Study of pharmacokinetics of prenylflavonoids and dynamics of estrogen action in sera following ingestion of epimedium using validated, ultra sensitive cell based bioassays

CHAPTER 2 MATERIALS AND METHODS 2.3.1 Establishment of ERα and ERβ stable cells 54 2.3.2 Optimization of ERα and ERβ bioassay parameters 55 2.3.3 Validation of stable ERα- and ERβ-drive

CHAPTER 2

MATERIALS AND METHODS

2.3.1 Establishment of ERα and ERβ stable cells 54 2.3.2 Optimization of ERα and ERβ bioassay parameters 55 2.3.3 Validation of stable ERα- and ERβ-driven reporter gene

2.3.4 Assay procedure for stably transfected ERα- and ERβ-responsive

2.5 Measurement of concentrations of estrone, estradiol, icaritin and

desmethylicartin and estrogenic activity in serum after oral

ingestion of a traditional Epimedium pubescens decoction or

estradiol valerate by human subjects

2.5.5 Liquid chromatography tandem mass spectrometry measurement

of estrone and estradiol in serum after oral ingestion of a

traditional Epimedium pubescens decoction or estradiol valerate

by human subjects

63

2.5.6 Statistical analysis for clinical trial 64 2.5.7 Measurement of icaritin and desmethylicaritin in serum after oral

ingestion of a traditional Epimedium pubescens decoction by

human subjects using gas chromatography–mass spectrometry

65

2.5.7.1 Extraction and preparation of serum samples 65

2.6 Development and validation of a liquid chromatography–tandem

mass spectrometry assay for simultaneous measurement of five

Epimedium prenylflavonoids in rat sera

Description Source

Gas

• Purified helium gas (purity 99.9999%) (Singapore) Soxal

Derivatizing agents

• bis- (trimethylsilyl) trifluoroacetamide Supelco

(Bellefonte, PA, USA)

• N-meth yl-N-(trimethylsilyl)

trifluoroacetamide (Rockford, IL, USA) Pierce

Cell culture reagents

• Eagle’s minimal essential medium

• pooled rat serum

• pooled male human serum

• sodium bicarbonate

• sodium pyruvate

• trypsin

Sigma (St Louis, MO, USA)

• fetal bovine serum

• Sulphatase from Acetobacter aerogenes

• β-Glucoronidase from Escherichia coli

Sigma (St Louis, MO, USA)

Cells

• HeLa cervical cancer cells

• MCF- 7 breast cancer cells American Type Culture Collection (Manassas, VA, USA)

Description Source

Equipment

• Glomax 96- well microplate luminometer

• API triple-quadrupole LC-MS/MS system

• Agilent 6890N GC coupled to a 5975 inert XL

mass selective detector

• Agilent 1200 LC system coupled to an

API3200 triple-quadrupole mass spectrometer

with Turbo V source and TurboIonSpray and

ABI- Sciex (Foster City, CA, USA)

Agilent Technologies (Santa Clara, CA, USA)

Phenomenex (Torrance, CA, USA)

J&W (Santa Clara, CA, USA)

A pplied Biosystems (Carlsbad, CA, USA)

Eppendorf (Hamburg, Germany) NanoDrop (Waltham, MA, USA)

Illumina (San Diego, CA, USA)

Description Source

Software

• GraphPad Prism version 4.0

• Microsoft Excel software

• Stata version 8 software

• Illumina BeadStudio software

• Genesifter software

• Analyst 1.4.2 software

GraphPad Software (La Jolla, CA, USA) Microsoft (Redmond, WA, USA)

Stata Corp (College Station, TX, USA)

Illumina (San Diego, CA, USA)

Geospiza (Seattle, WA, USA)

ABI-Sciex (Foster City, CA, USA) Assays & RNA/DNA Extraction Kits

• Luciferase assay system

• CyQUANT cell proliferation assay kit

• Illumina Sentrix HumanRef-8 V2 BeadChip

Arrays

• RNeasy Mini Kit

• Illumina TotalPrep RNA Amplification Kit

• Superscript III Reverse Transcriptase

Promega (Madison, WI, USA) Sigma Invitrogen (Carlsbad, CA, USA)

Illumina (San Diego, CA, USA) QIAGEN (Hilden, Germany)

Ambion (Carlsbad, CA, USA) Gibco Invitrogen (Carlsbad, CA, USA)

2.2 Cell lines and cell culture

HeLa cervical cancer cells and MCF-7 breast cancer cells were purchased and cultured as recommended by the American Type Culture Collection (ATCC) Cells were maintained in phenol red-free Eagle’s Minimal Essential Medium supplemented with sodium pyruvate, 2 mM L-glutamine, sodium bicarbonate, and 5% dextran-coated, charcoal-treated fetal bovine serum in humidified atmosphere of 5% carbon dioxide in air

at 37°C ERα and ERβ stable cells (described in Section 2.3 below) were maintained in same cell culture media that contains 100 µg/mL hygromycin B and 300 µg/mL G418

MCF-7 breast adenocarcinoma cells were maintained and grown as monolayer cultures in phenol red-free Eagle’s Minimal Essential Medium supplemented with 5% complete fetal bovine serum and 10 μg/mL insulin in a humidified atmosphere of 5% carbon dioxide in air at 37°C

2.3 ERα and ERβ cell-based bioassays

2.3.1 Establishment of ERα and ERβ stable cells (from Yap et al., 2005)

Determination of LD90of antibiotics in HeLa cells

To successfully generate stable cell lines expressing ER and ERE4 genes, the minimum concentration of hygromycin B and G418 required to kill the untransfected HeLa cells were determined HeLa cells plated in 96-well plate were allowed to adhere overnight Next, the cells were exposed to various doses of hygromycin B and G418 (0,

50, 100, 200, 300, 400 and 500 µg/mL) The selective media was replenished every 3 days and the viable cell counts were determined after 10 days of incubation The lethal dose of the antibiotics to kill 90% of untransfected cells (LD90) was determined

ERE4 stable transfection

HeLa cells were seeded in a 6-well tissue culture plate and stably transfected with ERE4 plasmid using lipofectamine Sixteen hours post-transfection, the cells were washed, trypsinized and split at ratio of 1 in 10, 1 in 100 and 1 in 1000 into selective media containing 100 μg/mL of hygromycin B The diluted cells were re-seeded onto a new 6-well plate and allowed to grow in selective cell culture media for 2 to 3 weeks The selective media were refreshed every 3 days, until hygromycin-resistant foci can be identified The selected clones were screened by transiently transfecting ERα or ERβ plasmid and exposed the cells to estradiol at 10 nM The positive clones were frozen in liquid nitrogen tank for future use

ERα/β stable transfection

The positive ERE4 clones were seeded in a 6-well plate and stably transfected with pGFP-N2-ERα or pGFP-N2-ERβ plasmids using lipofectamine Sixteen hours post-transfection, the cells were washed, trypsinized and split at ratio of 1 in 10, 1 in 100 and 1

in 1000 into selective cell culture media containing 100 μg/mL of hygromycin B and 300 μg/mlL of G418 The diluted cells were re-seeded onto a new 6-well plate and allowed to grow in selective cell culture media for 2 to 3 weeks The selective media were refreshed every 3 days, until hygromycin/neomycin-resistant foci can be identified The selected clones were screened by exposing the cells to estradiol at 10 nM The positive clones were optimized to achieve maximum fold induction and the good clones were frozen in liquid nitrogen tank for future use

2.3.2 Optimization of ERα and ERβ cell-based bioassay parameters

ERα and ERβ cells were developed by Yap et al., 2005 and optimization of

bioassay parameters was performed by seeding cells at various densities (0.25, 0.5, 1, and

2 × 104cells per well) in 96-well plates and incubated for various time intervals (18, 24,

36, and 48 h) to select assay conditions that will give the highest fold increase in estrogen-induced luciferase activity relative to vehicle The optimal cell plating density for ERα and ERβ cells was determined to be 1 × 104 cells and 0.5 × 104 cells per well, respectively The optimal incubation period was 30 h for ERα or 24 h for ERβ cells

2.3.3 Validation of stable ERα- and ERβ-driven reporter gene bioassays

Inter- and intra-assay coefficients of variance (CV) values were determined using 0.1 or 0.5 nM estradiol Intra-assay CV is the relative SD of the mean of eight determinations in a single assay Inter-assay CV is the relative SD from the mean of three determinations done on 10 different occasions The detection limit is the concentration of the ligand inducing luciferase activity equivalent to the mean plus 3 SD of the vehicle

value (Legler et al., 1999)

2.3.4 Assay procedure for stably transfected ERα- and ERβ-responsive cell lines

For the determination of estrogenic activity of compounds and botanical extracts, ERα- and ERβ-responsive cells were passaged, plated, and allowed to adhere overnight Culture medium was then removed and replaced with ligands dissolved in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum After an optimized incubation period, medium was decanted, and cells were washed with phosphate saline buffer and lysed with M-PER Mammalian Protein Extraction Reagent (Pierce) Luciferase activity was measured using the Luciferase Assay System (Promega)

on the Glomax 96-well Microplate Luminometer (Promega) Estrogenic activity was expressed as a percentage of maximal luciferase activity induced by estradiol Experiments were performed in triplicate over three different occasions and expressed as mean ± SEM

Sigmoidal curve-fitting and data analyses were performed using GraphPad Prism version 4.0 (GraphPad Software) Ligand concentrations were log transformed, and curve-fitting was performed using a sigmoidal dose–response model with variable slope The value for the concentration of ligand inducing 50% of the maximum activity (EC50) was generated based on the respective dose–response curves

For human and animal studies, test sera were thawed and diluted with Eagle’s Minimal Essential Medium to a final concentration of 20% ERα- and ERβ-responsive cells were exposed to this 20% test serum for 24 h After the incubation period, culture medium was removed, cells were lysed, and luciferase activity was quantified in a 96-well multiplate reader

To study combinatorial effects (additive, antagonistic or synergistic) of test compounds in the presence of estradiol, increasing concentrations of the test compound

were separately co-incubated with 0.1 nM or 0.5 nM of estradiol, with ERα or ERβ stable cells, respectively Estrogenic activity was expressed as a percentage of maximal luciferase activity induced by estradiol Experiments were performed in triplicate over three different occasions and expressed as mean ± SEM

2.3.5 Bioassay calibration curves

For human and animal studies, calibration standards for the construction of calibration curves were obtained by adding increasing doses of estradiol to dextran-coated, charcoal-treated commercially available pooled serum Calibration standards and QC samples containing low and high steroid concentrations of estradiol were equilibrated for

4 h at 37°C before use All serum samples were diluted to 20% with serum-free medium and were tested in duplicate The aromatase inhibitor, DL-aminoglutethimide (final concentration, 50 μM) was added to every sample to inhibit the conversion and inference from estrogenic products due to the aromatization of 4-androstene-3, 17-dione and testosterone that may be present in the sample Calibration standards and QC samples were included in every 96-well plate Calibration curves were fitted with the regression method that visually best fits the points within the range of the test samples and maximizes the correlation coefficient (R2) value Data were expressed as estradiol equivalent activity and obtained by interpolation from calibration curves in each plate Microsoft Excel software was used for curve fitting and interpolation Assays were accepted if four out of six QC samples had relative errors of < 30%

2.4 MCF-7 cell proliferation assay

For assay purposes, MCF-7 cells that were maintained in Eagle’s Minimal Essential Medium supplemented with 10% complete fetal bovine serum were switched to phenol red-free cell culture media containing 10% charcoal-stripped fetal bovine serum for a week, with media changes performed on every alternate day

For the determination of estrogenic activity of compounds and botanical extracts, MCF-7 cells were trypsinized, disaggregated, and plated at a density of 3,500 cells per well in 96-well plates Cells were allowed to adhere overnight On the day of assay, culture medium was removed and replaced with ligands dissolved in Eagle’s Minimum Essential Medium supplemented with 10% charcoal-stripped fetal bovine serum Test sera were removed and replaced with fresh ones every alternate day for a total of 6 days On day 6, culture media were removed Cell numbers were measured with a CyQUANT cell proliferation assay kit (Sigma Invitrogen) Induction of cell growth was plotted as fold induction over vehicle control Sigmoidal curve-fitting and data analyses were performed using GraphPad Prism version 4.0 (GraphPad Software)

For human and animal studies, test sera were thawed and diluted with Eagle’s Minimal Essential Medium to a final concentration of 20%, and 100 μL of this diluted test serum or calibration standard was added to MCF-7 cells

To study combinatorial effects (additive, antagonistic or synergistic) of test compounds in the presence of estradiol, increasing concentrations of the test compound were separately co-incubated with 50 pM of estradiol with MCF-7 cells On day 6, culture media were removed Cell numbers were measured with a CyQUANT cell proliferation assay kit (Sigma Invitrogen) Induction of cell growth was plotted as fold induction over vehicle control

2.5 Measurement of concentrations of estrone, estradiol, icaritin and desmethylicartin and estrogenic activity in serum after oral ingestion of a traditional

Epimedium pubescens decoction or estradiol valerate by human subjects

2.5.1 Subjects

To minimize variations due to endogenous estrogen production, healthy male volunteers were recruited Inclusion criteria include age between 21 and 65 years, body mass index between 19 and 29 kg/m2, and normal hepatic and renal laboratory tests

Subjects who were taking dietary supplements in the previous 3 months, current smokers, and those who consume more than 14 units of alcohol a week were excluded from this study Subjects were counseled to follow a phytoestrogen-free diet for 3 days prior to the first blood sampling and for the duration of the study

2.5.2 Preparation of the Epimedium pubescens decoction

The main study drug was a water decoction of Epimedium pubescens, obtained

from Sichuan Province, People’s Republic of China The herb was taxonomically authenticated by Dr Guo Baolin (Institute for Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China) and phylogenetic identity was confirmed by fluorescent-amplified fragment length polymorphism analysis

(Shen et al., 2007) Voucher specimens (SBG-EP02-060110) have been deposited in the Singapore Herbarium at the Singapore Botanic Gardens

were removed by filtration, and the decoction was reduced to 4 L by boiling The water extract was aliquoted into glass bottles and stored at 4°C overnight The chilled water extract (500 mL per subject) was administered to subjects the next morning No additives were added into the decoction To assess the estrogenicity of the decoction, an aliquot of the extract was evaporated to dryness, resuspended in culture medium, and tested directly

in the ERα bioassay

The dried water decoction (yield is about 12.6%) contained 2.00 mg/g icariin, 119 µg/g icaritin and 31 µg/g desmethylicaritin It dose-dependently increased ERα bioactivity up to 50% of the maximum observed with a saturating dose of the estradiol Each human subject in this study consumed 500 mL of decoction containing icariin: 12.6

mg, icaritin: 0.75 mg, and desmethylicaritin: 0.19 mg

A single dose of 50 g per subject was chosen according to calculations made on a

previous animal study performed by Yap et al (2007) The calculation method for human

equivalent dose (HED) spelled out in U.S Food and Drug Administration’s Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (2005) was used:

HED = animal dose in mg/kg x (animal weight in kg/human weight in kg)

Using data from Yap et al (2007) which used 500 mg extract per rat (mean weight: 0.250 kg), the human equivalent dose of Epimedium extract was calculated to be 7.14 mg/kg for

a 70 kg healthy male The dried extract used in this human study was 90.1 mg/kg and equivalent to 50 g dry leaves per subject and was a dose that was estimated to produce a detectable signal in the cell-based bioassays developed in this study Alcoholic extracts of

Epimedium using 3 to 200 g raw leaves are routinely given for various conditions (Tan &

Weng, 1998; Liao et al., 1995, Zhao, 2003) with no reported safety issues

2.5.3 Methodology

An open-label, two-period, randomized, crossover study with baseline assessment

of endogenous activity was conducted Subjects were admitted to the Lilly-National University of Singapore Center for Clinical Pharmacology and blood sampling was performed for baseline data over a 24 h period After an overnight fast, subjects were randomized to receive either a single dose of 2 mg estradiol valerate (Schering GmbH) supplied by the National University Hospital Pharmacy or a water decoction of

Epimedium pubescens between 0800 and 0900 h in the morning Following drug administration, blood sampling was performed over a 48 h period There was a 7-day washout period, after which subjects who received estradiol valerate were administered

freshly prepared Epimedium pubescens decoction, and vice versa, in the crossover arm of

the study Blood sampling in both arms of the study was performed in an identical manner over a 48 h period This study was approved by the Domain-Specific Review Board of National Healthcare Group of hospitals All participants provided written informed consent

The terminal elimination half-life of estradiol is about 15 hours (Zimmermann,

1998) The Epimedium decoction contains estrogenic flavonoids whose terminal

elimination half-lives are not available Studies that reported elimination half-lives of

flavonoids fell within a duration of 10 hours (Busby et al., 2002; Kanaze et al., 2007) A

7-day washout between study periods 1 and 2 was chosen to ensure that all estrogenic drugs are eliminated

2.5.4 Blood sampling

During the baseline sampling period, four hourly blood samples were collected to establish baseline levels of endogenous estrogenic activity In sequential study periods, blood samples were drawn at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h post-administration of

either estradiol valerate or Epimedium pubescens Serum was extracted from blood,

passed through a filter (pore size, 0.22 μm), and stored under sterile conditions at ─80°C for subsequent analysis

2.5.5 Liquid chromatography tandem mass spectrometry measurement of estrone

and estradiol in serum after oral ingestion of a traditional Epimedium pubescens

decoction or estradiol valerate by human subjects

Concentrations of estrone and estradiol were measured by LC-MS/MS (API quadrupole ABI-Sciex LC-MS/MS system) (ABI-Sciex) using d4-estrone and d5-estradiol

triple-as internal standards (Nelson et al., 2004) The concentrations of estrone and estradiol in

blood samples were quantified using a six-point calibration curve of peak area ratio for compound against the concentration internal standard The ranges of coefficients of variation for estrone and estradiol were 1.8–8.0% and 1.1–15.7%, respectively, within a linear range of 5–400 ng/L