The Steady-State Serum Concentration of Genistein Aglycone Is Affected by Formulation: A Bioequivalence Study of Bone Products

An FDA-regulated, prescription medical food (Fosteum; 27 mg natural genistein, 200 IU cholecalciferol, 20 mg citrated zinc bisglycinate (4 mg elemental zinc) per capsule) and an over-the-counter (OTC) supplement (Citracal Plus Bone Density Builder; 27 mg synthetic genistein, 600 mg elemental calcium (calcium citrate), 400 IU vitamin D3, 50 mg magnesium, 7.5 mg zinc, 1 mg copper, 75 μg molybdenum, 250 μg boron per two tablets) were compared to a clinically proven bone formulation (27 mg natural genistein, 400 IU cholecalciferol, 500 mg elemental calcium (calcium carbonate) per tablet; the Squadrito formulation) in an 8-day steady-state pharmacokinetic (PK) study of healthy postmenopausal women (n = 30) randomized to receive 54 mg of genistein per day. Trough serum samples were obtained before the final dose on the morning of the ninth day followed by sampling at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hrs. Total serum genistein, after β-glucuronidase/sulfatase digestion, was measured by time-resolved fluorometric assay. Maximal time (T max), concentration (C max), half-life (T 1/2), and area under the curve (AUC) were determined for genistein in each formulation. Fosteum and the Squadrito study formulation were equivalent for genistein T max (2 hrs), C max (0.7 μM), T 1/2 (18 ± 6.9 versus 21 ± 4.9 hrs), and AUC (9221 ± 413 versus 9818 ± 1370 ng·hr/mL). The OTC supplement's synthetically derived genistein, however, showed altered T max (6 hrs), C max (0.57 μM), T 1/2 (8.3 ± 1.9 hrs), and AUC (6474 ± 287 ng·hr/mL). Differences in uptake may be due to multiple ingredients in the OTC supplement which interfere with genistein absorption.


Introduction
Asian populations consume ∼25-50 mg of iso�avones daily with 10% consuming more than 100 mg per day [1]. Americans, on the contrary, consume ∼0.15-3 mg per day [2,3]. Much of the iso�avone consumed by Asian populations is in the form of aglycone from fermented soy product rather than glycoside forms consumed in mostly processed food in the USA. Many epidemiological studies of Asian women support an inverse relationship between iso�avone intake and bone loss as well as fracture rate. A large prospective study of 24,403 Chinese postmenopausal women, for example, demonstrated that ≥21 mg daily soy iso�avone consumption dramatically reduced subsequent fracture incidence over a 4.5-year period [4]. Most clinical trials, especially in the USA, are performed on extracted glucoside iso�avones from soy rather than aglycones forms which are found in fermented foods such as tofu, miso, and natto in the Asian diet. Recent clinical trials of 120 mg/day mixed glycoside iso�avones given to healthy postmenopausal women for 2 and 3 yrs, however, showed only modest effects on bone metabolism [5][6][7]. In a wellcontrolled dietary trial, natto, a fermented soy product containing 35 mg aglycone iso�avones enriched with 3.6 mg zinc given twice daily, showed statistically signi�cant increases bone formation and decreases bone resorption markers over natto alone [8]. To date, only genistein (aglycone), as a single entity, has been tested in well-controlled clinical trials for its effectiveness on building bone ( Figure 1) though studies have begun on S-equol, the intestinal bacterial conversion product of daidzein, for its effect on bone makers [9].
In a 12-month randomized, placebo-controlled clinical trial ( ), 54 mg of genistein administered daily showed equivalent increases in femoral neck and lumbar spine bone mineral density (BMD) (+3%) compared to the group given 1 mg of 17 -estradiol/0.5 mg noresthisterone acetate per day while the placebo group BMD declined [10]. All groups also received calcium carbonate (1000 mg) and cholecalciferol (800 IU) per day. is pilot study result was replicated in a larger ( ), long-term (24 months) study using the same amount of genistein compared to placebo [10]. A subcohort ( ) of this initial study extended to 36 months showed a similar rate of BMD increase (∼3%/yr to ∼9% over 3 yrs) at femoral neck and lumbar spine while the placebo BMD decreased by ∼10-11% at the femoral neck and lumbar spine. [11]. Markers of bone formation increased substantially while markers of bone resorption decreased signi�cantly for the genistein groups in these studies [10][11][12][13]. Bone quality assessed by quantitative ultrasound from the subcohort had statistically increased speed of sound, bone transmission time, and stiffness indices versus placebo [14]. In addition, a bone structural study in ovariectomized rats with established osteoporosis in which genistein was compared to alendronate, raloxifene, estradiol, and placebo showed superiority of genistein for all bone formation indices, fracture resistance, and histology (both trabecular and cortical bone) compared to all other therapies [15]. ese results have spawned the development of products for bone loss containing pure genistein but no comparative studies have been performed between these new commercial products.
Bioavailability comparisons can predict whether certain active ingredients in product formulations will show the same effect in clinical trials. It has been established that glycoside iso�avones are poorly absorbed in the intestine and that hydrolysis of the glycosidic bond by -glucosidases activates the aglycone for rapid absorption across the intestinal wall [16][17][18]. Most iso�avone bioavailability studies are performed in a food matrix using fermented or nonfermented products. Pure genistein and its glucoside, genistin, have been compared for uptake and the appearance in plasma as well as excretion of phase II metabolites in urine of healthy young women aer multiple doses [19]. is study showed that there were differences in total genistein max and AUC as well as several genistein metabolites. e addition of puri�ed components in combination with genistein or genistin is not well studied. One recent study showed that the continuous administration of fructooligosaccharide, a prebiotic, dramatically changed genistein and daidzein max and AUC obtained from a soy powder containing primarily genistin and daidzin [20]. With these data in mind, it is important to perform bioavailability comparisons for formulations containing puri�ed active ingredients and excipients which surround iso�avones before testing them clinically.
e FDA-regulated, prescription medical food [21], Fosteum, for the clinical dietary management of osteopenia and osteoporosis under physician supervision was formulated in collaboration with Squadrito and coworkers as previously described [11,12]. e OTC bone supplement, Citracal Plus Bone Density Builder, is based on a bone support formula already on the market (Citracal) and uses literature support to justify the addition of genistein [11,12]. Since the Squadrito formulation is the only mixture which contains genistein that has been clinically proven to build bone, the �rst step in determining whether Fosteum and/or Citracal Plus Bone Density Builder are bioequivalent is to test the bioavailability of genistein. erefore, the steady-state pharmacokinetics of 54 mg of genistein per day was compared for the Squadrito formulation to that of the Fosteum and Citracal Plus Bone Density Builder.  3.75 mg zinc (oxide salt), 0.5 mg copper (gluconate salt), 1 mg manganese (gluconate salt), 37.5 g molybdenum (amino acid chelate), 125 g sodium borate per tablet) (Citracal Plus Bone Density Builder, Bayer HealthCare LLC) genistein is synthetically produced. All mineral levels are expressed in elemental mass units. All three products purport genistein purity of ∼99%. e compositions and daily dosages of each formulation tested in the PK study are shown in Table 1. Products. In order to compare the purity of genistein and minor iso�avones in each product, HPLC analysis was performed [22]. Brie�y, samples were pulverized and then vortexed for 1 min in 2.5 mL of 1 : 1 : 1, hexane to methyl tert-butyl ether to methylene chloride extraction solvent. e samples T 1: Composition of three formulations for bone, a medical food indicated for osteopenia/osteoporosis, the Squadrito study formulation, and the OTC bone supplement. All minerals are given as elemental mass.

Constituent
Medical food were then vortexed gently for 15 min followed by a 10 min centrifugation at 3000 rpm to separate the aqueous and organic layers. e aqueous layer of each sample was then frozen at −80 ∘ C and the organic layer poured into a 10 mL glass conical screw cap tube where the sample was dried with nitrogen gas at 40 ∘ C. e dried extracts, as well as separate controls (genistein, daidzein, glycitein, and their glycosides), were reconstituted with 0.2 mL of 1 : 1, mobile phase buffer A (0.05% formic acid and 5 mM ammonium formate in distilled water) to mobile phase buffer B (0.05% formic acid and 5 mM ammonium formate in an 80 : 10 : 10 ratio, acetonitrile to methanol to distilled water). Samples were vigorously vortexed for 5 min and then centrifuged for 2 min at 1500 rpm to remove any insoluble material. e supernatants were removed and transferred to 0.25 mL polypropylene injection vials with caps for each chromatography run. Areas under curves were compared to standards to obtain purities.

Subjects.
Aer the Ethical Committee approved the study, a total of 30 participants were recruited among those reporting to the Center for Menopause in the Department of Obstetrical and Gynaecological Sciences at the University of Messina (Messina, Italy). All participants gave informed consent. All women were 50-65 yrs old, had been postmenopausal for at least 12 months at baseline and were in good general health. At the start of the study, a complete medical and family history was obtained. Exclusion criteria were the same of our previously published reports [12].
2.4. Diet. e intake of soy products, legumes, or other nutrient supplements which could contain iso�avones was prohibited for the 2 weeks before and during the study. e iso�avone intake before randomization as assessed by a foodfrequency questionnaire was 1 to 2 mg/day. is intake has been shown to be typical of Western populations.

Treatment
Protocol. e PK study was carried out at the laboratory of the Section of Pharmacology, Department of Clinical and Experimental Medicine and Pharmacology, University of Messina. Participants were randomly assigned to receive one of the following products for orally 8 days: 1 capsule twice daily (BID) of the medical food ( ); 1 tablet BID of the Squadrito study formulation ( ); or 2 tablets BID of the OTC supplement ( ). On the morning of the ninth day, trough serum samples (basal, 0 hr) were obtained following which subjects were given their �nal dose of study product. Blood samples were then collected using an intravenous cannula at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and �6 hrs aer �nal dosing. All other forms of calcium or vitamin D 3 were proscribed before and during the study.
e maximal plasma concentration ( max , nmol/L) and time to maximal plasma concentration ( max , hr) were obtained directly by the visual inspection of each subject's plasma concentration-time pro�le. e areas under the plasma concentration-time curve (AUC, ng⋅hr/mL) as well as half-life ( /2 , hrs) were determined for each formulation by using the PK Solutions 2.0 soware.

Plasma Genistein Levels.
Total genistein levels were measured in plasma samples by a time-resolved �uorometric assay following the manufacturer's instructions (TR-FIA test; Labmaster, Turku, Finland). Brie�y, 200 L of 100 mM acetate buffer (pH 5.0) containing 0.2 U/mL -glucuronidase and 2 U/mL sulfatase was added to 200 L serum. Samples were then incubated overnight at 37 ∘ C. Aer incubation, free genistein was extracted twice with 1.5 mL diethyl ether by mixing for 3 min. e water phase is frozen in dry ice-ethanol mixture, and the ether phase was transferred into a disposable glass tube. Aer thawing, the water phase was reextracted with ether, and the ether phases are combined and evaporated to dryness at 45 ∘ C water bath. en, 200 L assay buffer was added to each sample. A 20 L aliquot of this solution was used for time-resolved �uoroimmunoassay. e �uorescent signal was read using a Perkin-Elmer (Norwalk, CT) Victor 1420 multilabel counter.

Statistical Analysis.
Total plasma genistein concentrations were obtained at each time point in duplicate for each subject and PK analyses were performed. e primary variables of interest were max (the maximum observed concentration of total genistein), max (the elapsed time at which max was observed), /2 (the elapsed time at which genistein concentration was half of max ), and the imputed area under the curve (AUC) estimating the total body exposure to genistein over time. Area under the curve was computed by interpolating the concentrations of total genistein in the intervals between recordings using trapezoid calculations. Imputation was performed by using cubic spline estimation. Each of these variables was computed for each participant, and mean values and standard deviations were computed for the sample. Any value exhibiting 3 standard deviations ( 3) from the mean were removed from each analysis. A student's t-test was conducted for each measure to see if the observed difference in means was signi�cant. Descriptive statistics were presented for each of the primary outcome variables.

Genistein Content and Purity in Study
Products. e genistein in both the prescription medical food and the Squadrito study formulation are from natural sources, whereas the genistein in the OTC supplement is produced synthetically. e mineral content for all products was con�rmed by nutritional analysis (data not shown). HPLC analysis shows that the genistein molecules extracted from each formulation have equivalent purity with relative minor impurities of other iso�avone(s) amounting to <1% ( Figure  2). When the chromatograms are aligned and enlarged to compare the very small differences in genistein content between the two natural sources in the medical food ( Figure  2(a)) and the Squadrito study formulation (Figure 2(b)) to that of the synthetic source in the supplement ( Figure  2(c)), there are only small differences in the contaminating iso�avones of all products. Fosteum is contaminated by a small amount of glycitin, and daidzein, the Squadrito study formulation contains daidzin and genistin and the Citracal Plus Bone Builder supplement has a small amount of glycitin. No appreciable difference is seen in genistein purity in any of the three products with other aglycone impurities being less than 1%. Since the genistein levels are equivalent in all three products, a PK study of genistein should reveal any differences in uptake or excretion based on the surrounding vitamin, mineral, and excipient content in each formulation. us a bioavailability analysis can determine if genistein is bioequivalent in Fosteum and/or Citracal Plus Bone Density Builder compared to the Squadrito formulation which has been tested in clinical trials on bone.

Pharmacokinetic Comparison of Plasma Genistein in
Each Treatment Group. e PK pro�le for genistein obtained during the �rst 24 hours aer the last dose of each study product standardized to 54 mg per day aer 8 days intake is shown in Figure 3. Genistein from the medical food and the Squadrito study formulation were absorbed and excreted at approximately equal rates with statistically signi�cant higher concentrations at 1, 2, 5, and 12 hrs. e genistein contained in the supplement showed a much lower overall uptake by comparison. e PK analysis reinforced in this plot showed that the max of genistein for both the medical food and the Squadrito study formulation occurred 4 hrs earlier than that found in Citracal Plus Bone Density Builder supplement and the genistein max was also ∼23% higher at this point ( Table 2). e medical food and the Squadrito study formulation genistein peak serum concentrations are very similar with only nonstatistical differences in concentration at each time point over the course of the terminal half-lives for the products. is would represent the normal PK pro�le before a subsequent dose was consumed. e absorption and depletion pro�les of genistein from the medical food and the Squadrito study formulation exactly overlapped

Discussion
Health bene�ts of iso�avones are directly related to their bioavailability. Bioavailability is dependent upon an indi-vidual�s state of health, intestinal bacterial �ora, sex, age, food matrix in which iso�avones are consumed, the mix of iso�avones in products as well as host genetics [23]. e results of this PK analysis of three different bone formulations show genistein absorption is affected by speci�c ingredients formulated with the iso�avone which could have clinical implications on efficacy (Table 2; Figure 3). ere are a multitude of factors which could account for this difference. Normally, genistein is freely absorbed from the intestine and a large fraction is converted to 7 -O-glucuronide as it crosses the brush border and ultimately enters the portal vein [24]. Intestinal bacteria are known to in�uence glucuronidation and may also drive sulfonation [25,26]. Only a small percentage of the parent molecule remains as free genistein once it reaches the liver. Once in the liver, genistein undergoes additional biotransformation via CYP450-mediated BioMed Research International 5 T 2: e maximal plasma concentration ( max ), time to maximal plasma concentration ( max ), areas under curve (AUC) and half-life ( 1/2 ) aer steady administration of the medical food indicated for osteopenia/osteoporosis, the Squadrito study formulation, and the OTC bone supplement. hydroxylation [27] followed by glucuronidation and sulfation by UDP-glucuronosyl transferase and sulphotransferases, respectively [24]. A large majority of glucuronidated genistein undergoes efficient enterohepatic recirculation following biliary excretion. e preponderance of circulating genistein in serum has been found to be in the form of glucuronidate and sulfate conjugates [28].
Food, isolated nutrient molecules and binders, when coadministered with drugs, are known to affect their absorption, distribution in the body, metabolism in the lumen, liver and cells, and elimination [29]. is issue is so important that the FDA has issued guidance on oral administration of drugs with food, their bioavailability and need for bioequivalence studies to assure proper guidance for administration of therapeutic compounds [30]. Genistein is considered a class 2 compound with low solubility and high permeability by the FDA�s Biopharmaceutics Classi�cation System. ough there are no formal requirements for this type of analysis of medical foods or supplements, it is important that fasting and fed PK and bioequivalence studies be performed, especially since medical foods have a statutory requirement to be indicated for a speci�c disease and must be administered under the direction of a physician [21]. Indeed, a fasting and fed PK study of genistein has been performed on the medical food Fosteum indicated for osteopenia/osteoporosis suggesting a minor, nonstatistical effect of food on absorption [31]. ere is no published data on genistein bioavailability from the OTC Citracal Plus Bone Density Builder supplement. is steady-state PK study demonstrates that the medical food product for osteopenia/osteoporosis is equivalent to the clinically proven Squadrito study formulation for absorption and bioavailability of genistein, whereas the OTC supplement formulation dramatically and statistically affects the iso�avone absorption (Table 2; Figure 3).
Absorption of bioactive substances is in�uenced by several different factors such as the intestinal solubility and permeability [32]. Another factor that can affect absorption of bioactive molecules is viscosity induced by food additives, such as guar gum [33]. Citrate, an approved food additive, is also known to increase viscosity in the presence of collagen and �brous material [34] as well as change the water absorption pro�le in different parts of the small intestine [35]. It has been added to different oral rehydration formulations to modulate acidosis and glycemic index as a viscositypromoting agent [36][37][38]. Based on the above data, it is possible that normal dietary �ber in those randomized to the bone supplement group had increased gastrointestinal viscosity during the time of dosing which affected genistein uptake due to the dissociation of the citrate and calcium ions in the stomach. ough Fosteum contains citrated zinc bisglycinate, citrate along with glycine tightly coordinate zinc and is not ionized in the stomach. Preclinical studies have shown that the zinc from chelates is dissociated from the coordinating molecules on the lumen of the intestine [39]. Hence, the chance of the citrate portion of the chelate interacting with dietary �ber is minimal. Other mechanisms may also account of the lower level of genistein absorption from the supplement.
ATP-binding cassette (ABC) transport proteins are responsible, in part, for the transport of �avonoids, including iso�avones, into luminal intestinal cells for absorption [40]. Genistein speci�cally interacts with the ABCG2 receptor in a variety of cells including those in the intestinal lumen [41]. Calcium and magnesium ions are typically actively absorbed via transient receptor potential channel proteins (TRP) in the duodenum [42]. Vitamin D 3 is needed for calcium uptake through these channels while magnesium serves as a cofactor for ABC transport proteins in the uptake of �avonoid molecules. ere is no reported evidence that calcium, magnesium or other ions inhibit ABC receptors. Iso�avones, such as genistein, are also transferred from the intestine into the epithelial lumen by organic anion transport proteins (OATs) [43]. e OAT receptor family also serves to maintain anion balance throughout the body, including in the intestinal lumen [44] and is a subclass of a superfamily of proteins termed major facilitator superfamily (MFS) transporters [45,46]. Citrate is known to interact with both OAT receptors [44] as well as with members of the MFS called citrate-H+ symporter (CitA) [47] and Na+/citrate transporters [48]. Another OAT receptor, Mrp2, also known as canalicular multispeci�c organic anion transporter (cMOAT) and ABCC2 binds organic anions like citrate and gluconate [49]. Both ABCC2 and ABCG2 have extensive homology and exist together in the intestinal lumen having a broad range of nutrient transport capabilities. ese include the transport of organic anions, glucuronidated and sulfonated molecules, and a number of drugs [50]. ese receptors have been shown to have speci�c functional overlap in absorption of various molecules [51]. erefore, organic anions such as citrate, silicate, gluconate, and stearate present as counterions in the OTC Citracal Plus Bone Density Builder supplement formulation may directly compete with genistein for absorption on these receptors and transporters. is and the possibility that citrate increases viscosity, and hence slows gastric emptying, might explain the difference in uptake resulting in a lower max , lengthened max and decreased AUC compared to the medical food product and the Squadrito study formulation. e carbonate anion in the Squadrito study formulation is known to interact with the solute carrier family (SLC) of receptors [52], rather than ABC or OAT receptors. is may explain why calcium carbonate does not affect genistein absorption while the calcium citrate supplement formulation appears to do so.

Conclusion
e medical food for osteopenia/osteoporosis, Fosteum, and Squadrito study formulation tested for bone building in clinical trials are bioequivalent for absorption of genistein compared to that from bone supplement Citracal Plus Bone Density Builder which inhibits genistein uptake. Even with the 10% difference in AUC between the medical food and the Squadrito study formulation over the 96 hr period, one could expect similar genistein pharmacokinetic behavior from both products under usual conditions of use. e steady-state genistein concentration attained by dosing with the OTC Citracal Plus Bone Density Builder supplement, however, would presumably be signi�cantly lower compared to Fosteum and the Squadrito study formulation even over long periods of time. is difference could adversely affect overall efficacy on bone metabolism. Based on this evidence, care must be taken when combining bioactive substances like genistein with speci�c salts to prevent changes in viscosity or competition for receptors or transport proteins during intestinal absorption.