Pharmacokinetics, Tissue Distribution, Synergistic Activity, and Antitumor Activity of Two Isomeric

103 Pages·2016·5.1 MB·English

East Tennessee State University
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Tennessee State University
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12-2016
Pharmacokinetics, Tissue Distribution, Synergistic
Activity, and Antitumor Activity of Two Isomeric
Flavones
Crystal L. Whitted
East Tennessee State University
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Whitted, Crystal L., "Pharmacokinetics, Tissue Distribution, Synergistic Activity, and Antitumor Activity of Two Isomeric Flavones"
(2016).Electronic Theses and Dissertations.Paper 3167. https://dc.etsu.edu/etd/3167
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Pharmacokinetics, Tissue Distribution, Synergistic Activity, and Antitumor Activity of Two
Isomeric Flavones
_____________________
A dissertation
presented to
the faculty of the Department of Pharmacology
East Tennessee State University
In partial fulfillment
of the requirements for the degree
Doctor of Philosophy in Biomedical Science Pharmaceutical Science Concentration
_____________________
by
Crystal L. Whitted
December 2016
_____________________
Dr. Victoria Palau, Chair
Dr. Sam Harirforoosh
Dr. Zac Walls
Dr. Gary Wright
Dr. Koyamangalath Krishnan

Keywords: Flavone A, Flavone B, Pharmacokinetic, Colon cancer, Anticancer
ABSTRACT
Pharmacokinetics, Tissue Distribution, Synergistic Activity, and Antitumor Activity of Two
Isomeric Flavones
by
Crystal Whitted
Flavonoids are polyphenolic secondary metabolites found in plants that have bioactive properties
including antiviral, antioxidant, and anticancer. Two isomeric flavones were extracted from
Gnaphalium elegans and Achyrocline bogotensis, plants used by the people from the Andean
region of South America as remedies for cancer. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-
chromen-4-one (5, 7–dihydroxy- 3, 6, 8 trimethoxy flavone/ flavone A) and 3,5-dihydroxy-6,7,8-
trimethoxy-2-phenyl-4H-chromen-4-one (3, 5–dihydroxy-6, 7, 8–trimethoxy flavone/ flavone B)
have shown antineoplastic activity against colon cancer cell lines dependent upon their
differentiation status. Pharmacokinetic studies reported herein were used to determine dosing for
antitumor assays, as well as determine target tissue concentration. These included the
development of methods to extract the flavones from plasma or colon tissue and reverse phase
high performance liquid chromatography methods for quantification. Quantification methods
were linear (r2 e 0.99) with plasma calibration curves ranging from 250 - 2,500 ng/mL and 2,500
- 100,000 ng/mL for both flavones and colon calibration curves ranging from 250 – 100,000 ng/g
(flavone A) and 1,000-25,000 ng/g (flavone B). Intravenous administration of a 20 mg/kg dose in
rats yielded half-lives of 83.68 ± 56.61 and 107.45 ± 53.31 minutes with clearance values of
12.99 ± 13.78 and 80.79 ± 35.06 mL/min/kg for flavones A and B, respectively. Analysis of
colon tissue yielded concentrations of 1639 ± 601 ng/g (flavone A) and 5975 ± 2480 ng/g
(flavone B), suggesting both may be good candidate for individual or adjunct therapy for colon
2
cancer due to distribution to the target tissue. Preliminary studies in colon cancer cells CaCo 2
and HCT 116 using either flavone in combination with 5-fluorouracil (5-FU) suggested
synergistic activity of these compounds. The combination treatment increased induction of
apoptosis by enhancing the DNA damaging mechanism of 5-FU. In vivo, preliminary xenograft
experiments using HCT 116 cells showed smaller tumors in mice dosed with flavone B as
compared to the 5-FU or combination treatment. Further experiments are warranted to confirm
these observations.

3
TABLE OF CONTENTS
Page
ABSTRACT ....................................................................................................................................2
LIST OF TABLES ..........................................................................................................................8
LIST OF FIGURES ........................................................................................................................9

Chapter
1. INTRODUCTION ....................................................................................................................11
2. DEVELOPMENT OF REVERSED-PHASE HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY METHODS FOR QUANTIFICATION OF TWO ISOMERIC
FLAVONES AND THE APPLICATION OF THE METHODS TO PHARMACOKINETIC
STUDIES IN RATS .......................................................................................................................21
Abstract ............................................................................................................................21
1. Introduction ...................................................................................................................22
2. Experiments ..................................................................................................................23
2.1 Chemicals and standards ..................................................................................23
2.2 Stock solution and standards ...........................................................................32
2.3 Sample preparation .........................................................................................24
2.4 HPLC conditions and quantification ...............................................................24
2.5 Accuracy and precision ...................................................................................25
2.6 Recovery .........................................................................................................25
2.7 Stability ...........................................................................................................26
2.8 Animals and drug administration ....................................................................26
2.9 Pharmacokinetic analysis ................................................................................26
3. Results ...........................................................................................................................27
3.1 Specificity ........................................................................................................27
4
3.2 Linearity, lower limit, and precision ...............................................................30
3.3 Recovery .........................................................................................................35
3.4 Stability ...........................................................................................................36
3.5 Pharmacokinetics of flavone A and B ............................................................36
4. Discussion .....................................................................................................................39
5. Conclusion ....................................................................................................................40
Conflict of interest ............................................................................................................40
Acknowledgements ...........................................................................................................40
References .........................................................................................................................41
3. QUANTIFICATION OF TWO ISOMERIC FLAVONES IN RAT COLON TISSUE
USING REVERSE PHASE HIGH PERFORANCE LIQUID CHROMATOGRAPGHY ...........43
Introduction 43
Methods and results ..........................................................................................................44
Procedure to extract and purify flavones A and B ................................................44
Stock solution and standards .................................................................................44
Sample preparation ...............................................................................................44
HPLC conditions and quantitation ........................................................................45
Precision and accuracy ..........................................................................................48
Animals and drug administration ..........................................................................49
Discussion .........................................................................................................................50
Reference ..........................................................................................................................51
4. THE EFFECT OF TWO ISOMERIC FLAVONES, 5-FLUOROURACIL, AND
COADMINISTRATION AGAINST COLON CANCER ............................................................52
Introduction .......................................................................................................................52
Methods .............................................................................................................................54
5
Chemicals ..................................................................................................54
Cell culture ................................................................................................55
Cell viability assay ....................................................................................55
Apoptosis assay .........................................................................................56
Cell cycle assay .........................................................................................56
Comet assay ..............................................................................................57
Antibodies .................................................................................................57
Immunoblot ...............................................................................................58
Immunofluorescence .................................................................................58
Animal and drug administration ...............................................................59
Results ...............................................................................................................................60
Flavone A and flavone B have synergistic activity when used in
combination with 5-fluorouracil ...............................................................60
Combination treatment increases induction of apoptosis in CaCo 2 cells 61
Combination treatment increases induction of apoptosis in HCT 116
Cells ..........................................................................................................64
5-fluorouracil and the combination treatment increases cells cycle
shift to S phase ..........................................................................................66
The addition of flavone A or flavone B to 5-fluorouracil treatment causes
an increase induction of DNA damage .....................................................67
Flavone A and B enhances the mechanism of action of 5-fluorourail in
colon cancer cells ......................................................................................70
Both flavone A and flavone B increase cleavage of Caspase 3 when
combined with 5-fluorouracil ...................................................................75
6
5-fluorouracil and combination treatment resulted in large fluid filled
tumors .......................................................................................................77
Discussion .........................................................................................................................79
References .........................................................................................................................86
5. DISSCUSION ..........................................................................................................................89
REFERENCES .............................................................................................................................93
APPENDIX: Copyright ..............................................................................................................100
VITA ...........................................................................................................................................101

7
LIST OF TABLES
Table Page
2.1. Intra-day and inter-day precision and accuracy for 2500-100000 ng/mL flavone A
extracted from rat plasma ......................................................................................................33
2.2. Intra-day and inter-day precision and accuracy for 250-2500 ng/mL flavone A
extracted from rat plasma ......................................................................................................34
2.3. Intra-day and inter-day precision and accuracy for 2500-100000 ng/mL flavone B
extracted from rat plasma ......................................................................................................34
2.4. Intra-day and inter-day precision and accuracy for 250-2500 ng/mL flavone B extracted
from rat plasma ..................................................................................................................... 35
2.5. Recovery of flavone A extracted from rat plasma ................................................................ 35
2.6. Recovery of flavone B extracted from rat plasma ................................................................ 36
2.7. Stability of flavone A and flavone B in rat plasma after freeze/thaw cycle ......................... 36
2.8. Pharmacokinetic parameters of flavone A after administration of a 20 mg/kg dose
via intravenous injection .......................................................................................................38
2.9. Pharmacokinetic parameters of flavone B after administration of a 20 mg/kg dose via
intravenous injection .............................................................................................................38
3.1. Precision and accuracy for 250-100000 ng/g flavone A extracted from rat colon
tissue ......................................................................................................................................48
3.2. Precision and accuracy for 1000-25000 ng/g flavone B extracted from rat
colon tissue ............................................................................................................................49

8
LIST OF FIGURES
Figure Page
1.1.
9