α-DHT. Can be explained by significant differences. The latter circulation is almost bound to protein, but equol is 50% free. In general, a dose sufficient to produce a concentration of equol or an active fragment thereof in the recipient's blood stream of at least about 0.2 mg, preferably at least about 0.5 mg / kg equol, per kg of recipient body weight. . This dose can be dramatically increased to greater than about 10 mg / kg without incurring significant dose limiting side effects. Oral administration may be accomplished in microcapsule form, which can result in delayed or sustained release of the drug.

  Equols are lotions, ointments, foams (including shaving foam), nasal sprays, skin patches (such as those described in US Pat. No. 5,613,958, which is incorporated herein by reference in its entirety). Electromechanical devices including micropump systems, such as those described in US Pat. No. 5,693,018 and US Pat. No. 5,848,991, incorporated herein by reference in their entirety, and subcutaneous Be administered topically, transdermally, and subcutaneously in various forms, including implants, such as those described in US Pat. No. 5,468,501, which is incorporated herein by reference in its entirety. Good.

Experiments (a) Determination of equol enantiomers in “equol-producing” adults A urine sample from an adult consuming soy food that was previously identified as an “equol producer” was analyzed. Equol was isolated from urine (25 mL) by passage of the sample through a solid phase Bond Elut C18 cartridge. After washing the cartridge with water, isoflavones were recovered by elution with methanol (5 mL) and the methanol phase was collected and dried under a stream of nitrogen. Samples were subjected to enzymatic hydrolysis with Helix pomatia and then re-extracted with a Bond Elut C18 cartridge. The methanol extract was collected, dried under nitrogen gas, and redissolved in HPLC mobile phase (100 μL). The equol enantiomer was identified by HPLC using a Chiralcel OJ chiral phase column as described herein above. Detection of equol was achieved by selective ion monitoring with electrospray ionization mass spectrometry (ESI-MS). A mass chromatogram of urine from a pure standard of S-equol and from adults consuming soy food is shown in FIG. Similar results indicate that soy-derived isoflavones are converted to equol in the rat, which in turn confirms a rodent model of isoflavone metabolism.

  The retention index and mass chromatogram confirm that this is exclusively the S-enantiomer of equol excreted in human urine. This is because no detectable R-enantiomer of equol is found. Analysis of plasma from the same “equol producer” also revealed only the S-enantiomer of equol.

(B) Chemical synthesis of racemic equol. Daidzein (200 mg, 0.8 mmol) was dissolved in a mixture of glacial acetic acid (20 mL) and isopropanol (20 mL) and 55 p.p. on charcoal (150 mg) with 10% Pd. s. i. g. Reduce at 3.7 atm, measurement. At the end of the reaction (2 hours, TLC: isopropanol / n-hexane 1/4), the catalyst is filtered off and the filtrate is evaporated. The crude residue was purified by chromatography on a silica gel column using a mixture of isopropanol and n-hexane (1: 4 v / v) as eluent to give (±) equol pure product crystallized from n-hexane. Product (160 mg, yield: 82%). The product is colorless crystals, not hygroscopic, stable in air and does not decompose during the final filtration procedure. The product of this chemical synthesis was identical in all respects to the (±) equol standard sample (racemic equol). FIG. 4 shows GC-MS analysis of the synthesized product trimethylsilyl ether derivative as a single pure peak and mass spectrum consistent with the published electron ionization spectrum of the standard equol trimethylsilyl (TMS) ether derivative. Indicates. The expected molecular ion is m / z 470 and the base peak is m / z 234. The purified equol product had a purity greater than 99%, as confirmed by HPLC and mass spectrometer.

(C) Elution order of S-enantiomer and R-enantiomer by optical dichroism A racemic mixture of S-equol and R-equol is contained in hexane using a flow rate of 1.0 mL / min. Separation for 15 minutes according to the program shown in Table 1 by chiral chromatography on Chiralcel OJ Column using a gradient elution consisting of an initial mobile phase of% ethanol and an increase to 90% ethanol in hexane.

図５は、Ｓ−エクオルおよびＲ−エクオルのラセミ混合物のイオン記録（ｍ／ｚ２４１）のマスクロマトグラムを示す。

FIG. 5 shows a mass chromatogram of an ion record (m / z 241) of a racemic mixture of S-equol and R-equol.

  The first eluting material was named enantiomer-1 and the second eluting material was named enantiomer-2, which were collected separately. Each enantiomer was weighed and the weighed sample was dissolved in 1 mL of spectrometer grade ethanol. The optical rotation of each enantiomer was measured at 200 ° C. using light with a wavelength of sodium line D.

  Enantiomer-1 substance (exact weight 1.6 mg) has a first measurement and a second measurement, −0.023 and −0.022, which allows the equol S-enantiomer to A corresponding optical rotation of −14 [−0.0225 × 1000 / 1.6] occurred. Enantiomer-2 substance (exact weight 1.7 mg) has a first measurement and a second measurement, +0.023 and +0.023, thereby corresponding to the R-enantiomer of equol +13.5 [+ 0.023 × 1000 / 1.7] optical rotation occurred.

d) Determination of the receptor binding capacity of S-enantiomers and R-enantiomers In vitro binding studies were performed to determine the relativeities of S-enantiomeric and R-enantiomeric equol with the estrogen receptors ERα and ERβ. Affinity was tested.

  Synthesis of hormone receptor protein: hormone receptor in vitro using full-length rat ERα expression vector (pcDNA-ERα; RH Price UCSF) and ERβ expression vector (pcDNA-ERβ; TA Brown, Pfizer, Groton, Colo.) Here, a TnT-coupled reticulocyte lysis system (Promega, Madison, Wis.) Was used with T7-RNA polymerase for a 90 minute reaction at 30 ° C. The translation reaction mixture was stored at −80 ° C. until further use.

  Saturated isomer: In order to calculate and establish the binding affinity of S-equol and R-equol enantiomers for ERα and ERβ, 100 μL aliquots of reticulocyte lysate supernatant were incubated at optimal time and temperature Increasing concentrations (0.01-100 nm) of [3H] 17β-estradiol (E2) were used for 90 minutes at room temperature (ERβ) and 18 hours at 4 ° C. (ERα). These times were determined empirically and corresponded to optimal binding of the receptor to estrogen. Nonspecific binding was assessed in parallel tubes using a 300-fold excess of the ER agonist diethylstilbestrol. After incubation, bound and unbound [3H] E2 was separated by passing the incubation reaction through a 1 mL lipophilic Sephadex LH-20 (Sigma-Aldrich Co., Saint Louis, Mo) column. The column is TEGMD (10 mm Tris-Cl, 1.5 mm EDTA, 10% glycerol, 25 mm molybdate, and 1 mm dithiol according to a previously published protocol (Wanda et al., 1986; O'Keefe and Handa, 1990). Threitol, pH 7.4) was constructed by packing disposable pipette tips (1 mL; Labcraft, Curtin Matheston Scientific, Inc, Houston, Tex) using saturated Sephadex. For chromatography, the columns were re-equilibrated with TEGMD (100 μL) and the incubation reactions were added individually to each column and allowed to incubate on the column for an additional 30 minutes. After this incubation, 600 μL of TEGMD was added to each column, the flow-through was collected, 4 mL of scintillation fluid was added, and the samples were counted in a 2900 TR Packard scintillation counter (Packard Bioscience, Meriden, Conn.). (5 minutes each).

  Competitive binding studies were used to evaluate the estrogenic properties of the enantiomers of equol S-equol and R-equol. Based on the ability of S and R to compete with [3H] E2 for ER binding, the affinity for in vitro translated ER was shown to be very different for the two enantiomers. The S-equol enantiomer showed the greatest affinity for ERβ [Kd (nm) = 0.73 ± 0.2], but its affinity for ERα was relatively low [Kd ( nm) = 6.41 ± 1.01. The R-equol enantiomer possessed significantly lower affinity for both ERβ [Kd (nm) = 15.4 ± 1.31] and ERα [Kd (nm) = 27.38 ± 3.8]. As a reference, 17β-estradiol binds in this system with ERα at Kd (nm) = 0.13 and ERβ with Kd (rum) = 0.15.

  This study shows that only the S-equol enantiomer binds with sufficient affinity to the ER and has a potential relevance to the circulating equol values reported in humans. Compared to 17β estradiol, the relative binding affinity of the S-equol and R-equol enantiomers for ERα was 1/49 and 2/1 211, respectively. However, the S-equol enantiomer appears to be fairly ERβ selective with a relatively high affinity for ERβ, while the R-equol enantiomer binds with an affinity of about 1/100. Another and related decision is that S-equol is exclusively found in human plasma and urine is significant in terms of specificity in the binding of the two enantiomers.

e) R-equol bioavailability 20 mg of pure R-equol was orally administered to healthy adults after an overnight fast. Blood samples were then collected at regular intervals over the next 24 hours, and plasma concentrations of equol were determined by isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. A rapid appearance of equol was observed in the plasma and peak concentrations were observed after 8 hours. The terminal elimination half-life of R-equol was about 8 hours. Electrospray ionization mass spectrometry confirms that equol present in plasma is the R-equol enantiomer (data not shown but available on request), so that it is stable, intestinal It was confirmed that neither racemization nor further biotransformation occurred. FIG. 2A shows a plot of R-equol appearance / disappearance. 2B shows a plot (mean value) of the appearance / disappearance of S-equol and R-equol in plasma after oral administration of S-equol or R-equol in healthy human adults (KD Setchell). Et al., Am J. Clin. Nutr., 81: 1072-1091, 2005).

  These results confirm that R-equol is extremely bioavailable when administered as a pharmacological or nutritional preparation.

  Data were analyzed as needed by analysis of variance (ANOVA) followed by Newman-Keuls post hoc (post-hoc Neumann-Keuls) test. Significance was set at p <0.05. Curve fitting, scientific graphing and analysis were completed using GraphPad Software (GraphPad Prism 3.0, San Diego, Calif.).

Example 1
This example demonstrates the in vivo effects of equol on prostate size and hormone secretion. Male Sprague-Dawley rats (400-500 g) were obtained from Charles Rivers Laboratories (Wilmington, Mass., USA). Rats are placed in a pair in pairs and are kept on a 12 hour dark field 12 hour Akeno schedule (lighted at 07) with free access to food and water.

  One week after arrival, animals receive a subcutaneous (sc) injection of either dimethyl sulfoxide (DMSO) (vehicle control) or racemic equol (0.25 mg / kg) (once daily for 4 days). 18 hours after the final injection, the animals are decapitated and killed, and trunk blood and prostate are collected for analysis.

  A significant decrease in prostate weight is observed in intact males injected subcutaneously with equol compared to intact control males. Furthermore, luteinizing hormone (LH) in these same intact males is significantly increased in equol compared to control treated males. These findings are shown in FIGS. 6A and 6B, respectively. These effects are observed at relatively low levels of equol compared to 5α-DHT, which is that of equol (about 50% is free circulating) and 5α-DHT (most bound to serum proteins). This can be explained by a significant difference in protein binding.

(Example 2)
In addition to the effect of equol on the prostate, racemic equol blocks the effects of DHT in other tissues and reduces weight. One week after arrival, intact males are given subcutaneous injections of either DMSO (control) or equol (0.5 mg / kg) once daily for 7 days. After treatment, the animals are weighed and then decapitated and killed, and the tissues are collected (prostate, testis, epididymis and pituitary).

  Significant weight loss in 5α-DHT sensitive epididymis is observed in racemic equol-treated males compared to controls. However, racemic equol had no effect on testis weight or pituitary weight. Body weight during this relatively short treatment period does not differ significantly between racemic equol and control treatments. These results are shown in Table 2.

（実施例３）
成体の雄性Ｓｐｒａｇｕｅ−Ｄａｗｌｅｙラットを、３群に無作為に割り当てて、ＤＭＳＯ、エクオルのラセミ混合物（０．２５０ｍｇ／ｋｇ／日）、Ｒ−エクオル（０．２５０ｍｇ／ｋｇ／日）、またはＳ−エクオル（０．２５０ｍｇ／ｋｇ／日）のいずれかを毎日注射する。各々の注射の総容積は０．３ｃｃであって、ラットの首の襟首にｓｃ投与する。連続７日の処置の後、ラットを殺傷して、注射期間の体重増加を決定する。Ｒ−エクオルを注射したラットは、表３に示されるように、コントロールのラットに比較して体重増大が有意に少ない。

(Example 3)
Adult male Sprague-Dawley rats were randomly assigned to 3 groups to receive DMSO, a racemic mixture of equol (0.250 mg / kg / day), R-equol (0.250 mg / kg / day), or S- Either equol (0.250 mg / kg / day) is injected daily. The total volume of each injection is 0.3 cc and is administered sc into the neck collar of the rat. After 7 consecutive days of treatment, the rats are killed and the weight gain during the injection period is determined. Rats injected with R-equol, as shown in Table 3, have significantly less weight gain compared to control rats.

精巣および脳下垂体の重量は、処置によって有意に変化しない（データ示さず）。エクオル実験からの体重のわずかな低下（ほぼ１０％）は、Ｐｈｙｔｏ−Ｆｒｅｅの食餌（極めて低レベルの植物エストロゲンを含む食餌）対Ｐｈｙｔｏ−６００食餌（６００ｐｐｍのイソフラボンを含む植物エストロゲン豊富な食餌）を給餌された動物の間でみられたものと極めて類似している。ラセミのエクオルの注射（約３６％）での白色脂肪組織の有意な減少はまた、食餌処置研究由来のデータセットでみられたものに匹敵する。これらの知見によって、Ｒ−エクオルは、体重を調節して、白色脂肪の蓄積を有意に低下することが示唆される。

Testis and pituitary weights do not change significantly with treatment (data not shown). A slight loss in weight (approximately 10%) from the equol experiment resulted in a Phyto-Free diet (a diet containing very low levels of phytoestrogens) vs. a Phyto-600 diet (a plant estrogen-rich diet containing 600 ppm isoflavones). It is very similar to that seen among fed animals. The significant reduction in white adipose tissue with racemic equol injection (about 36%) is also comparable to that seen in the dataset from dietary treatment studies. These findings suggest that R-equol regulates body weight and significantly reduces white fat accumulation.

Example 4
This example shows equol binding to 5α-DHT. In the first binding competition study conducted to determine and establish equol binding affinity for AR, we found that [3H] DHT apparent binding was in the presence of equol rather than in the absence of equol. It was repeatedly observed that it was large. A slight modification in the protocol where AR was removed from the incubation tube (only [3H] DHT and equol remained) resulted in the elution of [3H] DHT into the eluent containing the [3H] DHT reaction complex. occured.

To further investigate this phenomenon, a 30 cm long Sephadex LH-20 column is used to identify the elution peak and confirm the binding of [3H] DHT to equol. As shown in FIG. 7, the [3H] DHT peak is evident in the elution fraction between 5-9 mL when [3H] DHT + equol column incubation is added. This peak is not present when only [3H] DHT is added to this column. Furthermore, when 5α-DHT or 5α-DHT + equol is incubated with prostate supernatant and then passed through a 30 cm column (FIG. 8A), two distinct binding peaks can be identified. The first peak of [3H] DHT indicates that it has bound to AR in the prostate. This is found in 4-5 ml elution fractions. In addition, there is a back peak (5-9 ml) consistent with [3H] DHT binding to equol. However, when [3H] DHT is allowed to incubate with prostate supernatant for 36 hours (until equilibration) prior to introduction of equol, [3H] DHT binding is not evident (FIG. 8B). Both [3H] DHT and [3H] DHT + equol (equol added after 36 hours) show a single peak in the elution of 4-5 ml, indicating that equol competes with 5α-DHT for AR. It also suggests that it does not bind to [3H] DHT that is already bound to the receptor. Furthermore, it should be noted that the binding of equol to 5α-DHT appears to be specific. Because similar competition and binding studies have been carried out with [3H] E2, [3H] T, [3H] DHEA, [3H] CORT and [3H] progesterone, and no appearance of binding to equol. (Data not shown). Saturation analysis of equol binding to [ ³ H] DHT shows the apparent Kd calculated at 1.32 ± 0.4 nM (FIG. 9).

(Example 5)
In this example, in addition to modulating the effect of 5α-DHT on prostate size, it is shown that equol binds to 5α-DHT in vivo and blocks negative effects on LH secretion. GDX males treated with DHT propionate (DHTP), a long acting analog of 5α-DHT, show a significant increase in prostate weight compared to vehicle treated GDX control rats. Simultaneous treatment with equol (DHTP + equol) blocks the effect of DHTP, but equol has no effect on the prostate alone as shown in FIG. 10A. Equol also blocks the negative feedback effect of 5α-DHT on LH. In GDX males, LH is significantly reduced by DHTP treatment compared to treatment with DMSO. Treatment with equol in combination with 5α-DHT blocks the negative feedback effect of DHTP on LH secretion. Equol alone has no effect on LH levels (shown in FIG. 10B).

(Example 6)
This example demonstrates the effect of racemic equol on androgen sensitive tissues. One week after arrival, the animals are gonadectomized (GDX) under isoflurane anesthesia and allowed to recover for 7 days. After recovery, animals are assigned to the following groups: 1) DMSO, 2) DHTP (2 mg / 1 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 decapitated and killed, and trunk blood and tissue are collected for analysis. Plasma 5α-DHT is measured (shown in FIG. 11).

  As expected, there was a significant increase in plasma 5α-DHT in animals treated with DHTP (GDX + DHTP, GDX + equol + DHTP group). Plasma 5α-DHT was further elevated but was not significant by co-treatment with equol. Tissue including prostate, testis and epididymis is removed from the animal, fat and connective tissue are removed, weighed and fixed by immersion in 4% paraformaldehyde, then cut to 15 μm with cryostat. Tissue sections were loaded on filled slides (Superfrost Plus, Fisher Scientific, Pittsburgh, Pa.) Preheated to 23 ° C., stained with hematoxylin and eosin (H & E), dehydrated with rising (gradual) alcohol, and xylene Purify. Histological sections are shown in FIGS. H & E stained prostate reflects changes due to both GDX and treatment. The prostate of the equol treated group in addition to control, equol and DHTP shows similar histology (FIGS. 12A, 12B, 12D). In these animals, the prostate is characterized by a tiny atrophic gland (small gland lumen volume). In DHTP-treated animals (FIG. 12), the glands show signs of cell proliferation. Lumen size is increased compared to GDX animals; epithelial tissue is of a long cylindrical shape (FIG. 12C). Compared to intact control animals (FIG. 12E), the equol-treated male prostate shows regression and is further composed of a narrow space atrophic gland (FIG. 12F). Compared to the control male, the histology of the equol-treated intact male epidermis shows an overall small conduit as evidenced by the contracted lumen (FIG. 13).

(Example 7)
Long-Evans male rats are treated with a phytoestrogen-rich diet (hereinafter referred to as the “Phyto-600” diet) containing 600 μg isoflavone per gram diet, ie 600 ppm isoflavone, or very low levels of isoflavone. Breed on any of the diets they contain (hereinafter referred to as “Phyto-Free” diets; containing about 10 ppm isoflavones) (lifetime from conception to time of sample collection).

  As shown in FIG. 14, male Long-Evans rats fed the Phyto-600 diet showed significantly lower body weight at 33, 55 or 75 days of age compared to animals fed the Phyto-Free diet. Tissue (cutting right above the testis from just below the kidney in the abdominal pelvic cavity) is measured in 55 or 75 day old males. At both ages, white adipose tissue is significantly greater in Phyto-Free fed males compared to Phyto-600 fed animals (FIG. 15). The decrease in body weight of Phyto-600 fed male is about 10-15%, but the decrease in white adipose tissue from the same animal is about 50-60% compared to Phyto-Free fed male It should be noted. This greater reduction in white adipose tissue relative to the weight of soy fed animals is also a common feature seen in humans consuming soy based diets (DB Allison et al., Eur J Clin Nutr, 2003). 57: 514-522). This particular result is based on the various data present in these datasets, regardless of age, sex, strain of rats or whether female rats have had their ovaries removed (which stimulate human post-menopausal conditions). Repeated throughout the experiment.

  When measuring food and water to determine whether these parameters can affect body and adipose tissue weight, Phyto-600 fed males are slightly but significantly less than Phyto-Free fed animals High food (Figure 16A) and water (Figure 16B) intake are shown. Thus, the reduction in body and adipose tissue weight cannot be explained by changes in food / water intake during dietary treatment.

  Since leptin is produced by adipose tissue (DA Sandoval et al., J Diabetes Complications, 2003, 17: 108-113), serum leptin levels are measured along with insulin levels to show changes in these metabolic hormones. decide. As shown in FIG. 17A, leptin levels are 33, 55, or 75 days of age, with Phyto600 fed males (corresponding to the reduction seen in adipose tissue weight from these same animals), Phyto-Free fed males Significantly lower than Insulin levels are also significantly reduced in Phyt 600 fed males versus Phyt-Free fed males, consistent with the advantage of protecting against insulin resistance associated with type 2 diabetes (V. Jayagopal et al., Diabetes Care). 2002, 25: 1709-1714).

  To show that circulating isoflavone values differ in male and female rats (75 days of age) fed with Phyto600 vs. Phyto-Free, serum isoflavones were obtained by GC / MS as previously done in our laboratory. Values are determined (see the method of KDR Setchell, Am J Clin Nutr 129: 1333S-1346S, 1998; and KDR Setchell et al., J Nutr 132: 3577-3854, 2002). ). In each case for different classes of isoflavones, Phyto-600 fed males show significantly higher isoflavone values compared to those of Phyto-Free feeding (shown in Table 4). More importantly, equol values in rats fed Phyto-600 account for about 78% of total phytoestrogens values.

他の代謝性ホルモンが食餌処理によって、または年齢によって変化されるか否かを決定するために、血清グルコースおよび甲状腺（Ｔ３）のレベルをアッセイする。グルコース値は、年齢および血液サンプルの供給源とは独立して、Ｐｈｙｔｏ−Ｆｒｅｅ給餌の値に比較してＰｈｙｔｏ６００給餌のオスではわずかに（ただし有意に）高い［動脈（ＡＲＴ）または静脈（ＴＲＵＮＫ）のいずれか］（図１８に示す）。しかし、Ｔ３値を定量するとき、Ｐｈｙｔｏ−Ｆｒｅｅ給餌動物に比較してＰｈｙｔｏ−６００食餌を供給された８０または１１０日齢の雄性Ｌｏｎｇ−ＥｖａｎｓラットでのＴ３血清値は有意に増大している（図１９に示す）。甲状腺のレベルが、その甲状腺医薬を減少させたかまたは、その食餌中のダイズベースの食物の消費で甲状腺処置を完全にやめた個体の事例証拠と一致して、ダイズ消費で甲状腺レベルが増強されるということが、これから示唆される。これはまた、ダイズ食品の消費後のヒトにおけるＴ３値の同様の増大という報告とも一致している（Ｗａｔａｎａｂｅ，Ｓら、Ｂｉｏｆａｃｔｏｒｓ ２０００：１２（１４）：２３３４１）。

Serum glucose and thyroid (T3) levels are assayed to determine whether other metabolic hormones are altered by dietary treatment or by age. Glucose levels are slightly (but significantly) higher in Phyto600-fed males compared to those from Phyto-Free feeding, independent of age and blood sample source [artery (ART) or vein (TRUNK) Any of] (shown in FIG. 18). However, when quantifying T3 values, T3 serum values in 80- or 110-day-old male Long-Evans rats fed a Phyto-600 diet were significantly increased compared to Phyto-Free fed animals ( (Shown in FIG. 19). Thyroid levels are enhanced by soy consumption, consistent with case evidence of individuals who have either reduced their thyroid medication or who have completely discontinued thyroid treatment due to consumption of soy-based food in their diet This is suggested from now on. This is also consistent with reports of a similar increase in T3 values in humans after consumption of soy food (Watanabe, S et al., Biofactors 2000: 12 (14): 23341).

(Example 8)
In this experiment, female Long-Evans rats are housed on either the Phyto-600 diet or the Phyto-Free diet (lifetime from conception to time of sample collection). As shown in FIG. 20, rats fed the Phyto-600 diet showed significantly lower body weight at 80 days of age compared to animals fed the Phyto-Free diet, This corresponds to about a 12% decrease in body weight in animals.

  As previously shown in male Long-Evans Phyto-600 fed rats, females fed the Phyto-600 diet also had a significant reduction in adipose tissue weight compared to females fed the Phyto-Free diet. (Up to about 68%) (shown in FIG. 21).

  Similar to the results in male rats, serum glucose levels are slightly (but significantly) higher at 80 or 110 days of age in female rats fed Phyto-600 compared to animals in the Phyto-Free diet treated group ( Shown in FIG. However, T3 values are significantly higher in females fed the Phyto-Phyto-600 diet compared to 110-day-old Phyto-Phyto-Free fed animals (shown in FIG. 23). The T3 and glucose values generated in females are very similar to those obtained in male rats exposed to the same dietary treatment, thus suggesting similar health benefits for both sexes. Samples collected at 100 days of age produce similar results in female Long-Evans rats (ie, significant reduction in body weight and adipose tissue weight with consumption of Phyto-600 vs. Phyto-Free diet) (data not shown) .

Example 9
Adult female rats were placed on a Phyto-600 or Phyto-Free diet treatment at 50-215 days of age. Before 50 days of age, you may be raised on a diet that contains approximately Phyto-200 ppm isoflavones, or on a diet similar to that used by animal suppliers. At 215 days of age, Phyto-600 fed females are significantly lighter than Phyto-Free fed females, corresponding to an approximately 7% decrease in body weight (shown in FIG. 24). White adipose tissue in 215-day-old Phyto-600 fed females is significantly reduced to about 30% compared to female white adipose tissue fed the Phyto-Free diet (shown in FIG. 25). Correspondingly, serum leptin values in phyto-600 fed females were significantly lower than those in phyto-free feeding (shown in FIG. 26). Insulin levels were reduced to the same extent as previously shown in Phyto-600 feeding versus Phyto-Free fed females but did not reach statistical significance (shown in FIG. 27) .

(Example 10)
This example shows the effect 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. In addition, OVX allows the subcutaneous injection of estrogen and progesterone to stimulate estrous behavior in rats, and determines the effects of a Phyto-600 or Phyto-Free diet. Adult ovariectomized rats are fed approximately 200 ppm isoflavone phytoestrogens (“Phyto-200”) until age 50 days (all animals are ovariectomized at about 40 days of age). The female rats are matched for age and body weight at 50 days of age and are served with two dietary treatments until 94 days of age: Phyto-600 (black bar) or Phyto-Free (white bar). Baseline weights were weighed at 50 days of age, after which the animals were placed in dietary treatment and again at 58 days (day 8 of dietary treatment), 92 days of age (prior to estradiol injection), and 94 days of age ( It is measured before progesterone injection and 6 hours after 94 days of age (after chemical induction of estrus behavior) (shown in FIG. 28).

  After consumption of the diet for 8 days, Phyto-600 fed rats show a slight but significant weight loss compared to Phyto-Free feeding (about 7%). This decrease in body weight is maintained before and during the chemical induction of estrous behavior by estrogen and progesterone (steroid) injections.

  White adipose tissue was measured at 94 days of age after chemical induction of estrous behavior, and Phyto-600 fed OVX rats compared to Phyto-Free fed PVX rats, consistent with the findings of Examples 9 and 10. Has approximately 50% less white adipose tissue mass (shown in FIG. 29).

  Serum leptin levels in Phyto-600 fed OVX rats are reduced to almost 30% compared to Phyto-Free fed rats (shown in FIG. 30), reflecting a decrease in white adipose tissue mass.

(Example 11)
Male and female Long-Evans rats are purchased from suppliers at 50 days of age. All animals are housed on a Phyto-200 diet (from conception to 50 days of age). Male and female rats at 50 days of age are randomly assigned to one of four dietary treatment groups: 1) AIN-76 diet containing less than about 5 ppm isoflavones, 2) Phyto-Free, 3) Phyto -200, or 4) Phyto-600 diet (described in previous examples). The AIN-76 diet contains very low concentrations of isoflavones and the formulation is quite different compared to the other three diets. For example, the sucrose content is very high (reaching almost 50% of the total dietary formulation) and has a dark white firmness that the rat cannot enjoy consumption like a normal plant-based ingredient diet (ie , Phyto-Free diet uses corn and wheat in its formulation with very low levels of isoflavones); Phyto-200 or Phyto-600 diet uses varying amounts of soy flour in the formulation . Male rats are raised on their assigned diet until 350 days of age (corresponding to middle age in humans). Female rats are kept on this diet until 279 days of age (reaching human middle age). Food and water intake is measured to determine the potential impact of these parameters on weight change. In any case, these factors do not contribute to weight loss upon consumption of isoflavone containing diets (ie, Phyto-200 and Phyto-600 diets; data not shown).

Male (male)
Body weight is recorded at 112 days of age (approximately 62 days of diet) (shown in FIG. 31). The males fed the Phyto-Free diet had the highest body weight and the males fed the Phyto-600 were the lowest, but the males on the AS-76 and Phyto-200 diets were in these two groups. Fit between values. Body weight fed Phyto-600 is significantly lower to about 14% than males fed Phyto-Free.

  Correspondingly, 279-day and 350-day-old male rats have a profile similar to that observed at 112 days of age (shown in FIGS. 32 and 33, respectively). Males fed the Phyto-Free diet showed the highest body weight, males fed the Phyto-600 diet showed the lowest body weight, while males on the AS-76 and Phyto-200 diets were It falls between the values of these two groups.

  Males fed with AIN-76 or males fed with Phyto-Phyto-Free show the highest white adipose tissue weight as measured at 350 days of age. Males on the Phyto-200 diet show a 19% insignificant reduction in white adipose tissue weight compared to AIN-76 or Phyto-Phyto-Free fed rats. Male rats fed the Phyto-600 diet have a significantly lower adipose tissue mass, approximately 40% reduction compared to AIN-76 or Phyto-Phyto-Free fed rats (shown in FIG. 34).

  Serum insulin and leptin levels are significantly reduced as a function of increasing concentrations of isoflavones in the dietary treatment, shown in FIGS. 35 and 36, respectively. For example, males fed a Phyto-200 or Phyto-600 diet have significantly lower insulin levels compared to AIB-76 fed males. Also, Phyto-600 fed males show an approximately 50% decrease in insulin levels compared to Phyto-Free fed males, and the serum leptin profile shows a pattern similar to that of insulin, where Phyto-200 or Phyto-600 fed males have a significant reduction in serum insulin levels compared to either AIN-76 or Phyto-Free fed males. Insulin levels in Phyto-600 fed males are 46% lower compared to Phyto-200 fed males. However, the difference between these two diet groups does not reach a significant difference (p <0.065).

Female (female)
To determine the effect of four dietary treatments on body weight in female rats, body weight is measured at 112 and 279 days of age. At 112 days of age, females fed the Phyto-Free and Phyto-200 diets had the highest body weight and females fed the Phyto-600 diet, while the AIN-76 diet group Falls between the other three groups of values shown in The weight of the Phyto-600 feeding group is significantly lower by about 10% compared to the Phyto-Free feeding and Phyto-200 feeding females.

  At 279 days of age, female rats have a profile similar to that of age-matched males for body weight changes as affected by dietary treatment (shown in FIG. 38). Females fed the Phyto-600 diet show the lowest body weight compared to the AIN-76 or Phyto-Free feeding groups. This significant decrease in body weight with the Phyto-600 female is approximately 15% among the dietary treatment groups tested.

(Example 12)
With Noble rats, inbred rats have changes in body weight and adipose tissue similar to that of outbred rats (eg, Long-Evans animals when fed an isoflavone-rich diet) Decided whether or not. Due to inbreeding, Noble rats are more vulnerable animals. For example, pregnancy does not always carry litters until delivery, and litters are frequently small. Noble rats have been used for over 20 years. Because they naturally develop tumors with age, especially in hormone-dependent organs of the reproductive tract. Therefore, Noble rats have been extensively studied in the area of cancer research (eg, RL Noble, “Prostate carcinoma of the Nblat in relation to homones” Int Rev Exp Path, 1982, 23: 113-159). ).

  Male and female Noble rats are fed either a Phyto-Free or Phyto-600 diet from conception to 145 days of age. Male Noble rats fed the Phyto-600 diet are significantly lower in weight at 145 days of age compared to age-matched males fed the Phyto-Free diet (shown in FIG. 39). As previously observed in Long-Evans rats, a significant decrease in body weight shows a slight but consistent decrease of about 8% compared to Phyto-Free fed males. Furthermore, the amount of white adipose tissue is significantly lower in Phyto-600 fed males compared to Phyto-Free feeding (shown in FIG. 40).

  Female Noble rats fed the Phyto-600 diet have a 6% decrease in body weight compared to females fed the Phyto-Free diet (shown in FIG. 41). White adipose tissue mass was significantly reduced in 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 the Phyto-Free fed rats. The decrease in white adipose tissue is similar to the decrease seen in Long-Evans rats.

(Example 13)
Before the start of the Phyto-Free diet period, male Long-Evans rats are fed a Phyto-200 diet as described in the previous examples. The rats are kept on a diet containing a Phyto-Free diet until about 52 days of age and are randomly assigned to three groups. Baseline weights after 14 and 21 days on the Phyto-Free diet for all rats are similar (shown in FIGS. 43 and 44, respectively). Starting at 73 days of age, rats are injected daily subcutaneously into a 0.1 cc vehicle (peanut oil), 1 milligram racemic mixture of equol in the vehicle (0.83 mg / kg body weight per day), or in the vehicle A 5 milligram racemic mixture of equol (4.2 mg / kg body weight / day) is given once every 3 days.

  At 80 and 88 days of age, there is a slight weight loss and average weight gain in both equol injection groups compared to controls, but these values are not significantly different from controls ( Shown in FIGS. 45 and 46, respectively).

  By the time the animals are 95 and 10 days old, the body weight decreases slightly, with a reduction of 5-9% in the equol treated group (shown in FIGS. 47 and 48). However, the average weight gain in equol-injected animals at both 95 and 101 days is significantly reduced compared to control values. The difference in body weight is not significant, and adipose tissue accumulation is significantly lower in the equol-treated group. Adipose tissue weight in 101-day-old rats injected with equol is reduced by approximately 33% compared to controls (shown in FIG. 49).

  Testis weight is quantified in these animals to determine if equol injection has a deleterious effect on the male reproductive organs. There is a significant change in testis weight with equol injection, which is essentially the same between the injection treatment groups shown in FIG.

(Example 14)
50-day-old Long-Evans males and females are individually placed in a cage and bred on a schedule of 10 hours dark field and 14 hours light field (1400 to 0400 lights up). Animals are randomly assigned to diet groups and have free access to one of the four diet treatment groups: 1) AIN-76, 2) Phyto-Free, 3) Phyto-200, or 4) Phyto-600 diet . Rats are maintained on this diet until middle age (approximately 300 days for males and approximately 330 days for females), when animals are tested in an elevated plus maze to quantify anxiety-related behaviors. Serum phytoestrogen levels are then quantified by GC / MS according to the method described by Coward L et al., J Agric Food Chem, 41: 1961-1967. Compare anxiety behavioral patterns to serum profiles of circulating isoflavone levels in the diet-treated group by gender.

  In males, there is a dose-dependent manifestation of anxiety-related behavior, with animals fed the highest concentration of isoflavones exhibiting the lowest anxiety parameters. In contrast, animals fed AIN-76 show the highest level of anxiety (shown in FIG. 51). When analyzing the percentage of time spent in an open arm, a similar pattern is seen for the number of times of entry into the open arm. In particular, Phyto-600 fed males are fed the AIN-76 diet, while the highest percentage of time elapsed in the open arm indicates the percentage, while the lowest percentage of time elapsed in the open arm is indicated. Phyto-free and Phyto-200 values fall between these maximal responses in a dose-dependent manner (shown in FIG. 52).

  Prior to testing in the elevated plus maze, the female is monitored with a 12-day continuous vaginal smear to ensure that there is no cycle that minimizes the effects of the estrous cycle. Female rats have a pattern of anxiety-related behavior similar to that observed in male rats. However, the effects of dietary isoflavones are not as robust as those seen in males. The highest rate of open arm entry (Fig. 53) and elapsed time (Fig. 54) is seen with the Phyto-600 female scalpel until the lowest rate of entry is seen with the AIN-76 female scalpel It is a staircase pattern that decreases.

  When behavioral testing is complete, serum phytoestrogens are determined and compared to anxiety-related behavioral patterns. In both males (FIG. 55) and females (FIG. 56), circulating isoflavone values correspond to the development of anxiety-related behavior, indicating an association between circulating isoflavone molecules and anxiety. These data indicate that the isoflavone content of the diet can have a significant effect on anxiety.

(Example 15)
Adult male Sprague-Dawley rats were treated with DMSO, racemic equol (0.250 mg / Kg / day), R-equol (0.250 mg / Kg / day), or S-equol (0.250 mg / Kg / day). Either daily injection is given by subcutaneous injection in a total volume of 0.3 cc DMSO. At the end of 7 consecutive days of treatment, animals are tested in an elevated plus maze to quantify anxiety related behavior. As shown in Table 5 below, males injected with racemic equol or R-equol show a significant decrease in the level of anxiety compared to control rats.

＊＝コントロール値に対する不安関連パラメータ（すなわち、迷路時間の中心領域またはオープン・アーム中で経過する時間またはオープン・アーム進入の回数）の有意な低下
＊＊＝コントロール値に対する不安関連行動（すなわち、オープン・アーム進入の回数）の有意な低下
ｎ＝８（１群あたりの動物）。

* = Significant decrease in anxiety-related parameters for control values (ie, central area of maze time or time spent in open arms or number of open arm entries) ** = Anxiety-related behavior for control values (ie, open) -Significant reduction in the number of arm entries n = 8 (animals per group).

  These findings are consistent with findings obtained using four different treatments containing different concentrations of isoflavones, and equol is associated with anxiety, as well as other neurological conditions such as a wide range of health benefits. It is shown to be a major factor in regulating mood and depression with clear ability.

(Example 16)
Twenty-nine adults with hypercholesterolemia are fed a diet containing 33 mg of total isoflavone daily for 5 weeks. FIG. 57 shows the changes observed in BMI for each of 29 individuals after 5 weeks that are strictly adjacent to a diet containing isoflavones. The average drop in BMI over this period is small but nevertheless significant (p = 0.01). These results suggest that a diet rich in phytoestrogens can affect human weight control. This study did not identify the component responsible for weight control or its mechanism. The average baseline BMI (n = 29) is 26.6 ± 0.8, and the average BMI (n = 29) is 26.2 ± 0.7 at 5 weeks.

  While various embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that further modifications and adaptations of the invention may occur. It should be clearly understood that such modifications and adaptations are within the spirit and scope of the present invention.

(Example 17)
Equol decreases depression and body weight in middle-aged female rats.

  Isoflavones have been shown to have beneficial effects on cardiovascular and hormone-dependent diseases, but little is known about their effects on the brain and behavior, particularly behavior such as depression and despair. In this study, middle-aged Long-Evans female rats were tested in this context.

  Age-related morphological and physiological changes in rodents are equivalent to humans using the following age ranges: middle-aged rats (ages 10-12 months) are up to 45-55 years old Although roughly equivalent to humans, older rats (21-23 months of age) correspond roughly to 65-75 years of age for humans (D. Mileusnic et al., Neurobiol. Aging, 20: 19-35, 1999). Furthermore, the middle-aged rat (12 months old) corresponds to the age of a postmenopausal woman about 55 years old. The average age of menopause in the United States is approximately 51 years old (National Institutes of Health, USA).

  All female rats in this study were examined for hormonal patterns characteristic of the estrous cycle. All animals in this study do not circulate in agreement with scientific reports using this aging animal model (CR AZalone et al., Biol. Reprod, 64: 1056-1062, 2001).

  Female rats were exposed to either phytoestrogen-rich (Phyto-600) or phytoestrogen-free (Phyto-free) diet (from the time of conception).

  At 12 months of age, animals were analyzed using the standard preclinical test, the Porsol forced swimming test (applied to humans to study the neurobiology of the effects of depression and antidepressants) (R D. Porsolt, M. Pichon and M. Jalfre, Nature 266: 730-732, 1997; 1995).

  The test is designed to force the rat to swim actively. Over time, some animals may become immobile, which is considered a state of behavioral despair. The total time an animal is stationary is an index of despair or depression. Other parameters include: a) total distance of movement, b) total time of movement (opposite of immobility index), c) number of diving attempts of animals showing escape behavior, and d) emotional level The number of bowel movements or vortices excreted during the test, which is an indicator (C. S. Hall, J. Comp. Psych., 18: 385-403, 1934), an increase in the number of boluses during the test is indicative of emotional behavior The emphasis is on. All of these parameters were recorded in this study and quantified by the AnyMaze® computer software, and the total time for the Porsol forced swim test was 480 seconds or 8 minutes.

  Thus, animals that show increased levels of diving with a decrease in total distance traveled, time traveled, and number of immobilities and excavations will exhibit low levels of despair or depression. Animals exhibiting behavior that opposes the above parameters exhibit increased levels of despair or depression.

  Animals were tested twice.

  Test 1 compared only food exposure, shown in FIGS. 58-69 and labeled “before equol injection”.

  In the second phase of the study, Phyto-free fed females were injected with equol for 8 days (5 mg / kg or 2.5 mg / kg by subcutaneous administration), while Phyto-600 fed females were treated with dimethyl sulfoxide. (DMSO) Vehicle control injections were given. (DMSO is used in medicine due to its ability to penetrate deeply through the skin and other membranes without damaging them and to carry compounds in the circulation).

  Test 2 evaluated depressive behavior after equol treatment, shown in FIGS. 58-69 and labeled “after equol injection”.

  In the first Porsolt test—indication A: Phyto-600 feeding female showed the following to be significantly greater: a) distance traveled (m), (see FIG. 58A); b) overall average Speed (m / s), (see FIG. 59A); c) total travel time (seconds), see FIG. 60A; and d) number of dives compared to Phyto-free female (see FIG. 61A) ) Conversely, Phyto-free females were shown to have significantly greater following compared to Phyto-600 females: a) total transit time (seconds) (see FIG. 62A), and b) excretion Boli (see Figure 63A).

  After 8 days of equol treatment, Phyto-free females showed values similar to those exhibited by Phyto-600 females for all parameters tested above. For example, a) the total travel distance was increased and was similar to the control value (see FIG. 58B); b) the average speed increase was similar to the control (see FIG. 59B); c ) With increase in total migration time (see FIG. 60B) (total migration time significantly increased in Phyto-free rats after equol injection compared to pre-equol injection values (see **) D) The number of dives was significantly reduced and comparable to the level of control (see FIG. 61B). Furthermore, the total travel time was significantly reduced and similar to the control (see FIG. 62B). Finally, as illustrated in FIG. 63B, the number of boli decreased after equol treatment, which was essentially the same as that of the control value.

  Also, the mean weight loss (in grams) at the end of the 8 day equol treatment revealed that equol significantly decreased body weight compared to controls (see Table 6), This corresponds to a 2.6-fold decrease in body weight over control after the 8 day treatment interval.

  Table 6. Weight loss in middle-aged female Long-Evans rats treated with equol.

＊＝エクオル処置した中年齢の雌性ラット対コントロールにおける体重の有意な減少値（ＳＥＭ＝平均の標準誤差）。

* = Significant decrease in body weight in equol-treated middle-aged female rats versus controls (SEM = standard error of the mean).

  These data suggest that exposure to equol (including short-term exposure) has a beneficial antidepressant effect in middle-aged female rats. The positive anxiolytic parameters seen in Phyto-600 fed animals are believed to be due to consuming an isoflavone (soy) enriched diet (which is then converted to equol in these rats). That rats produce very high levels of equol when fed a soybean-enriched diet (ie, Phyto-600 diet), and most of the levels of circulating isoflavone / isoflavone metabolites (70-90% of the total) ) Is well established by equol. Of note, when equol was administered (short term) to middle-aged female rats fed a Phyto-free diet, all Porsol test depression parameters (shown in B of FIGS. 58-63) were reversed. And similar to the Phy-600 group. These data sets are enhanced by the fact that Phyto-600 and Phyto-free animals themselves serve as controls (before and after equol injection). Therefore, the results of this study indicate that equol alone accounts for all or at least most of the positive effects of significantly reducing depression-like behavior. As such, the use of equol has considerable potential to prevent mood, despair and depression-related behaviors through potential food, nutrition and pharmaceutical applications.

(Example 18)
Equol decreases anxiety-related behaviors and body weight in adult male rats but does not alter hormone-sensitive brain areas Hypothalamic structures such as male and female dimorphic nuclei (SDN) and anterior ventricular circumferential nucleus (AVPV) is an important area involved in reproductive function / regulatory behavior that is sensitive to the effects of steroid hormones during birth and subsequent postnatal development in rats. The structure of SDN and AVPV has previously been shown to be sensitive to isoflavone effects in adult rats, and many reports generally demonstrate that phytoestrogens are neuroprotective (ED). Lepart et al., J. Steroid Biochem. Mol. Biol., 85: 299-309, 2003; ED Lepart et al., Curr.Neurovas.Res., 1: 455-464, 2004; Lepart, Neurosci. Lett., 385: 153-157, 2005). However, equol, the major isoflavone metabolite in rodents (and many other animal models), has been tested in brain studies.

  In this study, anxiety-related behaviors were examined along with hormone levels in adult Long-Evans male rats exposed to equol. Known and accepted animal behavioral tests for psychiatric disorders in humans, such as the elevated plus maze (EPM), were used for this purpose (P. Willner, in Behavioral Models in Psychopharmacology: Theoretical, Industrial and Clinical Perspectives, New York, Cambridge Univ. Press, 1991; MA Geyer & A. Markou, in Psychopharmacology: The Four Generation of Prog. 24: 1 9～158,2000).

  The EPM test relies on the inherent conflict between exploring a new environment and avoiding its disgusting features. Typically, an animal spends a little time in an open arm (new environment) compared to the closed arm (safe environment) of the maze and has less access to it. For example, anxiolytics or drugs increase the number of times that the maze is opened and the total time elapsed.

  50-day-old male rats were fed a diet low in phytoestrogens (10 ppm isoflavones) and remained on this diet until 215 days of age. At 150 days of age, rats were matched in age and body weight and then divided into control or equol-treated groups. Starting at 190 days, male rats were given daily subcutaneous (0.1 cc) injection of control vehicle (DMSO) or equol (2.5 mg / Kg) for 25 consecutive days.

  After 15 days of treatment, the animals were tested in EPM, where they were videotaped for 3 minutes, and the number of entries into the open arm and the total time elapsed on the open arm via the AnyMaze® computer software. The percentage of time was recorded later.

  At 215 days of age, animals are sacrificed to determine body weight / prostate weight, serum hormone and isoflavone values, as well as the volume of the hypothalamic area that is sensitive to hormones.

  A significant decrease in prostate weight and 5α-DHT with changes in serum testosterone, luteinizing hormone (LH) or 17β-estradiol levels in equol-treated male rats compared to control values is shown in US Patent Publication No. 2005 / 0245492A1 The content was previously reported in) in its entirety.

  The brain was processed via standard staining and analyzed via Bioquant® for morphological SDN and AVPV parameters by treatment. Serum equol values in equol-treated animals were equivalent to consuming phytoestrogen-rich soybean diet.

  As shown in FIG. 64, body weight was significantly reduced in equol-treated males compared to control animals. In EPM, equol-treated animals showed significantly less anxiety-related behavior compared to control values (ie, the number of times the maze entered the open arm and the time elapsed in the open arm was significant relative to the control). (See FIGS. 65 and 66, respectively).

  Exposure to equol for 25 consecutive days showed no significant changes in SDN or AVPV volumetric morphometric parameters (see FIG. 67 and Table 7).

  These results suggest that equol does not alter hormone-sensitive hypothalamic volume in rodents during adulthood, as previously reported in our laboratory. Contrary to the consumption of soybean (phytoestrogens) via the dietary route during equivalent exposure intervals. This further suggests that equol can be a safe and non-toxic drug and has potential neural applications to address brain / behavioral disorders and male / female health problems. Ultimately, these data suggest that the presence of equol during nutritional supplements, dietary supplements, or pharmaceutical applications is an effective treatment in weight control and modulates or reduces anxiety.

(Table 7: Hypothalamic structure of male rats treated with equol—morphological volume of male and female dimorphic nucleus (SDN) and anterior ventral perinuclear nucleus (AVPV) (expressed in mm ³ × 10 ⁻³ ))

局所の脳容積は、ｍｍ^３×１０^−３で表した。処置群の間にＳＤＮまたはＡＶＰＶにおいて有意な差はなかった（１群あたりｎ＝４）。

The local brain volume was expressed in mm ³ × 10 ⁻³ . There was no significant difference in SDN or AVPV between treatment groups (n = 4 per group).

(Example 19)
Equol is more potent than genistein in reducing anxiety levels through premenopausal and early postmenopausal treatment in male rat offspring tested as adults.

  Recently, isoflavones have been reported to have beneficial health benefits with respect to cancer (breast and prostate cancer), cardiovascular disease, osteoporosis and menopausal related symptoms. However, little information is known about the effects of isoflavones on the brain, especially behaviors such as anxiety. In this study, the long-term effects of genistein and equol (isoflavone metabolite) were examined for their ability to affect anxiety-related behavior in male offspring tested adults at EPM.

  Pregnant Long-Evans rats were treated via daily subcutaneous injections between 14-20 days of pregnancy and 2-25 days after birth, where 1) control-DMSO vehicle, or 2) genestein ( 3) equol (at 3 mg / day depending on treatment group). All animals were fed a low diet of phytoestrogens through these studies (10 ppm isoflavones). There was no significant difference in maternal body weight just before birth in the treatment group. At birth, body weight was slightly higher (but not significant) in genistein- and equol-treated animals compared to controls. Maternal and male offspring genistein and equol levels (25 days after birth) were parallel to the level of treatment administered.

  Approximately 90 day old male offspring were tested by treatment in EPM (control, genistein and equol), where they were videotaped for 3 minutes and the number of entries into the open arm as previously described And later recorded the percentage of total time elapsed on the open arm. As illustrated in FIG. 68, the equol-treated male showed significantly greater entry into the open arm compared to the control. However, open arm entry was not significantly different between genistein and the control group. In particular, as shown in FIG. 69, equol treated males showed significantly greater time in the open arm compared to genistein treated animals or controls. Genistein-treated males also showed significantly larger open arm time intervals compared to controls. The equol time interval was twice as large as the genistein value and the genistein value was about 9 times greater than the control.

  These data suggest the following: 1) equol is a more potent anxiolytic agent compared to genistein, and 2) equol, an isoflavone metabolite, is tested as an adult treated before birth. Has a long-term effect on anxiety-related behavior of the offspring. The findings of this experiment confirm the positive effect of equol seen in Example 17 above to alleviate depression-related behavior, probably due to circulating equol values but not genistein. The results of this study have application in the pharmaceutical regulation of food, nutrition and anxiety by various routes of administration.

  Accordingly, the foregoing detailed description is intended to be regarded as illustrative rather than limiting, and it includes the following claims, including all equivalents, that are intended to define the spirit and scope of this invention: It is understood that it is in the range.

Claims (19)

A method for preventing or ameliorating a neuropsychiatric or neurodegenerative disease or disorder in a subject comprising:
Administering a composition comprising equol in an amount sufficient to prevent or ameliorate said neuropsychiatric or neurodegenerative disease or disorder.   2. The method of claim 1, wherein the equol is a racemic mixture of R-equol and S-equol.   2. The method of claim 1, wherein the equol is enantiomerically enriched with R-equol.   2. The method of claim 1, wherein the equol is enantiomerically enriched with S-equol.   The neuropsychiatric or neurodegenerative disease or disorder is depression, anxiety, bipolar disorder, obsessive compulsive disorder, hyperactivity disorder, panic disorder, weight gain, obesity or bulimia. The method described.   2. The method of claim 1, wherein the neuropsychiatric or neurodegenerative disease or disorder is Alzheimer's disease or Parkinson's disease.   The method of claim 1, wherein the composition further comprises an excipient.   The method of claim 1, wherein the composition is in an oral formulation selected from the group consisting of tablets, capsules, powders, troches, buccal tablets and sublingual tablets.   The method of claim 1, wherein the composition is an oral formulation comprising at least about 0.25 mg equol.   The method of claim 1, wherein the composition is selected from the group consisting of food and beverages.   The method of claim 1, wherein the composition is a delayed or sustained release formulation.   2. The method of claim 1, wherein the neuropsychiatric or neurodegenerative disease or disorder is a perimenopausal or postmenopausal symptom.   13. The method of claim 12, wherein the perimenopausal or postmenopausal symptoms are depression, anxiety, fatigue, weight gain, obesity and polycystic ovarian disease.   The method of claim 1, wherein the equol is in an amount sufficient to bind free 5α-dihydrotestosterone and inhibit binding to the androgen receptor.   The method of claim 1, wherein the equol is in an amount sufficient to bind free 5α-dihydrotestosterone, inhibit binding to the androgen receptor, and bind to an estrogen receptor subtype.   A method of reducing obesity in a subject comprising administering a composition comprising a therapeutically effective amount of equol.   A method of alleviating depression in a subject comprising administering a composition comprising a therapeutically effective amount of equol.   A method of reducing anxiety in a subject comprising administering a composition comprising a therapeutically effective amount of equol. A method of providing personal treatment of a neuropsychiatric or neurodegenerative disease or disorder in a subject comprising:
Assessing the emotional, mental or endocrine health status of the patient;
Assessing the patient's equol producer status;
determining the optimally beneficial course of treatment selected from the group consisting of: a) mode of administration, b) dosage, and c) dosing interval.

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