Evaluation of Levothyroxine Bioavailability after Oral ...

DrugRes/2015-04-0999/23.6.2015/MPS

Original Article

Evaluation of Levothyroxine Bioavailability after Oral Administration of a Fixed Combination of Soy Isoflavones in Post-menopausal Female Volunteers

Authors

S. Persiani1, F. Sala1, C. Manzotti1, M. Colovic1, M. Zangarini1, Y. Donazzolo2, B. Barbetta1, C. Vitalini1, G. Giacovelli1, C. Benvenuti3, L. C. Rovati1

Affiliations

1

Key words ▶ interactions ● ▶ food supplement ● ▶ pharmacokinetics ● ▶ clinical study ● ▶ climacteric syndrome ●

Abstract

Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1555784 Published online: 2015 Drug Res © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Persiani, PhD, Director Translational Sciences and Pharmacokinetics Rottapharm Biotech S.r.l Via Valosa di Sopra 9 20900 Monza Italy Tel.: + 39/039/7390 396 Fax: + 39/039/7390 627 [email protected]

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Background: Post-menopausal women under treatment with levothyroxine for their medical conditions may take concomitantly dietary supplements containing soy isoflavones in combination to treat their post-menopausal symptoms. The aim of this study was to investigate the effect of a fixed combination of soy isoflavones on the oral bioavailability of levothyroxine in postmenopausal female volunteers. Methods: 12 healthy post-menopausal female, who were on stable oral levothyroxine as replacement/supplementation therapy for hypothyroidism, received a single recommended oral dose of a food supplement containing 60 mg of soy isoflavones ( > 19 % genistin and daidzin) concomitantly with (test) and 6 h later (reference) the administration of levothyroxine in a randomized, open label, crossover fashion. Plasma

Introduction

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Menopause is associated with estrogen deficiency and its related metabolic effects such as accelerated bone loss and atherosclerosis. Estrogen replacement therapy has been used for nearly 60 years to treat menopause-related conditions [1]. However, following the publication of the Women’s Health Initiative findings suggesting that the overall risks outweigh the benefits, the interest in alternative therapies for menopause and related symptoms was increased [2]. Soy-derived products with comparable benefits to estrogens but fewer side effects have been proposed [3, 4]. Their properties are due to the phytoestrogen components and include protection against coronary heart disease, breast cancer, osteoporosis and alleviation of hot flushes [5]. The most common forms of phytoestrogens are the soy isoflavones genistein and daidzein that

concentrations of levothyroxine and soy isoflavones (daidzin, daidzein, genistin, genistein, S-equol) were determined by LC-MS/MS. Pharmacokinetic (PK) parameters were determined by non-compartmental analysis. No effect of soy isoflavones was assumed if the 90 % confidence intervals (CIs) for the estimated ratio test/reference was included in the acceptance limits 0.80– 1.25 for PK parameters Cmax and AUCt. Results: The test/reference ratios Cmax and AUCt of levothyroxine were very close to unity (1.02 and 0.99, respectively) and the corresponding 90 % CIs (0.99–1.04 and 0.88–1.12, respectively) fell entirely within the acceptance bioequivalence limits. Conclusion: The combination of soy isoflavones used in the present investigation does not affect the rate and extent of levothyroxine absorption when administered concomitantly in post-menopausal women.

have both estrogenic and antiestrogenic effects, depending on the target tissue [6]. Based on their pharmacological profile, soy isoflavones appear as a possible alternative to ameliorate some climacteric symptoms (e. g., hot flashes) and to improve lipoprotein levels [7, 8], although clinical trials provided controversial results [9]. Since it has estimated that as many as 10 % of postmenopausal women in the United States may suffer from hypothyroidism [10] and that worldwide prevalence of subclinical hypothyroidism ranging from 1 to 10 % with the highest age- and sex-specific rates in women older than 60 years, approaching 20 % in some reports [5], it is possible that post-menopausal women under treatment with levothyroxine, take concomitantly dietary supplements containing soy isoflavones to treat their post-menopausal symptoms. It has been shown that food supplements containing soy might reduce the oral absorption of

Persiani S et al. Isoflavones Effect on Levothyroxine PK … Drug Res

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received 20.04.2015 accepted 11.06.2015

Rottapharm Biotech S.r.l., Monza, Italy Eurofins|OPTIMED Clinical Research, Gieres, France 3 Rottapharm S.p.A., Monza, Italy 2

levothyroxine [11], thus creating the potential for a clinically relevant interaction and resulting in a suboptimal control of the hypothyroidism due to a reduced bioavailability of levothyroxine. Therefore, the aim of this study was to investigate if a fixed combination of soy isoflavones1 that has demonstrated safety and efficacy in rigorously conducted clinical trials [12–15], reduces the oral absorption of levothyroxine in post-menopausal women who take levothyroxine as a replacement/supplementation therapy for hypothyroidism. In addition, to confirm that the subjects were exposed to soy isoflavones and thus to validate the obtained results, the plasma concentrations of daidzin and genistin, and their metabolites daidzein, genistein and S-equol were determined in this study.

Materials and Methods

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Materials

The analytical standards genistein and daidzein were purchased from Sigma-Aldrich (St. Louis, MO, USA). Genistin and daidzin were purchased from Interchim (Montluçon, FRANCE), and S-Equol, daidzein-D6 and genistein-D4 were purchased from TRC (Toronto, Canada). Levothyroxine was supplied by Cayman Chemical (Ann Arbor, MI, USA). Solvents were obtained from standard sources and were of HPLC grade.

Clinical study

The study was conducted according to a single centre, open label, randomized, single oral dose, 2-period crossover design in post-menopausal female volunteers on stable levothyroxine dose as a replacement/supplementation therapy. The study was carried out at Eurofins|OPTIMED Clinical Research (Gieres, France) in accordance with the Declaration of Helsinki as modified in Fortaleza (2013), with the recommendations on Good Clinical Practice and with any applicable local regulatory requirements. The study obtained the approval of the local Ethics Committee and of the national Health Authorities “ANSM”, Agence Nationale de Sécurité du Medicament et des produits de santé. Prior to initiation of study procedures, all volunteers gave their written informed consent.

Subjects

12 iodine-replete post-menopausal volunteers treated with levothyroxine aged between 40 and 60 years and with mean body mass index (BMI) between 18 and 30 Kg/m2 were included in the study. All subjects were in good health conditions on the basis of medical history, physical examination, electrocardiogram (ECG), and clinical laboratory measurements. Any consumption of food containing soy, cotton seeds meals, walnuts and dietary fiber was prohibited within one week from screening.

Treatment administration and plasma sampling

Subjects continued to receive levothyroxine at their prescribed dose (range 25–125 µg/day) throughout the study. In the test period they received their daily dose of levothyroxine concomitantly with soy isoflavones, whereas in the reference period they received soy isoflavones 6 h later their daily dose of levothyroxine, according to the randomization list. Treatment with soy isoflavones was at the recommended dose by administration of one 1

Estromineral Serena Plus; marketing authorization holder: Rottapharm S.p.A., a Meda Company, Monza (Italy)


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tablet containing 150 mg of Glycine max L. dry extract equivalent to 60 mg of soy isoflavones, Lactobacillus sporogenes (1 billion spores), 50 mg Magnolia officinalis dry extract equivalent to 0.75 mg of honokiol, 40 mg of Vitex agnus-castus L. dry extract equivalent to 0.2 mg agnuside, vitamin D3 (5 µg), magnesium (60 mg) and calcium (141 mg) (Estromineral Serena Plus, Rottapharm S.p.A., a Meda Company, Monza, Italy). The two study periods were separated by a washout of at least 3 days. All treatments were administered with 240 mL of tap water in sitting position and in fasting conditions that were maintained for 4 h after drug intake. The sampling times for determinations in plasma of levothyroxine were: pre dose, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18 and 24 h. Levothyroxine was determined both when it was administered alone (baseline) and when it was administered with soy isoflavones (test and reference period). When soy isoflavones were administered, blood samples were collected for the determination of genistin, daidzin, genistein, daidzein and S-equol at 2 and 6 h post dosing, to ensure compliance and exposure of the subjects to the soy isoflavones. Samples were kept at − 80 °C pending analysis.

Bioanalytical methods

Three different bioanalytical methods were developed and qualified at Eurofins|ADME BIOANALYSES (Vergeze, France) to determine the plasma concentrations of levothyroxine, daidzin, daidzein, S-equol, genistin and genistein. Levothyroxine plasma samples were spiked with 13C6-levothyroxine as internal standard (IS). The HPLC-MS/MS system consisted of Shimadzu SIL-20AC autosampler, SIL20AD pumps and CTO-20AC oven (Shimadzu Corporation, Kyoto, Japan) interfaced to a triple quadrupole mass spectrometer (API 4000 System, AB Sciex, Framingham, MA, USA). Chromatographic separation was obtained using a Supelcosil LC-18-DB (33 × 4.6 mm, 3 µm) column with the mobile phase A consisting of 5 mM ammonium acetate pH 4, and the mobile phase B consisting of methanol. The mass spectrometer was set to monitor levothyroxine (777.7 > 731.8 m/z) in the positive ion mode. Plasma samples collected for the determination of daidzin and its metabolites (daidzein or S-equol) were spiked with daidzeind6 (IS). The plasma protein precipitation was performed using methanol; the supernatant was evaporated to dryness, redissolved and injected into the HPLC-MS/MS system (Shimadzu and API5500 Qtrap System). The compounds were separated on a Phenyl hexyl column, 5 µm, 2 × 50 mm (Phenomenex, Torrance, CA, USA), with the mobile phase A consisting of 0.2 % acetic acid, and the mobile phase B consisting of acetonitrile. The MS/MS system was equipped with electrospray ionization (ESI) interface, monitoring in the negative ion mode. The monitored transitions were 415.2 > 252.0, 253.0 > 208.0, 241.0 > 118.9 and 259.1 > 92.9 m/z for daidzin, daidzein, S-equol and daidzein-d6, respectively. Plasma samples collected for the determination of genistin and its metabolite (genistein) were spiked with genistein-d4 (IS), and acetonitrile was added to obtain a plasma protein precipitation. The supernatant was evaporated to dryness, redissolved and injected into the HPLC-MS/MS system (Shimadzu and API5500 Qtrap System). The compounds were separated on a Phenyl hexyl column, 5 µm, 2 × 50 mm (Phenomenex, Torrance, CA, USA), and the mobile phases were 0.2 % acetic acid, and acetonitrile. The MS/MS system was equipped with electrospray ionization (ESI) interface and the mass spectrometer was set to


Original Article

DrugRes/2015-04-0999/23.6.2015/MPS

Original Article

Demography

Statistics

Sequence T-R (N = 6)

Sequence R-T (N = 6)

Overall (N = 12)

Female Caucasian Age (years)

N( %) N( %) Mean ± SD Min/Median/Max Mean ± SD Min/Median/Max Mean ± SD Min/Median/Max Mean ± SD Min/Median/Max

6(100.0) 6(100.0) 57.2 ± 3.3 51/58.5/60 160.17 ± 6.37 151.0/161.00/170.0 57.52 ± 12.32 44.3/54.55/74.6 22.38 ± 4.39 18.2/20.30/28.8

6(100.0) 6(100.0) 59.5 ± 3.0 56/60.0/63 162.00 ± 4.34 156.0/162.00/167.0 63.63 ± 9.48 52.4/65.80/73.6 24.20 ± 3.16 20.7/24.05/28.8

12(100.0) 12(100.0) 58.3 ± 3.3 51/59.0/63 161.08 ± 5.28 151.0/161.00/170.0 60.58 ± 10.96 44.3/59.70/74.6 23.29 ± 3.76 18.2/21.95/28.8

Height (cm) Weight (kg) BMI (kg/m²)

Table 1 Summary of demographic data for the subjects participating in the study.

T, soy isoflavones combination administered concomitantly with levothyroxine; R, soy isoflavones combination administered 6 h later levothyroxine; SD, standard deviation; BMI, body mass index

Pharmacokinetic analysis

Pharmacokinetic parameters of levothyroxine were determined from measured plasma concentrations using Kinetica version 4.3 (Thermo Electron Corporation, Philadelphia, USA) and applying a non-compartmental analysis. The maximum plasma concentration (Cmax) and the time to reach it (tmax) were obtained directly from the plasma concentration-time data. The area under the plasma concentration-time curve from time 0 to the last measurable time point (AUCt) was calculated according to the linear trapezoidal rule. The average plasma concentrations (Cav) were calculated for levothyroxine as AUC/24 h at baseline and after both the test and reference treatment periods.

Sample size determination and statistical analysis

Intra-subjects variability (CVintra %) were assumed not to exceed 15 % for Cmax and AUCt of levothyroxine, allowing the assessment of the effect of the fixed combination of soy isoflavones with a sample size of 12 subjects [16]. All statistical analyses were performed using SAS Versions 9.3 (SAS Inc., Cary, NC, USA). Log-transformed PK parameters Cmax and AUClast were analyzed using PROC MIXED (SAS statistical package, SAS institute – Version 9.3) with treatment, sequence, period and subject-within sequence as fixed terms. The 90 % confidence intervals (CIs) of the logarithmic difference between test (soy isoflavones concomitantly administered with levothyroxine) and reference (soy isoflavones administered 6 h after levothyroxine) treatment were constructed based on the Mean Square Error (MSE) from the ANOVA model. The CIs were then back transformed to obtain the CIs for the ratio on the original scale. According to Regulatory Guidelines [17], absence of effect on bioavailability of levothyroxine is established if the calculated 90 % CIs for the ratio of the geometric means between test and reference treatment, based on log-transformed data, is contained in the equivalence limits of 0.8–1.25 for both Cmax and AUCt. Differences in tmax between test and reference treatment were evaluated according to a non-parametric approach, by means of the Wilcoxon signed-rank test for paired samples. Statistical significance was set at p ≤ 0.05.

Table 2 Nominal calibration ranges, inter-assay precision and accuracy of the back-calculated concentrations for each calibration standard. Standard

Levothyroxine * Daidzin Daidzein S-Equol Genistin Genistein

Calibration

Inter-assay

ranges

precision

Accuracy

(ng/mL)

(CV %) N = 4

( %) N = 4

10–200 1–1 000 1–1 000 1–1 000 3–1 500 1–500

1.20–4.95 2.00–10.37 1.82–11.18 2.86–9.71 4.14–9.50 1.20–6.82

− 1.76 to 1.97 − 11.54 to 11.66 − 3.78 to 5.07 − 10.21 to 12.77 − 3.68 to 4.14 − 2.99 to 2.81

* N = 8; CV %, coefficient of variation

Results

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Subjects

12 iodine-replete postmenopausal volunteers completed the study according to the protocol and were included in the analysis. Demographic characteristics of the volunteers are summarized in ● ▶ Table 1. The dose regimen was well tolerated in all women. No serious adverse event was reported during the study. Two treatment emergent adverse events (TEAEs) were reported by 2 subjects during the study: one episode of painful legs and one episode of headache, both when soy isoflavones are concomitantly administered with levothyroxine. The painful legs was classified as mild in intensity and resolved spontaneously, whereas the headache was moderate in intensity and resolved spontaneously. Both TEAEs were considered not to have a reasonable causal relationship with the study products. Physical examination was normal at the end of the study in all subjects.

Bioanalytical methods

The ranges, precision and accuracy values for the calibration standards obtained during the methods qualification are presented in ● ▶ Table 2. Results obtained from the analysis of quality controls ranges were within the acceptable limits ( ± 20 %), validating the concentrations of levothyroxine, daidzin, daidzein, S-Equol, genistin and genistein determined in plasma. The specificity of the methods was tested by the analysis of 6 different individual batches of blank human plasma spiked with analytes at LLOQ (Lower Limit Of Quantification), and then processed and compared to the corresponding blank plasma samples. No interference peaks were detected. All samples were analysed within the long-term stability period.



monitor the compounds in the negative ion mode. The monitored transitions were 431.2 > 267.9, 269.0 > 133.0 and 273.0 > 137.2 m/z for genistin, genistein and IS, respectively. All the developed methods were qualified. The qualification comprised the determination of linearity on 3 different runs, the inter- and intra-run precision and accuracy for 4 concentration levels in sextuplicate, the evaluation of methods’ specificity and the assessment of storage stability.

DrugRes/2015-04-0999/23.6.2015/MPS

Original Article

( ± 12.9) ng/mL at baseline, 77.5 ( ± 15.2) and 77.5 ( ± 9.5) ng/mL in the test and reference treatment period, respectively. Mean values for genistin, daidzin, genistein, daidzein and S-equol at 2 and 6 h post dosing during the test and reference treatment indicated that these components were bioavailable when administered with levothyroxine, thus validating the study. Overall, the measured concentrations ranged from 1.1 to 13.2 ng/mL.

Fig. 1 Mean ( ± SD) plasma concentration–time profiles of levothyroxine administered 6 h before (○) or concomitantly (●) with soy isoflavones combination in post-menopausal females.

Table 3 Pharmacokinetic parameters of levothyroxine (Mean ± SD). Treatment Cmax (ng/mL) Baseline T (N = 12) R (N = 12)

AUCt (ng * h/mL)

90.4 ± 14 95.0 ± 17 93.4 ± 17

1 898 ± 311 1 861 ± 364 1 860 ± 229

tmaxa

(h)

Cav (ng/mL)

2.5 (1–8) 4 (1.5–12) 3.5 (1.0–12)

79.1 ± 13 77.5 ± 15 77.5 ± 10

SD, standard deviation; Cmax, maximum plasma concentration; AUCt, area under the concentration-time curve from time zero to the last measurable time point; tmax, time to maximum concentration; Cav, average plasma concentration; T, soy isoflavones combination administered concomitantly with levothyroxine; R, soy isoflavones combination administered 6 h later levothyroxine a

Value for tmax is median (range)

Table 4 ANOVA analysis on levothyroxine PK parameters. PK parameters

GMR

90 % CIs

CVintra %

Cmax AUCt

1.02 0.99

0.99–1.04 0.88–1.12

3.37 16.9

GMR, geometric mean ratio; CIs confidence interval; CVintra, intra-subjects variability; Cmax, maximum plasma concentration; AUCt, area under the concentration-time curve from time zero to the last measurable time point

Pharmacokinetic analysis

Mean concentration-time profiles of levothyroxine administered 6 h before or concomitantly with the fixed combination of soy isoflavones are shown in ● ▶ Fig. 1. The corresponding PK parameters are shown in ● ▶ Table 3. The plasma concentration-time profile of levothyroxine showed no significant differences between the two study periods. In all subjects included in the study, the measured levothyroxine plasma concentrations ranged 52.5–127.6 ng/mL and 53.0– 126.6 ng/mL when soy isoflavones were administered 6 h after the levothyroxine administration, or when they are administered concomitantly, respectively. The calculated mean Cmax was 93.4 ng/mL at 3.5 h (tmax) when the subjects received soy isoflavones 6 h after the administration of levothyroxine. A very similar Cmax value (95.0 ng/mL) reached at 4 h (tmax) was obtained when the subjects received soy isoflavones concomitantly with the dose of levothyroxine. The AUCt values calculated for these two study periods averaged 1 860 ng * h/ml and 1 861 ng * h/ml, respectively. The Cav ( ± SD %, standard deviation) of levothyroxine were 79.1


A summary of the statistical analysis results is reported in ● ▶ Table 4. For levothyroxine, test to reference geometric mean ratios (GMR) were very close to unit, resulting 1.02 and 0.99 for Cmax and AUCt, respectively. The relevant 90 % CIs fell entirely within the equivalence limit of 0.8–1.25 both for Cmax (0.99–1.04) and for AUCt (0.88–1.12). Levothyroxine intra-subjects variability (CVintra %) for Cmax and AUCt was 3.37 and 16.9 %, respectively. No significant differences were observed for tmax (p > 0.05).

Discussion

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Postmenopausal women wishing to ameliorate their symptoms such as hot flashes often assume combinations containing soy isoflavones [3, 4]. However, it has been speculated that soy foods and isoflavones may be contraindicated for certain population [10]. In particular, soy may interfere with the absorption of synthetic thyroid hormone resulting in therapeutic failure [11, 18] and, as it has been observed that a large portion of postmenopausal women suffer of hypothyroidism [10], the risk for an interaction between soy isoflavones and levothyroxine is considered possible [5, 11]. Therefore, the objective of the present study is relevant and, to our knowledge, it is one of the first study investigating the interaction of a fixed combination of soy isoflavones on the absorption of levothyroxine in humans. In agreement with the current guidelines on drug interactions and on bioequivalence [17, 19], the main PK parameters to quantify the magnitude of the mean effect of soy isoflavones on the absorption of levothyroxine were obtained from Cmax and AUCt. For the purpose of the analysis, since the absorption of levothyroxine is known to be completed in 3 h when given alone [20], no effect on levothyroxine is expected when the fixed combination of soy isoflavones is administered 6 h after the administration of levothyroxine. Therefore, in the present study, the administration of soy isoflavones 6 h after levothyroxine was considered the reference treatment and the concomitant administration of levothyroxine and soy isoflavones was considered the test treatment. The statistical analysis demonstrated that no differences in levothyroxine absorption were observed between these two treatments, being the 90 % CIs of geometric mean ratio of levothyroxine completely included within the predefined no-effect range of 0.8–1.25 for both Cmax and AUCt. In particular, the study demonstrated that in terms of levothyroxine Cmax the geometric mean ratio was very close to unity (1.02) and the 90 % CIs fell entirely within the equivalence limit (0.99–1.04). Similar results were obtained for the geometric mean ratio of levothyroxine AUCt (0.99) and for the corresponding 90 % CIs (0.88–1.12). The sample size of 12 evaluable subjects selected a priori in the present study proved to be sufficient and, based on the intrasubjects variability and the geometric mean ratio, should provide a power of 70 % for the conclusion of “no effect” for the PK parameters of levothyroxine [21].


Statistical analysis

In addition, being the levothyroxine Cav data very similar before and after treatment with the fixed combination of soy isoflavones, all the enrolled volunteers maintained their levothyroxine level within their baseline values indicating that their state of euthyroidism was maintained when the soy isoflavones combination was administered in both the test and reference treatment periods. This supports the conclusion that the combination used in the present investigation does not affect the absorption of levothyroxine in post-menopausal women. To further validate this lack of interaction, the plasma concentrations of soy isoflavones, daidzin, daidzein, S-equol, genistin and genistein, were determined in this study applying qualified bioanalytical methods. The data indicated that the enrolled volunteers were exposed to the soy isoflavones. The bioanalytical method for levothyroxine plasma determination was also qualified, and it had been developed to allow the inclusion of subjects treated with a wide range of levothyroxine dose, with the aim to generate data useful for the widest possible patient population treated for hypothyroidism. In conclusion, the present study is one of the few clinical studies rigorously designed to investigate a herb-drug interaction and demonstrates that the rate and extent of levothyroxine absorption is not significantly affected when administered concomitantly with the recommended dose of Estromineral Serena Plus in post-menopausal women. In addition, this study provides data, such as sample-size, intrasubjects variability and wash out period, that may be useful for the design of future and different clinical trials in post-menopausal women taking levothyroxine.

Conflict of Interest

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The authors declare that they have no conflicts of interest. Yves Donazzolo is the investigator of the clinical study. Claudio Benvenuti is Medical Consultant at Rottapharm S.p.A., the Marketing Authorization Holder of the study product.

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