RELATIVE BIOAVAILABILITY OF TWO DIFFERENT FORMULATIONS OF MILK THISTLE

By

WEN-YI LI

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY

UNIVERSITY OF FLORIDA

2018

© 2018 Wen-Yi Li

To my parents, sister, brother and family for their support and love

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ACKNOWLEDGMENTS

I would like to express my deepest gratitude and appreciation to my advisor

Dr. Reginald F. Frye for his training, guidance and support for my research work at the

University of Florida. I am very grateful to him for the great learning experiences and

professional development opportunities I was exposed to over the past three years.

Especially, I would like to thank him for giving me the opportunity to pursue my master’s

degree at the Department of Pharmacotherapy and Translational Research.

My sincerest indebtedness goes to my committee members, including Dr. John

S. Markowitz and Dr. Yan Gong for their help and guidance on my research project.

Specifically, I would like to thank Renee M. Hogan, ARNP, for her assistance on clinical

monitoring, data collection and conducting physical exams. I would like to express my

thankfulness to Rajesh Mohandas, MD, for providing clinical input and general medical

oversight to my study. Also, I am beholden to Guo Yu, PhD, a visiting scholar (Jiangsu,

China) in my lab who contributed to the PK data analysis and interpretation as well as

general encouragement to my research project. Lastly, I would like to thank Isagenix

International LLC (Gilbert, Arizona) for funding this project.

During my graduate studies, I worked as a graduate assistant for Dr. William

Cary Mobley and Dr. Heather R. Hardin. Here I want to thank them for their help and

advice on my work. In addition, I would like to thank all the people in the Department of

Pharmacotherapy and Translational Research for their great friendship and kindness

which created an enjoyable environment that I will bear in mind. Finally, I would like to

acknowledge the University of Florida, College of Pharmacy for supporting my graduate

work.

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Last but not the least, I would like to heartfeltly thank my parents, sister, brother

and other family members for their continuous inspiration, understanding and listening.

During my ups and downs, they are always the strongest source of care, support and

encouragement for me. I would like to dedicate this thesis to them for their endless love,

unlimited encouragement and unconditional support.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 8

LIST OF FIGURES .......................................................................................................... 9

LIST OF ABBREVIATIONS ........................................................................................... 10

ABSTRACT ................................................................................................................... 11

CHAPTER

1 INTRODUCTION .................................................................................................... 13

Dietary Supplements .............................................................................................. 13 Bioactivities of Milk Thistle in IsaGenesis and Product B ........................................ 15 Methods to Improve Silybin Bioavailability .............................................................. 17

Phytosomes ...................................................................................................... 17 Softgel Technology ........................................................................................... 18 Nanoparticles ................................................................................................... 19 Nanosuspensions ............................................................................................. 20 Nanoemulsions ................................................................................................. 21 Liposomes ........................................................................................................ 22

2 HYPOTHESIS ......................................................................................................... 27

Existing Problem in Herbal Supplements ................................................................ 27 Research Hypothesis .............................................................................................. 27 Objective ................................................................................................................. 28 Methods and Materials ............................................................................................ 28

Supplements and Chemicals ............................................................................ 28 Preparation of Stock Solutions, Calibrator Standards, and Quality Control

Samples ........................................................................................................ 29 Study Participants ............................................................................................ 29 Supplement Administration ............................................................................... 30 Sample Collection and Processing ................................................................... 30

Analytical Methods .................................................................................................. 31 Analysis of Silybin A and Silybin B from Oral Dosage Forms ........................... 31 Analysis of Silybin A and Silybin B from Human Plasma .................................. 31 Pharmacokinetic Analysis ................................................................................. 32 Statistical Analysis ............................................................................................ 33

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3 RESULTS ............................................................................................................... 36

Calibration and Linearity ......................................................................................... 36 Precision and Accuracy .......................................................................................... 36 Formulation Analysis............................................................................................... 36 Study Participants ................................................................................................... 36 Pharmacokinetic Analysis ....................................................................................... 37

4 DISCUSSION ......................................................................................................... 50

LIST OF REFERENCES ............................................................................................... 54

BIOGRAPHICAL SKETCH ............................................................................................ 60

8

LIST OF TABLES

Table page 1-1 Definition of dietary ingredients .......................................................................... 24

1-2 Formulation comparison and content ................................................................. 25

3-1 Intra- and inter-run precision (CV, %) and accuracy (RE, %) for quality control (QC) samples in human plasma. ............................................................ 39

3-2 Comparison of silybin A and silybin B amounts in Product B and IsaGenesis .... 40

3-3 Pharmacokinetic parameters of silybin A and silybin B after single oral doses of Product B and IsaGenesis. ............................................................................. 41

3-4 P values of log-transformed Cmax and AUClast of silybin A and silybin B after single oral doses of Product B and IsaGenesis between male (n=6) versus female (n=6) subjects. ........................................................................................ 42

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LIST OF FIGURES

Figure page 1-1 Structures of silybin A and silybin B .................................................................... 26

2-1 Overview of a single-dose, randomized crossover pharmacokinetic assessment. CRC: Clinical Research Center. .................................................... 34

2-2 Representative ion chromatograms of two products from the study sponsor (Isagenix International, LLC) and one well-known milk thistle extract Legalon® (Madaus, GH) ..................................................................................... 35

3-1 Mean plasma concentrations of silybin A versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 43

3-2 Mean plasma concentrations of silybin B versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 44

3-3 Mean plasma concentrations of silybin (A and B) versus time profiles after oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects. ............................................................................................................. 45

3-4 Box plots of AUClast/Dose (h*ng/mL/mg) of silybin A. ......................................... 46

3-5 Box plots of Cmax/Dose (ng/mL/mg) of silybin A. ................................................. 47

3-6 Box plots of AUClast/Dose (h*ng/mL/mg) of silybin B. ......................................... 48

3-7 Box plots of Cmax/Dose (ng/mL/mg) of silybin B. ................................................. 49

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LIST OF ABBREVIATIONS

ALT Alanine aminotransferase

AST Aspartate aminotransferase

AUC Area under the concentration-time curve

AUCinf AUC from time 0 to infinity

AUClast AUC from time 0 to the last detectable concentration

BMI Body mass index

CAM Complementary and alternative medicine

Cmax The maximum plasma concentration

CRC Clinical Research Center

DSHEA Dietary Supplement Health and Education Act

EGCG Epigallocatechin-3-gallate

FDA Food and Drug Administration

GI Gastrointestinal

IS Internal standard

LC-MS/MS Liquid chromatography-tandem mass spectrometry

LLOQ Lower limit of quantitation

MTBE Methyl tert-butyl ether

PC Phosphatidylcholine

PE Pharmaceutical equivalents

PK Pharmacokinetic

SLNs Solid lipid nanoparticles

t1/2 Elimination half-life

Tmax Time to maximum plasma concentration

λz Terminal elimination rate constant

11

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Master of Science in Pharmacy

RELATIVE BIOAVAILABILITY OF TWO DIFFERENT FORMULATIONS OF MILK THISTLE

By

Wen-Yi Li

December 2018

Chair: Reginald F. Frye Co-chair: John S. Markowitz Major: Pharmaceutical Sciences

As the popularity of herbal dietary supplements has increased, more and more

multi-herb combination products have appeared on the dietary supplement market.

Since the oral bioavailability of many phytochemicals is low, various dosage forms, such

as nanoemulsion formulations and phosphatidylcholine-complexed softgel formulations,

are being developed to improve the bioavailability of bioactive constituents. However,

there is scant clinical research on the effect of formulation on the bioavailability of

bioconstituents in multi-herb supplements in human subjects.

In the current study, we compared the bioavailability between two milk thistle-

containing dietary supplements, Product B, a standard powder-filled capsule and

IsaGenesis, a softgel formulation, by evaluating silybin A and silybin B as surrogate

markers for differences in absorption and bioavailability. For this randomized, crossover

PK study, 12 healthy study participants consumed a single-dose serving (2 capsules) of

each supplement separated by a washout period of at least one-week. Serial blood

samples were obtained at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h time points and analyzed by

Liquid chromatography-tandem mass spectrometry (LC-MS/MS).

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Both formulations exhibited rapid absorption and elimination of silybin A and

silybin B. However, the absorption, as indicated by mean Cmax values and mean plasma

AUC, were significantly higher for the IsaGenesis formulation. Relative to powder-filled

Product B, the IsaGenesis softgel formulation demonstrated 365% and 450% higher

mean Cmax, for silybin A and silybin B, respectively. On average, Tmax was reached at

least 1 h earlier with IsaGenesis relative to Product B for both silybin A and silybin B.

Our findings indicate that the IsaGenesis softgel formulation provides markedly higher

absorption and bioavailability of silybin A and silybin B in comparison with the powder-

filled capsule formulation, Product B.

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CHAPTER 1 INTRODUCTION

Dietary Supplements

In the “Dietary Supplement Health and Education Act (DSHEA)” of 1994, a

dietary supplement represents a product (other than tobacco) intended to supplement

the diet that contains one or more dietary ingredients [1]. DSHEA not only provides

regulations for dietary supplements, but also officially defines dietary ingredients as

shown in Table 1-1.

Over the past 15 years, dietary supplement use has grown steadily in the United

States and across the globe [2]. Herbal supplement sales accounted for nearly 20% of

the total dietary supplement sales in the U.S., and annual retail sales of these products

in the U.S. exceeded $7 billion in 2016 [3]. In developed countries, herbal supplement

or herbal medicine is generally referred to as a complementary and alternative medicine

(CAM). New CAM products are not required to undergo formal review like conventional

drugs regulated by the US Food and Drug Administration (FDA) [4, 5]. Accordingly,

many herbal supplements lack rigorous scientific evaluation relative to their safety and

effectiveness. Though CAM is regulated as food, consumers often regard herbal

supplements as medications for disease prevention and healthy aging [6]. For reasons

including desire for wellness, fitness, and disease prevention, as well as the perception

that natural alternatives are safer than conventional drugs, a substantial number of

individuals turn to herbal supplements.

As the popularity of herbal dietary supplements has increased, many more multi-

herb combination products have appeared on the dietary supplement market. But herbal

supplements usually differ in content of phytoconstituents from batch to batch and

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between different manufacturers. In addition, even with standardized active

constituents, there may be differences in other components, leading to variations in

bioactivity and bioavailability in humans [7]. A multi-herb supplement is a complex

mixture, and it cannot be assumed that individual components within a multi-herb

supplement will exert exactly the same effect(s) as they do in single-herb supplements.

In 2007, the Final Rule for Current Good Manufacturing Practices for Dietary

Supplements issued by the FDA provides standards to ensure the identity, purity,

quality, strength, and composition of dietary supplements, but it does not address

minimum requirements for quality and the inherent safety of the ingredients contained in

dietary products. Since dietary supplements or CAM products are regulated as food,

there is no guarantee that manufacturers register their commercial herbal products

before marketing [8, 9]. However, most of the evidence of positive effects of dietary

phytochemicals is inferred from in vitro models and most of the assessments of

bioactive constituents are conducted in single-herb supplements [10-14].

The bioavailability of many phytochemicals is low after oral administration,

therefore, different dosage forms, such as nanoemulsion formulations and

phosphatidylcholine-complexed softgel formulations, are being used to enhance the

bioavailability of bioactive constituents [15, 16]. To date, there is scant clinical research

on the effect of different formulations on the bioavailability of phytoconstituents in multi-

herb supplements in humans. Accordingly, the purpose of this study is to bridge the

knowledge gap regarding the influence of formulation on the bioavailability of individual

component in a multi-herb supplement.

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Bioactivities of Milk Thistle in IsaGenesis and Product B

In the current study, a softgel formulation, IsaGenesis, and a standard dry,

powder-filled formulation, Product B, are investigated to determine the impact of the

formulation on the bioavailability of bioactive constituents in these multi-herb

supplements. IsaGenesis and Product B both contain milk thistle extract as the main

ingredient, and both are mixtures of herbal extracts, vitamins and amino acids purported

to promote healthy aging and reduce oxidative damage. Besides milk thistle, other

herbal ingredients, such as green tea, horny goat weed, turmeric, and ashwagandha,

are incorporated into these two multi-herb dietary supplements (Table 1-2).

The milk thistle fruit has been utilized to treat liver and gallbladder disorders

since ancient times [17]. The crude extract obtained from crushed seeds of the plant

Silybum marianum, is termed silymarin, and contains a complex mixture of bioactive

constituents, including approximately 70% silymarin flavonolignans. These constituents

include silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B, and

silydianin, and silymarin flavonoids, including taxifolin and quercetin. Silymarin has

several pharmacological effects, including antioxidant, anticancer, and hepatoprotective

activity [18]. Among silymarin flavonolignans, silybin (silybin A and silybin B; Figure 1-1)

is the predominant and the most active constituent of silymarin. Silybin possesses

diverse bioactivities: hepatoprotection, antioxidant, chemopreventive and

neuroprotective effects, and it has been advocated for the treatment of alcohol-

associated liver diseases [19-21]. In animal experiments, silybin has been reported to

prevent the oxidative toxicities of alcohol, acetaminophen, and phalloidin [22]. These

findings imply that silybin, the predominant active constituent of silymarin, is a potent

liver protectant and an effective antioxidant in therapeutic applications. Given the

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chemical complexity of milk thistle extracts, Kroll, et al. addressed the importance of

proper nomenclature [23]. In this study, silybin represents two silybin diastereomers

(silybin A and silybin B), while silibinin is a roughly 1:1 ratio of silybin A and silybin B.

Since less than 50% of orally administrated silymarin (based on silybin) was

absorbed from the gastrointestinal (GI) tract due to its poor solubility in water and oil,

the oral bioavailability of silibinin (nearly 1:1 ratio of silybin A and silybin B) was

reportedly only 0.73% in rats [24-26]. Conspicuous PK variability of silymarin among

clinical trial outcomes has been reported, and low oral bioavailability of silymarin likely

leads to the inconsistency in clinical outcomes. Consequently, the oral bioavailability of

milk thistle-containing formulations is mostly considered limited and highly variable [27-

29]. Regarding low bioavailability of silybin, there are several possible causes: low

solubility, low permeability, extensive phase II metabolism, and rapid excretion [30].

Thus, methods to enhance the bioavailability of silybin have been pursued as the

popularity of herbal dietary supplements has increased. These methods to increase

silybin bioavailability include phytosomes, softgel technology, nanoemulsions, and

liposomes [22, 31, 32].

Since the ingredients contained in IsaGenesis and Product B have known

antioxidant properties, it is possible that these two plant-based supplements could

decrease oxidative stress and increase catalase concentrations in human subjects.

Catalase is the first identified antioxidant enzyme involved in cell defense against

oxidative damage by hydrogen peroxide (H2O2) and is the oldest known enzyme to

control cellular redox homeostasis [33].

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Sweazea et al. led a double-blind, placebo-controlled study to assess whether

the plant-based nutraceutical (Product B) can improve antioxidant status in healthy

subjects after 12 weeks [34]. Although markers of glucose and lipid regulation and most

biomarkers for antioxidant capacity were not significantly different, plasma catalase

levels were significantly augmented in the treatment group (+6.1 nmol/min/mL) as

compared to the control group (-10.3 nmol/min/mL).

Methods to Improve Silybin Bioavailability

Phytosomes

Since most bioactive constituents of plants are poorly absorbed and have limited

permeability, the clinical utility of these constituents is severely restricted [35]. A

phytosome is a biocompatible complex technology incorporating phytoconstituents or

standardized herbal extracts into phospholipids [36]. It has been observed that

complexation with phospholipid, mainly phosphotidylcholine, can significantly improve

the absorption and bioavailability of phytoconstituents [37]. Phytosomes serve as an

effective drug delivery system for enhancing therapeutic index and also can represent a

potent targeting agent to transport the encapsulated bioactive compounds to specific

sites for improving drug development. This phospholipid complex technique can also be

applied for conventional dosage forms and is generally known as a pharmacosome [37].

Because a phytosome possesses some advantages including enhanced

absorption, improved stability, and a lower therapeutic dose, its use has been a

noteworthy trend in newer drug delivery systems for bioconstituents and plant extracts

[22]. Silybin and phosphatidylcholine (PC) could form a 1:2 molecular complex-

Siliphos®, and the phytosme form was 4.6-times more bioavailable than non-phytosomal

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silymarin in healthy subjects [31]. Another study conducted in rats by Morazzoni et al.

found that the bioavailability of silybin phytosome was 10-times greater than that of

silybin alone, probably due to the effect of PC facilitating silybin movement across the

GI mucosa [38]. Moreover, after oral administration of silybin phytosome in

cholecystectomy patients, the mean area under the curve (AUC) for silybin phytosome

was over 4-fold greater than the mean AUC for non-phytosomal silybin [39].

Yan-yu et al. conducted a PK study in rats to compare the PK characteristics and

oral bioavailability of silybin phytosome complex and silybin-N-methylglucamine [40].

The PK parameter estimates of phytosomal silybin and silybin-N-methylglucamine in

rats were Cmax 126.72 and 104.29 ng/mL; Tmax 10 and 5 min; AUC0-inf 1020.33 and

235.81 ng/mL/h, respectively. After oral administration of silybin-phospholipid complex,

the oral bioavailability of phytosomal silybin in rats was much better than that of silybin-

N-methylglucamine because of the improvement of the lipophilicity of silybin phytosome.

These results indicate that silybin combined with phospholipids may augment the oral

bioavailability of silybin and improve its delivery into the liver.

Softgel Technology

Savio et al. performed a randomized, single-dose, cross-over study on silybin

bioavailability in a hard shell capsule and a softgel capsule in which silybin was mixed in

a 1:2 ratio with phosphatidylcholine [32]. After subjects consumed a softgel capsule, the

PK analysis showed that the mean values of Cmax and AUClast increased more than two-

fold in comparison to a hard shell capsule. The outcomes demonstrated that the soft

gelatin capsule could improve the bioavailability of silybin, probably because an oily

medium formulated softgel capsule worked as a physiological carrier to help silybin

cross the intestinal wall.

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Nanoparticles

Micronization and nanonization are two approaches to increase drug

bioavailability by increasing surface area. Particle size could be reduced to 2-5 µm in

micronization; whereas, nanonization is the process of minimizing the particle size to

10-1000 nm [16, 41]. Using nanonization, poorly soluble compounds could be made into

nanoparticles for increasing the dissolution rate and solubility. Due to the bioavailability-

improving ability of nanoparticles, they have been applied in the treatment of different

diseases.

Zhang et al. prepared silibinin-solid lipid nanoparticles (SLNs) by using stearic

acid (biodegradable lipid) and Brij-78 (surfactant) for slow release of silibinin. The in

vitro release results indicated that there was almost no drug release from their SLNs in

three days and that the SLNs with stearic acid and Brij-78 may possess extended-

release ability to avoid drug loss on delivering silibinin to the targeted liver cell [42].

Jun et al. studied the influence of SLNs on the oral bioavailability of silymarin

(based on silybin) in rats. Using three different particle sizes (150, 500, and 1000 nm) to

evaluate the impact of particle size on oral absorption, they found that the oral

bioavailability of the smallest SLNs (150 nm) was significantly higher than the other

SLNs. The AUC of 150 nm SLNs was 2.1- and 2.5-times larger than that of 500 and

1000 nm SLNs, respectively [43]. They also investigated the biodistribution of two kinds

of SLNs prepared by a hot and cold homogenization method. Based on the outcomes

from in vitro release experiments, they chose SLNs produced by cold homogenization

(cold-SLNs) for further biodistribution studies in mice. Compared with the milled

silymarin powder suspension, the relative bioavailability of cold-SLNs was 2.8-fold

higher. Apart from the kidneys, the exposure of cold-SLNs was higher in other organs

20

than that of the suspension. These results showed that silymarin prepared as SLNs can

enhance the oral bioavailability of silymarin (based on silybin) and might be a promising

drug delivery system for silymarin [44].

Nanosuspensions

Nanotechnology, such as nanosuspensions, has been employed to increase the

solubility of poorly soluble phytochemicals. Nanosuspensions are defined as sub-micron

colloidal dispersion systems which consist of pure drug particles and surfactants or

stabilizers with a mean particle size between 10 and 1000 nm [45, 46]. Compared with

polymeric nanoparticles or liposomes, nanosuspensions possess 100% drug loading

[47]. Nanosuspensions demonstrate several benefits, including dissolution velocity

improvement, particle size reduction, and various administration routes [48]. Since poor

solubility and low oral bioavailability are the main obstacles with the application of

silybin, nanosuspensions have been used to address these problems in a few studies.

Zheng et al. evaluated the in vitro antitumor effect of a silybin nanosuspension on

human prostatic carcinoma PC-3 cell line compared with silybin solution formulation

[49]. Though silybin nanosuspension did not change the mechanism of silybin-induced

apoptosis, findings from this study indicated that silybin nanosuspension exerted higher

inhibition rates to PC-3 cells in comparison to silybin solution at the same concentration.

This phenomenon may be attributed to the silybin nanosuspension adhering to the

mucus layer to prolong biological activity and facilitating silybin transfer across the

epithelial cells.

Wang et al. performed in vitro and in vivo studies on the influence of silybin

nanosuspensions [50, 51]. They found that nanosuspensions had a significant impact

on drug transport across the Caco-2 cell monolayer and the enhanced permeability was

21

due to the increased dissolution rate. After oral administration of silybin

nanosuspensions in beagles, the in vivo results showed that the silybin suspensions

significantly improved silybin’s bioavailability in comparison to the powdered

formulations.

Furthermore, they investigated the influence of particle size (637 ± 9.4 nm vs.

132 ± 4.8 nm) on the in vivo distribution and hepatoprotection. After intravenous

administration, the larger particle size was favorably targeted at liver and spleen. After

intravenous or oral administration, the silybin nanosuspensions demonstrated significant

hepatoprotective effects by lowering the levels of alanine transaminase (ALT), aspartate

transaminase (AST), and total bilirubin. When compared to the carbon tetrachloride

(CCl4) treated control group, hepatoprotective effects of the two silybin

nanosuspensions were confirmed by histopathological examination. These outcomes

suggested that the silybin nanosuspension could be used to improve the therapeutic

efficacy and drug target delivery of the silybin, and that silybin nanosuspensions with

smaller particle size exhibited a higher potential to increase oral bioavailability of silybin.

Nanoemulsions

An emulsion is a dispersion system constructed of oil phase, water phase, and

surfactants; the droplet size of the emulsion affects its target distribution [52]. Generally,

a nanoemulsion represents a droplet size from 10 to 100 nm [53]. Due to low oral

bioavailability of silymarin (based on silybin), incorporation of silymarin (based on

silybin) into a nanoemulsion formulation administered orally was employed to enhance

its solubility, oral bioavailability and therapeutic effects. A nanoemulsion could be

regarded as a sustained release formulation because the bioactive compound is

wrapped in the inner phase and avoids direct contact with the body [54]. Incorporation

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of herbal medicines into a nanoemulsion will not only sustain the effect of drugs and

increase the stability of herbal constituents, it may also enhance the penetrability of

drugs to the skin or mucous membranes for topical administration.

Parveen et al. evaluated the hepatoprotection of the nanoemulsion formulation of

silymarin against CCl4-triggered hepatotoxicity [55]. Through biochemical parameters

and histopathological properties, such as ALT, AST and liver histopathology,

hepatoprotective activity was assessed in Wistar rats. As compared to standard

silymarin, the nanoemulsion-treated rats demonstrated significant increase in total

albumin, globulin, and glutathione in tissues and decreased pyruvate transaminase,

glutamate oxaloacetate transaminase, and tissue lipid peroxides. The findings indicated

that a nanoemulsion formulation of silymarin-treated rats reversed liver toxicity induced

by CCl4 more effectively as compared to standard silymarin-treated rats. Though a

nanoemulsion formulation of silymarin appeared to be a promising way to improve the

effectiveness of silymarin in hepatoprotection, the PK properties of silybin within the

nanoemulsion formulation were not addressed in the study.

Liposomes

Liposomes are self-assembled, spherical particles featuring a lipid bilayer

characterized by amphiphilic properties [56]. Since polar lipids construct liposomal

bilayers, hydrophilic drugs are aggregated in the water section and lipophilic

compounds are entrapped in the lipid section. Therefore, hydrophilic or lipophilic

compounds can be encapsulated into liposomes without changing their chemical

structures. Given the unique characters, liposomes are able to boost the bioactivities of

drugs by augmenting compound solubility, enhancing bioavailability, and increasing

intracellular uptake [57, 58]. As a drug delivery system, liposomes are physiologically

23

inert because of the natural phospholipid composition and able to enhance the

therapeutic index and safety of incorporated drugs by maintaining drug concentrations

for a long period and by carrying them to the target site.

Maheshwari et al. used the ethanol injection method to make liposomal vesicles

of silibinin [59]. The liposomal systems were investigated for hepatoprotective activity in

mice against CCl4-induced hepatotoxicity. The results of the in vivo studies showed

55.6% hepatoprotection by silibinin in liposomes in comparison to 33.1%

hepatoprotection by non-liposomal silibinin and 24.2% hepatoprotection by plain

liposomes. Owing to the phospholipid content, plain liposomes can demonstrate

hepatoprotective effects.

Yan-yu et al. compared the oral bioavailability and PK parameters of silymarin

proliposome with plain silymarin in beagles [60]. In this in vivo study, PK characteristics

of silybin within silymarin proliposome and plain silymarin were Cmax 472.62 and 89.78

ng/mL, Tmax both 30 min; and AUC0-inf 2606.21 and 697 ng/mL/h, respectively. Based on

the results, this research demonstrated that silymarin proliposome improved the oral

bioavailability of silybin and increased GI absorption of silybin in comparison to plain

silymarin.

24

Table 1-1. Definition of dietary ingredients Clause Description A a vitamin B a mineral C an herb or other botanical D an amino acid E a dietary substance for use by man to supplement the diet by increasing the

total dietary intake F a concentrate, metabolite, constituent, extract, or combination of any

ingredient described in clause A, B, C, D, or E

25

Table 1-2. Formulation comparison and content IsaGenesis (2 softgel capsules) Product B (2 capsules)

Ingredient Amount Ingredient Amount Milk Thistle (Silybum marianum PE 55% silymarin) 320 mg

Milk Thistle (Silybum marianum PE 80% silymarin) 250 mg

Green Tea (35% EGCG) 64 mg Green Tea (98% polyphenols) 35 mg Horny Goat Weed (5:1 PE) 60 mg Horny Goat Weed 30 mg Turmeric (95% curcuminoids) 64 mg Turmeric (95% curcuminoids) 20 mg Pomegranate Juice (4:1 PE) 30 mg Pomegranate (punica granatum PE) 29 mg Ashwagandha (Withania somnifera 4:1 PE) 27.5 mg

Ashwagandha (Withania somnifera 4:1 PE) 50 mg

Grape Seed (80% oligomeric proanthocyanidins) 20 mg

Grape Seed (80% oligomeric proanthocyanidins) 20 mg

Bacopa (Bacopa monnieri whole plant 25:1 PE) 10 mg Bacopa (Bacopa monnieri PE 50%) 9 mg Knotweed root 62 mg - R-α-Lipoic Acid 12 mg - Black Tea (5:1 PE) 4 mg Black Tea (PE 50% polyphenols) 4 mg White Tea (30% EGCG) 4 mg White Tea (PE 90% polyphenols) 4 mg Blueberry 18 mg Blueberry 7 mg Raspberry 10 mg Red Raspberry 7 mg N-Acetyl Cysteine 18 mg - L-Glutathione 10 mg - Quercetin 8 mg - Bilberry 14 mg Bilberry 11 mg

Panax Ginseng (25:1 PE) 10 mg Panax Ginseng (Root PE 80% ginsenosides) 12 mg

Mucuna Pruriens (4:1 PE) 6 mg - Harada (Terminalia chebula fruit) 8 mg Harada (Terminalia chebula fruit) 8 mg Boswellia (20:1 PE) 18 mg Boswellia (PE 65% boswellic acid) 7 mg Maca 6 mg Maca 7 mg Shilajit 6 mg Shilajit (20% fulvic acid) 5 mg - Resveratrol 28 mg Ascorbic Acid 13 mg Ascorbic Acid 7.5 mg Acacia Bark (12:1 PE) 4 mg - Goldthread (5:1 PE) 6 mg - d-α-tocopherol 5 IU - Vitamin B12 12 mg - Canola Lecithin 10 mg - Vitamin A 2 mg - Flaxseed Oil 250 mg - Lutein 1 mg - Zeaxanthin 0.33 mg - Phosphatidylcholine 200 mg - PE=Pharmaceutical Equivalents EGCG=epigallocatechin-3-gallate

26

Figure 1-1. Structures of silybin A and silybin B

27

CHAPTER 2 HYPOTHESIS

Existing Problem in Herbal Supplements

Though herbal supplement use has been an emerging trend over the last several

decades, there are few studies conducted in multi-herb supplements at this time [61,

62]. Soare et al. performed a randomized, single-blind controlled trial to evaluate the

metabolic and cardiovascular effects of consumption of 10 dietary supplements each

day over 6 months. The clinical study showed that endothelial function, blood pressure,

insulin and inflammatory cytokines in healthy subjects are not influenced after a 6-

month use of these supplements. The low oral bioavailability of phytochemicals

contained in these dietary supplements might be a likely explanation for the lack of

therapeutic effects. Since many bioactive phytochemicals demonstrate relatively low

oral bioavailability, numerous biopharmaceutical approaches, including complexation

with phospholipids and nanotechnology-based drug delivery systems, have been

employed to enhance the bioavailability of bioactive phytochemicals contained in dietary

supplements.

Research Hypothesis

Owing to low oral bioavailability of many bioactive phytochemicals, softgel

formulations are considered promising dosage forms to enhance bioavailability of poorly

soluble phytochemicals. A softgel formulation, IsaGenesis, and a standard dry-

powdered formulation, Product B, have been chosen for the present study. We

hypothesized that the softgel formulation, IsaGenesis, can augment the bioavailability of

the selected bioactive constituents, silybin A and silybin B.

28

Objective

Since milk thistle extract is the most predominant ingredient in these two

formulations chosen for study, and since silybin A and silybin B are the most abundant

flavonolignans of milk thistle extract, the two silybin diastereomers have been chosen as

surrogate PK biomarkers for elucidating differences in bioavailability with the respective

formulations. The purpose of this study is to test our hypothesis by evaluating the

relative bioavailability of silybin A and silybin B with the respective formulations in

healthy study participants.

Methods and Materials

Supplements and Chemicals

Two milk thistle-containing oral dosage forms, Product B (Lot #041403615) and

IsaGenesis (Lot #025400816), were provided by the study sponsor (Isagenix

International, LLC). Product B containing 125 mg milk thistle extract/capsule (80%

silymarin) was manufactured by Arizona Nutritional Supplements (Chandler, AZ),

whereas IsaGenesis containing 160 mg milk thistle extract/softgel capsule (55%

silymarin) was manufactured at Best Formulations (City of Industry, CA). Compared

with the powder-filled Product B, a notable pharmaceutical difference in IsaGenesis is

the incorporation of flaxseed oil and phosphatidylcholine to enhance the absorption and

bioavailability of phytochemicals.

Authentic analytical reference standards of silybin A and silybin B were

purchased from Phytolab GmbH & Co. (Vestenbergsgreuth, Germany). Naringenin, the

analytical internal standard (IS), was obtained from SAFC Supply Solutions (St. Louis,

MO). Liquid chromatography-mass spectrometry (LC-MS) grade ammonium acetate,

29

methanol, formic acid, and methyl tert-butyl ether (MTBE) were all purchased from

Sigma-Aldrich (St. Louis, MO).

Preparation of Stock Solutions, Calibrator Standards, and Quality Control Samples

Stock solutions of silybin A, silybin B and the IS naringenin were prepared in

methanol at concentrations of 1 mg/mL and were used to prepare calibration standards

and quality control (QC) samples. All stock and working solutions were stored at -20°C

until use. Calibrator solutions were prepared by adding the desired amount of working

solutions to 180 µL of blank human plasma. The calibrator concentrations were 1, 5, 10,

25, 50, 80, and 100 ng/mL plasma for silybin A and silybin B. The lower limit of

quantitation (LLOQ) was considered to be the lowest calibration standard concentration.

QC samples were similarly prepared at concentrations of 1 (LLOQ), 3, 15, and 75

ng/mL. All calibration standards and QCs were stored at -20°C until use.

Calibration standards over the concentration range of 1-100 ng/mL were

analyzed in duplicate for three runs. The standards were considered acceptable if both

the precision, expressed as the coefficient of variation (CV), and the accuracy,

presented as the percent relative error (RE), were within ± 15%. The LLOQ was

acceptable if the CV and RE were within ± 20%.

Study Participants

Twelve healthy participants (6 male and 6 female) were recruited in the study.

Each participant granted written IRB-approved informed consent and underwent a

health assessment. The inclusion criteria were healthy nonsmokers within the age

interval of 18 and 50 years, as well as body mass index (BMI) between 18.5 and 28

kg/m2. All participants were required to have no clinically significant diseases or history

30

of a medical condition. To avoid pregnancy during the entire protocol, female subjects

were required to have negative urine pregnancy test before enrollment and during study

participation. No prescription or over-the-counter medications, and no botanical,

nutritional supplements (vitamins and energy drinks) were allowed during study

participation.

Supplement Administration

Two study visits were scheduled and conducted at the UF Clinical Research

Center (CRC). The washout period was at least 7 days between the two visits (Figure 2-

1). Based on the randomization sequence, participants were administrated two capsules

of Product B or two capsules of IsaGenesis with 240 mL water at room temperature.

Depending on product labeling, two capsules represent a recommended serving of the

two respective formulations. After administration of each formulation, participants

remained fasting for an additional 4 hours to minimize any food effect on absorption. On

both study visits, subjects were provided the same meal to minimize any potential food

influence on these multi-herb supplements.

Sample Collection and Processing

Blood samples were collected via indwelling venous catheters and a total of nine

blood samples were obtained over an 8-hour period. Blood collection occurred

immediately prior to formulation administration (0 h) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8 hrs.

All samples were drawn in heparinized tubes (BD Vacutainer) and stored on ice until

centrifugation at 4°C. Because the stability of phytoconstituents varies within biological

matrices, the samples were not kept on ice for longer than 15 minutes before

centrifugation. Plasma samples were obtained by centrifugation of blood samples. The

resulting plasma was acidified with 10 µL of 1M acetic acid/mL plasma as a

31

preservative, transferred to labeled storage tubes, and frozen immediately at -70°C until

LC-MS/MS analysis.

Analytical Methods

Analysis of Silybin A and Silybin B from Oral Dosage Forms

A comparison of silybin A and silybin B content of the two respective formulations

was performed before the PK study. The content of two capsules (1 serving) of each

formulation was weighed, placed into a 10 mL volumetric flask, and dissolved in pure

analytical grade methanol. A 1 mL aliquot of the resulting solution was removed and

further diluted to an appropriate concentration suitable for LC-MS/MS analysis.

Analysis of Silybin A and Silybin B from Human Plasma

A high-performance liquid chromatographic tandem mass spectrometric assay

was utilized in our laboratory for simultaneously analyzing the free (nonconjugated)

flavonolignans: silybin A and silybin B. This method was mainly based on our previously

published method for simultaneously detecting flavonolignans in human plasma [29,

63].

In short, 200 µL of plasma, 100 ng/mL naringenin and 2 mL of MTBE were

added. The samples were shaken at 200 cycles/min for 10 min and then centrifuged at

3000 g for 10 min at room temperature. The organic phase was then transferred to

clean glass tubes. The supernatant was evaporated to dryness under a gentle stream of

nitrogen at 40°C, and the remaining residue was reconstituted with 100 µL of mobile

phase, 20 µL of which were injected for analysis.

The LC-MS/MS system consisted of a Shimadzu HPLC system (Shimadzu,

Tokyo, Japan) and an Applied Biosystems-Sciex API 3000 quadrupole mass

spectrometer (Foster City, CA, USA). The analytical column was Phenomenex Luna 5µ

32

C18 column (100A 250 mm x 2 mm, 5 μm, Torrance, CA) and C18 guard column (4 mm x

20 mm, SecurityGuard, Torrance, CA). The mobile phase was 50% methanol and 50%

water containing 0.1% formic acid and 10 mM ammonium acetate and delivered at a

flow rate of 0.25 mL/min. The MS was operated in negative ion mode using turbo

electrospray ionization. In the multiple reaction monitoring mode, the following

transitions were monitored: M/Z: 481.2→125.0 for silybin A and silybin B, and M/Z:

271.1→151.0 for naringenin (internal standard) in this method. Measurement of

individual silymarin constituents (i.e., silybin A and silybin B) was achieved by using the

following parameters: curtain gas, 8 psi; nebulizer gas (gas 1), 12 psi; CAD gas, 6 psi;

TurboIonSpray (IS) voltage, -4500 V; entrance potential, -10 V; collision cell exit

potential, -6 V; declustering potential, -80 V; collision energy, 40 eV for m/z: 481.2>125

and 26 eV for m/z: 271.1>151.0; source temperature, 400 °C; and dwell time, 200 ms.

Data was analyzed by AB Sciex Analyst software, version 1.4.2 (AB Sciex, Toronto,

Canada). Representative ion chromatograms of IsaGenesis and Product B from the

study sponsor (Isagenix International, LLC) and one well-known milk thistle extract

Legalon® (Madaus, GH) are shown in Figure 2-2.

Pharmacokinetic Analysis

Plasma concentrations of silybin A and silybin B were determined in 12 healthy

participants completing the entire protocol of a single dose, randomized crossover

clinical trial. The PK parameters for silybin A and silybin B were estimated by a non-

compartmental analysis using WinNonlin 6.1 (Pharsight, Mountain View, CA). The

maximum plasma concentration (Cmax) and time to maximum plasma concentration

(Tmax) were obtained directly from the plasma concentration-time data. According to the

linear trapezoidal rule, the area under the plasma concentration-time curve from time 0

33

to infinity (AUCinf) and AUC from time 0 to the last detectable concentration (AUClast)

were estimated. The terminal elimination rate constant (λz) was calculated by linear

least-squares regression of the terminal portion of the plasma concentration time curve,

and the elimination half-life (t1/2) was then estimated using the formula t1/2 = 0.693/ λz.

Regarding relative bioavailability, the following calculation: relative bioavailability (F) =

(AUClast /Dose) IsaGenesis / (AUClast /Dose) Product B *100%, was used to estimate relative

bioavailability between these two formulations.

Statistical Analysis

For this crossover trial, G*Power software 3.1 was used for a sample size

calculation [64]. Based a two-tailed paired t-test with alpha level of 0.05, in order to

detect an effect size of 1 with 80% power, a total sample size of 10 is needed.

Therefore, our sample size (twelve particpants) would provide enough power to identify

effect sizes that are smaller than one in PK parameter differences between Product B

and IsaGenesis. Results are presented as median and interquartile range for Tmax and

mean ± SD for other PK parameters. To evaluate potential differences in peak and

systemic exposure of silybin diastereomers, paired sample t-test is applied to assess

the log-transformed Cmax and AUClast of silybin A and silybin B after oral administration

of either Product B or IsaGenesis. Statistical analysis was performed by SAS 9.4 (Cary,

NC) and a P-value ≤ 0.05 is considered statistically significant.

34

Figure 2-1. Overview of a single-dose, randomized crossover pharmacokinetic

assessment. CRC: Clinical Research Center.

Single Dose Randomized Crossover Pharmacokinetic Assessment

Screening/ Informed Consent

28 Days CRC

(1 Day) Sample collection

CRC (1 Day)

Sample collection Wash-out (7 Days)

IsaGenesis (capsule x 2)

Product B (capsule x 2)

Product B (capsule x 2)

IsaGenesis (capsule x 2)

35

Figure 2-2. Representative ion chromatograms of two products from the study sponsor

(Isagenix International, LLC) and one well-known milk thistle extract Legalon® (Madaus, GH)

36

CHAPTER 3 RESULTS

Calibration and Linearity

Calibration curves determined by plotting the analyte-to-IS peak area ratio vs.

concentration were linear over the range of 1-100 ng/mL for silybin A and silybin B. The

range of concentrations was selected based on anticipated concentrations of analytes in

human plasma within the two study visits. The correlation coefficients (r2) from three

independent experiments were ≥ 0.99 for silybin A and silybin B.

Precision and Accuracy

The intra- and inter-run precision (CV, %) and accuracy (RE, %) assessed for

silybin A and silybin B are shown in Table 3-1. The estimated precision and accuracy

met the acceptance criteria for silybin A and silybin B.

Formulation Analysis

The results of Product B and IsaGenesis capsule analysis are shown in Table 3-

2. These values were used to correct overall silybin exposure to the research

participants. In Table 3-2, both formulations contain roughly equivalent amounts of

silybin A and silybin B.

Study Participants

Twelve healthy subjects (six males and six females) completed the entire

protocol, and the mean age and weight of these participants is 24.1 ± 3.7 years and

67.9 ± 11.6 kg (mean ± SD), respectively. All participants tolerated the two formulations

well; none of them experienced any unanticipated adverse events during the entire

clinical trial.

37

Pharmacokinetic Analysis

After a single oral dose of either Product B or IsaGenesis, the PK results showed

that the two surrogate markers, silybin A and silybin B, were rapidly absorbed and

eliminated. The corresponding PK parameter estimates of silybin A and silybin B

evaluated in this study are presented in Table 3-3. Figures 3-1 and 3-2 depict mean

plasma concentrations versus time profiles after oral administration of silybin A and

silybin B, respectively; mean plasma concentrations versus time profiles of silybin

(silybin A and silybin B) are presented in Figure 3-3.

Because the total amounts of silybin A and silybin B are unequal in Product B

and IsaGenesis, a dose correction was made by multiplying by a factor of 0.88 (176/200

based on different silymarin exposure per serving) to compare directly data with

IsaGenesis to the powdered formulation-Product B (Table 1-2). This adjustment was

made assuming the absorption and disposition of silybin A and silybin B are linear. To

provide further comparison between the two respective formulations, the mean plasma

concentrations versus time profiles after dose adjustment of IsaGenesis are presented

in Figures 3-1 to 3-3.

Over the 8-hour blood-sampling period, the silybin A and silybin B PK profiles

differed markedly between IsaGenesis and Product B, as depicted in Figures 3-1 and 3-

2. The IsaGenesis softgel formulation displayed a more rapid rise in plasma

concentration and achieved higher peak concentrations (Cmax) of silybin A and silybin B

over the first 6-hour period in comparison with Product B. Furthermore, IsaGenesis

manifested an earlier median time to maximum plasma concentration (Tmax). For silybin

A and silybin B, the Tmax values presented in Table 3-3 were 2 h for the IsaGenesis

softgel formulation versus 3 h for the Product B formulation. Although the content of

38

silybin is similar, the softgel formulation resulted in a 365% and 450% larger Cmax, for

silybin A and silybin B, respectively, when compared with the Product B formulation.

Additionally, the dose-normalized Cmax for silybin A was 1.17 ± 0.67 ng/mL/mg versus

0.32 ± 0.23 ng/mL/mg and for silybin B was 0.72 ± 0.57 ng/mL/mg versus 0.16 ± 0.14

ng/mL/mg, respectively for IsaGenesis versus Product B. The values of relative

bioavailability (F) shown in Table 3-3 demonstrate that absorption was higher with

IsaGenesis compared with Product B by 284% for silybin A and 334% for silybin B.

Regarding inter-individual differences and variability in concentration-time curves,

Figures 3-4 through 3-7 are box plots exemplifying the discrepancy in these two

formulations in connection with dose-normalized AUClast and Cmax of both silybin

isomers. The minimum, first quartile, median, third quartile, and maximum values of

AUClast per dose and Cmax per dose are shown in these box plots. Regarding intra-

individual differences, our study design did not permit an evaluation because each

volunteer received each formulation only once. Notably, participants who demonstrated

lower overall absorption and lower concentrations of silybin A and silybin B did so with

the two respective formulations indicating some physiologic differences in

gastrointestinal tract rather than formulation issues [65, 66].

In view of reported gender differences in silybin A and silybin B

pharmacokinetics, we found no statistically significant difference in Cmax and AUClast of

silybin A and silybin B between male and female participants in this study (P > 0.05;

Table 3-4). However, gender differences in the pharmacokinetics of silybin A and silybin

B cannot be determined or excluded given the limited sample size and large inter-

individual variability in the present study.

39

Table 3-1. Intra- and inter-run precision (CV, %) and accuracy (RE, %) for quality control (QC) samples in human plasma.

Analyte Nominal concentration (ng/mL)

Intra-run Inter-run Precision

(%) Accuracy

(%) Precision

(%) Accuracy

(%)

Silybin A 1 (LLOQ) 4.99 7.10 9.77 6.08

3 (Low QC) 2.48 1.28 6.46 -2.61

15 (Medium QC) 3.89 4.00 8.21 -0.22

75 (High QC) 3.47 5.56 10.75 0.09

Silybin B 1 (LLOQ) 2.92 11.67 12.82 7.93

3 (Low QC) 1.94 -0.17 3.41 -2.72

15 (Medium QC) 2.76 -0.22 5.43 -3.33

75 (High QC) 7.17 1.00 7.37 3.36

40

Table 3-2. Comparison of silybin A and silybin B amounts in Product B and IsaGenesis

Formulation Silybin A content

(mg) Silybin B content

(mg) Silybin A and B content

(mg) IsaGenesis 55.1 87.5 142.6 Product B 48.7 80.3 129.0

Each sample is taken as 1/10 from the mixture of two capsules (1 serving) and diluted (10,000-fold) to a concentration in LC-MS/MS detection range.

41

Table 3-3. Pharmacokinetic parameters of silybin A and silybin B after single oral doses of Product B and IsaGenesis.

Pharmacokinetic Parameter

Silybin A for Product B

Silybin A for IsaGenesis

Silybin B for Product B

Silybin B for IsaGenesis

Dose (mg) 48.7 55.1 80.3 87.5 Tmax (h) 3.0 (1.5-6) 2.0 (1.0-4.0) 3.0 (1.5-4.0) 2.0 (0.5-4.0) t1/2 (h) 1.98 ± 1.64 1.30 ± 0.32 1.82 ± 1.20 1.28 ± 0.34 Cmax (ng/mL) 15.7 ± 11.4 64.4 ± 37.1 12.9 ± 11.4 62.8 ± 49.8 Cmax/Dose (ng/mL/mg) 0.32 ± 0.23 1.17 ± 0.67 0.16 ± 0.14 0.72 ± 0.57

AUClast (ng×h/mL) 37.2 ± 30.3 118.8 ± 55.9 30.2 ± 31.6 111.0 ± 74.3

AUClast/Dose (ng×h/mL/mg) 0.76 ± 0.62 2.16 ± 1.01 0.38 ± 0.39 1.27 ± 0.85

AUCinf/Dose (ng×h/mL/mg) 0.96 ± 0.62 2.24 ± 1.04 0.52 ± 0.40 1.31 ± 0.84

F (IsaGenesis/Product B)* 284.21% 334.21%

The Tmax values are expressed as median (range) and others are mean ± S.D. (n=12). *Relative bioavailability (F) values in Table 3-2 of 284% (silybin A) and 334% (silybin B) represent the relative bioavailability of the respective silybin isomers in IsaGenesis compared with that in Product B.

42

Table 3-4. P values of log-transformed Cmax and AUClast of silybin A and silybin B after single oral doses of Product B and IsaGenesis between male (n=6) versus female (n=6) subjects.

Parameter Silybin A for Product B

Silybin A for IsaGenesis

Silybin B for Product B

Silybin B for IsaGnesis

log (Cmax) 0.30 0.55 0.40 0.43 log (AUClast) 0.53 0.54 0.60 0.48

43

Figure 3-1. Mean plasma concentrations of silybin A versus time profiles after oral

administration of single doses of Product B and IsaGenesis in 12 healthy subjects.

44

Figure 3-2. Mean plasma concentrations of silybin B versus time profiles after oral

administration of single doses of Product B and IsaGenesis in 12 healthy subjects.

45

Figure 3-3. Mean plasma concentrations of silybin (A and B) versus time profiles after

oral administration of single doses of Product B and IsaGenesis in 12 healthy subjects.

46

Figure 3-4. Box plots of AUClast/Dose (h*ng/mL/mg) of silybin A.

47

Figure 3-5. Box plots of Cmax/Dose (ng/mL/mg) of silybin A.

48

Figure 3-6. Box plots of AUClast/Dose (h*ng/mL/mg) of silybin B.

49

Figure 3-7. Box plots of Cmax/Dose (ng/mL/mg) of silybin B.

50

CHAPTER 4 DISCUSSION

To evaluate biological activities and to interpret prospective health-promoting

benefits of dietary supplements in humans, it is crucial to know the bioavailability of

phytochemicals administrated as dietary supplements. This information is important to

discover whether promising in vitro findings are likely to be achieved in vivo based on

relative exposure. In other words, the putative bioconstituents must be sufficiently

penetrated or delivered to their target site of biological activity.

To date, a comprehensive evaluation of biodisposition is lacking for most dietary

supplements and relatively little clinical PK research has been conducted. In view of the

fact that significant PK differences among many milk thistle-containing supplements

have been reported, it is essential to perform a comparative PK study before marketing.

Additionally, the bioavailability of most oral formulations is usually considered low and

highly inconsistent [29]. Major reasons for the poor bioavailability of silymarin

constituents include low aqueous solubility, extensive pre-systemic metabolism, and

poor permeability across intestinal cells.

In order to compare the pharmacokinetics of two multi-herb dietary supplement

formulations, a single-dose crossover PK study was completed. Based on the

randomization sequence, participants were administered either two capsules of Product

B or two softgel capsules of IsaGenesis on alternate clinic visits. In the present study,

two flavonolignans of silymarin, silybin A and silybin B, served as surrogate PK

biomarkers for characterizing the absorption and relative bioavailability of these two

formulations. As with previous clinical PK studies on silybin, the peak concentration

(Cmax) and systemic exposure (AUCinf) were significantly higher for silybin A compared

51

with silybin B [27, 29]. The observed variability results from the known stereoselective

metabolism of silybin isomers with faster metabolism of silybin B [67].

The PK evaluation suggested that on average, silybin A and silybin B were

rapidly absorbed and eliminated with the two respective formulations. The absorption of

silybin A and silybin B was greater with IsaGenesis compared with Product B. Indeed,

after dose normalization of silybin A and silybin B, the IsaGenesis formulation exhibited

365% and 450% higher mean Cmax values, respectively, compared with the Product B

formulation. Moreover, the Cmax values of IsaGenesis for silybin A fitted the range of

values from our previous investigation on a well characterized silymarin formulation [29].

Regarding Tmax, both silybin A and silybin B achieved the Cmax at least 1 hour earlier

with IsaGenesis compared with Product B (Table 3-3). The estimated t1/2 of silybin A

and silybin B appeared to differ between the two formulations; both silybin A and silybin

B have a shorter t1/2 with the IsaGenesis formulation compared with the Product B

formulation (Table 3-3), the reasons for which are unclear.

Although the systemic concentrations of silybin A and silybin B achieved after

oral administration are relatively low compared to the in vitro concentrations typically

used to assess biological activities, there remains the possibility that these silybins may

exhibit their bioactivities at relatively low systemic concentrations.

Additionally, inter-subject PK differences were observed with both formulations.

In view of box plots in Figures 3-4 to 3-7, there seems to be a greater amount of inter-

individual variability in the silybins with the IsaGenesis formulation.

There are a few limitations to the current study. First, the respective milk thistle-

containing supplements were not single herbal extract formulations because they

52

included multiple herbal constituents from different plant sources. Secondly, there is no

potential effect of concomitant drugs on the results because of exclusion criteria for

participants. Thirdly, the two dosage forms did not contain the same amounts of

silymarin content (silybin A and silybin B) as well as other botanical extracts. But, the

uneven amounts of silybin A and silybin B administered in IsaGenesis and Product B

were normalized by dose for PK comparisons.

Regarding the pharmacokinetics of the silybins administered as part of a multi-

herb supplement in humans, there is scant research at this time and the possible

influence of other included bioconstituents on the disposition of silybin A and silybin B is

still an open question. The potential of synergistic or competitive effects on absorption

and metabolism among various bioconstituents administered as part of multi-herb

formulation versus the administration of a single extract or extract component from a

natural product has been documented [61, 68, 69]. Concerning silybin A and silybin B

systemic exposure, the IsaGenesis formulation obviously improved the absorption of the

two silybin isomers, however, it must be emphasized that these findings cannot simply

be projected to other constituents incorporated into these multi-herb supplements

without directly assaying plasma for representative bioactive constituents.

In the present study, flaxseed oil and phosphatidylcholine were excipients of

IsaGenesis but not Product B. As an n-3 fatty acid source, flaxseed oil was a potential

diluent for the fat-soluble bioconstituents but was not included with the particular

intention to improve bioavailability. With respect to the phosphatidylcholine content, the

IsaGenesis softgel formulation was not the same as a silybin-phosphatidylcholine

complex for some primary silymarin formulations used in previous studies [70].

53

However, recognizing the plenitude of phosphatidylcholine in cellular membranes and

the established role of phospholipids as excipients for different pharmaceutical

applications including as emulsifiers and solubilizers, its inclusion through a micro-

coating manufacturing process may enhance the absorption of lipophilic bioconstituents

[30]. Although IsaGenesis appeared to provide greater overall exposure to silybin A and

silybin B, whether this resulted from phosphatidylcholine inclusion remains speculative

at this time and requires further study.

Comprehensively, the IsaGenesis softgel formulation showed greater overall

systemic exposure to silybin A and silybin B compared with the Product B formulation.

Further study is needed to characterize the pharmacokinetics of other phytoconstituents

(e.g., withaferin A, withanolide A, bacopaside I, and icariin) in IsaGenesis and to

investigate the potential herb-drug interactions of taking a multi-herb supplement in

healthy subjects.

54

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BIOGRAPHICAL SKETCH

Wen-Yi Li was born in Taiwan. He received his master’s degree from the

University of Florida in the fall of 2018. Before he joined the clinical pharmaceutical

graduate program in the Department of Pharmacotherapy and Translational Research

at the College of Pharmacy in 2015, he received his master’s degree in pharmaceutical

sciences at National Defense Medical Center and then worked as a pharmacist and an

adjunct lecturer for several years.