WO2013050930A1 - Développement d'un produit de phytoestrogène de trèfle des prés pour la prévention ou le traitement de l'ostéoporose - Google Patents

Développement d'un produit de phytoestrogène de trèfle des prés pour la prévention ou le traitement de l'ostéoporose Download PDF

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WO2013050930A1
WO2013050930A1 PCT/IB2012/055277 IB2012055277W WO2013050930A1 WO 2013050930 A1 WO2013050930 A1 WO 2013050930A1 IB 2012055277 W IB2012055277 W IB 2012055277W WO 2013050930 A1 WO2013050930 A1 WO 2013050930A1
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composition
biochanin
formononetin
genistein
daidzein
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PCT/IB2012/055277
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English (en)
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Yun Kau Tam
Yi-Chan James Lin
Brian Duff Sloley
Chih-Yuan Tseng
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Sinoveda Canada, Inc,
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Publication of WO2013050930A1 publication Critical patent/WO2013050930A1/fr
Priority to US14/242,981 priority Critical patent/US9333192B2/en
Priority to US15/149,183 priority patent/US10307451B2/en
Priority to US16/376,044 priority patent/US11123389B2/en
Priority to US17/479,139 priority patent/US20220000955A1/en
Priority to US18/232,551 priority patent/US20230381260A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/2027—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2072—Pills, tablets, discs, rods characterised by shape, structure or size; Tablets with holes, special break lines or identification marks; Partially coated tablets; Disintegrating flat shaped forms
    • A61K9/2086—Layered tablets, e.g. bilayer tablets; Tablets of the type inert core-active coat
    • A61K9/209—Layered tablets, e.g. bilayer tablets; Tablets of the type inert core-active coat containing drug in at least two layers or in the core and in at least one outer layer
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/30—Prediction of properties of chemical compounds, compositions or mixtures

Definitions

  • HRT hormone replacement therapy
  • red clover isoflavones
  • coumestrols and lignans are classes of bioactives in red clover: isoflavones, coumestrols and lignans (Thompson, Boucher et al. 2007).
  • the quantity of coumestrols and lignans is small; therefore, their contribution to the overall activity is likely minimal.
  • the major isoflavones in red clover are biochanin A and formononetin (Liu, Burdette et al. 2001; Overk, Yao et al. 2005; Booth, Overk et al. 2006).
  • Genistein and daidzein are present in minute quantities.
  • Biochanin A and formononetin are precursors of their respective active moieties, genistein and daidzein. The conversion takes place in the intestine by intestinal flora and liver, although the relative significance has not been established. Daidzein is converted by bacteria in the colon to form a more estrogenic metabolite, equol. In Red clover, a significant quantity of Biochanin A and Formononetin is in the form of glycosides. The glycosides in soy and red clover are converted to their respective aglycones by the intestinal flora before absorption (Setchell and Cassidy 1999).
  • Metabolism of isoflavones is mainly mediated by Phase II enzymes in the enterocytes and hepatocytes. Although metabolism of individual isoflavones in rats has been well characterized (Jia, Chen et al. 2004; Chen, Lin et al. 2005; Chen, Wang et al. 2005), interaction between components has not been evaluated.
  • the present invention discloses a process of developing optimized red clover products using pharmaceutical platform technology.
  • the product contains 5 to 200 mg of red clover extract formulated with a-cyclodextrin.
  • the product is an immediate release dosage form containing 5 to 200 mg of Red clover extract.
  • the product comprises a blend of the immediate and sustained release dosage form containing 5 to 200 mg of Red clover extract.
  • a product contains Biochanin A, Daidzein, Equol and Genistein.
  • the product is an immediate release dosage form containing a 5 to 200 mg of total phytoestrogens.
  • Figure 1 shows a typical LC/MS chromatogram showing the composition of a Red clover extract.
  • Figure 2 shows the metabolism of the isoflavone mixtures by human fecal bacteria.
  • Figure 3 shows a dissolution profile of two PROMENSIL ® tablets, which contain 40 mg of total phytoestrogens per tablet. The content of Biochanin A and Formononetin was not completely released.
  • Figure 4 shows the plasma concentrations of pure formononetin released at different locations.
  • Figure 5 shows the metabolism of the isoflavone mixtures by human intestinal microsomes.
  • Figure 6 shows the metabolism of the isoflavone mixtures by human hepatocytes.
  • Figure 7 shows a Histogram of dose ratio distribution for individual isoflavones in the 121 mixtures. The solid line shows the fitted power law distribution and parameters are shown in the inset.
  • Figure 8 shows the ability for the 121 combinations (open square) to form osteoblast (ALP/XTT) and inhibit osteoclast formation (ACP/XTT) is shown on the upper panel.
  • the lower panel shows osteoblast forming activity (mean ⁇ SE) of pure isoflavone measured at 1 ⁇ , where "Bio” stands for Biochanin A, “For” is Formononetin, “Dai” is Daidzein, “Equ” is Equol and “Gen” is Genistein.
  • Figure 9 shows a histogram of five isoflavones dose ratio in four quadrants defined in Table 2. The number inside round bracket is the number of data points in this quadrant. The abbreviations in the legend are defined in Figure 7.
  • Figure 10 shows a comparison of the dissolution profiles of PROMENSIL and the combination formula developed in this invention.
  • the present invention describes a method of using the pharmaceutical platform technology (PPT) (Tarn and Tuszynski 2008) to fill in the knowledge gap in pharmacokinetic and pharmacodynamics of key ingredients and their metabolites of Red clover. Using this set of information, products are designed for optimal delivery.
  • PPT pharmaceutical platform technology
  • the major application of PPT is to predict individual component time course after a mixture is administered (Tarn and Tuszynski 2008).
  • a mixture is an extract of red clover.
  • the backbone of PPT is the construction of a set of physiologically based pharmacokinetic and pharmacodynamic models for describing the time course of concentration and effect in different animal species including human.
  • Parameters describing these models are derived from physiological data for a specific animal species; and kinetic and activity parameters are derived from in vitro studies.
  • Physiological parameters include but not limited to gastric emptying and intestinal transit time, hepatic blood and cardiac blood flow rate, etc.
  • Component specific parameters include but not limited to intestinal permeability, rate of intestinal and hepatic metabolism, plasma protein binding, tissue distribution and dose-response relationship.
  • the unique feature of PPT is its ability to estimate pharmacokinetic and pharmacodynamic activity of individual components and interaction between these components in a mixture without purification of these individual components.
  • a number of key parameters are identified from a Red clover extract.
  • the bioactives or precursors to bioactives in Red clover are identified.
  • the bioactive in Red clover is Biochanin A.
  • the precursors to bioactives are Biochanin A and Formononetin.
  • Genistein is the metabolite of Biochanin A.
  • Daidzein and Equol are active metabolites derived from Formononetin.
  • these active metabolites are mostly formed in the lower part of the intestine.
  • active Phase I metabolites: Genistein, Daidzein and Equol are not formed in human liver.
  • the solubility of Formononetin and Biochanin A is low in products that are currently available on the market.
  • Biochanin A, Daidzein, Equol and Genistein are active physiologically. In another embodiment, these compounds act synergistically for osteoblast formation and prevent osteoclast resorption.
  • the plasma levels of the precursors and active metabolites can be adjusted.
  • an immediate release dosage form comprising these four phytoestrogens and cyclodextrin.
  • the two major aglycones of Red clover are highly insoluble in the gastrointestinal fluids. Regardless of the ratio of the two, relative solubility, absorption and metabolism by the intestinal flora will dictate the consistency of the blood levels of these components and their metabolites.
  • an immediate-release dosage form is designed to release the two isoflavones: Formononetin and Biochanin A in the stomach. The intention is to promote solubility and absorption of the two isoflavones. Thus, the absorption of the two components will be more consistent.
  • a sustained-release dosage form is designed to release a drug in the lower portion of the intestine.
  • the intention is to expose the major isoflavones: Formononetin and Biochanin A to intestinal flora to promote conversion to the bioactive metabolites: Daidzein, Equol and Genistein. These metabolites have been shown to be bioactive and their potencies on cell differentiation are fairly similar.
  • the phytoestrogens are complexed with a-cyclodextrin to promote solubility.
  • the release of Red clover extract formulated with Kollidon has sustained release properties.
  • a dosage form comprising the immediate -release and sustained- release components is designed.
  • the purpose of this design is to provide optimal ratios of Formononetin, Biochanin A, Daidzein and Genistein in order to achieve optimal efficacy.
  • the present invention provides an immediate release composition
  • a-cyclodextrin and one or more of these isoflavones: biochanin A, daidzein, equol and genistein, wherein said composition enhances the bioavailability of the isoflavones.
  • a sustained release composition comprising one or more of biochanin A, formononetin, daidzein, equol and genistein, wherein said composition enhances the formation of active metabolites of formononetin and biochanin A in the distal part of the intestinal tract.
  • a composition comprising (i) a first composition comprising ⁇ -cyclodextrin and one or more of these isoflavones: biochanin A, daidzein, equol and genistein, wherein said first composition enhances the bioavailability of formononetin and biochanin A, and (ii) a second composition comprising one or more of biochanin A, formononetm, daidzein, equol and genistein, wherein said second composition enhances the formation of active metabolites of formononetm and biochanin A in the distal part of the intestinal tract.
  • the ratio of formononetm to biochanin A in the above compositions ranges from 0:5 to 5: 1.
  • biochanin A comprises between 10 to 100% of the composition
  • formononetm comprises between 0 to 90% of the composition.
  • the composition comprises between 5 to 200 mg of total isoflavones.
  • the present invention also provides uses of the compositions disclosed herein for treating or preventing osteoporosis.
  • compositions disclosed herein are obtained through a synthetic source or natural source.
  • compositions disclosed herein are obtained by a method comprising the steps of (a) obtaining a red clover (Beck, Unterrieder et al. 2003) extract comprising a plurality of components which are the aglycone forms of phytoestrogens; (b) inputting parameters into in silico models that will generate outputs to predict pharmacokinetics and pharmacodynamics properties of the components of the red clover extract and their metabolites in vivo, said parameters comprise (i) parameters describing the rate of metabolism of said components and their metabolites in a plurality of mammalian tissue systems, and (ii) parameters describing distribution of said components and their metabolites in a plurality of mammalian tissue systems; and (c) using said outputs to produce a composition comprising said components.
  • entropic component analysis comprises: (a) assigning probabilistic models to all possible combinations of variables and (b) determining the ranking scheme of these models.
  • determining the rate of metabolism in gastrointestinal tract comprises in vitro assays.
  • such assays comprise artificial gastric or intestinal juice, intestinal flora, intestinal microsomes, or permeability studies using cultured cells or intestinal tissues (e.g. Caco-2 cells or MDCK cells).
  • determining the rate of metabolism in liver comprises assays using freshly harvested hepatocytes, cryopreserved hepatocytes, hepatic microsomes, hepatic cytosol or S-9 fractions.
  • the determination of distribution in blood or plasma comprises determining one or more of binding to plasma protein, binding to blood protein, pKa, log P, log D, and volume of distribution of a component.
  • the above method uses a model comprising r « r + ⁇ w i (d i -3 ⁇ 4) 2 + ⁇ w. . ⁇ d t - d i )(d j - 3 ⁇ 4)
  • component i and ' and J are average dose of the ith and jth component, J is the weight of
  • A a 0 + ⁇ ⁇ . ⁇ . + ⁇ fi .x.X ;
  • B- ⁇ x x - j is the activity coefficient of the interacting pair, ' and j , wherein said equation is able to predict an optimized composition of the extract to achieve maximum possible potency.
  • the objective of this study is to track the events that occur in the lumen of the gastrointestinal tract.
  • the goals are to identify the stability of Red clover components, their physical and enzymatic stability, solubility and absorbability.
  • Formononetin, Genistein, Daidzein and their glucosides, along with other minute quantities of coumestrol and lignans have been prepared either using solvent extraction or a variety of cultivars.
  • the aerial portion of red clovers, leaves, stems or leaves and stems were dried powdered.
  • the plant material was extracted with 50% ethanol at 50 °C for 1 hour.
  • the resultant sample was centrifuged and the ethanolic component was removed and dried.
  • Fresh human fecal samples were collected from 4 volunteers. Five grams of each were pooled together and mixed well with 30 mL BHI culture medium. The fecal suspension was centrifuged at 200 g for 5 min and supernatant was decanted and centrifuged at 5,000 g for another 30 min. The resultant precipitate was re-suspended with 10 mL BHI medium to produce intestinal micro flora solution. [0050] As The biotransformation of drugs by human intestinal bacteria was determined in a 5 mL incubation system containing 250 intestinal microflora solution, 50 stock solution in DMSO in BHI medium.
  • the incubation system was anaerobically incubated at 37 °C in a GasPakTM EZ Anaerobe Pouch System for 0 h, 24 h, 72 h, and 120 h for red clover isoflavones (the final concentrations for Biochanin A, Daidzein, Equol, Formononetin, and Genistein were 100 ⁇ each).
  • Zero-minute incubations served as controls. Reactions were stopped by extracting samples with 15 ml of ethanol twice. The two ethanol extractions were combined, dried and resuspended in 80% methanol for HPLC/MS analysis.
  • Red clover isoflavones are shown to be metabolized extensively by human intestinal microflora ( Figure 2).
  • Figure 2 When Biochanin A was incubated with intestinal microflora, dihydrobiochanin A, Genistein, and dihydrogenistein were formed. Daidzein was metabolized into dihydrodaidzein, and Equol. Equol was the most resistant to bio-transformation. At the end of 120 hours, there was still over 60%> of equol left in the incubation media, while there were less than 5% of Biochanin A, Daidzein, and Genistein left. The bio-transformed products of Equol were not identified in this study.
  • Formononetin was bio-transformed into dihydroformononetin, dihydrodaidzein, Daidzein, and Equol. At the end of 120-hour incubation, there was over 20% of formononetin remained. Genistein was bio-transformed into dihydrogenistein.
  • Red clover extracts were subjected to permeability measurements using Caco-2 and
  • An optimal extract of Red clover should consist of the aglycones only.
  • An enzymatic or chemical conversion of the glucosides to their respective aglycones prior to extraction will be desirable. This can be accomplished using the literature methods (Tsao, Papadopoulos et al. 2006).
  • Daidzein and Genistein was measured in artificial gastric and intestinal juice.
  • the solubility of these four compounds, in general, is low.
  • Formononetin has the lowest solubility, which is approximately 4 ⁇ g/ml at 37 °C in artificial intestinal juice.
  • Biochanin A has a higher solubility, -23 ⁇ g/ml.
  • the solubility of daidzein and genistein is quite a bit higher, ranging between 80 to 100 ⁇ in buffer at 37 °C. This set of data suggests that solubility instead of absorption is a huge issue in terms of bioavailability.
  • the objective of this example is to evaluate the effects of first-pass gut metabolism on the bioavailability of the major phytoestrogens.
  • the permeability data produced as described in Example 1 and the regional difference in the metabolism of Biochanin A and Formononetm published by Jia et al. (Jia, Chen et al. 2004) are used to estimate regional bioavailability.
  • Jia et al. Jia, Chen et al. 2004
  • Figure 4 The estimated bioavailability of formononetm is five times higher when it is administered directly to colon as compared to that of oral. Similar observations are expected for biochanin A.
  • the objectives of this example are to evaluate the metabolism of absorbable components of Red clover extracts.
  • Human liver microsomes, intestinal microsomes, and hepatocytes of human female origin were purchased from XenoTech. All chemicals were purchased from Sigma-Aldrich. Isoflavones (biochanin A, daidzein, equol, formononetin, and genistein) were first dissolved in DMSO and then mixed according to a randomized table, consisting of 60 samples. The final DMSO in buffer or media was kept at 0.1%. Protocols supplied by Xenotech Inc., the supplier, were used for glucuronidation with microsomal incubation, and hepatocyte incubation. Samples were analyzed using LC/MS.
  • FIG. 5 shows that metabolism of the mixtures by human intestinal microsomes: biochanin A (5.41E-4 ml/min/mg protein) > genistein (4.28E-4 ml/min/mg protein) > equol (1.07E-
  • Figure 6 shows the rate of metabolism of the mixtures by human hepatocytes.
  • the rates are: equol (1.21E-5 ml/min/million cells) > biochanin A (8.88E-6 ml/min/million cells) > genistein (5.14E-6 ml/min/million cells) > daidzein (4.07E-6 ml/min/million cells) > formononetin (3.45E-6 ml/min/million cells).
  • TNF fibroblast growth factor
  • FGF fibroblast growth factor
  • calcitonin fibroblast growth factor
  • Estrogen is known to enhance the growth and differentiation of osteoblasts (bone growth), while parathyroid hormone to the growth and differentiation of osteoclasts (bone resorption).
  • Osteoblasts stimulated by estrogen can produce osteoproteoglygan (OPG) to regulate the differentiation of osteoclasts (Eriksen 2010).
  • Isoflavones are plant materials with estrogen-like structure and activity. Numerous animal/human studies using plant extracts, particularly red clover extracts, showed that isoflavones have beneficial effects on the condition of osteoporosis (Setchell and Lydeking-Olsen 2003; Nelson, Vesco et al. 2006). In order to understand the effect of red clover extract on bone growth, individual isoflavones were purified and test for their effects in in vitro studies.
  • Isoflavones like Formononetin, Biochanin A, Daidzein, and Genistein are able to enhance differentiation and mineralization of osteoblast cell lines in vitro (Sugimoto and Yamaguchi 2000; Sugimoto and Yamaguchi 2000; Chen, Garner et al. 2003; Wende, Krenn et al. 2004; Lee and Choi 2005; Dong, Zhao et al. 2006; Ji, Zhao et al. 2006).
  • the effective concentration varied among studies, even for the same isoflavones.
  • the major drawback of these published results is that the culture media used contained estrogen or substances with estrogen activity.
  • ALP/XTT and ACP/XTT ratios are used to quantify osteoblast and osteoclast activities.
  • the upper panel of Figure 8 shows a plot of the normalized ACP vs. ALP of the 121 combinations. The lower panel is the activity of pure isoflavone measured at 1 ⁇ .
  • the strategy is divide the data presented in Figure 8 into four quadrants: Ql, Q2, Q3 and Q4. The division is achieved using the average values of ACP and ACP ratios (dotted lines of Figure 8).
  • Qi has data of 29 mixtures. These mixtures have the highest ALP and minimum ACP values.
  • the optimal mixture(s) is present in this quadrant.
  • Q 4 has data of 23 mixtures. These mixtures have the least favorable for bone formation and resorption.
  • Q 2 has 37 mixtures. These mixtures, like that of Qi, have the highest bone forming activity, but they also least activity against bone resorption.
  • Q 3 has 32 mixtures.
  • Equol have been identified as active moieties.
  • the ultimate goal of this study is to search for optimal combination(s), which is most effective in preventing/treating osteoporosis. All possible combinations were studied in order to cover potential interactions induced by each isoflavone (Figure 7).
  • the optimal candidates should possess the highest osteoblast production (highest ALP XTT ratios) and the lowest osteoclast activity (lowest ACP/XTT ratios).
  • Figure 8 is constructed to facilitate the identification of the optimal candidates. The figure is divided into four quadrants. Candidates in Qi have the highest ALP and lowest ACP ratios, suggesting that the optimal candidates are present in this quadrant. The most ineffective candidates are found in Q 4 where candidates have the lowest osteoblast activity and highest osteoclast counts. Q 2 candidates have the highest osteoblast and osteoclast activity, not ideal as optimal candidates. Q 3 contains candidates that are most effective for osteoclast inhibition, but these candidates have the lowest osteoblast forming ability.
  • Genistein was identified to be the most potent among the five isoflavones in bone formation ( Figure 8, lower panel). The activity for bone formation was not significantly different among the rest of the four isoflavones, although equol has a higher mean value. Data presented on Figure 8 show that candidates in Qi have a higher potency than genistein alone, suggesting synergistic interactions. [0085] A detailed analysis of the distribution of individual isoflavones in the combinations
  • Genistein is the most potent compound in its pure form, it is included in the mixture of combinations.
  • Example 6 The objective of this example is to design dosage forms for Red clover extracts that are highly soluble and absorbable; and promote conversion to active metabolites in the lower intestinal tract.
  • An immediate release dosage form was designed to enhance the solubility of the two major aglycones: biochanin A and formononetin of Red clover extract. This is accomplished by incubating a 1 : 1 ratio of Red clover extract and a-cyclodextrin in methanol.
  • the Red clover extract used for this example contains >40% of total phytoestrogens.
  • the ratio of Formononetin and Biochanin A is 4: 1.
  • the resulting complex was dried at room temperature.
  • An immediate release tablet is prepared with typical excipients like microcrystalline cellulose, disintegrants and lubricants. A tablet prepared using this method releases 100% of its actives: Biochanin A and Formononetin within 60 minutes using a typical USP dissolution test.
  • a sustained release dosage form was designed to solubilize the major components of a Red clover extract: Biochanin A and Formononetin; and to promote metabolic conversion to active metabolites in the lower portion of the intestine.
  • a sustained release tablet contains two parts of Kollidon and one part of Red clover extract.
  • a typical dissolution test shows that the release of Formononetin over 4 hours is 80% and Biochanin A 70%.
  • a combination of immediate release and sustained release tablet has been designed to achieve optimal solubility, absorption and conversion to active metabolites.
  • about 50% of the actives in the combination tablet was release within 30 to 60 minutes and about 70% of the total dose will be released in 4 hours.
  • the purpose of this example is to establish optimal ratios of the isoflavones so that a product can be designed to produce maximal effect for preventing/treating osteoporosis.
  • the preferred combinations consist primarily of daidzein, equol, small amount of Biochanin A and Genistein. Dose ratio may not be an important issue, however, Formononetin should be avoided.
  • 40 % Daidzein, 40 % Equol and 20 % Genistein generate high osteoblast (ALP/XTT: 0.67 ⁇ 0.03 and 0.7 ⁇ 0.02) and low osteoclast (ACP/XTT: 0.66 ⁇ 0.07 and 0.67 ⁇ 0.06) values.
  • Biochanin A Daidzein, Genistein and Equol. Since this combination does not produce Equol in a high proportion and since two third of the population does not produce equol, an ideal produce is a combination of these four phytoestrogens.
  • the source of these compounds could be synthetic or purified from natural sources.

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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

Selon l'invention, à l'aide d'une modélisation par ordinateur et d'une analyse entropique (la technologie de plateforme pharmaceutique), diverses compositions pharmaceutiques issues d'extraits de trèfle des prés (Trifolium pratense) comprenant des isoflavones ont été développées. Lesdites compositions comprennent une formulation à libération immédiate comprenant de l'alpha-cyclodextrine, une formulation à libération prolongée qui comprend de la polyvinylpyrrolidone comme excipient et une formulation combinée comprenant la composition à libération immédiate susmentionnée et la composition à libération prolongée susmentionnée. Les isoflavones intéressantes comprennent la biochanine A, la formononétine, la daidzéine, l'équol et la génistéine. Les compositions pharmaceutiques ainsi obtenues sont utiles dans le traitement et la prévention de l'ostéoporose.
PCT/IB2012/055277 2010-02-15 2012-10-02 Développement d'un produit de phytoestrogène de trèfle des prés pour la prévention ou le traitement de l'ostéoporose WO2013050930A1 (fr)

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US14/242,981 US9333192B2 (en) 2010-02-15 2014-04-02 Phytoestrogen product of red clover and pharmaceutical uses thereof
US15/149,183 US10307451B2 (en) 2010-02-15 2016-05-09 Phytoestrogen product of red clover and pharmaceutical uses thereof
US16/376,044 US11123389B2 (en) 2010-02-15 2019-04-05 Phytoestrogen product of red clover and pharmaceutical uses thereof
US17/479,139 US20220000955A1 (en) 2010-02-15 2021-09-20 Phytoestrogen product of red clover and pharmaceutical uses thereof
US18/232,551 US20230381260A1 (en) 2010-02-15 2023-08-10 Phytoestrogen product of red clover and pharmaceutical uses thereof

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US201161542253P 2011-10-02 2011-10-02
US61/542,253 2011-10-02

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US14/069,740 Continuation US20140057005A1 (en) 2010-02-15 2013-11-01 Development of a pytoestrogen product for the prevention or treatment of osteoporosis using red clover
US14/069,740 Continuation-In-Part US20140057005A1 (en) 2010-02-15 2013-11-01 Development of a pytoestrogen product for the prevention or treatment of osteoporosis using red clover

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