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Preformulation and Topical Penetration Studies
Item Type text; Electronic Dissertation
Authors Aodah, Alhassan
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/624589
PREFORMULATION AND TOPICAL PENETRATION STUDIES
by
Alhassan Hamood Aodah
_________________________________________________
Copyright © Alhassan Hamood Aodah 2017
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF PHARMACEUTICAL SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2017
2
THE UNIVERSITY OF ARIZONA
GRADUATE COLLEGE
As members of the Dissertation Committee, we certify that we have read the dissertation
prepared by Alhassan Hamood Aodah, titled Preformulation and Topical Penetration
Studies and recommend that it be accepted as fulfilling the dissertation requirement for the
Degree of Doctor of Philosophy.
______________________________________________________Date: (April 18, 2017)
Dr. Paul B. Myrdal
_____________________________________________________ Date: (April 18, 2017)
Dr. Samuel H. Yalkowsky
______________________________________________________Date: (April 18, 2017)
Dr. Michael Mayersohn
______________________________________________________Date: (April 18, 2017)
Dr. Heidi M. Mansour
Final approval and acceptance of this dissertation is contingent upon the candidate’s
submission of the final copies of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and
recommend that it be accepted as fulfilling the dissertation requirement.
________________________________________________ Date: (April 18, 2017)
Dissertation Director: Dr. Paul B. Myrdal
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an
advanced degree at The University of Arizona and is deposited in the University Library
to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided
that accurate acknowledgment of source is made. Requests for permission for extended
quotation form or reproduction of this manuscript in whole or in part may be granted by
the head of the major department or the Dean of the Graduate College when in his or her
judgment the proposed use of the material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED: Alhassan Hamood Aodah
3
4
ACKNOWLEDGMENTS
I would like to thank my advisor, Dr. Paul B. Myrdal, for providing the opportunity that
allowed me to get the degree of doctor of philosophy in Pharmaceutical sciences. I am
grateful to him for all of his time, effort, advices and chances he provided to me while
helping me develop my scientific thought and knowledge.
I would like to thank my committee members, Dr. Samuel Yalkowsky, Dr. Michael
Mayersohn, Dr. Heidi Mansour for their scientific advises and directions. I would like to
thank all my friends and colleagues in the department for their support and motivations.
I am grateful to my father Dr. Hamood Aodah, my mother Mrs. Aziza, brothers and
sisters for supporting and encouraging me. I am practically grateful to my wife, Basma
Ezzi and my kids Rand, Faris, Jood and Roaa for strong support and patience which
withstand all obstacles I met.
5
DEDICATION
To my parents and my family.
6
TABLE OF CONTENTS
page
List of figures…………………………………………………………………… 9
List of tables …………………………………………………………………… 11
ABSTRACT 12
CHAPTER 1 PREFORMULATION STUDIES ON PIPERLONGUMINE….... 15
Introduction………………………………………………………………………. 15
Materials…………………………………………………………………………. 17
Methods…………………………………………………………………………... 18
1- HPLC analysis. ………………………………………………………… 18
2- Solubility studies. ………………………………………………………... 18
3- Partitioning studies……………………………………………………… 19
4- Solid state characterizations. …………………………………………...... 19
5- Aqueous stability studies. …………………………………………….. 20
6- Photo-stability. …………………………………………………………... 20
7- Additional stability studies. ……………………………………………... 21
Results and Discussion…………………………………………………………... 22
1- Chromatography…………………………………………………………. 22
2- Solubility studies………………………………………………………… 24
3- Partitioning study………………………………………………………… 27
4- Solid-state characterization………………...……….……….…….……... 27
5- Stability studies………………………………………………………….. 30
6- Photo-stability……………………………………………………………. 37
7- Stability with antioxidants……………………………………………….. 38
Conclusion……………………………………………………………………….. 40
CHAPTER 2 PREFORMULATION AND EVALUATION OF RESATORVID
FOR TOPICAL DELIVERY …………………………………...
42
Introduction……………………………………………………………………... 42
Materials ……………………………………………………………………... 44
7
Methods ………………………………………………………………………... 44
1- UV spectrum …………………………………………………………...... 44
2- HPLC analysis ………………………………………………………....... 45
3- Melting point determination …………………………………………….. 45
4- Solubility studies………………………………………………………..... 45
5- Photo-stability…………………………………………………………..... 46
6- Chemical stability studies ……………………………………………….. 47
7- Ex- vivo penetration studies…………………………………………….... 47
8- Drug extraction method development…………………………………..... 49
9- In-vivo penetration studies……………………………………………….. 51
Results and discussion………………………………………………………….. 52
1- UV spectrum……………………………………………………………... 52
2- Chromatography……………………………………………………...…... 54
3- Solid state characterizations……………………………………………... 56
4- Solubility studies…………………………………………………………. 58
5- Photo-stability……………………………………………………………. 60
6- Degradation as a function of pH and temperature……………………...... 60
7- Ex- vivo penetration studies……………………………………………... 64
8- In- vivo penetration studies…………………………………………..…... 68
Conclusion……………………………………………………………………….. 71
CHAPTER 3 CORRELATION OF DRUG PENETRATION BETWEEN
STRAT-MTM MEMBRANES AND MURINE SKIN FOR VARIOUS DRUGS.
72
Introduction……………………………………………………………………..... 72
Materials………………………………………………………………………...... 75
Methods…………………………………………………………………………... 75
1- HPLC method development……………………………………………... 75
2- Method development for drug extraction from Strat-MTM and skin. ……. 77
3- Strat-MTM membrane and mice skin preparation……………………….... 78
4- Strat-MTM membrane washing and drug extraction method…………....... 79
5- Skin segment stripping and drug extraction……………………………… 79
6- Calculations………………………………………………………………. 80
8
o Drug depletion from the donor…………………………………... 80
o Flux……………………………………………………………... 80
Results and discussion 83
1- HPLC method development…… ………………………………………... 83
2- Drug extraction method development from Strat-MTM membrane: …... 86
3- Drug extraction method development from murine skin………………… 87
4- Penetration studies……………………………………………………...... 88
o Drug distribution………………………………………………..... 100
o Interesting correlations………………………………………........ 106
Conclusion………………. ……………………………………………………… 109
References………………………………………………………………………... 110
9
LIST OF FIGURES
page
Figure 1.1 The chemical structure for piperlongumine. ………………………… 15
Figure 1.2 HPLC chromatograms for the separation of piperlongumine (peak at
6.9 minutes) in different stability conditions. (a) Sample at pH 8. (b) Sample at
pH 5. (c) Sample at pH 3. (d) Photo-stability sample………………………....
23
Figure 1.3 Solubility of piperlongumine in different surfactant and complexing
systems…………………………………………………………………………
26
Figure 1.4 Solubility of piperlongumine in different cosolvent systems………... 26
Figure 1.5 Photomicrographs of piperlongumine (400X). (a) Original crystals,
(b) Crystalline precipitant. ………………………………………………………
27
Figure 1.6 DSC thermogram of piperlongumine………………………………. 29
Figure 1.7 Pattern of X-ray diffraction of piperlongumine crystals……………... 29
Figure 1.8 Degradation of piperlongumine as a function of pH at 56°C………... 31
Figure 1.9 Arrhenius plots for piperlongumine degradation at different pH
values. ……………………………………………………………………………
31
Figure 1.10. Piperlongumine pH degradation rate profiles. All samples were at
an ionic strength of ~0.2 M. Buffer types are: citrate for pH 3 and 4, phosphate
for pH 5, 6 and 7, borate for pH 8 and 9………………………………………….
34
Figure 1.11. Effect of ionic strength (IS) on stability of piperlongumine at pH 3,
5 and 9 at 26°C…………………………………………………………………...
36
Figure 1.12. Major degradation products of piperlongumine……………………. 36
Figure 1.13. Stability of piperlongumine with the addition of different
excipients. (a) pH 7 at 26°C. (b) pH 3 at 56°C………………………………….
39
Figure 2.1 Resatorvid chemical structure………………………………………... 44
Figure 2.2 Franz cell components……………………………………………... 48
Figure 2.3 Resatorvid UV absorbance intensity, light spectrum wavelengths … 53
Figure 2.4. Chromatogram of (a) resatorvid, (b) the chromatogram of blank
adhesive tape, (c) the chromatogram of control murine skin extract.….…………
55
Figure 2.5 DSC thermogram for resatorvid……………………………………… 57
10
Figure 2.6 Photomicrographs of resatorvid raw material (400 X). ……………… 57
Figure 2.7 Resatorvid pH-solubility profile. …………………………………... 59
Figure 2.8 Comparison of resatorvid solubility in a different cosolvents systems 59
Figure 2.9 Degradation of resatorvid over 17 days as a function of pH at 25, 48
and 65°C………………………………………………………………………...
61
Figure 2.10 Arrhenius plots for resatorvid degradation at different pH value…... 63
Figure 2.11 Resatorvid pH degradation rate profiles. …………………………... 63
Figure 2.12 Flux of resatorvid through mice skin using acetone and PBS
formulations (a), drug percentage of distribution into Franz cell (b). …………...
67
Figure 2.13 Resatorvid dermal concentration after single dose application on
mice. ……………………………………………..……………………………...
70
Figure 3.1: Illustration of skin stripping procedure……………………………... 78
Figure 3.3: Illustration curve for donor drug depletion calculation……………… 82
Figure 3.4: Illustration curve for flux calculation ……...………………………... 82
Figure 3.5: Curves of drug distribution during 8 hours of in-vitro release using
Franz penetration cells with Strat-MTM membrane and murine skin………….
103
Figure 3.6: Correlation of Strat-MTM membrane and hairless murine skin. … 107
Figure 3.7: Correlation of percent of drug dose deposition between murine skin
and Strat-MTM membrane for (a) all studied drugs, and (b) with excluding
M-PABA. ………………………...………………......................................
108
11
LIST OF TABLES
page
Table 1.1 Summary of stability results…………………………………………. 33
Table 1.2 Summary of photo-stability studies of piperlongumine……………... 37
Table 1.3. Stability comparisons for piperlongumine with different excipients... 40
Table 2.1: Results of extraction method development of resatorvid from (a)
adhesive tape, and (b) skin.…………………………………………………...
50
Table 2.2: Summary of resatorvid light stability………………………… 60
Table 2.3: Results of in-vitro penetration study for resatorvid, acetone and PBS
formulations. ………………………………………………………………...
66
Table 2.4: Summary of resatorvid flux study. ………………………………....... 68
Table 2.5: Resatorvid distribution after topical application on hairless mice of
dose (241.21 ug/ spot). ………………………………………………….............
69
Table 3.1 Chemical structures and physiochemical properties for the compounds
used in this study…………………………………………………....
74
Table 3.2 Summary of HPLC methodology. ……………………………………. 76
Table 3.3 Separation HPLC chromatograms of selected drugs, blank Strat-MTM
membrane and blank murine skin. …………………………...…………...
85
Table 3.4 Summary of extraction method development from Start M membrane. 86
Table 3.5 Summary of extraction method development from murine skin…….. 87
Tables 3.6 Penetration studies of resatorvid, M-PABA, diclofenac sodium,
salicylic acid and hydrocortisone…………………………………………………
99
Table 3.7 Total percent of recovery of drugs in each Franz cell, where A, B and
C represent individual experiments…………………………………...………….
100
Table 3.8 Summary of drug depletion curves slope from donor chamber and
corresponded percentages of drug retention ……………………………………..
104
Table 3.9 Summary of data from penetration studies.……………………….... 105
12
ABSTRACT
Chapter I: preformulation studies on piperlongumine
Piperlongumine is a natural alkaloid extracted from piper plant which has been used
traditionally for the treatment of certain diseases. This compound shows interesting in-vitro
pharmacological activity such as selective anticancer activity and higher cytotoxicity than
methotrexate, cyclophosphamide and adriamycin on breast, colon, and osteosarcoma
cancers, respectively. However, the physicochemical properties of this compound have not
been well characterized. In this research, preformulation studies for piperlongumine have
been performed to determine factors which influence solubility and stability which, in turn,
can be used to assist future formulation development. The solubility of piperlongumine in
water was found to be approximately 26 μg/ml. Using 10% polysorbate 80 as a surfactant
resulted in a 27 fold increase in solubility. Cosolvents and cyclodextrins afforded
concentrations of 1 mg/ml and higher. The pH degradation rate profile for piperlongumine
at various temperatures shows significant instability of the drug at pH values 7 and 3, and
maximum stability around pH 4. It was estimated that it would take approximately 17
weeks for piperlongumine to degrade by 10% at 25°C, pH 4. Additionally, piperlongumine
showed marked photo-degradation upon exposure to an ultraviolet light source, especially
in aqueous media.
Chapter II: preformulation and evaluation of resatorvid topical delivery
Resatorvid is a small molecule shows interesting anti- inflammatory biological activity.
The clinical trial was conducted for sepsis-induced cardiovascular and respiratory failure,
but it was terminated due to low efficacy. Further researches show a different biological
13
activity of resatorvid such as its activity against UV-induced skin cancer. The goal of this
study is to determine some important physiochemical properties of resatorvid, as well as
intrinsic penetration criteria through the murine skin, either ex-vivo or in-vivo.
Intrinsic water solubility of resatorvid was found to be 95.32± 1.75 ug/ml and could be
duplicated by using 10 % ethanol as cosolvent. The pH solubility profile shows the acidic
pka value of resatorvid is around 8-8.1. Photo-stability results indicate more stability in
non-aqueous more than aqueous medium. Resatorvid pH degradation rate profiles indicate
the maximum stability between pH3 -5 and maximum instability at pH 8 and 9 at all
experimental temperatures over 26 days. T90 at 25 °C was 648 days in pH 3 versus 11 days
in pH 9.
Ex-vivo penetration evaluation for resatorvid through hairless murine skin was evaluated
using acetone and phosphate buffer formulations. The flux values were 0.82 and 0.36 for
acetone and phosphate buffer formulation respectively. The percent of drug retention in the
dermis layer of skin were also evaluated and found to be 37.99% for the acetone
formulation and 21.13 % for phosphate buffer formulation. The in-vivo penetration
evaluation study was performed by topically applying of resatorvid in acetone solution.
Skin biopsy from the site of application was analyzed one, three, eight and 24 hours post-
application. The analysis was performed by tape stripping of the stratum corneum of the
skin segment. It was found that percent of resatorvid at the dermis layers 5.92, 1.47, 0.45
and 0.23 % for 1, 3, 8 and 24 hours post-application respectively. The percent of resatorvid
retention in dermis layer from the in-vivo study are not in compliance with the result of the
ex-vivo study, which could refer to possible enzymatic degradation of resatorvid in the live
animal skins.
14
Chapter III: correlation of drug penetration between Strat-MTM membranes and murine
skin for various drugs.
Strat-MTM is a synthetic model for transdermal diffusion testing made by EMD Millipore
and was marketed as a new skin mimetic membrane. It has been reported to be predictive
of diffusion in human skin. Independent researchers had evaluated this membrane and
compared it with animal and human skin and other polymeric membranes. Yet, there are
not a published research to correlate the animal skin and Strat-MTM based on the amount
of drug retained after topical application, which is one of the critical criteria for dermal
drug delivery system.
In this research, five compounds, with various physiochemical properties, were selected to
perform this correlation. Resatorvid, Methyl Para aminobenzoate (M-PABA), Diclofenac
sodium, Salicylic acid and hydrocortisone, each one was dissolved in phosphate buffer
saline (pH 7.4) in concentrations of 60 ug/ml for resatorvid and 100-120 ug/ ml for others.
All experiments were uniform in the setting and made in triplicate.
As a conclusion from the results, the number of tested compounds were shorted to reflect
the correlation in flux or permeability coefficient between murine skin and Strat-MTM
membrane. With exception of M-PABA, there is a trend of correlation with the percentage
of drug retained in dermis layers; however, the number of compounds still low to reflect a
real correlation.
15
CHAPTER 1
PREFORMULATION STUDIES ON PIPERLONGUMINE
Published by: Alhassan Aodah, Aaron Pavlik, Kelly Karlage, Paul B. Myrdal
In PLOS ONE 11(3): e0151707. doi:10.1371/journal. pone. 0151707 March 16, 2016
Introduction
Piperlongumine, or piplartine, 1-[(2E)-3-(3, 4, 5-trimethoxyphenyl) prop-2-enoyl]-1, 2, 5,
6- etrahydropyridin-2-one, (Figure1.1), is a historically interesting natural alkaloid
compound as piper plants have been used in traditional Ayurvedic system medicine to treat
some tumors and other diseases such as malaria, gonorrhea, bronchitis, asthma, and cough
(1). First isolated from the piper plant in 1961, the chemical structure was elucidated in
1963 (2).
Figure 1.1 The chemical structure for piperlongumine.
16
Piperlongumine has become a compound of interest in recent years as it exhibits an array
of promising anticancer activities. This compound shows selective cytotoxicity toward
cancer cells (3), as well as demonstrating higher cytotoxic activity on breast, colon, and
osteosarcoma cancer cells of patient derived cell line growth in comparison with other
anticancer drugs like methotrexate, cyclophosphamide and adriamycin (4). While the
molecular mechanisms behind piperlongumine’s cytotoxicity are still being elucidated, it
has been shown to induce cell death by enhancing reactive oxygen species and DNA
damage for several types of cancer i.e. head and neck (5), pancreatic (6), and melanoma
(7), as well as inhibiting CRM1, a major nuclear exporter protein (8). Additionally,
piperlongumine has been used to inhibit signal transducer and activation of transcription 3
(STAT3) in order to induce anoikis in anoikis resistant melanoma and pancreatic cells lines
[9, 10]. As published in Nature in 2011, piperlongumine also manifests antiangiogenic
effects via its ability to reduce vascular endothelial growth factor (VEGF) and CD31
kinases in cancer cells, which in turn inhibits the formation of new blood vesicles for tumor
cells, as well as its ability to work as an antimetastatic agent for the cancer cells (11). As
well as being a successful single agent, several studies have explored the use of
piperlongumine in combination with other agents such as docetaxel (12), cisplatin (5), and
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (13), further
demonstrating the utility of this compound as an anticancer agent.
In addition to promising anticancer activities, other pharmacological activities include
inhibition of the formation of artherosclerosis plaque in-vivo, (14) and in-vitro antibacterial
activity on Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus
(15). A recent review by Bezerra et al. highlights many of these activities as well as
17
piperlongumine’s ability to act as an inhibitor of platelet aggregation, anxiolytic and
antidepressant, and antidiabetic (16).
These activities, especially anticancer, makes piperlongumine an appealing compound for
further development. However, to this date, very few studies have focused on the
preformulation and/or formulation of this drug. In 2016, Fofaria et al. published their
findings for the preparation of nanoemulsion formulations of piperlongumine (17).While
this study provides some solubility data for piperlongumine, more preformulation work is
warranted.
In this current research, preformulation studies have been conducted to determine the
solubility of piperlongumine in water and other systems including cosolvents, surfactants
and complexing agents, which are commonly used in various pharmaceutical formulations.
Moreover, the aqueous stability of piperlongumine as a function of pH, temperature and
ionic strength has been extensively studied for more than 18 months. Photo-stability studies
and solid-state characterization were also performed. The results of these essential
preformulation studies will aid in the future formulation development for this drug.
Materials
Piperlongumine was supplied by Research Products International (Mt. Prospect, IL, USA).
HPLC grade acetonitrile (ACN) was obtained from Spectrum Chemical Manufacturing
Gardena, CA, USA). USP grade ethanol was obtained from Decon Laboratories Inc. (King
of Prussia, PA US). Cremophor Rh 40 was obtained from BASF Aktiengesellschaft,
(Ludwigshafen, Germany). Sulfobutyl ether β-cyclodextrin sodium salt research grade
(Captisol1) was obtained from CyDex Inc. (Lenexa, Kansas, USA). Hydroxypropyl-β-
18
cyclodextrin (Cavasol1) was obtained from Wacker Chemie AG (Burghausen, Germany).
Polyethylene glycol 400, propylene glycol, glycerin, polysorbate 80 (Tween 80), n-octanol,
ascorbic acid, ethylenediaminetetraacetic acid (EDTA) disodium salt, sodium bisulfite,
monopotassium phosphate, disodium phosphate, disodium citrate and boric acid were
obtained from Sigma Aldrich (St. Louis, MO, USA). Phosphate buffered saline was
obtained from Amresco, Inc. (Solon, OH, USA).
Methods
1. HPLC analysis.
The HPLC system consisted of a Waters 2690 separation module (Waters, Milford, MA,
USA) coupled with a Waters 996 Photodiode Array (PDA) detector. Analysis was
performed by a reverse phase HPLC assay, using a 150 mm × 2.1 mm, Alltima C18 5 μm
column (Grace Davison Discovery Sciences, Deerfield, IL), maintained at 30 ± 2°C.
Ultraviolet detection was done at 328 nm. Mobile phase conditions were 40:60 (v/v) ACN:
H2O at a flow rate of 0.3 ml/min. The injection volume was 5 μl. The retention time for
piperlongumine was ~6.9 min. Quantification was determined using peak area and
calculated from a five-point standard curve. Standards were prepared by volumetric
dilution in acetonitrile and stored at 4°C, protected from light.
2. Solubility studies.
Solubility of piperlongumine was determined in purified water and the following cosolvent
and surfactant systems: 5, 15, 30% (v/v) acetonitrile; 5, 15, 30, 50, 100% (v/
v) of polyethylene glycol 400 (PEG 400), ethanol, glycerin and propylene glycol; 3, 7, 10%
(w/v) of Tween 80; 2.5, 5, 10% (w/v) of Cremophor Rh 40; and a solution composition of
19
10% ethanol: 40% PEG 400 (w/v). Additionally, the complexing agents, sulfobutyl ether
β- cyclodextrin sodium salt and hydroxyl-propyl-β-cyclodextrin, were examined at
concentrations of 5, 10, 20, 40% (w/v). An excess quantity of raw drug crystals were placed
in a known volume of each medium in closed, light protected glass vials and allowed to
agitate for at least 24 hours at ambient temperature. Samples were visually inspected to
ensure that solid drug was still in excess and then filtered through a 0.2 μm PTFE filter.
The filtrate was assayed via the previously described HPLC method and the concentration
of piperlongumine in solution was determined.
3. Partitioning studies.
Piperlongumine crystals were placed in a small glass bottle containing equal volumes of n-
octanol and water. The bottle was shaken for 24 hours, after which time, the bottle was
allowed to equilibrate without mixing for an additional 24 hours to ensure full separation
between the n-octanol and water layers. A sample from each layer was carefully withdrawn
and analyzed by HPLC.
4. Solid state characterizations.
Thermal analysis was performed with a Q1000 series differential scanning calorimeter
(DSC) (TA Instruments, New Castle, DE, USA). Indium was used for the calibration of
the DSC. Samples of 4 to 5 mg were weighed out and placed in an aluminum pan and
crimped with an aluminum lid. Samples were equilibrated and isothermally heated at 30°C
for 5 min, followed by heating at 2°C/min up to 150°C. A nitrogen purge was used at 40
ml/min for each sample.
20
Thermogravimetric analysis (TGA) was performed on a TA Instruments Q50 TGA.
Samples weighing 4 to 5 mg were placed in an empty aluminum pan, covered with an
aluminum lid, crimped and pin perforated. The pan was then heated at 5°C/min up to
150°C. Weight loss as a function of temperature was analyzed under a nitrogen purge at
60 ml/minute.
Powder X-ray diffraction patterns were obtained with a Philips PANalytical X’pert Pro
MPD instrument equipped with an X’Celerator detector. Cu Kα radiation was used (λ =
1.5418 Å). Powdered samples were smoothed onto a zero background silicon wafer and
rotated at 1 revolution/8 seconds during data collection. The scan time was 1.5 hours.
5. Aqueous stability studies.
Aqueous stability studies for piperlongumine were conducted in three buffer systems:
citrate (pH 3, 4, and 5); phosphate (pH 5, 6, and 7); and borate (pH 8 and 9). The ionic
strength for the solutions was also varied, utilizing approximately 0.2 or 0.5 M. Each
buffered sample was prepared with 10% acetonitrile as a cosolvent, then sealed and
protected from light. Samples were stored at four different temperatures: 4°C (for samples
at pH 3, 8 and 9 only), 26°C, 56°C and 67°C. Aliquots were regularly withdrawn for
analysis. Changes in pH, if any, were also monitored. Stability studies were carried out for
18 months or until complete degradation occurred.
6. Photo-stability.
Photo-stability was examined by preparing four groups of samples, each containing a
triplicate sample and one reference (protected from light). These groups covered high and
low drug concentrations in the following compositions:
21
• 150 μg/ml of drug in 100% acetonitrile
• 150 μg/ml of drug in 15:85 acetonitrile: water
• 30 μg/ml of drug in 100% acetonitrile
• 30 μg/ml of drug in 15:85 acetonitrile: water
Each sample was prepared in a transparent glass vial and closed without any entrapped air.
A reference sample was utilized for each group by covering one of the vials with aluminum
foil to protect it from the light exposure. All vials were exposed for 165 min using Sun 340
sunlamps yielding UV emission between 295 and 390 nm to include UVA and UVB
wavelengths. Samples were placed in an area where the UV intensity was measured to be
1.57 mw/cm2. After the exposure time, samples were analyzed using the HPLC method
described previously.
7. Additional stability studies.
The addition of antioxidants such as ascorbic acid and sodium bisulfite and the chelator
EDTA were evaluated with the drug at pH 3 (citrate buffer) and pH 7 (phosphate buffer).
Samples in phosphate buffer were maintained at 26°C while the samples in citrate buffer
were kept at 56°C. All three agents were used at a concentration of 0.05% w/v. Aliquots
were withdrawn on a regular basis and pH and drug concentration were monitored.
Piperlongumine stability was also examined after the addition of 1.5% w/v hydrogen
peroxide in pH 3 citrate buffer at room temperature. LC-MS analysis. Stability samples
which showed degradation, as evidenced by new peaks appearing in the HPLC
chromatograms, were submitted for LC-MS analysis, in order to identify the masses of the
degradation products. Samples were separated on an Altima HP C18, 5 μm, 150 x 2.1 mm
22
column (Grace Davison Discovery Sciences, Deerfield, IL) using a Paradigm MS4B—
multi-dimensional separations module (Michrom BioResources, Inc., Auburn,
CA). The mobile phase (40% ACN: H2O) was delivered isocratically at a flow rate of 300
μl/ min. A 20 μl injection loop was used to introduce 2 μl of sample diluted with 18 μl of
the mobile phase. The AB SCIEX API 3000 triple-quadrupole mass spectrometer (Applied
Biosystems, Foster City, CA) was controlled by Analyst 1.5.1 and used in-line with the
HPLC. Mass spectrometric analysis was performed using both MS (full scan) and MS/MS
(product ion) type in positive mode with an APCI source. Instrument-specific parameters
were as follows: source temperature, 350°C; declustering potential (DP), 20 V; entrance
potential (EP), 10 V; collision energy (CE), 10 V.
Results and Discussion
1. Chromatography
The isocratic HPLC method developed eluted piperlongumine within a 10 minute run time,
giving a Gaussian shaped peak for the drug at ~6.9 minutes. Furthermore, utilizing this
method, piperlongumine was completely resolved from the degradation products which
resulted from the stability studies as seen in Figure 1.2. The method was shown to be linear
over the working concentration range of 0.5 to 120 μg/ml. The same HPLC method was
used to analyze all samples from solubility, thermal and photo-stability studies.
23
Figure 1.2 HPLC chromatograms for the separation of piperlongumine (peak at 6.9
minutes) in different stability conditions. (a) Sample at pH 8. (b) Sample at pH 5. (c)
Sample at pH 3. (d) Photo-stability sample.
24
2. Solubility studies
Experimental measurement of the aqueous solubility of piperlongumine indicated that the
drug has an intrinsic solubility of 26 ± 2.9 μg/ml. This experimental solubility value is
about ten times less than that determined by Fofaria et al (17).Water solubility does not
change as a function of pH. Solubilization studies were also performed utilizing
surfactants, cyclodextrins and cosolvents. The results are shown in Figures 1.3 and 1.4.
The cyclodextrin and surfactant systems demonstrate linear solubilization profiles (Figure
1.3), while the cosolvent systems followed predominantly log-linear profiles (Figure 1.4).
It was found that 10% Tween 80 was capable of enhancing piperlongumine solubility up
to 27 fold (solubility of approximately 700 μg/ml). A 10% (w/v) Cremophore Rh 40 system
solubilized approximately 550 μg/ml. Hydroxypropyl- β-cyclodextrin and sulfobutyl ether
β-cyclodextrin both solubilized about 1 mg/ml at a 20% (w/v) concentration. While
acetonitrile was the most efficient cosolvent, with 30% improving the drug solubility by
63 fold (1.6 mg/ml), its utility is limited to analytical uses. Of the cosolvents utilized,
ethanol and PEG 400 were the best cosolvents for piperlongumine. At lower concentrations
the solubilization was similar for both ethanol and PEG 400. At 50% cosolvent, ethanol
solubilizes nearly twice as much as PEG 400 (~ 2.3 mg/ml and ~1.1 mg/ml, respectively).
Interestingly, in pure solvent, piperlongumine has better solubility in PEG 400 than in
ethanol (~22 mg/ml and ~11 mg/ml, respectively). Fofaria et al. (17) also observed that
piperlongumine is more soluble in PEG 400 as compared to ethanol. By utilizing a 10%
ethanol: 40% PEG 400 (w/v) composition, a solubility of approximately 1.7 mg/ml was
obtained.
25
Three piperlongumine prototype formulations were evaluated for precipitation upon
dilution. These formulations were a 500 μg/ml in a 10% (w/v) Tween 80 formulation, a 1
mg/ml in a 10% ethanol: 40% PEG 400 (w/v) formulation, and a 1 mg/ml in a 20% (w/v)
hydroxypropyl-β-cyclodextrin formulation. All three were serially diluted from a 1:1 to a
1:10 by adding either 1 ml of water or 1 ml of phosphate buffered saline (PBS) to the
formulation.
After the addition of each ml of either water or PBS, the mixture was evaluated for
precipitation. This was repeated for each sequential addition. No precipitation was
observed for any of the dilutions for any of the three formulations. However, a small
amount of precipitation was observed after 24 hours for dilutions with the 10% ethanol:
40% PEG 400 (w/v) formulation.
26
Figure 1.3 Solubility of piperlongumine in different surfactant and complexing
systems.
Figure 1.4 Solubility of piperlongumine in different cosolvent systems.
27
3. Partitioning study
Piperlongumine exhibits lipophilic properties when evaluated in an n-octanol/water
partition coefficient study. The partition coefficient (log P) was experimentally determined
to be 2.37 ± 0.12 at room temperature.
4. Solid-state characterization
It was observed upon microscopic evaluation that piperlongumine crystals were shaped
like rectangles (Figure 1.5a). Using hot stage microscopy, these crystals start melting at
123°C, and are completely melted at 126°C. Examination of different re-crystallized forms
of piperlongumine such as: precipitate of the drug in water, ethanol, methanol and
acetonitrile, demonstrated that all conditions give the same irregular needle like crystal
morphology (Figure 1.5b).
Figure 1.5 Photomicrographs of piperlongumine (400X). (a) Original crystals, (b)
Crystalline precipitant.
28
Analysis of piperlongumine crystals by DSC gave an endothermic peak with an onset of
109.6°C and a maximum of 123.3°C (Figure 1.6). TGA analysis for piperlongumine
crystals showed no weight loss occurred until the melting point was reached.
Piperlongumine precipitated from supersaturated solutions in water, ethanol, methanol and
acetonitrile was examined by DSC. Although the recrystallized crystals had different
crystal habits, they had the same melting point as the original drug crystals.
Analysis of crystals utilizing X- ray powder diffraction supports that piperlongumine exists
as a specific crystalline solid (Figure 1.7). The X-RPD chart shows long range molecular
order. This result is in accordance with the results of the calculated single crystal data
published by Banerjee and Chaudhuri in 1985 (18).
29
Figure 1.6 DSC thermogramof piperlongumine.
Figure 1.7 Pattern of X-ray diffraction of piperlongumine crystals.
30
5. Stability studies
Figure 1.8 plots the logarithmic percentage of drug remaining verses time at 56°C and
shows first order degradation kinetics of thermal degradation of piperlongumine for several
pH conditions. Apparent first order degradation was also obtained for the 4, 25 and 67°C
degradation profiles. Degradation rate constants, k-values, for piperlongumine were
calculated using the slopes of the trend lines of each profile.
An Arrhenius plot of log k versus the reciprocal of the temperature (Figure 1.9) was utilized
to determine the length of time it takes for piperlongumine to degrade 50% (T50) and 10%
(T90).
31
Figure 1.8 Degradation of piperlongumine as a function of pH at 56°C.
Figure 1.9 Arrhenius plots for piperlongumine degradation at different pH values.
32
For a given pH the Arrhenius equation was utilized:
Log k = log A – (Ea/2.303 RT)
Where:
o A is the Arrhenius factor.
o Ea is the activation energy (J/mole).
o R is the gas constant = 8.314 J/mol· K
o T is the temperature in Kelvin.
Arrhenius activation energies at different pHs were calculated from the slope of each linear
curve and by using the Arrhenius equation. For all pHs, the activation energies were within
the range of 88–95 kJ/mol. All calculated values are presented in Table 1.1.
Figure 1.10 shows the overall degradation rate vs. pH profile of piperlongumine at 26°C,
56°C and 67°C. From the stability profiles it can be observed that piperlongumine has
maximum stability around pH 4; the T90 at this pH is about 17 weeks at 25°C.
33
pH Buffer
type
Ionic
strength
K values (weeks-1) Ea
(kJ/mol)
T50 at 25
°C
(weeks)
T90 at 25
°C
(weeks)
4 °C 26 °C 56 °C 67 °C
3 citrate 0.2 * 0.0110 0.3057 0.7808 88.5 69.6 10.6
3 citrate 0.5 * 0.0101 0.3413 0.8383 92.3 75.4 11.5
4 citrate 0.2 --- 0.0067 0.1739 0.3813 84.8 112.9 17.2
5 phosphate 0.2 --- 0.0076 0.2201 0.5741 89.9 101.5 15.4
5 phosphate 0.5 --- 0.0090 0.1764 0.5741 84.8 88.7 13.5
5 citrate 0.2 --- 0.0090 0.2045 0.6125 86.7 87.6 13.3
6 phosphate 0.2 --- 0.0108 0.2919 1.1652 95.0 75.5 11.5
7 phosphate 0.2 --- 0.0506 1.3607 5.1370 94.0 16.0 2.4
8 borate 0.2 0.0232 0.5120 7.2924 20.933 83.0 2.0 0.3
9 borate 0.2 0.1182 2.4375 --- --- 94.8 0.3 0.05
9 borate 0.5 0.1171 2.4372 --- --- 95.1 0.3 0.05
*Not determined. Rates were not significantly different than zero over the course of the
study.
Table 1.1 Summary of stability results.
34
Figure 1.10. Piperlongumine pH degradation rate profiles. All samples were at an
ionic strength of ~0.2 M. Buffer types are: citrate for pH 3 and 4, phosphate for pH
5, 6 and 7, borate for pH 8 and 9.
35
The effects of ionic strength and buffer species were also studied. As can be seen from the
data presented in Table 1.1 and shown in Figure 1.11, an increase in ionic strength from
approximately 0.2 M to 0.5 M had nominal effects (and likely not significant) on the
stability half-life of piperlongumine at pH 3 and pH 5. No difference in degradation rate
was observed between the 0.2 M to 0.5 M ionic strength samples at pH 9 in a borate buffer.
Two different buffers, citrate and phosphate, were evaluated at pH 5. As can be seen in
Table 1.1, the degradation rates are similar for both buffer systems.
Degradation products were elucidated from stability samples at pH 3, 5 and 8. It was found
that 3,4,5- trimethoxycinnamic acid, Figure 1.12, is a major degradation product. Pure
3,4,5- trimethoxycinnamic acid, externally spiked into stability samples, confirmed that it
was the same molecule (retention time approximately 3.1 min). A molecular mass
corresponding to what is consistent with piperlongumic acid (see Figure 1.12) was also
isolated (retention time approximately 2.04 min). These findings are in agreement with
Chatterjee and Dutta, in which they state that piperlongumine alkaloid undergoes imide
hydrolysis in ethanolic alkali to give 3,4,5-trimethoxycinnamic acid as well as
piperlongumic acid (19).
36
Figure 1.11. Effect of ionic strength (IS) on stability of piperlongumine at pH 3, 5
and 9 at 26°C.
Figure 1.12. Major degradation products of piperlongumine.
37
6. Photo-stability
The results from photo-stability studies (Table 1.2) indicate that piperlongumine undergoes
degradation after exposure to UV light. This was determined by comparing the percentage
of drug remaining in the non-light protected samples with their light protected controls.
After the exposure duration the concentration of piperlongumine in the control samples
remained at 100% of the initial concentration. Interestingly, the greatest degradation
occurred in the pure organic solvent, acetonitrile; 60% degradation as compared with
approximately 20% occurring in the 15:85 acetonitrile: water samples. Photo-degradation
does not appear to be significantly reliant on initial drug concentration, although the
samples with the higher initial concentration degraded less than those at the lower initial
concentration.
Drug conc. (µg/ml) % Acetonitrile: water Average % of drug remaining ± SD
150 100:00 44.5 ± 0.2
150 15:85 82.3 ± 0.6
30 100:00 39.5 ± 0.03
30 15:85 79.0 ± 0.1
Table 1.2 Summary of photo-stability studies of piperlongumine.
38
7. Stability with antioxidants
The addition of ascorbic acid and EDTA to a pH 7 (phosphate buffer) solution did not have
a significant effect on the stability profile upon comparing with drug alone at the same
condition. This can be seen from the slopes of the graphs in Figure 1.13a and k values in
Table 1.3. However, the addition of sodium bisulfite degrades piperlongumine very
rapidly, within a few minutes, at pH 7. Although sodium bisulfite is routinely used as an
antioxidant in pharmaceutical formulations, is has been shown on occasion to decrease the
stability of some drugs (20). Interestingly, the addition of sodium bisulfite with the drug in
a pH 3 citrate buffer, at 56°C, did not affect the degradation rate of the drug upon
comparing it with drug alone under the same condition (see Figure 1.13b and Table 1.3).
This finding could indicate that partial ionization of the sodium bisulfite molecule, as
would occur at pH 7, plays a part in the rapid degradation of piperlongumine. This pH
effect is similar to what has been reported by Scheiner et al. about the stability of thiamine
with sodium bisulfite, in which thiamine shows better stability with sodium bisulfite at
lower pH (pH 5), but destruction at higher pH (pH 7) (21). Finally, the addition of ascorbic
acid at pH 3 also did not readily affect the degradation of piperlongumine.
39
Figure 1.13. Stability of piperlongumine with the addition of different excipients. (a)
pH 7 at 26°C. (b) pH 3 at 56°C.
40
Buffer
type
pH °C Additional substance to
the drug
k values
(weeks-1)
T90
weeks
Citrate 3 56 None 0.3107 0.3
Citrate 3 56 Ascorbic acid 0.3706 0.3
Citrate 3 56 Sodium bisulfite 0.3860 0.3
Citrate 3 56 EDTA 0.3266 0.3
Citrate 3 26 Hydrogen peroxide 0.3369 0.3
Phosphate 7 26 None 0.0494 2.1
Phosphate 7 26 Ascorbic acid 0.0459 2.3
Phosphate 7 26 Sodium bisulfite 3069.8950 0.0
Phosphate 7 26 EDTA 0.0559 1.9
Table 1.3. Stability comparisons for piperlongumine with different excipients.
The addition of an oxidizing agent like hydrogen peroxide does significantly increase
degradation. The T90 with hydrogen peroxide at 26°C is 0.34 weeks, (Table 1.3). Whereas
the T90 without hydrogen peroxide would be about 9.5 weeks (Table 1.1).
Conclusion
Preformulation studies on piperlongumine have been performed. Although it has poor
water solubility (0.026 mg/ml), solubility can be increased to 1 mg/ml by using a 20%
(w/v) hydroxypropyl- β-cyclodextrin solution or a 10% ethanol: 40% PEG 400 (w/v)
formulation. Piperlongumine is more stable in acidic conditions. The T90 at pH 4 is about
41
17 weeks at 25°C, however, it is only a few days at pH 7 and above. The major hydrolysis
degradation products of piperlongumine are 3,4,5-trimethoxycinnamic acid and
piperlongumic acid. Moreover, it was noted that piperlongumine does undergo photo-
degradation. These preformulation studies on the fundamental physicochemical
characterizations of piperlongumine can be utilized to assist in the further drug
development of this promising therapeutic agent.
42
CHAPTER 2
PREFORMULATION AND EVALUATION OF RESATORVID FOR TOPICAL
DELIVERY
Introduction:
Resatorvid (TAK-242), shown in Figure 2.1, is a small sulfonamide molecule developed
by Takeda Pharmaceutical Company Limited (Takeda). It was identified as an active
pharmaceutical agent from a screening of small molecule inhibitors of inflammatory
mediator production. It is a novel cyclohexene derivative containing a phenylsulfamoyl
and ethyl ester group and showed inhibition of the production of nitrous oxide, TNF-R,
and interleukin-6 (IL-6). In-vitro IC50 values are less than one micromolar. Moreover,
intraperitoneal administration of 10-30 mg/kg to mice, showed significant protective
effects in a mouse endotoxin sepsis model (22).
After successful preclinical efficacy and safety studies on resatorvid, clinical trials were
initiated to find the safety and efficacy in patients with sepsis-induced cardiovascular and
respiratory failure. Phase 3 clinical studies using resatorvid were conducted in Japan, the
U.S., and Europe. Unfortunately, the clinical studies were terminated at phase 3 due to low
efficacy (23). In 2009, Takeda announced the discontinuation of clinical development for
its investigational compound TAK-242 for treatment for severe sepsis.
Although resatorvid was not marketed as a sepsis drug, other research has found that it may
be applicable for other clinical conditions. Resatorvid has shown specific inhibition of Toll-
like receptor (TLR4) signaling via binding to the cys747 (binding site) in the intercellular
43
domain of TLR4. This inhibition leads to inhibit the production of lipopolysaccharide
induced inflammatory mediators (24).
Since resatorvid was accepted to inhibit TLR4 mediated pathway, further study by Zhang
et al. was conducted to investigate the effect of resatorvid as neuroprotective after traumatic
brain injury in mouse model (25). Zhang’s results showed a significant reduction of the
levels of TAK1, p-TAK1, TNF- and IL-1b proteins after treatment with resatorvid. These
proteins are clinically observed after traumatic brain injury, suggesting that targeting TLR4
inhibition by resatorvid may be clinically useful in treating traumatic brain injury (25).
In a recent study by Janda et al., it was found that resatorvid has activity against UV-
induced skin cancer (26). Resatorvid was shown to block UV-induced NF-jB and MAP
kinase/AP-1 activity and cytokines Il-6, I1-8, and Il-10 expression after topical treatment
on the murine skin as well as in cultured keratinocytes. Thus the finding that resatorvid can
suppress UV induced skin signaling may present a useful treatment modality for non-
melanoma skin cancer prevention (26).
While resatorvid has shown diverse biological activities, limited information has been
published about the preformulation and formulation properties of resatorvid. The goal of
this work is to investigate some of the physio-chemical properties of resatorvid, including
solubility, stability and solid state properties. Moreover, with the potential application for
the topical delivery of resatorvid, a particular emphasis will be on evaluating skin
penetration. Ex-vivo and in-vivo skin penetration studies using murine models will be
evaluated. Rodents skins from mice, rats, and guinea pigs are commonly used for such kind
of studies due to small animal size, availability and low coast. Hairless strains of these
rodents have been validated to mimic human skin more than those that are hairy (27).
44
Figure 2.1 Resatorvid chemical structure
Materials:
Resatorvid (TAK-242) was supplied by Chem Scene (Monmouth Junction, NJ, USA).
HPLC grade acetonitrile (ACN) was obtained from Spectrum Chemical Manufacturing
(Gardena, CA, USA). USP grade ethanol was obtained from Decon Laboratories Inc. (King
of Prussia, PA US). Trifluoroacetic acid, Polyethylene glycol 400, propylene glycol,
monopotassium phosphate, disodium phosphate, Sodium chloride and potassium chloride
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Methods:
1. UV Spectrum:
A Lambda 365 UV/Vis spectrophotometer, PerkinElmer (MA, USA) was used to
determine the UV spectrum. A soluble sample of resatorvid in acetonitrile at a
45
concentration 60 µg/ml was placed in a glass cuvette and a UV scan was done from
wavelength 200 through 800.
2. HPLC analysis:
The HPLC system consisted of a Waters 2690 separation module (Waters, Milford, MA,
USA) coupled with a Waters 2487 Dual wavelength detector. The analysis was performed
by a reverse phase HPLC assay, using a 150 mm × 2.1 mm, Symmetry C18 5 μm column
(Waters, Milford, MA, USA), maintained at 25 ± 2°C. Ultraviolet detection was done at
254 nm. Mobile phase conditions were 40:60 (v/v) ACN: Trifluoroacetic acid (0.1% v/v)
at a flow rate of 0.3 ml/min. The injection volume was 10 μl. The retention time for
resatorvid was ~11 min. Quantification was determined using peak area and calculated
from a five-point standard curve. Standards were prepared by volumetric dilution in
acetonitrile and stored at 4°C, protected from light.
3. Melting point determination:
Thermal analysis was performed with a Q2000 series differential scanning calorimeter
(DSC) (TA Instruments, New Castle, DE, USA). Indium was used for the calibration of
the DSC. Samples of 4 to 5 mg were weighed out and placed in an aluminum pan and
crimped with an aluminum lid. Samples were heated at 5°C/min up to 250°C. A nitrogen
purge was used at 40 ml/min for each sample.
4. Solubility studies:
Solubility studies. The solubility of resatorvid was determined in purified water at the
following buffers and solubilizing agents: Citrate buffer (pH 3 and 4), phosphate buffer
46
(pH 5.1 and 6), phosphate buffer saline (pH 7.4), and borate buffer (pH 8). Also, 10% (v/v)
of each of propylene glycol, polyethylene glycol, ethanol, and acetonitrile.
An excess quantity of raw drug crystals was placed in a known volume of each medium in
closed, light protected glass vials and allowed to agitate for at least 48 hours at ambient
temperature (22±1ºC). Samples were visually inspected to ensure that solid drug was still
in excess and then filtered through a 0.2 μm PTFE filter. The filtrate was assayed using the
HPLC method and the concentration resatorvid in solution was determined. All samples
were done in triplicate.
The partition coefficient (log P) was determined by calculating the logarithm of the octanol/
water resatorvid saturated concentrations ratio. Equal volumes of octanol and water were
used in triplicate and performed at room temperature.
5. Photo-stability:
Photo-stability was examined by preparing two groups of samples, each containing four
samples and two references (protected from light). These groups are as following
compositions:
• 80.6 μg/ml of drug in 100% acetone
• 4.9 μg/ml of the drug in water.
Each sample was prepared in a transparent HPLC glass vial and closed without any
entrapped air. The two reference samples were utilized for each group by covering the vials
with aluminum foil to protect it from the light exposure. Sun 340 sunlamps yielding UV
emission between 295 and 390 nm (to incorporate UVA and UVB wavelengths) were used.
Samples were placed in an area where the UV intensity was measured to be 1.57 mW/cm2
47
and a single dose was given over 165 min. A single dose of exposure was calculated to be
50 kJ UVA/m2 for UVA and 2.4 kJ UVB/m2 for UVB. Half of the vials were exposed to
the solar simulated light for one dose of UVA/UVB radiation (Dose 1x). The second set of
vials were exposed to a second dose (Dose 2x). After the exposure, samples were analyzed
using the HPLC method described previously.
6. Chemical Stability Studies:
Aqueous stability studies for resatorvid were conducted in three buffer systems: citrate
(pH 3), phosphate (pH 5.2, 7.4 and 7.4 PBS); and borate (pH 8 and 9). Each buffered
sample was prepared with 5% acetonitrile as a cosolvent, then sealed and protected from
light. Samples were stored at three different temperatures: 25°C, 48°C, and 65°C. Aliquots
were regularly withdrawn for analysis. Changes in pH, if any, were also monitored.
Thermal stability of resatorvid in organic solvent i.e. acetone was conducted at three
temperature: 0°C, 4°C, and 25°C.
7. Ex-vivo penetration studies:
Resatorvid penetration into and through murine skin was tested ex-vivo by using acetone
and phosphate buffer saline (pH 7.4) formulations. For this test, hairless p85DN mice were
used. First, the mouse was euthanized and the skin was extracted from the dorsal region
from shoulder to flank. Any underlying adipose tissues were removed without stretching
or damaging to the skin. Then the skin segment was sandwiched between the receiver and
donor chamber of vertical Franz diffusion cell (Figure 2.2) with care taken to avoid any
entrapped air. The stratum corneum side of skin was faced up.
48
Figure 2.2 Franz cell components (28)
The resatorvid concentration used for the bulk acetone formulation was 691.34 ug/ml, and
from this formulation 66 ul was placed on the skin sample in the donor side. The solution
of resatorvid in phosphate buffer saline was 47.12 ug/ml and the amount added to the donor
side was 1 ml. The experiments were conducted 32±1 °C and monitored over 8 hours.
Sampling from the receiver solution was performed through the horn of the cell and was
taken from the middle of receiver solution using a long needle. After each sampling, an
equal volume of fresh receptor fluid was immediately replenished. The receptor fluid was
stirred at 600 rpm by a magnetic stir bar. The Franz cell receiver volume was approximately
4 ml and the top contact surface area was approximately 0.9 cm2.
49
8. Drug extraction method development:
Resatorvid extraction methods were developed to extract the drug from adhesive tape
(which is used to strip off the stratum corneum) and from murine skin. Briefly, the drug of
each formulation was spiked on a piece of 3M’s No. 810 adhesive tape (Scotch™ Brand
Magic Tape, 3M Co., Minneapolis, MN) and allowed to dry. Then 2ml of acetonitrile was
added to the tape and sonicated for 30 min following by centrifugation for 10 min at 1400
rpm. The resulted supernatant was collected and filtered before HPLC analysis.
For murine skin, each skin segment was tape stripped 12 times to remove the first 12 layers
of stratum corneum. Then a known amount of drug for each formulation was spiked onto
the stripped skin segment and allowed to dry. Drug extraction from the skin started by
shredding the skin segment by scalpel into tiny cubes (around 0.5 mm3 each). Then 2 ml
of acetonitrile was added to the minced skin and homogenized for 5 min using Ultra-
Turrax® homogenizer in the case of phosphate buffer formulation. For the acetone
formulation, a probe sonicator (VIRSONIC 100®) Ultrasonic Homogenizer (VirTis,
Gardiner NY) was used for homogenization and set for 6 minutes on tune 6. Each
homogenized sample was centrifuged for 10 minutes at 1400 rpm. The supernatant was
collected and filtered prior to HPLC analysis.
As shown in Table 2.1a, the extraction method out of the tape provides essentially a 100%
recovery. Whereas, the extractions from the murine skin dermis layers have a 48.3 and 63.5
% for the formulation of phosphate buffer and acetone, respectively (see Table 2.1b. All
values from the experiments were normalized to these percentages.
50
Recovery of resatorvid from tape strips #1 #2 #3 Average ± std.
Spiked amount of drug in tape sample (ug) 37.50 37.50 37.50
Amount of drug recovered from tape
sample (ug)
39.41 38.11 36.24 37.92±1.59
% of recovery 105.08 101.62 96.63 101.11±4.25
a)
Recovery of resatorvid from phosphate
buffer formulation in murine skin
#1 #2 #3 Average ±
Std.
Spiked drug content in the sample (ug) 10.52 21.51 6.29
Washing skin with 5 ml PBS for 20 times
(ug)
0.67 1.27 0.39
Washing confirmation with 1 ml PBS (ug) 0.03 0.03 0
Skin extraction in 2 ml acetonitrile (ug) 4.66 10.29 3.32
% of recovery 44.29 47.81 52.77 48.29±4.26
Recovery of resatorvid from acetone
formulation in murine skin
#1 #2 #3 Average ±
Std.
Spiked drug content in the sample (ug) 15.00 15.00 15.00
Skin extraction in 2 ml acetonitrile (ug) 10.47 7.57 10.51 9.52±1.68
% of recovery 69.81 50.46 70.07 63.45±11.24
b)
Table 2.1: Results of extraction method development of resatorvid from (a) adhesive
tape, and (b) skin.
51
9. In-vivo penetration studies:
In-vivo mice skin model has been used to evaluate the topical delivery of resatorvid
molecule. In this study, hairless p85DN mice have been used for evaluating the in-vivo
drug penetration. Approximately 66 ul of an acetone formulation of resatorvid, having a
concentration of 10 mmol (3.618 mg/ml), was applied topically over on three distinct spots
(≈1.8 cm2 each) on the backs or flanks of the hairless mice. One animal was euthanized at
1, 3, 8 and 24 hours post application. Skin biopsies of 80-110 mg were removed from the
site of application. For each of the skin biopsies, the SC was progressively removed by the
serial application of adhesive 12 tape strips. Layers 1 to 3, layers 4-12 were analyzed
separately as well as the dermis layer.
Tape stripping was performed using 3M’s No. 810 (Scotch™ Brand Magic Tape, 3M Co.,
Minneapolis, MN) tape, a cellulose-backed adhesive tape. Each group of tapes was
dissolved in 5 ml of acetonitrile and sonicated for 40 min. After that, a sample was
centrifuged at 14000 rpm for 10 min. Supernatants were collected and filtered before
analyzed by the HPLC.
Drug extraction from the dermis layers was initiated by shredded the skin segment, by
scalpel, into tiny cubes (around 0.5 mm3 each). Then, 2 ml of acetonitrile was added to the
ground skin and sonicated for 6 min using ultrasonication.
After homogenization, the resultant suspension was transferred to Eppendorf vial to be
centrifuged for 10 min at 14000 rpm. The clear supernatant was collected and quantified
by HPLC using the same method of analysis for each drug.
52
Results and discussion:
1. UV Spectrum:
The UV spectrum for resatorvid is given in Figure 2.3a. The UV scan for resatorvid shows
that the molecule has significant UVA absorption below 250 nm and a minor lambda max
at 280 nm with decreasing absorption until approximately 300 nm. Figure 2.3b illustrates
that UVA and UVB are defined as the wavelengths between 290 to 400 nm. Since exposure
to the UVA and UVB range has been shown to induce skin carcinogenesis, compounds
applied topically that absorb in these wavelengths can have a sunscreen effect. Upon
consideration of the UV absorbance spectrum for resatorvid, it can be concluded that it will
not have a significant sunscreen effect when applied topically.
53
a)
b)
Figure 2.3 (a) Resatorvid UV absorbance intensity and (b) light spectrum wave-
lengths (29).
54
2. Chromatography:
The developed HPCL method was able to elute resatorvid within 14 minutes of run time
and gives a predominately Gaussian shaped peak at approximately 11.1 minutes. This
method resolves resatorvid from possible degradation products and buffer peaks. The
method also affords the separation of drug from skin or adhesive tape extracts as seen in
Figure 2.4. The method was shown to be linear over the concentration range of 0.125 to
145 ug/ml. This method was used to analyze all samples of solubility, photostability and
penetration studies.
55
a)
b)
c)
Figure 2.4. Chromatogram of (a) resatorvid, (b) the chromatogram of blank
adhesive tape, (c) the chromatogram of control murine skin extract.
56
3. Solid state characterizations:
DSC results indicate a relatively low melting point for the resatorvid crystals. A sharp
endothermic peak at 65°C indicates the melting point of the resatorvid. The thermogram
does not show any glass or phase transitions or decomposition up to 225C, Figure 2.5.
Microscopic examination of resatorvid raw material show square to irregular birefringent
crystals (Figure 2.6).
57
Figure 2.5 DSC thermogram for resatorvid
Figure 2.6 Photomicrographs of resatorvid raw material (400 X).
58
4. Solubility studies:
The pH solubility profile is shown in Figure 2.7. The intrinsic solubility is approximately
48.29 ± 3.18ug/ml, based on the solubilities from pH 3.0 to pH 6.5. Resatorvid has an
acidic pKa pH between 8.0 - 8.1. The sulfonamide functional group likely produces the
acidic hydrogen.
Figure 2.8 shows the intrinsic solubility in water compared to the solubility in different
10% v/v cosolvent systems. Unbuffered water solubility was found to be 95.32 ± 1.75
ug/ml. The data shows that using of 10% v/v ethanol can double the water solubility and is
more soluble than 10% v/v polyethylene glycol or propylene glycol systems.
The octanol/water partition coefficient (log P) for resatorvid was experimentally
determined to be 2.22 ± 1.34.
59
Figure 2.7 Resatorvid pH-solubility profile.
Figure 2.8 Comparison of resatorvid solubility in different cosolvent systems.
60
5. Photo-stability:
For topically applied compounds, it is important to understand the impact of UV exposure
on chemical stability. For resatorvid, UV exposure produced more degradation in water
than acetone. After two doses of exposure, 74.7% of the drug remain in water sample versus
91% in acetone sample. This could suggest a good stability for the drug in the organic base
formulations. Table 2.2.
Vehicle Sample ID Dose (kJ UVA/m2;
kJ UVB/m2)
Avg. Conc.
(mcg/mL)
% of
remaining
Wate
r
Control protected 5.06 100.00
Dose 1x 50; 2.4 3.78 74.67
Dose 2x 100; 4.8 3.12 61.75
Ace
ton
e Control protected 86.18 100.0
Dose 1x 50; 2.4 80.61 93.54
Dose 2x 100; 4.8 78.42 91.00
Table 2.2: Summary of resatorvid light stability
6. Degradation as a function of pH and temperature:
The stability of resatorvid was evaluated as a function of pH and temperature. Figure 2.9
shows the log % resatorvid remaining as a function of time for 25, 48 and 65ºC at various
pH values. Based on the elevated temperatures, it was found that plotting the logarithmic
percentage of resatorvid remaining versus time (days), gives a linear negative slope (first
order degradation). Degradation rate constants, k-values, for resatorvid at each pH and
temperature were calculated using the regressed slopes of the data shown in Figure 2.9.
61
Figure 2.9 Degradation of resatorvid over 26 days as a function of pH at 25, 48 and
65°C
62
Moreover, an Arrhenius plot of log k versus the reciprocal of the temperature (Figure 2.10)
was utilized to determine the length of time it takes for resatorvid to degrade 10% (T90). It
was determined that T90 for resatorvid in aqueous media at 25ºC are: 648, 98, 55, 21, 11
days for pHs 3, 5.2, 7.4. 8 and 9 respectively.
Figure 2.11 shows the overall degradation rate vs. pH profile of resatorvid at 25°C, 48°C
and 65°C. From the stability profiles; it can be observed that resatorvid has the best stability
at pH 3 and progressively gets less stable as pH increases.
63
Figure 2.10 Arrhenius plots for resatorvid degradation at different pH values.
Figure 2.11 Resatorvid pH degradation rate profiles.
64
7. Ex-vivo penetration studies:
Ex-vivo penetration evaluation of resatorvid from both formulation indicates that acetone
solution has a higher flux than phosphate buffer solution. The lag time values were
calculated from the flux curve at the steady state linear area of the curve. Interestingly, the
lag times were 5.7 and 3.02 hours for acetone and phosphate buffer formulation
respectively, Figure 2.12a, and Table 2.4. There was a higher percentage of the resatorvid
dose accumulated after 8 hours in the skin in the case of acetone formulation (38%) as
compared to the phosphate buffer solution (21.13%), Table 2.3.
Figure 2.12b shows the percent of initial dose distribution after 8 hours run time. There
were two samples that had > 50% of the dose in the dermis for the acetone formulations
and there was a consistent ~21% of the total dose for the phosphate buffer formulation.
This finding is very encouraging and supports that resatorvid may be useful for topical
treatment and targeting the skin dermis layers, which is critical in the case of skin cancer
treatment.
65
Acetone formulation
Dose= 45.63 ug
PBS formulation
Dose= 47.12 ug
Flux
cumulative amount (ug)/cm2 % of dose released cumulative amount (ug)/cm2 % of dose released
Hr. Cell
# A
Cell
# B
Cell
# C
Avg. ±
std.
Cell
# A
Cell
# B
Cell
# C
Avg.
± std.
Cell
# D
Cell
# E
Cell
# F
Avg. ±
std.
Cell
# D
Cell
# E
Cell
# F
Avg.
± std. 0 0 0 0 0 0 0 0 0 0 0 0 0±0 0 0 0 0±0
1 0 0 0 0 0 0 0 0 0.13 0 0 0.04 ±
0.07
0.24 0 0 0.08 ±
0.13
2 0 0 0 0 0 0 0 0 0.20 0.12 0 0.11 ±
0.10
0.38 0.23 0 0.20 ±
0.19
3 0 0 0 0 0 0 0 0 0.35 0.23 0.09 0.22 ±
0.13
0.65 0.44 0.18 0.43 ±
0.24
4 0 0 0 0 0 0 0 0 0.48 0.40 0.17 0.35 ±
0.16
0.90 0.74 0.35 0.66 ±
0.28
5 0 0 0 0 0 0 0 0 0.91 0.84 0.47 0.74 ±
0.24
1.70 1.56 0.94 1.40 ±
0.40
6 0 0.49 0 0.16 ±
0.28
0 1.07 2.14 0.35 ±
0.61
1.16 1.45 0.63 1.08 ±
0.41
2.16 2.71 1.27 2.04 ±
0.72
7 1 1.41 0.98 1.13 ±
0.24
2.19 3.09 0 2.47 ±
0.54
1.20 1.78 0.49 1.16 ±
0.64
2.25 3.32 0.99 2.19 ±
1.16
8 2.81 2.50 0.11 1.81 ±
1.47
6.16 5.48 0.24 3.96 ±
3.23
1.25 1.49 0.78 1.17 ±
0.36
2.33 2.78 1.57 2.23 ±
0.61
Skin analysis
5- SC layers 1-3
Amount of drug (ug) % of drug Amount of drug (ug) % of drug 1.57 1.47 7.67 3.57±
3.54
3.45 3.23 16.82 7.83±
7.77
66
2.56 2.33 3.04 2.64±
0.36
5.60 5.11 6.66 5.79±0.
79
7- Extraction of dermis layers (Drug amount ug) 17.35 15.09 0.55 11.00±
9.11
38.03 33.07 1.22* 24.11±
19.97
4.80 4.80 4.95 4.86±
0.08
10.18 10.23 10.50 10.30 ±
1.7
8- Extraction of dermis layer normalized to % of recovery in method of extraction development 27.35 23.79 0.87 17.34±
14.36
59.93 52.13 1.92* 37.99±
31.48
9.94 9.98 10.24 10.05±
0.16
21.09 21.18 21.74 21.13 ±
0.06
*Accedental loss to part of the sample during extraction proccess.
Table 2.3: Results of in-vitro penetration study for resatorvid, acetone and PBS formulations.
6- SC layers 4-12
67
Figure 2.12 Flux of resatorvid through mice skin using acetone and PBS
formulations (a), drug percentage of distribution into Franz cell (b).
68
Acetone formulation Phosphate buffer saline pH
(7.4) formulation
Flux (Jss) (ug/cm2/hour) 0.82 0.36
Lag time (hours) 5.74 3.02
% of resatorvid dose accumulated
in dermis over 8 hours
37.99±31.48 21.13 ± 0.06
Table 2.4: Summary of resatorvid flux study.
8. In-vivo penetration studies:
Analysis of mice skin biopsy after topical application of resatorvid in acetone formulation,
on live mice, shows a fast penetration of resatorvid to the lower layer of skin (dermis
layers). Around 6 % of the dose of each spot was found in dermis layer one hour post-
application, Table 2.5 and Figure 2.13. Also, in 3 hours post- application, this percentage
decreased to around 1.5 %. However, a small percent of the resatorvid does remain in the
dermis layers over the time from 8 to 24 hours post application (0.45% and 0.23 % at 8th
and 24th, respectively).
69
Amount /spot (ug) Percent of dose/ spot (%)
n SC layers
1-3
Average
drug in
SC layers
4-12/spot
Average
drug in
dermis
(ug)/spot
Drug in
dermis
normalized
to % of
analysis
recovery
SC layers
1-3
Average
drug in
SC layers
4-12/spot
Average
drug in
dermis
(ug)/spot
Drug in
dermis
normalized
to % of
analysis
recovery
Hours post
application
Average ±
Std.
Average ±
Std.
Average ±
Std.
Average ±
Std.
Average ±
Std.
Average ±
Std.
Average ±
Std.
Average ±
Std.
1 6 66.74±26.11 3.46±4.04 9.07±6.27 14.30±9.88 27.67±10.82 1.43±1.67 3.76±2.59 5.93±4.09
3 3 49.93±20.44 0.00±00 2.26±0.42 3.56±0.66 20.70±8.47 0.00±00 0.94±0.17 1.48±0.27
8 6 4.71±3.89 0.82±0.89 0.69±0.32 1.09±0.51 1.95±1.61 0.34±0.37 0.29±0.13 0.45±0.21
24 3 1.15±0.94 2.11±0.27 0.35±0.04 0.55±0.06 0.48±0.39 0.87±0.11 0.14±0.01 0.23±0.02
Table 2.5: Resatorvid distribution after topical application on hairless mice of dose (241.21 ug/ spot).
70
Figure 2.13 Resatorvid dermal concentration after single dose application on mice
71
Interestingly, the ex-vivo results show relatively higher resatorvid concentrations retained
in the lower dermal layer after 8 hours, when compared to the in-vivo study. This could be
due to the intrinsic properties of live animal blood circulation, grooming or enzymatic
degradation of resatorvid. Additional studies are needed to replicate these results and to
look at ex-vivo dermis concentrations at earlier time points than 8 hours.
Conclusion:
Preformulation studies for resatorvid were helpful in determining the physicochemical
properties such as solubility, solubilization, and thermal stability which are essential for
further formulation.
The ex-vivo intrinsic penetration studies of resatorvid show that resatorvid has the ability
to penetrate the murine skin with a lag time 3 and 5.7 hours for phosphate buffer and
acetone formulation, respectively. The percentage of drug accumulated in the dermis from
both formulations is encouraging, and suggests resatorvid can be delivered via topical
application to the lower layers of skin.
The in-vitro study does not show a one to one correlation with the ex-vivo results, with
respect to dermal amounts as a function of time. However further studies are needed to
look at ex-vivo concentrations as a function of time and possibly metabolism in the skin.
72
CHAPTER 3
CORRELATION OF DRUG PENETRATION BETWEEN STRAT-MTM
MEMBRANES AND MURINE SKIN FOR VARIOUS DRUGS.
Introduction:
Synthetic membranes have been widely investigated as surrogates for actual skin in drug
penetration studies. Some of the synthetic membranes include cellulose acetate, nylon,
polysulfone, polycarbonate, and Teflon. Recently, Strat-MTM, a synthetic model for
transdermal diffusion testing made by EMD Millipore (Merck Millipore, USA), was
marketed as a new skin mimetic membrane (30). Strat-MTM is composed of a multilayer of
polyethersulfone, polyolefin and synthetic lipid post-treatment (31). It is a non-animal
based model for transdermal diffusion testing that has been reported to be predictive of
diffusion in human skin without lot-to-lot variability, safety and storage limitations (31).
The thickness of the membrane is 0.3 mm. Recently, independent researchers had evaluated
this membrane and compared it with animal and human skin and other polymeric
membranes. Uchida et al. evaluated the Strat-MTM membrane by comparing the
permeability of thirteen compounds through Strat-MTM, human and animal skin (30). They
concluded a direct relationship between a compound’s octanol/water partition coefficient
and the logarithm of permeability (log P) in all skins as well as Strat-MTM membrane. They
also found a correlation in log P (permeability) between Strat-MTM and human and hairless
murine skin, which lead them to suggest the use of Strat-MTM membrane as an alternative
to animal or human skin for in-vitro tests (30).
73
Another study by Bignon et al., found that a long-term application of film-forming
occlusive formulation on Strat-MTM membrane showed agreement with the ex-vivo human
skin (32). Also, Strat-MTM has wettability similar to the human skin, which was determined
via measuring water and oil contact angels (32).
Studies of skin penetration are an essential step in the process of drug selection for the
dermal or transdermal application. Moreover, clinical efficacies of topical skin products
are correlated with the amount of drug which penetrates the stratum corneum and reaches
the dermis layers. Thus, research efforts have been made to increase drug retention in skin
to avoid or minimize the systemic side effects for drug products that are not intended or
desired to be absorbed systemically (33, 34).
In-vitro studies have been performed to evaluate the amount of drug penetrated into skin
(human or animal), as well as artificial membranes using Franz penetration cell (35).
However, there is lack of such studies using Strat-MTM membrane.
In this study, hairless mouse skin has been used to as a comparator to the Strat-MTM
membrane. In assessing Strat-MTM, drug penetration, permeability coefficient, diffusivity,
lag time as well as drug mass distribution between donor, membrane or skin and receiver
chamber from Franz cell are investigated. The goal of this study is to investigate any
quantitative correlation between skin and Strat-MTM membrane on drug mass distribution
and to inquire if Strat-MTM, can be useful for the screening, not only for transdermal
delivery but for specifically epidermal/topical delivery as well. Five compounds having
various physiochemical properties are evaluated in both ex-vivo murine skin as well as in-
vitro using Strat-MTM. Table 3.1 shows some important physicochemical properties of the
compounds used in this study.
74
Compound Chemical
structure
pKa LogP Solubility in
water (ug/ml)
Solubility in
PBS pH 7.4
(ug/ml)
Melting
point
(ºC)
Molecular
weight (g/mol)
M-PABA 11.53 1.37 3820 1384 110 151.163
Diclofenac
Sodium
4.15 4.51 2370 > 3000 284 296.148
Resatorvid 7.9 3 95.3 60.77 65 361.812
Salicylic Acid 2.97 2.26 2480 > 3000 158.6 138.121
Hydrocortisone 12.58 1.61 320 362 218 362.466
Table 3.1 Chemical structures and physiochemical properties of the compounds used in this study
75
Materials:
Resatorvid (TAK-242) was purchased from Chem Scene (Monmouth junction, NJ, USA).
HPLC grade acetonitrile (ACN) was obtained from Spectrum Chemical Manufacturing
(Gardena, CA, USA). USP grade ethanol was obtained from Decon Laboratories Inc. (King
of Prussia, PA, US). Diclofenac, Methyl para-aminobenzoic acid (M-PABA), salicylic
acid, trifluoroacetic acid (TFA), Polyethylene glycol 400, monopotassium phosphate,
disodium phosphate, Sodium chloride and potassium chloride were obtained from Sigma-
Aldrich (St. Louis, MO, USA). Hydrocortisone was obtained from LKT laboratories Inc.
(St. Paul, MN, USA). Strat-MTM was purchased from Merck Millipore (Billerica, MA,
U.S.A)
Methods:
1. HPLC method development:
High-performance liquid chromatography methods for quantification were developed for
each drug separately. Each method was developed not for just quantification from aqueous
solution but also to separate the drug from skin extract, membrane extract and adhesive
tape extract.
The HPLC system consisted of a Waters 2695 separation module (Waters, Milford, MA,
USA) coupled with a Waters 2487 Dual absorbance detector. Quantification was
determined using peak area and calculated from a five-point standard curve. Standards
were prepared by volumetric dilution in various solvents and stored at 4°C, and protected
from light. Table 3.2 summarize the developed analytical methods.
76
Resatorvid Diclofenac M-PABA Salicylic
Acid
Hydro-
cortisone
Standard
solution
vehicle
ACN ACN: water
50:50
ACN: water
50:50
Ethanol: PBS
50:50
Methanol
Column Waters®,
Symmetry®
C18, 5um,
2.1x150 mm
Phenomenex®,
Synergi 4µ,
polar-RP, 80A
4.6x150 mm
Waters®,
Symmetry®
C18, 5um,
2.1x150 mm
Grace®,
Apollo C18,
5u
4.6x150 mm
Grace®,
Apollo C18,
5u
4.6x150 mm
Column
temperature
25 °C 25 °C 25 °C 25 °C 25 °C
Mobile phase 0.1% (v/v)
TFA in
water: ACN
60:40
0.1% (v/v)
TFA in water:
ACN 40:60
0.1% (v/v)
TFA in
water: ACN
90:10
ACN: water
30:70
Methanol:
water 60:40
Flow rate 0.6 ml/min 1.2 ml/min 0.6 ml/min 0.5 ml/min 1 ml/min
Absorbance
wavelength
230 nm 280 nm 230 nm 300 nm 247 nm
Retention
time
10.5 min 3.8 min 4.2 min 2.6 min 5.6 min
Injection
volume
10 ul 5 ul 10 ul 10 ul 10 ul
Table 3.2 Summary of HPLC methodology.
77
2. Method development for drug extraction from Strat-MTM and murine skin.
A method for rinsing drug from the membrane surface, as well as extraction from the
membrane or skin, was developed. A sample of each donor solution was mounted on a
piece of Strat-MTM membrane and incubated at 32±1 °C for several hours. Then 5 milliliters
of phosphate buffer saline pH 7.4 was used to rinse the drug residue off the membrane at
the end of each run by using a dropper to run the solution 20 times.
After rinsing the membrane, the membrane was transferred to scintillation vial with 2
milliliters of acetonitrile and subjected to ultrasound sonication for 30 minutes. All
collected samples were analyzed by the same HPLC method for each drug.
The method of drug extraction from skin started by tape stripping a piece of skin from the
back of the mouse 15 times, as illustrated in Figure 3.1. Then a sample of the drug in
phosphate buffer saline was added to the top face of stripped skin and incubated for several
hours at 32 ±1 °C. After that, a rinsing process was performed by receiver fluid in the same
way used for Strat-MTM membrane.
Drug extraction from the skin began by shredded the skin segment by scalpel into tiny
cubes (around 0.5 mm3 each). Then, 2 ml of acetonitrile was added to the ground skin and
homogenized for 5 min using Ultra-Turrax® homogenizer.
After homogenization, the resultant suspension was transferred to Eppendorf vial to be
centrifuged for 10 min at 14000 rpm. The clear supernatant was collected and analyzed by
HPLC using the method of analysis for each drug, as defined above.
78
Figure 3.1: Illustration of skin stripping procedure (36)
3. Strat-MTM membrane and mouse skin preparation:
Strat-MTM membranes were cut by a whole punch having the diameter of the Franz cell
chambers. According to the usage instructions of this membrane, the membrane does not
have to have any hydration before the use.
Skin segments were obtained from hairless p85DN mice. Both male and female mice were
used and all were aged 3-7 weeks. Each mouse was euthanized with carbon dioxide 30 min
before the experiment. Dorsal skin was removed carefully from the mouse and any
underlying adipose tissues were removed without any stretching or damaging to the skin.
The skin was then cut to the diameter of the Franz cell.
Sampling from the receiver was performed through the horn of the cell and was taken from
the middle of receiver solution using a long needle. After each sampling, an equal volume
of fresh receptor fluid was immediately replenished. Additionally, samplings from the
donor solution were performed carefully to avoid any disruption of the membrane or the
79
skin. The donor sample was returned to the donor chamber right after an aliquot was
injected into the HPLC system to minimize the loss of donor drug content.
The Franz cell has a receiver volume around 4 ml and the top surface area around 0.9 cm2.
A magnetic stir bar in the receiver chamber was set at 600 rpm. Each receiver cell was
filled with phosphate buffer saline at pH 7.4. The Strat-MTM membrane or skin segment
was mounted in between the donor and receiver chambers of the cell avoiding any
entrapped air. The shiny side of the membrane was faced the donor chamber. Similarly, the
stratum corneum side of skin was faced towards the donor side. The drug was dissolved in
phosphate buffer saline pH 7.4 and a volume of 1 ml with specific concentration was loaded
on the donor chamber and sealed from the top to avoid evaporation.
Penetration studies were performed in fixed temperature 32±1 °C. Samples were taken over
8 hours. Three vertical Franz penetration cells were used for each experiment (n=3).
4. Strat-MTM membrane washing and drug extraction method:
At the end of each penetration study using Strat-MTM membrane, the Franz cell was
carefully disassembled and the membrane was rinsed then extracted with the same method
mentioned in the method development section above.
5. Skin segment stripping and drug extraction:
After the skin was rinsed, the Franz cell was carefully disassembled and the skin segment
removed and washed with the same method mentioned on method development.
Then, skin segment was kept over the bench for 20 minutes to dry from the excess rinsing
solution. Next the skin was spread over a flat surface and the stratum corneum side was
80
adhesive film tape stripped for 15 times. The tape stripping reveals a shiny skin layer with
more bright blood vessels. The weight of skin segment was recorded before and after the
stripping process. This method of stripping can remove 15 layers of stratum corneum
Figure 3.2.
6. Calculations:
Drug depletion from the donor:
In these studies the drug concentration as a function of time in the donor solution was
measured for each experiment. The percentage of drug remaining in the donor chamber
was been plotted versus time in hours. In most cases, a linear curve was resulted and the
slope has been obtained mathematically. Figure 3.3 illustrates the slope calculation of drug
depletion within donor solution.
Flux:
Flux is the amount of drug crossing the membrane per time. It is given in units of mass/area/
time. Since all experiments in this study have been performed with a finite dose of the drug
the flux could be calculated by the formula J = Q/(A•t) where Q is the quantity of compound
traversing the membrane in time t, and A is the area of exposed membrane in cm2. Flux
values have been calculated as it is reported in many references of transdermal and dermal
drug delivery using the curve of cumulative drug penetration (ug/cm2) versus time (hr.).
The flux value is the slope in steady state region before the cure gets to the plateau in which
drug does start depletion. Maximal flux (Jmax) was calculated from the slope of the graph
determined after equilibrium was reached, i.e. when the substance penetration rate became
81
constant and maximum. The intercept of this slope with the x-axis corresponded to the
apparent lag-time (36).
82
Figure 3.3: Illustration curve for donor drug depletion calculation
Figure 3.4: Illustration curve for flux calculation (35)
83
Set of equations used to calculate other important parameters useful in describing the drug
permeability:
• J = P·C
• Pe=K·D/h
• J= K·D·C/h
• tlag= h2/6D or D= h2/6 tlag
• K= (J·h)/ (D·C)
Where:
J= Flux of the drug.
Pe= effective permeability coefficient.
K= partition coefficient of the drug between formulation and skin or membrane.
D=Diffusion coefficient for the drug in the skin or membrane.
h= thickness of membrane or skin.
Results and discussion:
1. HPLC method development:
All chromatograms of each drug separation from Strat-MTM membranes and skins samples
are presented in Table 3.3. All developed HPLC methods were successful in separating
drug from skin or Strat-MTM extracts. Drug retention times for resatorvid, M-PABA,
diclofenac sodium, salicylic acid, and hydrocortisone were 11.2, 4, 3.8, 2.6 and 5.6 minutes
respectively. The HPLC methods separated each drug from interfering drug and extractions
peaks. In addition, acceptable accuracy and drug recovery, and good linearity within the
84
working standard curve concentrations was achieved. The standard curves concentrations
were selected to include the minimum detectable drug concentrations.
85
Chromatogram of drug Chromatogram of blank Strat-MTM Chromatogram of control murine skin
Res
ato
rvid
M-P
AB
A
Dic
lofe
nac
sod
ium
Sali
cyli
c
Aci
d
Hyd
ro-
cort
ison
e
Table 3.3 Separation HPLC chromatograms of selected drugs, blank Strat-MTM membrane, and blank murine skin.
86
2. Drug extraction method development from Strat-MTM membrane:
Resatorvid Diclofenac Sodium M-PABA Salicylic Acid Hydrocortisone
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3
Theoretical drug
content in the sample
(ug)
3.48 5.77 4.19 6.51 6.51 6.51 271.32 271.32 271.32 5.75 5.75 5.75 5.86 5.86 5.86
Rinsing membrane
with 5 ml PBS for 20
times (ug)
0 0 0 1.43 6.24 5.39 40.63 49.12 52.26 5.83 5.96 0.88 1.27 1.13 2.93
Rinsing confirmation
with iml of PBS (ug)
0 0 0 0.05 0 0.05 0.52 0.50 0.69 0 0 0 0 0 0
Extraction in 2 ml
acetonitrile (ug)
3.42 5.46 3.96 5.73 0.54 1.08 206.80 212.79 202.68 0 0 0 4.44 4.59 4.55
% of recovery 98.50 94.54 94.50 110.59 104.17 100.11 91.39 96.719 94.21 101.43 103.61 97.56 97.67 127.75
Average ± Std. 95.85 ± 2.29 104.96±5.28 94.10 ±2.66 102.53± 1.54 107.66±17.39
Table 3.4 Summary of extraction method development from Start-MTM membrane.
87
3. Drug extraction method development from murine skin:
Resatorvid Diclofenac Sodium M-PABA Salicylic Acid Hydrocortisone
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3
Theoretical drug
content in the
sample (ug)
10.52 21.51 6.29 6.51 6.51 6.51 95.45 95.45 95.45 5.75 5.75 5.75 5.86 5.86 5.86
Rinsing membrane
with 5 ml PBS for
20 times (ug)
0.67 1.27 0.39 4.80 3.09 3.67 23.39 22.20 21.67 -- -- -- -- -- --
Rinsing
confirmation with
iml of PBS (ug)
0.03 0.03 0 0 0 0 0.30 0.27 0.29 -- -- -- -- -- --
Skin extraction in 2
ml acetonitrile (ug)
4.66 10.29 3.319 1.62 2.80 2.19 73.03 73.24 74.52 0.58 3.36 1.29 1.03 0.70 1.40
% of recovery 44.29 47.81 52.77 98.71 90.49 89.92 101.34 100.27 101.08 10.19 58.44 22.49 17.62 11.89 23.97
Average ± Std. 48.29±4.26 93.04 ±4.91 100.90±0.55 30.38± 25.07 17.82±6.04
Table 3.5 Summary of extraction method development from murine skin.
88
4. Penetration studies
❖ Resatorvid
Strat-MTM membrane Murine skin
Donor solution: 51.019 ug/ml saturated solution of PBS pH 7.4 47.12 ug/ml saturated solution of PBS pH 7.4
Frans cell number# #1 #2 #3 #4 #5 #6
Area Cm2 0.89 0.89 0.89 0.88 0.88 0.95
Volume of receiver’s fluid (ml) 4.17 4.17 4.17 4.24 4.38 4.24
Drug depletion from donor chamber:
Amount (ug) % of released drug Amount (ug) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 51.01 51.02 51.02 51.02
±5.42
100 100 100 100 ± 0 47.12 47.12 47.12 47.12±
8.70
100 100 100 100 ± 0
1 31.27 31.58 29.97 30.94±
5.21
61.30 61.90 58.74 60.65 ±
1.68
39.48 39.84 44.20 41.17±
2.62
83.78 84.55 93.80 87.38 ±
5.57
2 21.92 24.50 19.73 22.05±
5.05
42.97 48.03 38.67 43.22 ±
4.68
39.80 40.32 42.87 41.00±
1.64
84.48 85.57 90.98 87.01 ±
3.48
3 16.02 16.65 13.84 15.50±
4.92
31.39 32.63 27.13 30.39 ±
2.88
38.12 37.97 40.11 38.73±
1.19
80.91 80.57 85.11 82.20 ±
2.53
4 10.99 11.50 8.97 10.48±
4.75
21.53 22.54 17.57 20.55 ±
2.62
36.46 35.25 38.25 36.65±
1.50
77.37 74.82 81.17 77.79 ±
3.19
5 8.02 7.65 6.72 7.46±
4.62
15.72 15.00 13.18 14.63 ±
1.31
35.32 33.58 36.42 35.10±
1.42
74.95 71.27 77.29 74.51 ±
3.03
6 6.32 6.08 5.36 5.92±
4.51
12.39 11.91 10.50 11.60 ±
0.97
32.98 32.13 34.44 33.18±
1.17
69.99 68.17 73.09 70.42 ±
2.49
7 5.66 4.96 4.49 5.03±
4.47
11.09 9.72 8.80 9.87 ±
1.15
31.65 29.16 33.83 31.55±
2.34
67.17 61.88 71.80 66.95 ±
4.96
89
8 4.60 4.09 4.64 4.44±
4.38
9.024 8.01 9.09 8.71 ±
0.60
30.55 27.81 31.66 30.01±
1.98
64.83 59.02 67.19 63.68 ±
4.20
Receiver drug flux:
Cumulative amount (ug/cm2/hr.) % of released drug Cumulative amount (ug/cm2/hr.) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0±0
1 0 0 0 0 0 0 0 0 0.13 0 0 0.04
±0.07
0.24 0 0 0.08±
0.13
2 0 0 0 0 0 0 0 0 0.20 0.12 0 0.11±
0.10
0.38 0.23 0 0.20±
0.19
3 0 0 0 0 0 0 0 0 0.35 0.23 0.09 0.22±0.1
3
0.65 0.44 0.18 0.43±
0.24
4 0 0 0 0 0 0 0 0 0.48 0.40 0.17 0.35±0.1
5
0.90 0.74 0.35 0.66±
0.28
5 0 0 0 0 0 0 0 0 0.91 0.84 0.47 0.74±0.2
3
1.70 1.56 0.94 1.40±
0.40
6 0 0 0 0 0 0 0 0 1.16 1.45 0.63 1.08±0.4
1
2.16 2.71 1.27 2.04±
0.72
7 0 0 0 0 0 0 0 0 1.20 1.78 0.49 1.16±0.6
4
2.25 3.32 0.99 2.19±
1.16
8 0 0 0 0 0 0 0 0 1.25 1.49 0.78 1.17±0.3
6
2.33 2.78 1.57 2.23±
0.61
Membrane/skin analysis:
ug/ membrane % of drug ug/ skin % of drug
#1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
Drug deposition in membrane Drug deposition in dermis
38.02 41.64 42.61 39.83±
2.55
74.52 81.62 83.51 79.88±
4.74
4.80 4.82 4.95 4.84 ±
0.07
10.18 10.23 10.50 10.30 ±
1.7
Extraction normalized to method development
90
39.67 43.45 44.45 41.56 ±
2.67
77.75 85.16 87.13 83.35±
4.94
9.94 9.98 10.24 10.05±
0.16
21.09 21.18 21.74 21.13 ±
0.06
❖ M-PABA
Strat-MTM membrane Murine skin
Donor solution: 103.94 ug/ml saturated solution of PBS pH 7.4 103.94 ug/ml saturated solution of PBS pH 7.4
Frans cell number# #1 #2 #3 #4 #5 #6
Area Cm2 0.84 0.9 0.95 0.91 0.88 0.88
Volume of receiver’s fluid (ml) 4.2 4.33 4.26 4.07 4.29 4.35
Drug depletion from donor chamber:
Amount (ug) % of released drug Amount (ug) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 103.94 103.94 103.94 103.94±
0
100 100 100 100± 0 103.94 103.94 103.94 103.94±
0
100 100 100 100± 0
1 69.13 70.41 71.69 70.41±
1.28
66.51 67.75 68.98 67.75±
1.23
93.81 95.46 90.35 93.21±
2.60
90.26 91.84 86.93 89.67±
2.50
2 66.54 67 65.76 66.43±
0.63
64.02 64.46 63.27 63.92±
0.60
89.53 90.86 90.18 90.19±
0.66
86.13 87.42 86.76 86.77±
0.64
3 60.89 60.89 63.09 61.62±
1.27
58.59 58.58 60.70 59.29±
1.22
86.32 87.87 83.88 86.02±
2.00
83.05 84.54 80.71 82.77±
1.93
4 58.15 56.17 58.37 57.56±
1.21
55.95 54.05 56.15 55.38±
1.16
83.55 79.08 78.74 80.46±
2.68
80.39 76.09 75.76 77.41±
2.58
5 54.77 54.35 55.58 54.90±
0.62
52.69 52.29 53.47 52.82±
0.60
82.44 78.14 76.79 79.12±
2.94
79.31 75.18 73.88 76.13±
2.83
91
6 53.54 51.65 52.46 52.55±
0.95
51.51 49.69 50.47 50.56±
0.91
80.77 75.18 78.88 78.28±
2.84
77.71 72.33 75.89 75.31±
2.73
7 50.55 50.78 49.84 50.39±
0.48
48.64 48.85 47.95 48.48±
0.47
76.52 69.96 72.70 73.06±
3.29
73.62 67.31 69.94 70.29±
3.17
8 50.09 48.83 49.00 49.30±
0.68
48.19 46.98 47.15 47.44±
0.65
77.08 73.87 70.99 73.98±
3.04
74.16 71.07 68.30 71.18±
2.92
Receiver drug flux:
Cumulative amount (ug/cm2/hr.) % of released drug Cumulative amount
(ug/cm2/hr.)
% of released drug
Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 0 0 0 0±0 0 0 0 0 0 0 0 0±0 0 0 0 0±0
0.5 0 0 0.14 0.05±
0.08
0 0 0.13 0.04±
0.07
0.45 0.24 0.09 0.26±
0.18
0.40 0.21 0.07 0.23±
0.16
1 0.83 0.30 0.62 0.58±
0.27
0.67 0.26 0.57 0.50±
0.21
1.20 0.42 0.30 0.64±
0.48
1.05 0.36 0.25 0.55±
0.43
1.5 2.11 0.93 1.83 1.63±
0.62
1.71 0.81 1.68 1.40±
0.51
2.41 1.06 1.09 1.52±
0.76
2.11 0.90 0.92 1.31±
0.69
2 3.18 1.53 2.72 2.47±
0.85
2.57 1.32 2.48 2.12±
0.69
3.45 1.68 1.80 2.31±
0.99
3.02 1.42 1.52 1.99±
0.89
3 5.52 2.79 5.18 4.50±
1.48
4.46 2.42 4.73 3.87±
1.26
4.83 2.57 2.72 3.37±
1.26
4.23 2.18 2.30 2.90±
1.15
4 6.63 4.18 7.38 6.06±
1.67
5.35 3.62 6.74 5.24±
1.56
6.36 4.55 3.96 4.95±
1.24
5.56 3.85 3.35 4.26±
1.16
5 7.55 5.66 9.11 7.44±
1.73
6.10 4.90 8.33 6.44±
1.74
7.46 5.79 4.72 5.99±
1.37
6.53 4.90 4.00 5.14±
1.28
6 7.64 6.36 10.86 8.29±
2.32
6.18 5.51 9.93 7.20±
2.38
8.55 7.14 5.50 7.06±
1.52
7.48 6.04 4.66 6.06±
1.41
7 7.98 6.84 11.59 8.81±
2.48
6.45 5.93 10.59 7.66±
2.55
9.26 7.34 5.95 7.52±
1.66
8.11 6.21 5.04 6.45±
1.54
8 7.90 7.38 12.76 9.35±
2.97
6.38 6.39 11.67 8.148±
3.04
9.49 6.97 6.10 7.52±
1.76
8.31 5.90 5.16 6.46±
1.64
Membrane/skin analysis:
92
ug/ membrane % of drug ug/ skin % of drug
#1 #2 #3 Average
± Std.
#1 #2 #3 Averag
e ± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
Membrane wash SC layers 1-3 in 5 ml (1 water + 4 acetonitrile)
4.71 2.05 1.521 2.76±
1.71
4.54 1.97 1.46 2.65±
1.64
1.17 0.88 0.60 0.88±
0.28
1.13 0.85 0.58 0.85±
0.27
SC layers 4-12 in 5 ml (1 water + 4 acetonitrile)
1.41 2.40 1.70 1.84±
0.51
1.35 2.31 1.64 1.77±
0.48
Drug deposition in membrane Drug deposition in dermis
42.08 43.23 37.06 40.79±
3.28
40.48 41.60 35.66 39.24±
3.15
0.31 0.53 0.22 0.35±
0.15
0.30 0.51 0.22 0.34±
0.14
Extraction normalized to method development
44.71 45.94 39.38 43.35±
3.48
43.02 44.20 37.89 41.70±
3.35
0.31 0.53 0.22 0.35±
0.15
0.30 0.51 0.22 0.34±
0.14
❖ Diclofenac Sodium:
Strat-MTM membrane Murine skin
Donor solution: 122.53 ug/ml saturated solution of PBS pH 7.4 122.53 ug/ml saturated solution of PBS pH 7.4
Frans cell number# #1 #2 #3 #4 #5 #6
Area Cm2 0.9 0.88 0.88 0.91 0.88 0.88
Volume of receiver’s fluid (ml) 4.2 4.18 4.29 4.07 4.29 4.35
Drug depletion from donor chamber:
Amount (ug) % of released drug Amount (ug) % of released drug
93
Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 122.53 122.53 122.53 122.53±
0
100 100 100 100± 0 122.53 122.53 122.53 122.53±
0
100 100 100 100±
1.74
1 107.60 107.51 108.99 108.03±
0.83
87.82 87.74 88.95 88.17±
0.67
115.41 111.71 115.46 114.19±
2.15
94.19 91.17 94.23 93.20±
1.75
2 108.15 106.62 108.92 107.90±
1.17
88.27 87.01 88.89 88.06±
0.95
111.86 107.85 111.91 110.54±
2.32
91.29 88.02 91.33 90.22±
1.89
3 101.12 107.80 105.58 104.83±
3.40
82.52 87.98 86.17 85.56±
2.77
109.97 109.78 112.17 110.64±
1.32
89.75 89.60 91.55 90.30±
1.08
4 102.88 97.11 104.98 101.66±
4.07
83.97 79.26 85.67 82.97±
3.32
101.90 106.64 111.39 106.64±
4.74
83.16 87.03 90.91 87.03±
3.87
5 103.78 104.89 106.71 105.13±
1.48
84.69 85.60 87.09 85.80±
1.21
107.07 107.91 110.74 108.57±
1.92
87.38 88.07 90.37 88.61±
1.56
6 103.05 100.62 102.08 101.92±
1.21
84.10 82.12 83.31 83.18±
0.99
106.73 108.65 107.47 107.62±
0.92
87.11 88.67 87.71 87.83±
0.78
7 103.65 103.78 100.81 102.75±
1.67
84.59 84.69 82.28 83.85±
1.36
106.99 103.27 107.90 106.05±
2.45
87.31 84.28 88.06 86.55±
2.00
8 100.15 110.80 110.14 107.03±
5.96
81.73 90.43 89.89 87.35±
4.87
114.74 114.51 118.49 115.91±
2.23
93.64 93.46 96.70 94.60±
1.82
Receiver drug flux:
Cumulative amount (ug/cm2/hr.) % of released drug Cumulative amount
(ug/cm2/hr.)
% of released drug
Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 0 0 0 0±0 0 0 0 0±0 0 0 0 0±0 0 0 0 0±0
0.5 0.02 0.12 0.07 0.07±
0.04
0.01 0.08 0.05 0.05±
0.03
0 0 0 0±0 0 0 0 0±0
1 0.14 0.15 0.15 0.14±
0.00
0.10 0.11 0.11 0.10±
0.00
0.12 0.066 0.134 0.11±
0.03
0.09 0.05 0.09 0.08±
0.02
1.5 0.28 0.29 0.22 0.26±
0.03
0.21 0.21 0.16 0.19±
0.02
0.06 0.07 0.09 0.08±
0.01
0.04 0.06 0.06 0.06±
0.00
2 0.37 0.48 0.35 0.40±
0.07
0.27 0.34 0.25 0.29±
0.04
0.16 0.16 0.17 0.16±
0.01
0.12 0.13 0.12 0.12±
0.00
94
2.5 0.40 0.58 0.42 0.47±
0.10
0.30 0.42 0.30 0.34±
0.07
0.11 0.19 0.17 0.15±
0.07
0.08 0.14 0.12 0.11±
0.03
3 0.53 0.70 0.62 0.61±
0.08
0.39 0.46 0.45 0.43±
0.03
0.04 0.48 0.35 0.29±
0.22
0.03 0.37 0.24 0.21±
0.17
4 0.71 1.11 0.87 0.90±
0.20
0.52 0.80 0.63 0.65±
0.14
0.37 0.47 0.40 0.41±
0.04
0.28 0.36 0.28 0.30±
0.05
5 1.09 1.23 1.05 1.12±
0.09
0.80 0.88 0.76 0.81±
0.06
0.57 0.70 0.50 0.59±
0.10
0.42 0.54 0.34 0.43±
0.10
6 1.26 1.38 1.25 1.30±
0.07
0.93 0.99 0.90 0.94±
0.04
0.69 0.79 0.63 0.70±
0.08
0.51 0.61 0.43 0.52±
0.09
7 1.47 1.67 1.42 1.52±
0.13
1.08 1.20 1.02 1.10±
0.09
0.68 0.85 0.74 0.76±
0.08
0.51 0.66 0.51 0.57±
0.08
8 1.74 1.98 1.68 1.80±
0.16
1.28 1.42 1.20 1.30±
0.11
0.69 0.844 0.78 0.77±
0.07
0.52 0.65 0.54 0.57±
0.07
Membrane/skin analysis:
ug/ membrane % of drug ug/ skin % of drug
#1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
Membrane wash SC layers 1-3 in 5 ml (1 water + 4 acetonitrile)
0.24 0.37 0.11 0.24±
0.12
0.20 0.30 0.09 0.20±
0.10
0 0 0 0±0 0 0 0 0±0
SC layers 4-12 in 5 ml (1 water + 4 acetonitrile)
0 0 0 0±0 0 0 0 0±0
Drug deposition in membrane Drug deposition in dermis
6.95 7.16 7.43 7.18±
0.23
5.67 5.84 6.06 5.86±
0.19
0.52 0.57 0.47 0.52±
0.05
0.42 0.46 0.38 0.42±
0.041
Extraction normalized to method development
6.95 7.16 7.43 7.18±
0.23
5.67 5.84 6.06 5.86±
0.19
0.55 0.61 0.50 0.55±
0.05
0.45 0.50 0.41 0.45±
0.04
❖ Salicylic Acid:
95
Strat-MTM membrane Murine skin
Donor solution: 112.88 ug/ml saturated solution of PBS pH 7.4 112.88 ug/ml saturated solution of PBS pH 7.4
Franz cell number# #1 #2 #3 #4 #5 #6
Area Cm2 0.88 0.91 0.88 0.9 0.84 0.95
Volume of receiver’s fluid (ml) 4.37 4.09 4.22 4.26 4.23 4.27
Drug depletion from donor chamber:
Amount (ug) % of released drug Amount (ug) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 112.88 112.88 112.88 112.88±
0
100.00 100.00 100.00 100.00±
0
112.88 112.88 112.88 112.88±
0
100.00 100.00 100.0 100.00±
0
1 100.30 100.80 102.2 101.11±
0.99
88.86 89.30 90.56 89.57±
0.87
102.50
13131
102.59 102.33 102.47±
0.13
90.81 90.89 90.65 90.78±
0.11
2 98.02 102.33 99.96 100.10±
2.16
86.83 90.66 88.55 88.68±
1.91
106.21 101.97 108.51 105.56±
3.32
94.09 90.33 96.13 93.52±
2.94
3 103.09 100.75 99.91 101.25±
1.64
91.33 89.26 88.51 89.70±
1.45
105.09 107.14 110.00 107.41±
2.46
93.10 94.918 97.45 95.16±
2.18
4 101.14 104.93 101.35 102.48±
2.13
89.60 92.96 89.79 90.79±
1.88
109.46 107.88 108.42 108.59±
0.79
96.97 95.58 96.05 96.20±
0.70
5 102.84 107.10 111.76 107.23±
4.46
91.11 94.88 99.01 95.00±
3.95
110.85 113.51 111.53 111.97±
1.37
98.21 100.56 98.81 99.19±
1.22
6 104.16 103.40 101.58 103.05±
1.32
92.28 91.61 89.99 91.29±
1.17
108.21 106.17 109.13 107.84±
1.51
95.87 94.06 96.68 95.54±
1.33
7 103.69 105.41 106.58 105.23±
1.45
91.86 93.38 94.42 93.22±
1.28
113.62 113.27 112.85 113.25±
0.38
100.65 100.35 99.98 100.33±
0.33
8 107.81 108.29 113.22 109.77±
2.99
95.51 95.94 100.30 97.25±
2.65
117.95 115.14 116.18 116.42±
1.42
104.50 102.00 102.92 103.14±
1.26
Receiver drug flux:
96
Cumulative amount (ug/cm2/hr.) % of released drug Cumulative amount (ug/cm2/hr.) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 0 0 0 0 0 0 0 0 0.27 0.18 0.22 0.22±
0.04
0.21 0.13 0.18 0.18±
0.04
Membrane/skin analysis:
ug/ membrane % of drug ug/ skin % of drug
#1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
Membrane wash SC layers 1-3 in 5 ml (1 water + 4 acetonitrile)
2.42 4.80 4.17 3.80±
1.23
2.14 4.26 3.69 3.36±
1.09
0.10 2.99 3.32 2.14±
1.77
0.09 2.65 2.94 1.89±
1.56
SC layers 4-12 in 5 ml (1 water + 4 acetonitrile)
2.82 7.56 7.59 5.99±
2.74
2.50 6.70 6.72 5.31±
2.43
Drug deposition in membrane Drug deposition in dermis
0.19 0.05 0.21 0.15±
0.08
0.17 0.04 0.19 0.13±
0.07
0.19 0.18 0.11 0.16±
0.04
0.17 0.16 0.10 0.14±
0.03
Extraction normalized to method development
0.19 0.05 0.21 0.15±
0.08
0.17 0.04 0.19 0.13±
0.07
0.64 0.58 0.38 0.53±
0.13
0.56 0.52 0.34 0.47±
0.11
97
❖ Hydrocortisone:
Strat-MTM membrane Murine skin
Donor solution: 116.07 ug/ml saturated solution of PBS pH 7.4 116.07 ug/ml saturated solution of PBS pH 7.4
Frans cell number# #1 #2 #3 #4 #5 #6
Area Cm2 0.88 0.88 0.9 0.84 0.95 0.91
Volume of receiver’s fluid (ml) 4.33 4.3 4.209 4.18 4.3 4.116
Drug depletion from donor chamber:
Amount (ug) % of released drug Amount (ug) % of released drug Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 116.07 116.07 116.07 116.07±
0
100 100 100 100± 0 116.07 116.07 116.07 116.07±
0
100 100 100 100± 0
1 107.34 103.45 106.98 105.92±
2.15
92.47 89.13 92.16 91.25±
1.85
109.75 109.34 109.86 109.65±
0.27
94.55 94.20 94.65 94.46±
0.23
2 104.75 103.95 104.67 104.46±
0.44
90.25 89.55 90.18 89.99±
0.38
109.09 107.24 108.86 108.39±
1.00
93.98 92.39 93.78 93.38±
0.86
3 105.03 110.41 103.17 106.20±
3.75
90.48 95.12 88.88 91.50±
3.23
104.95 105.13 113.09 107.72±
4.65
90.41 90.57 97.43 92.80±
4.00
4 99.82 102.63 102.81 101.75±
1.67
86.00 88.42 88.57 87.66±
1.44
108.17 106.44 106.81 107.14±
0.90
93.19 91.70 92.01 92.30±
0.78
5 105.25 102.05 106.20 104.50±
2.17
90.68 87.92 91.49 90.03±
1.87
107.73 106.52 107.17 107.14±
0.60
92.81 91.76 92.33 92.30±
0.52
6 99.52 97.89 99.26 98.89±
0.87
85.74 84.33 85.51 85.19±
0.75
107.53 104.23 107.53 106.43±
1.90
92.64 89.79 92.64 91.69±
1.64
7 100.05 101.25 99.17 100.16±
1.04
86.19 87.23 85.44 86.29±
0.89
102.40 107.38 106.35 105.38±
2.62
88.22 92.51 91.62 90.78±
2.26
8 97.61 98.12 96.16 97.29±
1.01
84.09 84.53 82.84 83.82±
0.87
104.66 105.06 107.33 105.69±
1.44
90.17 90.51 92.47 91.05±
1.24
98
Receiver drug flux:
Cumulative amount (ug/cm2/hr.) % of released drug Cumulative amount
(ug/cm2/hr.)
% of released drug
Hr. #1 #2 #3 Average
± Std.
#1 #2 #3 Average
± Std.
#4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
0 0 0 0 0±0 0 0 0 0±0 0 0 0 0±0 0 0 0 0±0
0.5 0.03 0.04 0.01 0.03±
0.01
0.02 0.03 0.01 0.02±
0.01
0 0.01 0.03 0.01±
0.01
0 0.01 0.03 0.01±
0.01
1 0.009 0.03 0.03 0.02±
0.01
0.01 0.02 0.02 0.02±
0.01
0.03 0.03 0.02 0.03±
0.00
0.02 0.02 0.02 0.02±
0.00
1.5 0.06 0.03 0.02 0.04±
0.01
0.04 0.02 0.02 0.03±
0.01
0.02 0.02 0.03 0.02±
0.01
0.015 0.02 0.02 0.02±
0.00
2 0.03 0.03 0.03 0.03±
0.00
0.025 0.03 0.02 0.02±
0.00
0.05 0.05 0.03 0.04±
0.01
0.04 0.04 0.03 0.03±
0.00
3 0.02 0.08 0.02 0.04±
0.03
0.02 0.06 0.02 0.03±
0.02
0.06 0.05 0.02 0.04±
0.01
0.04 0.04 0.02 0.03±
0.01
4 0.08 0.06 0.07 0.07±
0.01
0.06 0.04 0.06 0.05±
0.00
0.12 0.09 0.09 0.10±
0.01
0.09 0.07 0.07 0.08±
0.01
5 0.13 0.08 0.06 0.09±
0.03
0.096 0.06 0.04 0.07±
0.02
0.11 0.07 0.08 0.09±
0.02
0.08 0.06 0.07 0.07±
0.01
6 0.07 0.12 0.12 0.10±
0.03
0.05 0.09 0.10 0.08±
0.02
0.14 0.15 0.11 0.13±
0.02
0.10 0.12 0.08 0.10±
0.01
7 0.10 0.12 0.11 0.11±
0.00
0.08 0.09 0.09 0.08±
0.00
0.22 0.22 0.07 0.17±
0.08
0.16 0.18 0.06 0.13±
0.06
8 0.15 0.09 0.04 0.09±
0.05
0.11 0.07 0.03 0.07±
0.03
0.19 0.22 0.17 0.19±
0.02
0.14 0.18 0.13 0.15±
0.02
Membrane/skin analysis:
ug/ membrane % of drug ug/ skin % of drug
#1 #2 #3 Average
± Std.
#1 #2 #3 Average ± Std. #4 #5 #6 Average
± Std.
#4 #5 #6 Average
± Std.
Membrane wash SC layers 1-3 in 5 ml (1 water + 4 acetonitrile)
99
0.71 0.69 1.57 0.99±
0.50
0.62 0.60 1.36 0.86± 0.43 0.03 0.07 0.03 0.04±
0.02
0.03 0.06 0.02 0.04±
0.02
SC layers 4-12 in 5 ml (1 water + 4 acetonitrile)
0 0 0 0 0 0 0 0
Drug deposition in membrane Drug deposition in dermis
8.24 7.90 7.62 7.92±
0.31
7.10 6.80 6.56 6.82±0.26 0.14 0.59 0.05 0.26±
0.28
0.12 0.50 0.04 0.22±
0.24
Extraction normalized to method development
8.24 7.90 7.62 7.92±
0.31
7.10 6.80 6.56 6.82±0.26 0.80 3.29 0.27 1.45±
1.61
0.69 2.83 0.23 1.25±
1.38
Tables 3.6 Penetration studies of resatorvid, M-PABA, diclofenac sodium, salicylic acid and hydrocortisone.
100
• Drug Distribution:
A single dose in the donor chamber will distribute according to the physiochemical
properties of the drug molecule in phosphate buffer saline (pH 7.4) and its permeability
through membrane or skin. For these studies, all experiments have a high percentage of
recovered drug at the end of each run. Typically, more than 91% of the total drug was
recovered with Strat-MTM Franz cell experiments and more than 80% of the drug was
recovered in the Franz cell experiments with skin. As can be seen in Table 3.7, all
experiments have modest variations. The table shows the additive percentage of drug
recovered from the donor chamber, receiver chamber and the membrane or skin after
normalization to the recoverable percentage in extraction method development.
Total percent of recovery of drug at end of penetration experiment
A B C Average ± Std.
Resatorvid Strat-MTM 86.78 93.17 96.22 92.05±4.82
Skin 88.26 82.98 90.49 87.24±3.86
M-PABA Strat-MTM 102.13 99.55 98.17 99.95±2.01
Skin 85.25 80.64 75.90 80.60±4.67
Diclofenac Strat-MTM 88.89 98.00 97.25 94.71±5.06
Skin 94.61 94.61 97.65 95.62±1.75
Salicylic Acid Strat-MTM 97.83 100.23 104.18 100.75±3.21
Skin 103.37 109.99 110.19 107.85±3.88
Hydrocortisone Strat-MTM 91.92 92.00 90.80 91.57±0.67
Skin 91.02 93.59 92.87 92.49±1.33
Table 3.7 Total percent of recovery of drugs in each Franz cell, where A, B and C
represent individual experiments.
Figure 3.5, displays all experiments drug mass distribution, as notice, there is a clear direct
correlation between the percent of drug penetrates (flux curve) and the amount of drug
deposition on the membrane or skin. It is obvious in the case of resatorvid which has the
101
highest percent of drug deposition either on skin or the membrane. However, M-PABA
with murine skin has a deviation of this since it has similar flux with membrane but a very
low percent of deposition on the skin compared with Strat-MTM.
The curves of drug concentration depletion from the donors, are shown in Figure 3.5. The
data in Figure 3.5 often give a straight line with a negative slope which could be described
as zero-order kinetic drug depletion from the donor, except the case of high-affinity drug
like resatorvid with Strat-MTM membrane which gives a first order kinetic depletion.
From Figure 3.5 and Table 3.8, we conclude that there is a direct correlation between the
values of negative slopes of drug depletion from the donors and the percent of drug
deposition on murine skin of Strat-MTM membranes. The only exception to this conclusion
is the case of M-PABA and murine skin.
102
103
Figure 3.5: Curves of drug distribution during 8 hours of in-vitro release using
Franz penetration cells with Strat -MTM membrane and murine skin
104
Strat-MTM Murine skin
Slope of drug
depletion
from donor
chamber
% of drug
retention in
the membrane
Slope of drug
depletion
from donor
chamber
% of drug
retention in
the dermis
layer
Resa
torv
id
A -9.8557 39.670 -3.7573 21.089
B -10.2402 43.447 -4.6008 21.179
C -9.7290 44.453 -4.0142 21.737
Average -9.9417 41.558 -4.1241 21.335
M-P
AB
A
A -4.8630 43.019 -2.8975 0.2977
B -5.0765 44.204 -3.8137 0.5061
C -5.1219 37.892 -3.4381 0.2169
Average -5.0205 41.705 -3.3831 0.3402
Diclo
fenac
sod
ium
A -1.4818 5.6752 -0.9463 0.4534
B -0.9927 5.8422 -0.7841 0.4974
C -1.1784 6.0642 -0.6686 0.4091
Average -1.2176 5.8605 -0.7997 0.4533
Salicy
lic
Acid
A 0.0288 0.1715 0.9365 0.5638
B 0.0585 0.0420 0.8248 0.5183
C 0.4364 0.1869 0.7020 0.3371
Average 0.1746 0.1335 0.8211 0.4731
Hyd
ro-
cortiso
ne
A -1.5219 7.0986 -0.9766 0.6890
B -1.4202 6.8039 -0.7835 2.8332
C -1.5922 6.5622 -0.7763 0.2357
Average -1.5114 6.8216 -0.8455 1.2526
Table 3.8 Summary of drug depletion curves slope from donor chamber and
corresponded percentages of drug retention.
105
Am
ount o
f dru
g
in d
onor (u
g)
Mem
bran
e
% left o
n d
onor
% o
f released at en
d
of ru
n
% in
SC
3 u
pper lay
ers
% in
SC
low
er layers
% o
f dru
g in
derm
is
% in
derm
is norm
alized
to ex
traction m
ethod
% in
Strat-M
TM
mem
bran
e
% n
orm
alized
to ex
traction
Flu
x (J) (u
g/cm
2/hr.)
t-lag (h
r.)
D (cm
2/hr.)
K
Pe (cm
/hr.)
Resatorvid
51.02 Strat-
MTM
8.71 0 --- --- --- --- 79.88 83.35 0 N/A N/A N/A N/A
47.12 Murine
skin
63.68 2.49 14.03 *Total SC back
calculation 10.30%
21.13 --- --- 0.365 3.02 4.53x
10-4
0.513 7.746x
10-3
Diclofenac
Sodium
122.53 Strat-
MTM
87.35 1.30 --- --- --- --- 5.86 5.86 0.247 0.44 6.614x
10 -5
0.915 2.017x
10-3
122.53 Murine
skin
94.60 0.57 0 0 0.42 0.45 --- --- 0.143 1.04 1.554x
10-4
0.225 1.169x
10-3
M-PABA
103.94 Strat-
MTM
47.44 8.15 --- --- --- --- 39.25 41.70 1.729 0.56 8.442x
10-5
5.911 1.663x
10-2
103.94 Murine
skin
71.18 6.46 0.85 1.77 0.34 0.34 --- --- 1.276 0.32 4.796x
10-5
7.680 1.228x
10-2
Salicylic
acid
112.88 Strat-
MTM
97.25 0 --- --- --- --- 0.13 0.13 0 90 0.0135 0 0
112.88 Murine
skin
93.52 0.18 1.89 5.31 0.14 0.47 --- --- 0.221 7 1.05x
10-3
0.056 1.962x
10-3
Hydro-
cortisone
116.07 Strat-
MTM
83.82 0.07 --- --- --- --- 6.82 6.82 0.013 0.89 1.341x
10-4
0.025 1.14x
10-4
116.07 Murine
skin
91.05 0.15 0.04 0 0.22 1.25 --- --- 0.024 0.18 2.721x
10-5
0.225 2.04x
10-4
Table 3.9 Summary of data from penetration studies.
Table 3.9 summarize the resulted calculations from penetration studies. All values on the
table are an average of at least 3 experimental penetration cell studies.
Several plots have been made in trying to identify interesting correlations between these
data and some of the physiochemical properties of these compounds like melting point,
partition coefficient, molecular weight, etc. Some of the correlations investigated are given
in Figure 3.6.
• Interesting correlations:
The previous study by Uchida et.al (30) got to the conclusion of correlation between Start-
M TM membrane and hairless rat skin and human skin. In evaluating similar correlations
in this study, permeability, diffusion coefficient, partition coefficient between donor
formulation and membrane or skin, and flux results were evaluated (Figure 3.6). These
results do not show a notable and significant correlation. One likely explanation for this is
due to the low number of compounds used.
The correlation in percent of drug retention between Strat-MTM membrane and murine
skin is shown in Figure 3.7. M-PABA was observed to be somewhat of an outlier. After
excluding M-PABA data, the correlation becomes stronger with slope 1.8 and R2 = 0.989.
Again due to the small sample size this correlation is interesting but may not be accurate.
More compounds that have higher skin depositions should be evaluated.
107
Figure 3.6: Correlation of Strat-MTM membrane and hairless murine skin.
108
Figure 3.7: Correlation of percent of drug dose deposition between murine skin and
Strat-MTM membrane for (a) all studied drugs, and (b) with excluding
M-PABA
109
Conclusion:
Using the result of these selected drugs, Strat-MTM membrane could be helpful in the used
for assisting the drug properties for deposition of skin dermis layers. Correlation of drug
percent deposition (with the exception on M-PABA) between the murine hairless skin and
Start-M TM membrane is notable. For future work, this study could be extended with more
compounds with other physiochemical properties. Also, it is suggested that using non-
aqueous and/or aqueous formulations including different pH values to examine ionization
effects, could elucidate intrinsic properties affecting drug retention in the Strat-MTM
membrane.
110
References
1. Kumar S, Kamboj J, Suman, Sharma S. Overview for Various Aspects of the Health
Benefits of Piper Longum Linn. Fruit. J Acupunct Meridian Stud. 2011; 4 (2):134–
140. doi: 10.1016/S2005-2901(11) 60020-4 PMID: 21704957.
2. Mishra SS, Tewari JP. Phytochemical Investigation of Piper chaba. J Pharm Sci.
1964; 53, (11): 1423– 1424.
3. Adams DJ, Dai M, Pellegrino G, Wagner BK, Stern AM, Shamji AF, et al.
Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs.
Proceeding of the National Academy of Sciences of the United States of America.
2012; 109 (38): 15115–15120.
4. Lee SW, Mandinova A. Patent application title: Methods for the treatment of cancer
using piperlongumine and piperlongumine analogs 2009. WO 20090312373.
5. Roh J-L, Kim EH, Park JY, Kim JW, Kwon M, Lee B-H. Piperlongumine
selectively kills cancer cells and increases cisplatin antitumor activity in head and
neck cancer. Oncotarget. 2014; 5 (19): 9227–9238. PMID: 25193861
6. Dhillon H, Chikara S, Reindl KM. Piperlongumine induces pancreatic cancer cell
death by enhancing reactive oxygen species and DNA damage. Toxicol Reports.
2014; 1: 309–318.
7. Fofaria NM, Kim S-H, Srivastava SK. Piperine causes G1 phase cell cycle arrest
and apoptosis in melanoma cells through checkpoint kinase-1 activation. PLOS
One. 2014; 9 (5): e94298. doi: 10.1371/ journal.pone.0094298 PMID: 24804719
111
8. Niu M, Xu X, Shen Y, Yao Y, Qiao J, Zhu F, et al. Piperlongumine is a novel
nuclear export inhibitor with potent anticancer activity. Chemico-Biological
Interactions. 2015; 237: 66–72. doi: 10.1016/j.cbi.2015. 05.016 PMID: 26026911.
9. Fofaria NM, Srivastava SK. Critical role of STAT3 in melanoma metastasis
through anoikis resistance. Oncotarget. 2014; 5 (16): 7051–7064. PMID: 25216522
10. Fofaria NM, Srivastava SK. STAT3 induces anoikis resistance, promotes cell
invasion and metastatic potential in pancreatic cancer cells. Carcinogenesis. 2015;
36 (1): 142–150. doi: 10.1093/carcin/ bgu233 PMID: 25411359
11. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Selective killing of
cancer cells by a small molecule targeting the stress response to ROS. Nature. 2011;
475: 231–234. doi: 10.1038/ nature10167 PMID: 21753854
12. Patel K, Chowdhury N, Doddapaneni R, Boakye CH, Godugu C, Singh M.
Piperlongumine for enhancing oral bioavailability and cytotoxicity of docetaxel in
triple-negative breast cancer. J Pharm Sci. 2015; 104: 4417–4426. doi:
10.1002/jps.24637 PMID: 26372815
13. Li J, Sharkey CC, King MR. Piperlongumine and immune cytokine TRAIL
synergize to promote tumor death. Nature Sci Rep 2015; 5, 9987; doi:
10.1038/srep09987
14. Son DJ, Kim SY, Han SS, Kim CW, Kumar S, Park BS, et al. Piperlongumine
inhibits atherosclerotic plaque formation and vascular smooth muscle cell
proliferation by suppressing PDGF receptor signaling. Biochemical and
Biophysical Research Communications. 2012; 427: 349–354. doi: 10.1016/j.
bbrc.2012.09.061 PMID: 22995306
112
15. Naika R, Prasanna KP, Sujan Ganapathy PS. Antibacterial activity of
piperlongumine an alkaloid isolated from methanolic root extract of Piper Longum
L. Pharmacophore. 2010; 1 (2): 141–148.
16. Bezerra DP, Pessoa C, de Moraes MO, Saker-Neto N, Silveira ER, Costa-Lotufa
LV. Overview of the therapeutic potential of piplartine (piperlongumine). Eur J
Pharm Sci. 2013; 48: 453–463. doi: 10.1016/ j.ejps.2012.12.003 PMID: 23238172
17. Fofaria NM, Qhattal HSS, Liu X, Srivastava SK. Nanoemulsion formulations for
anti-cancer agent piplartine- Characterization, toxicological, pharmacokinetics and
efficacy studies. Intl J Pharm. 2016; 498: 12–22.
18. Banerjee T, Chaudhuri S. The crystal and molecular structure of N-(3,4,5-
trimethoxycinnamoyl) -Δ3- piperidine- 2- one, an amide alkaloid
(piperlongumine), C17 H19 NO5. Can. J. Chem. 1986; 64: 876– 880.
19. Chatterjee A, Dutta CP. Alkaloids of Piper Longum linn-I structure and synthesis
of piperlongumine and piperlonguminine. Tetrahedron, pergamon press. 1967; 23:
1769–1781.
20. Amidon GL, Connors KA, Kennon L. Chemical stability of pharmaceuticals. A
handbook for pharmacist. 1st ed. John Wiley & sons; 1979. ISBN 0-471-02653-0.
P 97.
21. Scheiner JM, Araujo MM, DeRitter E. Thiamine destruction by sodium bisulfite in
infusion solutions. Am. J. Hosp. Pharm. 1981 Dec; 38(12):1911–3. PMID: 7325171
22. Yamada, M., Ichikawa, T., Ii, M., Sunamoto,, M., Itoh, K., Tamura, N., & Kitazaki,
T. Discovery of Novel and Potent Small-Molecule Inhibitors of NO and Cytokine
Production as Antisepsis Agents: Synthesis and Biological Activity of Alkyl 6-(N-
113
Substituted sulfamoyl) cyclohex-1-ene-1-carboxylate. J. Med. Chem, 2005; 48:
7457-7467.
23. Clinical trials Website. Available at:
https://clinicaltrials.gov/ct2/results?term=resatorvid&Search=Search. Accessed
March 5, 2017.
24. Matsunaga M, Tsuchimori N, Matsumoto T, and Ii M. TAK-242 (Resatorvid), a
Small-Molecule Inhibitor of Toll-Like Receptor (TLR) 4 Signaling, Binds
Selectively to TLR4 and Interferes with Interactions between TLR4 and Its Adaptor
Molecules. Mol Pharmacol 2011; 79:34–41.
25. Zhang D, Li H, Li T, Zhou M, Hao S, Yan H, Yu Z, Wei Li, Li K, Chunhua Hang.
TLR4 inhibitor resatorvid provides neuroprotection in experimental traumatic brain
injury: Implication in the treatment of human brain. Neurochemistry International
2014; 75:11–18.
26. Janda J, Burkett N, Blohm-Mangone K, Huang V, Curiel-Lewandrowski C, Alberts
D, Petricoin III E, Calvert V, Einspahr J, Dong Z, Bode A, Wondrak G and
Dickinson S. Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4
Blocks UV-induced NF-jB and AP-1 Signaling in Keratinocytes and Mouse Skin.
Photochemistry and Photobiology 2016; 92: 816–825.
27. Simon G, Maibach H. Relevance of hairless mouse as an experimental model of
percutaneous penetration in man. Skin Pharmacol Physiol. 1998;11(2):80–86.
28. Particle Sciences drug development services, technical brief 2009, volume 10.
Available at:
114
http://www.particlesciences.com/news/technical-briefs/2009/in-vitro-release-
testing-methods.html
29. Sunlight & Ultraviolet Radiation. Retrieved May 01, 2017,
Available at:
https://mycpss.com/skin/sunscreens/sunlight-ultraviolet-radiation/
30. Uchida T, Kadhum W, Kanai S, Todo H, Oshizaka T, Sugibayashi K. Prediction of
skin permeation by chemical compounds using the artificial membrane, Strat-M™.
European Journal of Pharmaceutical Sciences. 2015; 67: 113–118.
31. Strat-M® Membrane for Transdermal Diffusion Testing Website. Available at
https://www.emdmillipore.com/US/en/product/Strat-M-Membrane-for-
Transdermal-Diffusion-Testing,MM_NF-C112892. Accessed March 1, 2017.
32. C_ecile Bignon, Sonia Amigoni, Thierry Devers, Fr_ed_eric Guittard. Barrier
cream based on CeO2 nanoparticles grafted polymer as an active compound against
the penetration of organophosphates. Chemico-Biological Interactions 267 (2017)
17e24.
33. ZHANG, J.; SMITH, E. Percutaneous permeation of betamethasone 17-valerate
incorporated in lipid nanoparticles J. Pharm. Sci., v.100, n.3, p.896-903, 2011.
34. Luís Antônio Dantas Silva, Stephânia Fleury Taveira, Eliana Martins Lima,
Ricardo Neves Marreto. In vitro skin penetration of clobetasol from lipid
nanoparticles: drug extraction and quantitation in different skin layers. Brazilian
Journal of Pharmaceutical Sciences vol. 48, n. 4, oct./dec., 2012.
115
35. Fritz P. Schmook, Josef G. Meingassner, Andreas Billich. Comparison of human
skin or epidermis models with human and animal skin in in-vitro percutaneous
absorption. International Journal of Pharmaceutics 215 (2001) 51–56.
36. Benson, H. A., & Watkinson, A. C. (Eds.). (2012). Transdermal and Topical Drug
Delivery Principles and Practice. Hoboken, New Jersey: A John Wiley & Sons,
Inc., Publication. doi:978-0-470-45029-1
