Repeated Oral Once Daily Intake of Increasing Doses of the Novel Synthetic Genistein Product Bonistein™ in Healthy Volunteers

• Abstract
• Introduction
• Materials and Methods
• Ethics
• Subjects
• Study medication
• Study design
• Assessment procedures
• Bonistein™ plasma concentrations
• Pharmacokinetic analysis
• Statistical analysis
• Results
• Discussion
• References

Abstract

Bonistein™ is a new product consisting of > 99.5 % synthetic genistein, an isoflavone with phyto-oestrogenic properties, which might be a safe and efficacious alternative for the prevention of post-menopausal bone loss to the traditional hormone replacement therapy. A randomised, open-labelled and sequential-group phase I study was performed to assess safety, tolerability and pharmacokinetic characteristics of oral administrations of Bonistein™. Thirty healthy volunteers received in three subsequent groups 30, 60 or 120 mg once daily for 14 days. For the pharmacokinetic profiles of Bonistein™, blood samples were taken on study Days 1 (after first dose) and 14 (steady state). Repeated intake of Bonistein™ was well tolerated. A total of 33 adverse events were reported, mainly of mild intensity. No relevant changes in clinical laboratory or vital signs were observed. The pharmacokinetic characteristics of Bonistein™ revealed comparable results for extent and rate of absorption on Days 1 and 14. Both AUC and C_max values of Bonistein™ increased in proportion with the dose.

Introduction

In elderly women oestrogens and their combinations with other hormones are used to treat conditions of inadequate oestrogen production, menopausal complaints and to prevent postmenopausal osteoporosis. The risk-benefit relation of the hormone replacement therapy (HRT) is presently under discussion. Used for decades, recent findings from clinical studies raised concerns about its safety with regard to an increased risk for certain cancers and cardiovascular events [1], [2]. As a consequence, increasingly more women and physicians are seeking for alternatives.

In this search for alternatives to the classical HRT, phytochemicals with oestrogenic activity received great attraction [3], [4]. Phyto-oestrogens induce biological responses in vertebrates that mimic or modulate the actions of endogenous oestrogens usually by binding to oestrogen receptors [3], [5]. The most chemically efficacious and structurally similar to 17β-oestradiol are the isoflavones and among them genistein. Genistein seems to be able to ease menopausal symptoms without creating oestrogen-related problems and may also be beneficial in preventing osteoporosis [6], [7], [8], [9]. A recently published review intended to elucidate the bone-sparing effects of dietary isoflavones revealed enough conclusive positive results from in vitro and preclinical animal research [10], [11]. In epidemiological surveys a positive correlation between the amount of dietary phyto-oestrogens and bone mineral density was found consistently. However, the results of prospective, controlled clinical trials are still conflicting.

DSM plans to investigate the effects of synthetic genistein on postmenopausal bone loss. Therefore, a new product named Bonistein™ consisting of 99.6 % synthetic, unconjugated genistein was developed and tested extensively in preclinical studies including in vitro and in vivo models for bone metabolism [18]. For the evaluation of safety, long-term toxicological studies (52 weeks) were conducted providing no-observed-adverse-effect levels (NOAELs) of 50 mg/kg body weight/day in rats and 150 mg/kg body weight/day in dogs [12; data on file].

In healthy volunteers oral single doses from 30 to 300 mg were safe and well tolerated [13]. The present study investigated safety and pharmacokinetics (PK) of Bonistein™ as response to repeated oral administration of 30, 60, or 120 mg in healthy volunteers.

Materials and Methods

Ethics

This study was conducted in accordance with the revised Declaration of Helsinki 2002, the German Drug Law and the ICH GCP at the Institute for Clinical Pharmacology Bobenheim GmbH (IKP GmbH), Grünstadt, Germany. The study protocol and the procedure for informed consent were approved by the competent ethics committee of the Chamber of Physicians in Rhineland-Palatinate, Germany. Each volunteer received full written and verbal information and gave written informed consent to participate in this trial prior to start of the study.

Subjects

A total of 30 healthy Caucasian volunteers of either sex were enrolled into this study. Age for inclusion was 18 to 45 years and a body mass index between 18 and 30 kg/m² was allowed (minimum body weight 50 kg). The term ”healthy” was defined as ”no clinically relevant deviations from normal”, assessed by medical examinations and medical history, a broad range of clinical laboratory tests and vital signs measurements. The demographic data of the participants are summarised in Table [1]. Only non-smokers and non-vegetarians were selected.

Subjects were excluded from study participation if they had a history of food allergies, specifically a hypersensitivity to genistein, isoflavones, soy or peanuts. Other exclusion criteria included: history of diseases, gastrointestinal surgery or any other condition that was likely to interfere with the absorption or excretion of Bonistein™, genistein screening value above 50 ng/mL, subjects on a weight reduction program or medically supervised diet or unexplained weight loss/gain of more than 5 kg in the month prior to the study. Subjects were not eligible for this study if they had donated blood, had taken enzymatic inductors or inhibitors, any drugs, OTC products or dietary supplements in the 3 months prior to the study. All volunteers had to agree in writing to practice reliable contraception for at least 7 days or one hormonal cycle prior to and after the study as well as during the study itself. Subjects were excluded during the study if they took any of the aforementioned products in addition to Bonistein™ (drugs were only allowed for the treatment of adverse events), failed to comply with the study protocol or were tested positive for drugs or alcohol.

Study medication

Bonistein™ was produced at DSM Nutritional Products in Sisseln, Switzerland. Hard-gelatine capsules containing 10 mg or 50 mg of Bonistein™ were blister-packed and analysed for content uniformity by SwissCo Services AG, Sissach, Switzerland. All procedures were compliant with GMP and European Pharmacopoeia requirements.

Study design

A prospective multiple, escalating dose study was conducted according to an open-label, sequential group design. The subjects were randomly allocated to each of the 3 dose groups (3 × N = 10) receiving 30, 60 or 120 mg of Bonistein™ once daily for 14 days. For safety reasons the study started with the lowest dose and increase was not performed before all safety parameters of the previous dose were available and evaluated by a physician.

Assessment procedures

Each volunteer was confined to the study site from 12 h before first intake until 26 h after last administration of Bonistein™. Study medication was swallowed with 400 mL tap water after an overnight fast. The volunteers remained fasting for up to 4 h post administration. Thereafter they had a standardised and genistein/isoflavones-free diet. The time of capsules intake was defined as T₀ ¹ and T₀ ¹⁴, respectively. On study Days 1 (after first dose) and 14 (steady state) 24-hours PK profiles were measured. Blood was withdrawn for determination of genistein plasma concentrations pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16 and 24 hours after T₀. In addition on study Days 12, 13 and 14 trough values were measured to confirm that the steady state was reached. Blood samples were immediately centrifuged and the resulting plasma was stored at -80 °C pending analysis.

Tolerability of Bonistein™ was assessed by adverse event inquiry using a set of standardised questions. Safety assessments included clinical laboratory tests (haematology, clinical chemistry, coagulation, hormones, urine analysis) and vital signs measurements (blood pressure, ECG).

Bonistein™ plasma concentrations

Plasma samples were assayed for genistein by means of a liquid chromatography-mass spectrometry method (LC-MS). The analytes were extracted with ethyl acetate, the organic phase was evaporated and the residue was redissolved in the mobile phase. For the determination of the total genistein content, the plasma was treated with β-glucuronidase from Helix promatia to hydrolyse conjugated genistein (glucuronides and sulphates). HPLC separation was done with a gradient system on a C₁₈ phase and the mass spectrometric detection run after electrospray ionisation in the negative mode under SIM conditions. Analytical determinations of genistein were performed according to GLP standards.

Pharmacokinetic analysis

Non-compartmental PK analysis for total genistein was done using WinNonlin^® Professional software (version 3.2, Pharsight Co., Mountain View, CA, US). The maximum plasma concentration (C_max) and the time to reach C_max (T_max) were read directly from the observed concentration-time profiles. Non-linear regression of a single (Ce^-Kel × ^t) exponential function in the terminal phase of the log-transformed plasma concentration versus time profile, where Kel is the terminal rate constant, was performed using the method of non-linear least squares. The apparent terminal elimination half-life (t_œ,z) was then calculated as (ln2)/Kel = 0.693/Kel. The area under the plasma concentration versus time curve was calculated according to the linear trapezoidal rule (AUC_(0-inf.) and AUC_{(0- τ)}). Concentrations reported as ‘below the lower limit of quantification’ were set to zero before calculating the AUC_{(0- τ)}. The area under the curve from 0 h to infinity (AUC_(0-inf.)) was extrapolated as follows: AUC_(0-inf.) = AUC_{(0- τ)} + C(t)/Kel, where C(t) is the last quantifiable concentration above the lower limit of quantification. The accumulation factor (R) was assessed by AUC _{(0- τ)} study Day 14/AUC _(0-inf.) study Day 1. To facilitate estimation of dose-proportionality C_max, AUC_{(0- τ)} and AUC_(0-inf.) (after first dose and steady state) were dose-normalised by multiplying the original characteristics with the subjects body weight and dividing them by the corresponding dose. They are marked by the postfix ”norm”.

Statistical analysis

The PK analysis was performed on those randomised subjects who received the study drug and completed the study per protocol; safety analysis was performed on all the randomised subjects (intention-to-treat analysis). To detect differences an ANOVA model was applied including terms for treatment, gender, body weight and intersubject residual error terms. For T_max the Kruskal-Wallis test for unpaired data was used. A log-normal distribution was assumed for AUC_(0-inf.) and C_max. To test for dose proportionality AUC_(0-inf.) and C_max were subjected to one-way ANOVA of the natural logarithm transformed AUC_(0-inf.)/dose (AUC_{(0-inf.,norm)}) and C_max/dose (C_max,norm) data. In addition these logarithmically transformed data were subjected to linear regression of the dose-normalised response variables versus dose and testing of slopes for being equal to zero. Statistical analyses were performed using SAS^® software version 8.0 (Statistical Analysis System, SAS Institute, North Carolina, USA).

Results

Safety and tolerability of Bonistein™ were good. Except of one subject, who dropped out for private reasons, all volunteers completed the study per protocol. No serious adverse events occurred. Sixteen subjects experienced a total of 33 adverse events (AEs). Six subjects in the 30 mg group reported 13 AEs, four subjects exposed to 60 mg experienced five AEs, whereas six subjects treated with 120 mg Bonistein™ had 15 AEs. All AEs were of mild intensity except for two which were moderate.

Fourteen AEs were judged as likely Bonistein™-related: 4 × headache, 2 × feeling of swollen face, 3 × increased pancreas lipase, 4 × increased pancreas amylase and 1 × feeling of peripheral oedema. The following AEs were documented and judged as not related to the treatment: 1 × tachycardia, 4 × headache, 2 × decreased haemoglobin, 1 × tiredness/asthenia, 1 × increased bilirubin, 1 × pain in both knees, 1 × increased iron, 1 × vivid dreams, 1 × constipation, 1 × increased creatinine kinase, 2 × increased thyroid stimulating hormone, 2 × decreased luteinising hormone and 2 × decreased follicle stimulating hormone. The AEs were equally distributed between male and female volunteers.

Vital signs and ECGs were not relevantly influenced by the different doses. No treatment-emergent findings were observed during the physical examinations. There were seven ongoing AEs in five subjects at the post-study examination: lipase (2 × ) and amylase (2 × ) above NR, blood iron, luteinising hormone and low follicle-stimulation hormone below NR.

Plasma concentration-time profiles for Bonistein™ together with the respective mean curves for each dosage group on study Day 1 (after first dose) and 14 (steady state), are given in Fig. [1] a and [1] b. A group summary of the PK characteristics is given in Table [2] a and [2] b.

Genistein plasma concentrations started to increase within 0.5 hours after ingestion of the hard-gelatine capsules in all subjects independently from dose and study day. Concentrations ascended in a mono-exponential manner up to C_max, which was reached after 5 to 6 hours (T_max). Average maximum plasma concentrations (C_max) for the different Bonistein™ dose levels increased dose-dependently (Day 1 : 297, 802 and 1015 ng/mL, Day 14 : 334, 787 and 1119 ng/mL), showing a trend (not statistically significant) to level off at the highest dose (either on day 1 and 14). In the later phases, when excretion and distribution dominated, genistein blood levels decreased multi-phasically. In the 60 mg and 120 mg groups the declining part of the curves presented a second peak occurring approximately 10 hours after T₀ ¹⁴. In the terminal elimination phase the geometric means of half-lives (t_1/2) were calculated to be 8.1, 6.7, and 9.0 hours on Day 1 and did not show remarkable changes with increasing dose or during steady state (Day 14 : 7.4, 8.3 and 9.6 hours).

The extent of systemic exposure (geometric means of AUC_{(0- τ)}) were 3244, 8273 and 13 110 ng·h/mL on study Day 1 and 3546, 8308 and 14513 ng·h/mL on study Day 14. Mean trough values (± SD) on study Days 12, 13 and 14, reflecting the systemic equilibrium of genistein at steady state, were 62.1 (± 48.4) ng/mL, 71.6 (± 59.0) ng/mL and 66.6 (± 57.7) ng/mL in the 30 mg group, 222.6 (± 197.0) ng/mL, 186.0 (± 143.0) ng/mL and 183.0 (± 156.5) ng/mL in the 60 mg group and 224.9 ng/mL (± 109.6), 383.1 ng/mL (± 236.7) and 355.0 ng/mL (± 337.8) in the 120 mg group. The calculated accumulation factors were close to 1 in all dosage groups. To facilitate the estimation of dose-proportionality between dose groups and over time, the dose-normalised values are given in Table [3]. Except for a weak statistically significant difference in the C_max values on day 1 for the 60 mg group compared to the 120 mg group, no differences were detected. The ANOVA testing of the gender effect together with the main variables (treatment and study day), carried out using logarithmically (ln) transformed and dose-normalised data, was weakly significant for C_max,norm (p = 0.045). This might suggest that women exert slightly higher maximum plasma concentrations than men, albeit the overall systemic exposure (AUC) is not statistically different.

The results of the linear regression of AUC_(0-inf.) for the across-treatment comparison [slope = dose, logarithmic (ln) data were used] showed high correlation coefficients (0.894 - 0.946) and no significant difference from zero (p > 0.05). This indicates not only dose proportionality for Bonistein™ doses up to 120 mg, but serves also as an additional indication that neither accumulation nor relevant re-absorption occurs. Overall variability as demonstrated by the coefficient of variations (CVs) superimposed on the arithmetic means profiles appears to be moderate to high, increasing in parallel with dose escalation. Neither age or body weight influenced rate and extent of absorption assessed by repeated ANOVA.

Discussion

Bonistein™ consists of nearly 100 % synthetic, unconjugated genistein and is, due to its broad toxicological testing and GMP production process, allowed for human use. After the preclinical development, first clinical studies are intended to investigate its tolerability and pharmacokinetics (PK). In this present study healthy human subjects received three ascending dose levels of Bonistein™ (30, 60 and 120 mg). PK were compared after single dose application to steady state; overall exposure was in lowest dose group 420 mg/14 days (30 mg/d) and in the highest 1680 mg/14 days (120 mg/d).

Volunteers took their trial supplies under fasting conditions and remained fasting up to 4 hours to minimise food interaction. A parallel-group study design was chosen instead of a cross-over design, as no reliable data on the half-life or tolerance of synthetic genistein were available at the time of designing this study and in order to increase the number of subjects who could be assessed for safety. Subjects of either sex were selected primarily for safety reasons; secondarily, to gain orienting PK data in men, because men might also be a target population which could benefit from the presumed bone-sparring effects of genistein.

The lag time of zero across all dosage groups reflects the immediate dissolution of the hard-gelatine trial supplies (15 minutes according to specifications of the European Pharmacopoeia) and, subsequently, rapid intestinal absorption of Bonistein™. The plasma concentration-time profiles showed a smooth increase in the invasion-dominated phase, and also a moderate decrease during elimination and late distribution phase. A ”hill-shaped” curve emerged. It is known that genistein undergoes enterohepatic circulation, which contributes to the prolonged elimination resulting in a slight plateau phase between 6 and 16 hours and a second peak of the concentration-time curve. Elimination half-life was assessed at approximately 8 hours. Bonistein™ showed to be dose proportional up to 120 mg/d, both after single dose and steady state.

Of all reported adverse events (AE) 46 % were judged as Bonistein™-related (AE definition and reporting followed the ICH GCP guideline E6 [14]). None of the AEs were serious or unexpected (unexpected means, published as AE in a human intervention trial with a comparable, genistein-containing product). Most of the AEs were of mild intensity, except of two, which represents 6 %. The most common AE was headache and slight fluctuation of the pancreas enzymes lipase and amylase. Both are well-know effects of highly concentrated, purified isoflavones and were reported in other clinical trials [15], [16]. Nearly all other AEs were unspecific and might reflect the usual observation of complaints in clinical studies with healthy volunteers and (for laboratory and vital signs parameters) the scattering around the normal ranges. The incidence of AEs was not correlated to systemic exposure of Bonistein™ or sex of the volunteers.

Three subjects reported AEs of special interest during Bonistein™ intake: vivid dreams, swollen face and peripheral oedema. These symptoms could not be verified by the investigator and were partly assigned as ”feelings of”. Therefore, it remained unclear whether these AEs had to be attributed to CNS sensations, allergy or skin. Comparable AEs have been occasionally reported in other isoflavone intervention trials (mostly reported as unrelated to the treatment) [15], [16], [17].

In conclusion, Bonistein™ up to 120 mg/d administered over two weeks to healthy volunteers is well tolerated. Systemic exposure is proportional to the given dose and did not accumulate significantly. Bonistein™ was safe and well tolerated.

References

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  Search in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 18 Ulmann U, Bendik I, Flühmann B. Bonistein™ (synthetic genistein), a food component in development for a bone-health nutraceutical.  J Physiol Pharmacol. 2005;  56 (Suppl1) 79-96
  PubMedSearch in Google Scholar

Uwe Ullmann, MD

DSM Nutritional Products

R&D Human Nutrition & Health

4303 Kaiseraugst

Switzerland

Phone: +41-61-68-88798

Fax: +41-61-68-89684

Email: uwe.ullmann@dsm.com

References

• 1 Rossouw J E, Anderson G L, Prentice R L, LaCroix A Z, Kooperberg C, Stefanick M L, Jackson R D, Beresford S A, Howard B V, Johnson K C, Kotchen J M, Ockene J (Writing Group for the Women's Health Initiative Investigators). Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomized controlled trial.  JAMA. 2002;  288 321-33
  CrossrefPubMedSearch in Google Scholar
• 2 Beral V (Million Women Study Collaborators). Breast cancer and hormone-replacement therapy in the Million Women Study.  Lancet. 2003;  362 19-427
  PubMedSearch in Google Scholar
• 3 Adlercreutz H, Mazur W. Phyto-oestrogens and Western diseases.  Ann Med. 1997;  29 95-120
  CrossrefPubMedSearch in Google Scholar
• 4 Andersson J J, Anthony M S, Cline J M, Washburn S A, Garner S C. Health potential of soy isoflavones for menopausal women.  J Public Health Nutr. 1999;  2 489-504
  PubMedSearch in Google Scholar
• 5 Kuiper C G, Lemmen J G, Carlsson B, Corton J C, Safe S H, van der Saag P T, van der Burg B, Gustafsson J A. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.  Endocrinology. 1998;  139 4252-63
  CrossrefPubMedSearch in Google Scholar
• 6 Suetsugi M, Su L, Karlsberg K, Yuan Y C, Chen S. Flavone and isoflavone phytoestrogens are agonists of estrogen-related receptors.  Mol Cancer Res. 2003;  13 981-91
  PubMedSearch in Google Scholar
• 7 Morabito N, Crisafulli A, Vergara C, Gaudio A, Lasco A, Frisina N, D’Anna R, Corrado F, Pizzoleo M A, Cincotta M, Altavilla D, Lentile R, Squadrito F. Effects of genistein and hormone-replacement therapy on bone loss in early post-menopausal women: a randomized double-blind placebo-controlled study.  J Bone Mineral Res. 20 002;  10 1904-10
  PubMedSearch in Google Scholar
• 8 Albertazzi P, Pansini F, Bonaccorsi G, Zanotti L, Forini E, De Aloysio D. The effect of dietary soy supplementation on hot flushes.  Obstet Gynecol. 1998;  91 6-11
  CrossrefPubMedSearch in Google Scholar
• 9 Kreijkamp-Kaspers S, Kok L, Grobbee D E, de Haan E H, Aleman A, Lampe J W, van der Schouw Y T. Effect of soy protein containing isoflavones on cognitive function, bone mineral density, and plasma lipids in postmenopausal women: a randomised controlled trial.  JAMA. 2004;  292 65-74
  CrossrefPubMedSearch in Google Scholar
• 10 Setchell K DR, Lydeking-Olsen E. Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studies.  Am J Clin Nutr. 2003;  78 593S-609S
  PubMedSearch in Google Scholar
• 11 Messina M, Ho S, Alekel D L. Skeletal benefits of soy isoflavones: a review of the clinical trial and epidemiologic data.  Curr Opin Clin Nutr Metab Care. 2004;  7 649-58
  PubMedSearch in Google Scholar
• 12 McClain R M, Wolz E, Davidovich A, Pfannkuch F, Bausch J. Subchronic and chronic safety studies with genistein in dogs. Food and Chemical Toxicology in press
  Search in Google Scholar
• 13 Ullmann U, Metzner J, Frank T, Cohn W, Riegger C. Safety, tolerability and pharmacokinetics of single ascending doses of synthetic Genistein (Bonistein™) in healthy volunteers.  Advances Therap. 2005;  22 65-78
  PubMedSearch in Google Scholar
• 14 ICH 1996 (E6). Guideline for good clinical practice. CPMP/ICH/135/95. 
• 15 Bloedon L T, Jeffcoat A R, Lopaczynski W, Schell M J, Black T M, Dix K J, Thomas B F, Albright C, Busby M G, Crowell J A, Zeisel S H. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women.  Am J Clin Nutr. 2002;  76 1126-1137
  PubMedSearch in Google Scholar
• 16 Busby M G, Jeffcoat A R, Bloedon L T, Koch M A, Black T, Dix K J, Heizer W D, Thomas B F, Hill J M, Crowell J A, Zeisel S H. Clinical characteristics and pharmacokinetics of purified soy isoflavones: single-dose administration to healthy men.  Am J Clin Nutr. 2002;  75 126-36
  PubMedSearch in Google Scholar
• 17 Lydeking-Olsen E, Beck-Jensen J E, Setchell K D, Holm-Jensen T. Soymilk or progesterone for prevention of bone loss - a 2 year randomized, placebo-controlled trial.  Eur J Nutr. 2004;  43 246-57
  PubMedSearch in Google Scholar
• 18 Ulmann U, Bendik I, Flühmann B. Bonistein™ (synthetic genistein), a food component in development for a bone-health nutraceutical.  J Physiol Pharmacol. 2005;  56 (Suppl1) 79-96
  PubMedSearch in Google Scholar

Uwe Ullmann, MD

DSM Nutritional Products

R&D Human Nutrition & Health

4303 Kaiseraugst

Switzerland

Phone: +41-61-68-88798

Fax: +41-61-68-89684

Email: uwe.ullmann@dsm.com