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Optical characteristics of Deuteporfin (Deuxemether), a photodynamic therapy sensitizer

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Optical characteristics of Deuteporfin (Deuxemether), a photodynamic therapy sensitizer
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Duffy, Michael Charles ( author )
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Laboratory results on some of the optical properties of Deuteporfin, a relatively new photosensitizing drug that has been in clinical trials in China since around 2009, is discussed. The drug was characterized on the basis of one photon absorption and fluorescence emission for providing data for proper drug applications and dosimetry. In addition, the effects of photobleaching were investigated to characterize decay kinetics. The results of this research on this photosensitizer were also compared against HMME Hematoporphyrin monomethyl ether (HMME) (Hemoporfin) key characterization data which includes Q-band absorption to compare peak wavelengths and fluorescence intensity to show that Deuteporfin has similar absorption profile to HMME while it has superior fluorescence characteristics. The findings help to support the manufacturer’s claim that Deuteporfin can be an effective photosensitizer for tumor treatment.
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Thesis (M.S.)-University of Colorado at Denver.
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Includes bibliographic references
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by Michael Charles Duffy.

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Full Text
OPTICAL CHARACTERISTICS OF DEUTEPORFIN (DEUXEMETHER),
A PHOTODYNAMIC THERAPY SENSITIZER
By
MICHAEL CHARLES DUFFY
B.S., University of Colorado Denver, 2004
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Science
Electrical Engineering
2016


This thesis for the Master of Science degree by
Michael Charles Duffy
has been approved for the
Electrical Engineering Program
by
Tim Lei, Chair
Yiming Deng
Hamid Fardi


Duffy, Michael Charles (M.S., Electrical Engineering)
Optical Characteristics of Deuteporfin (Deuxemether), a Photodynamic Therapy
Sensitizer
Thesis directed by Associate Professor Tim C. Lei
ABSTRACT
Laboratory results on some of the optical properties of Deuteporfin, a relatively new
photosensitizing drug that has been in clinical trials in China since around 2009, is
discussed. The drug was characterized on the basis of one photon absorption and
fluorescence emission for providing data for proper drug applications and dosimetry. In
addition, the effects of photobleaching were investigated to characterize decay kinetics.
The results of this research on this photosensitizer were also compared against HMME
Hematoporphyrin monomethyl ether (HMME) (Hemoporfin) key characterization data
which includes Q-band absorption to compare peak wavelengths and fluorescence
intensity to show that Deuteporfin has similar absorption profile to HMME while it has
superior fluorescence characteristics. The findings help to support the manufacturers
claim that Deuteporfin can be an effective photosensitizer for tumor treatment.
The form and content of this abstract were approved. I recommend its
publication.
m
Approved: Tim Lei


DEDICATION
I dedicate this work to Raquel Duffy, whose infinite love and support helped
made this possible.
To all who have the desire to succeed in their goals, it is truly possible with hard
work and determination.
The three great essentials to achieve anything worth while are: Hard work,
Stick-to-itiveness, and Common sense. Thomas A. Edison
IV


ACKNOWLEDGMENTS
I would like to thank Dr. Tim Lei, for his support and encouragement has guided
me beyond what I thought possible in making this thesis a reality. Zheng Huang MD
PhD for supplying the research materials and advising me through the laboratory process.
Gregory F. Glazner, without whose inspiration, foundation research and laboratory
support at the University of Colorado Anschutz Medical Campus this work would not be
possible.
A big thank you to Brian Atkinson, for your classes, advisement and camaraderie;
keeping me focused on my undergraduate career while making it enjoyable no matter the
challenges that the outside world at the time had to offer.
I would also like to thank posthumously Dr. Carl T. A. Johnk, whose instruction,
enthusiasm, advice and friendship enabled me to step outside my comfort zone to
consider this research. His Electromagnetic Fields classes and TA experience he
requested on me were instrumental in my path leading to this point in my academic
career.
Last, and certainly not least, I thank my family. My amazing wife Raquel, who
never complained about the countless hours and sleepless nights of work, along with her
steadfast support helped me persevere to fulfill this research. My wonderful daughters
Gwyneth and Breanna, who keep the child in me alive with enthusiasm in everything I do
no matter the tasks I have in front of me. Thank you.
v


TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION...............................................................1
Deuteporfin Drag Description.............................................1
PDT Concept and Treatment................................................3
Light Administration used in PDT.........................................7
Surface Illumination..................................................8
Intraluminal Illumination............................................10
Interstitial Illumination............................................15
Bronchial Tumor Interstitial PDT.................................16
Prostate Cancer Interstitial PDT.................................19
Objective...............................................................24
II. MATERIALS AND METHODS......................................................25
Process.................................................................25
Preparation of Deuteporfin Solutions....................................25
One-Photon Absorption Measurements......................................26
Absorption Measurement Instrumentation and Theory....................26
Absorption Spectra...................................................29
One-Photon Fluorescence Measurements....................................30
Fluorescence Measurement Instrumentation and Theory..................30
Fluorescence Emission Spectra........................................32
Photobleaching Experiment...............................................33
Laser Photobleaching.................................................33
Solar Photobleaching.................................................33
Experiments to Compare Properties of Deuteporfin to HMME................34
vi


One Photon Absorption Spectra......................................34
Fluorescence Emission Spectra......................................34
III. RESULTS.................................................................36
One-Photon Absorption Profile of Deuteporfin..........................36
Fluorescence Properties of Deuteporfin................................38
Photobleaching Kinetics of Deuteporfin................................41
Comparison of Deuteporfin to HMME.....................................44
IV. DISCUSSION...............................................................49
Measurement Observations..............................................49
One Photon Absorption..............................................49
Fluorescence Properties and Photobleaching Kinetics................50
Deuteporfin and HMME Comparison Observations.......................51
Considerations for Future Analysis....................................52
Photobleaching Quantum Yield.......................................52
Fluorescence Quantum Yield.........................................54
Photodynamic Diagnosis.............................................55
In Vivo Characterization Utilizing Various Light Sources/Configurations.... 56
Two Photon Excitation..........................................56
Light Source Delivery..........................................58
Other Concerns.....................................................59
V. CONCLUSION...............................................................61
Deuteporfin in the PDT Arsenal........................................61
Final Thoughts........................................................62
REFERENCES...............................................................63
vii


LIST OF TABLES
Table
1.1 Photofrin Treatment Regimen for High-Grade Dysplasia in Barrett's Esophagus from
the Prescribing Information......................................................13
1.2 Fiber Optic Power Outputs and Treatment Times Required to Deliver 130 J/cm of
Diffuser Length Using the Centering Balloon per Length of Barretts Mucosa to be
treated..........................................................................13
1.3 Short Fiber Optic Diffusers to be Used Without a Centering Balloon to Deliver 50
J/cm of Diffuser Length at a Light Intensity of 400 mW/cm........................14
viii


LIST OF FIGURES
Figure
1.1 Chemical structure of MHD and DMD, the main components of Deuteporfin..........2
1.2 Simplified Jablonski Energy Level diagram for photosensitizer excitation leading to
singlet oxygen production..........................................................4
1.3 BLU-U Model 4170 Blue Light Photodynamic Therapy Illuminator (DUSA
Pharmaceuticals, Inc.@)..............................................................9
1.4 Diomed PDT 630 laser system and close-up view of an illuminated
OPTIGUIDE cylindrical fiber optic diffuser (Pinnacle Biologies, Inc.).............11
1.5 PDT to treat precancerous changes in Barretts esophagus (the dark red, smooth
areas), process descriptions below.................................................15
1.6 Intrinsic airway compression following photodynamic therapy....................17
1.7 Combined rigid and flexible bronchoscope used in interstitial bronchoscopic
photodynamic therapy................................................................18
1.8 Example of interstitial procedure of tumor mass in the right upper lobe........18
1.9 Illustration of prostate with interstitial fiber placement in PDT treatment....19
1.10 Illustration of calculated treatment light fluence being delivered through multiple
optical fibers positioned inside the prostate gland.................................20
1.11 Geometry description of prostate relative to the urethra and rectum..........20
1.12 Example of an interstitial procedure of a prostate with a tumor mass in the right
upper lobe..........................................................................23
II. 1 Cary 100 UV/Vis Spectrophotometer.............................................26
11.2 Cary 100 Top Level Schematic Diagram..........................................27
11.3 ISS PCI Steady-State Photon Counting Spectrofluorimeter.......................30
Figure II.4 ISS PCI Top Level Schematic Diagram....................................31
III. 1 Absorption Spectra of Deuteporfin in Various Solvents at 22 +2C............37
111.2 Deuteporfin Emission Spectra in the Range 610710 nm at 22 2C..............38
111.3 Deuteporfin in DMSO standard dilution curves..................................39
IX


III.4 Emission chart of Deuteporfin lpg/ml in DMSO at 22 2C.....................40
IH5 Photobleaching kinetics of Deuteporfin in DMSO................................42
IE.6 Solar bleaching of Deuteporfin in DMSO.......................................43
111.7 Chemical structure of HMME..................................................45
111.8 Absorption Q-band comparison of Deuteporfin and HMME in various solutes at
100 pg/ml.........................................................................46
111.9 Emission comparison of Deuteporfin and HMME.................................48
IV. 1 FRET process as applied to secondary sensitizer use in PDT.................57
IV.2 Schematic representation of the CQD-P conjugate illustrating indirect excitation of
the protoporphyrin IX upon two-photon excitation of the CQD........................58
x


LIST OF ABBREVIATIONS
DI H20 Deionized water (H20)
DMSO Dimethyl Sulfoxide
DP Deuteporfin, also known as Deuxemether
DXM Deuxemether, also known as Deuteporfin
FDA Food and Drug Administration
HMME Hematoporphyrin Monomethyl Ether, also known as Hemoporfiif
HpD Hematoporphyrin Derivative
MRI Magnetic Resonance Imaging
NP H20 Nanopure (18.3MQ/cm) water (H20)
o2 Oxygen
PMT Photomultiplier Tube
PDD Photodynamic Diagnosis
PDT Photodynamic Therapy
PBS Phosphate Buffered Saline
PBS/FBS Phosphate Buffered Saline/ Fetal Bovine Serum
PS Photosensitizer
ROS Reactive Oxygen Species
102 Singlet oxygen
TPE Two-photon Excitation
TRF Time-Resolved Fluorescence
US United States
UV Ultraviolet
xi


CHAPTER I
INTRODUCTION
Deuteporfin Drug Description
Deuteporfin (DP), also known as Deuxemether (DXM), is a relatively new
photosensitizing drag used in Photodynamic therapy (PDT), which has been in clinical
trials in China since around 2009. The photosensitizer (PS) is manufactured by Shanghai
Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd., which also manufactures
Hematoporphyrin monomethyl ether (HMME) (Hemoporfin), which has been also been
researched for its optical properties at the University of Colorado Denver. The primary
indication of Deuteporfin is for oncological (tumor) reduction therapy, where PDT
treatment can be administered either externally or internally to the site with delivery of
the light source to the affected area for activation of the drug being the only limitation to
treatment. Deuteporfin is a Hematoporphyrin Derivative (HpD) preparation which
mainly consists of 3(or 8)-(l-methoxyethyl)-8(or 3)-(l-hydroxyethyl)-deuteroporphyrin
IX (MHD), 3,8-di(l-methoxyethyl)-deuteroporphyrin IX (DMD), 3(or 8)-(l-
methoxyethyl)-8(or 3)-vinyl-deuteroporphyrin IX (MVD), 3(or 8)-(l-hydroxyethyl)-8(or
3)-vinyldeuteroporphyrin IX (HVD) and small portion of protoporphyrin IX [1], The
quantities of the components are proprietary, and because of this the molecular weight is
not available. Estimations on the two primary active components of the drug have been
given to be that MHD and DMD comprise of greater than 85% of the composition of
Deuteporfin, with the proportion of photosensitive porphyrin that has poor tumor
selectivity is much lower than that in other HpDs such as Photofrin, and a preclinical
1


study on rats has shown good tolerance to the as well as favorable elimination
characteristics that support the prospects of being a favorable PDT drug [2],
The core structure, a porphyrin ring (porphine), defined as a heteroaromatic
compound characterized by a tetrapyrrolic structure consisting of four pentagonal
pyrroles linked by four methylene bridges [3], The chemical structure for MHD and
DMD is shown in Figure 1.1.
Figure 1.1 Chemical structure of MHD and DMD, the main components of
Deuteporfin.
MHD: R1=CH3CH (OCH3) and R2= CH3CHOH or Rl=CH3CHOH and
DMD: R2=CH3CH (OCH3) or R1=CH3CH (OCH3) and R2=CH3CH (OCH3).
2


Because the molecular weight of Deuteporfin is not known, various analyses
involving molecular quantities cannot be performed without either having the weight be
supplied by the manufacturer or by extrapolation which is a time consuming process.
Further details into the analyses involved will be explained the discussion of this study.
Research has also been recently performed to test its effectiveness for malignant
tumor detection by Photodynamic Diagnosis (PDD). This procedure is common practice
but is currently in the early stages of research for Deuteporfin and has not been
performed in vivo to date [4],
PDT Concept and Treatment
Although this not a study on photochemistry or photobiology, a brief explanation
into PDT reaction principles and cell response are required to gain an understanding into
the need for optical characterization of photosensitizers. PDT occurs through a
photochemical reaction initiated by three non-toxic components; a photosensitizer (PS),
oxygen and light. However when the photosensitizer is excited by the appropriate
wavelength of light combined with oxygen supplied at the site the resulting reaction
generates Reactive Oxygen Species (ROS), which include singlet oxygen (102), which is
cytotoxic meaning toxic to cells. Singlet oxygen is more toxic to malignant cells but is
less harmful to healthy tissue.
This reaction occurs when a light source typically in the range of 400 700 nm,
irradiates the PS where a molecule in turn absorbs a photon which in turn excites the
molecule from the singlet So to Si state through a process called internal conversion (IC).
Si has a short lifetime in the nanoseconds; molecules can then either relax back to the So
ground state via IC generating heat or by fluorescence. However, there are some
3


molecules that cross over to the triplet Ti state through an intersystem crossing (ISC); a
state that has a significantly longer lifetime the micro to millisecond range. The time
advantage for molecules in this triplet state is the ability to efficiently transfer energy,
and when this transfer to naturally occurring triplet oxygen (3C>2) occurs the result is the
generation of 'CT. This process is shown in the energy level diagram in Figure 1.2 [5, 6]
Figure 1.2 Simplified Jablonski Energy Level diagram for photosensitizer excitation
leading to singlet oxygen production.
A light source irradiating the photosensitizer, or PS at the singlet So state which in turn a
molecule of the PS absorbs a photon promoting it to the excited singlet state Si by
internal conversion (IC). While many molecules convert the light to heat through IC or
by fluorescence when returning to So, some molecules through the intersystem crossing
(ISC) enter the triplet Ti state which in turn an energy transfer between the exited PS
molecules and naturally occurring triplet oxygen produces reactive singlet oxygen.
4


For PDT to be effective, the PS ideally has to be possess the following
characteristics:
be chemically pure and of known composition,
have minimal dark toxicity and only be cytotoxic in the presence of light,
be preferentially retained by the target tissue, with high contrast to the
neighboring normal tissue,
be able to be rapidly excreted from the body to provide low systemic
toxicity,
have a high quantum yield for the photochemical event, such as the
generation of singlet oxygen ('CF) to effectively kill cancerous tissue,
high photostability (low photobleaching) by prevention of degradation of
the PS, again for optimal 102 production to promote consistent and
thorough destruction of malignant cells,
high quantum yield of natural fluorescence for PDD by fluorescence
spectroscopy and for optical dosimetry measurements, and
have strong absorbance with a high extinction coefficient in the 600-800
nm range where tissue penetration of light is at a maximum and where the
wavelengths of light are still energetic enough to produce singlet oxygen
[7, 8],
The PS is typically administered by intravenous injection and therefore is
dispersed throughout the body; however the ideal scenario is to have PS localization at
the treatment site. Many photosensitizers have the property of being retained for longer
periods in malignant or target tissue versus healthy tissue, so this can be used to an
5


advantage for treatment [8], Furthermore, PDT activation occurs only where light is
applied, thereby it is possible to minimize damage to healthy tissue, but at the same time
to have the ability to properly illuminate the volume of target tissue in order to effectively
damage and kill the population of target cells is the goal. This delicate balance of drug
and light administration in combination with an adequate oxygen source to for the energy
transformation to !02 at a level appropriate for successful PDT treatment requires
analysis of the PS characteristic properties. What is the minimal concentration of PS
required to generate the proper quantity of 'CT to effectively treat the site? At what time
after delivery of the PS should PDT be administered for vascular or tumor treatment? At
what wavelength(s) of light can the PS be activated? How far can the light penetrate into
tissue before being absorbed to the point where it does not effectively activate the PS? Is
density of tissue a factor on light administration? Determining the optimal wavelength
and intensity for a specific PS activation, examining photobleaching characteristics and
PS concentration effects are just some of the factors in prescribing the proper PDT drug
dosage and light administration techniques [9], This can be a complicated evaluation that
could be researched to a level of extensive chemical, optical en vitro and en vivo
experiments that could easily be the subject of a doctoral dissertation. While the
experiments performed in this paper are but a subset of which can and should be
explored, the research that has been completed to date is a substantial step in fully
understanding Deuteporfins optical properties.
6


Light Administration used in PDT
Taking from a phrase, not all PSs and treatment regimens are alike, and because
of this different methods of light administration are used depending on the medical
condition and drug being utilized for treatment. Light regimen is as important as drug
regiment in order for a treatment to be successful. This is due to the PSs particular
characteristics such as absorption spectra and singlet oxygen quantum yield, and
therefore have different PDT treatment methodologies to best treat a particular medical
condition [10]. This section will address three methodologies in use that could ultimately
be utilized in Deutepofrin PDT administration.
Three examples of PS drugs which are approved by the FDA and actively used for
PDT treatment in the United States (US) include:
porfimer sodium injection (Photofrin), for treatment of esophageal
cancer, endobronchial cancer and high-grade dysplasia in Barretts
esophagus,
aminolevulinic acid HC1 for Topical Solution, 20% (Levulan) to treat
actinic keratosis, and,
vertiporfin injection (Visudyne) for treatment of macular degeneration; to
seal leaks at the center of the macula without damaging the central
vision [10].
For purposes of describing treatment methodologies that Deuteporfin would be
considered, Photofrin and Levulan treatment will be discussed but Visudyne will not be
explored as Deuteporfin has not been indicated in this type of macular degeneration
surface treatment. What follows is an introduction to the three types of light
7


administration (surface, intraluminal and interstitial) and how it could ultimately be
utilized in DP drug utilization. These examples are intended for describing light dosage
techniques only and not the chemical mechanisms of these PDT drugs.
Surface Illumination. This is a superficial methodology primarily used for
treatment of epidermal conditions such as acne, psoriasis, actinic keratosis or melanoma
[11]. Since these are surface related conditions PDT can easily administered by using a
topical solution ointment drug formulation and a light of either broad spectrum or
particular wavelength be applied based on the Q-band absorption spectra. An FDA
approved PS for this application used in the US is aminolevulinic acid HC1 for Topical
Solution, 20% (Levulan, DUSA Pharmaceuticals, Inc.) to treat actinic keratosis which
is a patch of moderate to thick scaly or crusty UV damaged skin (usually facial) that is
potentially 10% pre-cancerous leading to squamous cell carcinoma [12], As noted in the
manufacturer prescribing information, the drug is first applied topically to the affected
area and surrounding tissue to ensure all pre-cancerous subject tissue is treated and then
left to be absorbed by the tissue for 14 hours, which during that time the patient has to be
careful to not be exposed to bright indoor light or sunlight. After the time period has
elapsed there is a two hour window of opportunity to perform PDT treatment. Surface
illumination via blue light (400-450 nm) lamp exposure is administered for a period of
1,000 seconds (16 minutes, 40 seconds) in order to completely activate the PS with a total
light dose of 10 J/cm2. After treatment the treated lesions will become red and inflamed
with possibly some scaling, and hypersensitivity to light with pain and tingling to the
treated area may occur. The lesions will typically heal after approximately four weeks
8


after treatment. A recommended light source which is also manufactured by the drug
manufacturer that is shown in Figure 1.3.
Figure 1.3 BLU-U Model 4170 Blue Light Photodynamic Therapy Illuminator
(DUSA Pharmaceuticals, Inc.).
9


Levulan PDT treatment can be repeated twice in an eight week period per the
drag manufacturer to optimize results. This method of light delivery is the simplest to
administer compared to using laser light to accomplish PDT from a procedural
standpoint, but obviously this is limited to external applications. Deuteporfin is not
indicated for epidermal PDT, but this procedure could possibly be explored if a topical
formulation of the PS is developed in a dosimetry favorable for this application in the
future.
Intraluminal Illumination. The term intraluminal seems obvious grammatically
but needs to be properly explained in order to understand the full connotation of the
word. Oxford English Dictionary (2015) defines Lumen in physics as The SI unit of
luminous flux, equal to the amount of light emitted per second in a unit solid angle of one
steradian from a uniform source of one candela; however in anatomical terms it is the
central cavity of a tubular or other hollow structure in an organism or cell. The
esophagus, trachea, bronchial passages, arteries and veins, and intestines would be
considered to have luminous cavities. In the field of PDT, these areas if affected by a
malignancy could conceivably be treated by PDT via use of superficial intraluminal light
administration. The colon and lower gastrointestinal tract, esophagus and bronchial
passages are good candidates for this methodology. An FDA approved drag with a
proven success rate in intraluminal applications in PDT is Porfimer sodium (Photofrin,
Pinnacle Biologies, Inc.), which has been prescribed for treatment of endobronchial and
esophageal cancers as well as High-Grade Dysplasia (HGD) of Barretts Esophagus (BE)
[10]. BE is a precancerous condition that if left untreated has a 59% risk of developing
into adenocarcinoma of the esophagus which has a 5-year survival rate of less than 10%,
10


thus early detection and treatment of this condition can be the difference between life and
death [13]. The following scenario describes the process utilizing intraluminal 630 nm
laser light delivery to activate Photofrin to treat HGD in BE
As with DUSA who manufactures Levulan and the BLU-U illuminator, Pinnacle
Biologies also manufactures light delivery equipment for intraluminal PDT treatment.
The Diomed PDT 630 laser system is a recommended light source, and per the
manufacturers prescribing information alternate laser systems must be approved for
delivery of a stable power output at a wavelength of 630 3nm for use; however the
OPTIGUIDE cylindrical fiber optic diffusers for use in intraluminal therapy are
required by the manufacturer to ensure required tissue illumination to activate the PS
[14], The laser system and associated diffuser is shown in Figure 1.4.

/
Figure 1.4 Diomed PDT 630 laser system and close-up view of an illuminated
OPTIGUIDE cylindrical fiber optic diffuser (Pinnacle Biologies, Inc.).
11


According to the manufacturers prescribing information, the first step is to
administer the PS drug intravenously, in a dosage of 2 mg/kg of body weight in 3 to 5
minute period 40-50 hours prior to light treatment [14], This will enable the Photofrin to
be absorbed and selectively retained in the affected lumen tissue to be treated, while at
the same time healthy tissue will have the ability to excrete the drug clear of the PS in the
allotted wait time prior to treatment. At the time of treatment, an endoscope aides in
placement of a clear windowed centering balloon that is of a predetermined length of the
Barrets mucosa (tissue) to be treated through the lumen and to the treatment site,
followed by a complementary length cylindrical fiber optic diffuser which is placed into
the center channel of the balloon. The balloon is used to surround the fiber tip as an aid
in centering the light source in the area to be treated. A 630 nm laser light source is then
applied to deliver a required light dose of 130 J/cm of diffuser length where an acceptable
intensity of 200-270 mW/cm of diffuser length is recommended. To calculate the proper
dose the following equation is used:
Power Output From Diffuser (W) x Treatment Time (s)
Light Dose (J/cm)---------------------Diffuser Length (an)--------------------
Tables are provided in the prescribing information for treatment regimen and to account
for different diffuser lengths and power output combinations to successfully treat an area
for a required minimal time, as represented below. Table 1 is the procedure regimen in
the prescribing information HGD of BE treatment, and Table 2 lists the light dosimetry at
480 seconds at a light intensity of 270 mW/cm, with an option at a light intensity of
200mW/cm for lower power lasers with a total output up to 2.5W. Table 3 applies to the
optional short fiber 50 J/cm skip area treatment at a light intensity of 400 mW/cm.
12


Table 1.1 Photofrin Treatment Regimen for High-Grade Dysplasia in Barrett's
Esophagus from the Prescribing Information
Procedure Study Day Light Delivery Devices Treatment Intent
PHOTOFRIN Injection Day 1 NA
Laser Light Application Day 3a 3, 5 or 7 cm balloon Photoactivation
(130 J/cm)
Laser Light Application Day 5 Short (<2.5 cm) Treatment of
(Optional) fiber optic diffuser "skip" areas only
(50 J/cm)
a Discrete nodules will receive an initial light application of 50 J/cm (using a short fiber optic diffuser
without balloon) before the balloon light application.
NA: Not Applicable.
Table 1.2 Fiber Optic Power Outputs and Treatment Times Required to Deliver 130
J/cm of Diffuser Length Using the Centering Balloon per Length of Barretts
Mucosa to be treated.
Treated Balloon Fiber Optic Light Required Treatment Time
Barretts Window Diffuser Intensity Power
Mucosa Length Length (mW/cm) Output from (sec) (min:sec)
Length (cm) (cm) Diffuser3
(cm) (mW)
1-3 3 5 270 1350 480 8:00
4-5 5 7 270 1900 480 8:00
6-7 7 9 270 2440 480 8:00
200 1800 480 10:50
a As measured by immersing the diffuser into the cuvette in the power meter and slowly increasing the
laser power.
Note: No more than 1.5 times the required diffuser power output should be needed from the laser. If more
than this is required, the system should be checked.
13


Table 1.3 Short Fiber Optic Diffusers to be Used Without a Centering Balloon to
Deliver 50 J/cm of Diffuser Length at a Light Intensity of 400 mW/cm
FiberOptic Required Power Treatment Time
Diffuser Output from , , , . ,
r (sec) (min:sec)
Length (cm) Diffuser3 (mW)
1.0 400 125 2:05
1.5 600 125 2:05
2.0 800 125 2:05
2.5 1000 125 2:05
a As measured by immersing the diffuser into the cuvette in the power meter
and slowly increasing the laser power.
Note: No more than 1.5 times the required diffuser power output should be
needed from the laser. If more than this is required, the system should
be checked.
An initial BE treatment length of 7 cm is performed per the dosimetry. A secondary light
treatment may be applied immediately after up to five days from the PS injection to
address areas that visually appear as skipped or did not properly respond to treatment in
the initial procedure. A follow up full dose treatment can be performed to address
additional lengths of BE greater than 7 cm or to retreat the original area 90 days after the
initial PDT. For HGD of BE no debridement, or removal of dead tissue, is necessary
after treatment as compared to lung cancer procedures where residual dead tumor tissue
can potentially block the airways and does not naturally dissipate which in turn impedes
the healing process.
14


a
#
c
Figure 1.5 PDT to treat precancerous changes in Barretts esophagus (the dark red,
smooth areas), process descriptions below.
(a) View before therapy.
(b) View one day after PDT showing destruction of the esophageal mucosa (the
innermost layer of the wall of the esophagus, where the disease arises).
(c) View one month after PDT showing regeneration of the normal lining of the
esophagus.
(d) Shows the balloon encapsulating a cylindrical fiber for light delivery to the
esophagus. The balloon is first inserted over a guide wire positioned endoscopically at
the treatment site. The wire is then removed and replaced by a diffuser laser fiber, which
can be seen as a thin red line in the center of the balloon [15].
In the case of Deuteporfin, this would be one of the preferred methods of light
delivery and treatment of precancerous and malignant tissues, and could be a potential
alternative to Photofrin in PDT. Clinical trials would have to be completed in order to
determine its efficacy.
Interstitial Illumination. Interstitial implies that the optical fibers are directly
inserted into the tissue being treated such as a malignant tumor, which is normally dense
to where superficial light would not be able to penetrate to the center of the tumor. This
is the most invasive process used in PDT, and great care has to be used in this process of
treatment. Interstitial light treatment is approved in the US for lung cancer on
15


noncircumferential endobronchial tumors that are soft enough to penetrate the tissue, as
detailed in the Photofrin manufacturers prescribing information, but no evidence of pre
or post clinical trial results or use in this application could be found in the prescribing
information or elsewhere for reporting in this study [14], However, international
publications have documented this procedure as well as for other malignant tumor
interstitial techniques [16, 17]. There are also ongoing interstitial treatment regimen
studies that are in phase II US clinical trials which are expected to complete in 2017, as
documented in the ClinicalTrials.gov website database, that involve candidates who have
recurring malignant tumors of the head and neck.
The following presents two examples of the application of interstitial illumination
in PDT cancer treatment. The first which is authorized in the US utilizes the FDA
approved photosensitizer Photofrin, prescribed in the same drug and light dosimetry as
intraluminal PDT but the light delivery method is now more direct in that a small
cylindrical optical diffuser is directly placed in the tumor tissue. Next to be described is a
procedure that is being used in trials internationally in treating prostate cancer through
the same interstitial illumination method using multiple illumination fibers which has
also evidence of promising outcomes.
Bronchial Tumor Interstitial PDT. In the treatment of endobronchial cancer,
Photofrin is administered per the same manufacturers prescribed dosimetry regimen used
in the BE intraluminal procedure [14], After 40-50 hours the interstitial procedure is
performed as determined by the size and geometry of the tumor. Two to three days after
treatment, debridement, which is the process of removing necrotic tissue which can
obstruct the airways, inhibit lesion healing, and potentially contribute to secondary
16


infection needs to be performed. The added benefit of debridement from a secondary
PDT treatment standpoint is the remaining malignant live tissue is now exposed surface
area making light treatment more effective should additional intraluminal treatment be
prescribed. Secondary spot treatment can be administered between 96-120 hours after
the initial Photofrin injection. Additional PDT treatment can be administered a minimum
of 30 days after the initial therapy, and up to three courses (each separated by a minimum
of 30 days) can be given as needed.
tumor tissue
Figure 1.6 Intrinsic airway compression following photodynamic therapy.
Endoscopic view of left lower lobe of a patient with intrinsic compression related to
endobronchial carcinoid upon completion of photodynamic therapy (PDT).
(a) View during therapy, using a rigid bronchoscope containing thin laser fiber to deliver
633nm interstitial illumination. Fiber is placed in the center of the tumor in order to
evenly illuminate throughout the tumor to ensure even PS activation.
(b) Debridement of the tissue would occur 2-3 days after light administration to prevent
intrinsic airway compression (obstruction) due to necrotic debris.
(c) Post debridement, laser cauterization of the tumor base (Hemostasis) may be required
to minimize bleeding [18].
Devitalization of
vascularized
Debridement of
necrotic tissue
Hemostasis of
the tumor base
17


Figure 1.7 Combined rigid and flexible bronchoscope used in interstitial
bronchoscopic photodynamic therapy.
Note the cylindrical optical fiber with its end diffuser protruding through the flexible
biopsy channel of the fiber optic bronchoscope for illumination [16].
Figure 1.8 Example of interstitial procedure of tumor mass in the right upper lobe.
(a) Cylindrical diffuser in the tumor and (b) with illumination in progress [16].
18


Prostate Cancer Interstitial PDT. A more complex treatment regimen is
required for prostate cancer due to the physiology of the tumor and surrounding tissues.
The prostate gland surrounds the urethra and is in close proximity to the rectum. This
makes any treatment processes susceptible to complications from collateral tissue
necrosis that can effect quality of life, such as Erectile Dysfunction (ED) or incontinence
because isolating radiation treatment to the gland is very difficult given the close
proximity to the urethra and rectum [19]. This is where interstitial PDT shows promise in
that light levels can be adjusted via fiber placement in the organ along with tailored light
dosimetry as shown in Figures 1.9 through 1.11 below.
Figure 1.9 Illustration of prostate with interstitial fiber placement in PDT
treatment.
For localized prostate cancer interstitial treatment, cylindrical fiber optic diffusers can be
positioned and quantity adjusted to deliver PDT to either a portion of or to the entire
gland determining on the nature of the treatment [19].
19


Figure 1.10 Illustration of calculated treatment light fluence being delivered
through multiple optical fibers positioned inside the prostate gland.
Two different instances during the same treatment session, where the isosurfaces indicate
tissue that has received at least the threshold dose. Note the urethra in the central region
of the gland and the rectum just below the prostate [17].
Figure 1.11 Geometry description of prostate relative to the urethra and rectum.
3 dimensional rendition of the geometry of the prostate, urethra and rectum are indicated
(in red, green and blue, respectively). The template grid is shown on the x, y-plane. The
maximum projection of the prostate and cross sections of the urethra and rectum at the
same height are superimposed on the template, with rectal surface up to y > -1 observed
for determine the correct PDT exposure [20],
20


As seen in the three figures above, to effectively treat the cancerous tissue without
impacting the urethra and rectum is a difficult problem to solve, since both have also
been exposed to the photosensitizer meant to treat solely the prostate and all associated
cells are highly susceptible to PDT damage upon exposure to the threshold light
activation dosage. Therefore a threshold light dose has to be determined in each
individual case that ensures the minimum exposure required in order to activate the PS in
the treatment region only in order to prevent unintended cell death is paramount in
preventing post procedure medical complications and retaining optimal quality of life for
the patient post treatment.
A simplified example in three steps can be described for determining light dose
[17]. First using the light distribution, or fluence rate, and taking the PS concentration
into account as shown in the process below:
o
Where:
-Dpdt = PDT dose
T = treatment time
e = extinction coefficient of the PS
[P.V] = PS concentration
O = fluence rate, light distribution'
21


Next, interstitial treatment [PS] and O needs to be addressed three dimensionally.
Assuming for simplicity that the PS is homogeneously distributed in the target volume a
fluence dose can be determined:
Where: ^Fluence f ^ J 0 £>Fiuence = fluence dose T = treatment time O = fluence rate
Using calibrated fiber optic probes can aid in determining the fluence rate. Through
integration of the signal for a specified treatment time a fluence dose can be found for a
particular fiber location. Third, to develop a spatial map for the entire treatment volume,
the photon propagation is theoretically calculated and optical properties of the tissue
undergoing treatment are determined, which is aided by prior ultrasound and/or Magnetic
Resonance Imaging (MRI) procedures taken from prior examinations, and an imaging
procedure with the fibers in place before treatment. A photon propagation model is then
used, such as the theoretical model for the analytical solution to the diffusion equation:
Where: P 4>(r)=4n:£>reXp( '"r) (r) = radial fluence rate P = power (watts) D = diffusion coefficient (cm) //eir = effective attenuation coefficient of the light in the treated tissue (cm-1) r = radial distance from the point source (cm)
22


z = 0.6 cm
z = 1.2 cm
z = 1.8 cm
1000
0 1 2 3 4 5
z = 2.4 cm
0 1 2 3 4 5
x [cm]
Figure 1.12 Example of an interstitial procedure of a prostate with a tumor mass in
the right upper lobe.
Fluence rate map for 7 diffusers, with perr = 2.5/cm. The red line corresponds to the
prostate, the green to the urethra, and the blue to the rectum. The black lines are contour
levels at 100 (inner curve) and 62.5 (outer curve) arbitrary dose units [20],
As observed from this interstitial procedure, there are many variables that have to
be accounted for in order for this form of PDT treatment to be successful, but at the same
time the benefit can far outweigh the potential risks when you take into consideration
other treatment options. Modalities such a radiation therapy or brachytherapy have a
high incidence of side effects (incontinence, ED) which is why interstitial PDT is being
explored in preventing these outcomes. Photofrin is one PS that has been explored in this
PDT procedure, and Deuteporfin could be a potential candidate as well [17].
23


Objective
Little has been published to date in regards to Deuteporfin optical properties,
which is the purpose behind this research. The results from this initial optical
characterization of this compound can aid in developing treatment regimen and dosimetry
recommendations in regards to excitation methodology. The goal of this thesis is to
document preliminary laboratory analysis data and evaluate the characterization of
Deuteporfin using techniques including one-photon absorption and fluorescence series
dilution and photobleaching measurements in various solvents. Additionally, a
comparison of key Deuteporfin characterization results will be compared against HMME
data to reinforce previous published statements noting the potential effectiveness of this
photosensitizer for use in tumor PDT treatment.
24


CHAPTER II
MATERIALS AND METHODS
Process
Preparation and experiments with Deuteporfin require careful processes to ensure
that drug light exposure be eliminated/minimized wherever possible to ensure not to skew
laboratory results. In preliminary measurements to determine the correct concentration
for absorption and fluorescence it was noticed that the slightest variation can have drastic
changes in experiment outcomes, and experiments rerun to address anomalies confirmed
this scenario was usually the case. Processes to ensure accuracy and repeatable outcomes
were developed and adhered to ensure that experiments were repeatable and verifiable.
These processes are detailed in the following sections.
Preparation of Deuteporfin Solutions
Sample Deuteporfin was obtained in drug grade powder form and kept
refrigerated at 4C. Deuteporfin powder is similar to HMME in appearance however the
powder is lighter in consistency and tends to disperse in a fine film shortly after
transferring a sample to a plastic test tube. As such a base stock solution is required to
have readily available liquid form of the drug for experiments. The base stock solution
was made at lOmg/ml Deuteporfin with Dimethyl Sulfoxide (DMSO) for later use in
target dilutions for experiments. The stock solution was diluted with the solvents to be
evaluated prior to measurement to obtain lOOpg/ml concentration for Q-band absorption
measurements, 8 pg/ml for Soret absorption/emission measurements and 1 pg/ml for
fluorescence measurements unless otherwise noted. These dilutions were based on the
25


sensitivity range of the instruments used for measurement. All Deuteporfin solutions
were kept in light safe containers at all times up to measurement.
Absorption Measurement Instrumentation and Theory. One-photon
absorption spectra measurements were obtained to determine absorption peaks in the
Soret and Q-bands for the purpose of checking for optimal conditions for tissue light
absorption as higher wavelength light is more preferable for tissue absorption. A Cary
100 UV/Vis spectrophotometer (Agilent Technologies, Inc., Santa Clara CA, USA) was
used to measure absorbance, which is shown in Figure II.l.
One-Photon Absorption Measurements
Figure II. 1 Cary 100 UV/Vis Spectrophotometer.
26


Figure II.2 Cary 100 Top Level Schematic Diagram.
The step by step process which a spectrophotometer operates is described as
follows. A light source comprised of UV and visible light lamps are used to provide the
190 900nm instrument range which the bounds can be adjusted to a specific
measurement. The light enters a monochromator to select a particular wavelength of
light for evaluation, which then is sent through a series of mirrors to a beam diverting
chopper which either blocks, reflects or allows light to pass straight through depending
on the cell absorbance to be measured, ensuring that either the reference or sample beam
is aligned for accurate reproducible measurement. In the case of this example the light
will pass through to the path leading to the sample cell and continue until it is reflected at
a second chopper where the absorbance light is received by the Photomultiplier Tube
detector (PMT) for measurement.
27


In the case of the Cary 100, there are two ways to compensate for light absorption
discrepancies from the cuvette and solvent properties that can be subtracted from the
sample substance measurement. A reference cell of the same material composition can
be filled with the solute used for dilution and measured in the reference path along with
the sample cuvette and systematically subtracted out to produce the final measurement, or
the reference cuvette with solute can be measured first in a process called zero/baseline,
where the system software can record the reference measurements through a range and
calculations to compensate for the cuvette and solute can be performed during the final
measurement. The advantage of the second process is that the same cuvette can be used
for the reference and sample measurements removing any minimal discrepancies of
cuvettes composed of the same material that may have slightly different optical
properties, and also this process keeps the choppers in the same position which further
removes any variances that could occur due to chopper position anomalies.
The purpose behind performing absorption measurements is to confirm linearity
of concentration versus absorbance levels of a solution, to determine absorption intensity
peak wavelengths for the Soret and Q-band, to compare absorbance levels of the PS
against various solutes and compare levels against other compound/solute measurements,
in this case HMME. This measurement is based on the Beer-Lambert law, which states
that the concentration of a solute is directly proportional to the absorbance of a solution
[6], First by defining Transmittance as:
Is Is
T = j- and %T = 100Xj-
Ir Ir
Where: Is = Sample Intensity
Ir = Reference Intensity of cuvette with solvent
28


The Beer-Lambert law then defines Absorbance A as:
A = eel = Logl0 (£) = Log, (i) = Log (100 o/tZorption)
Where: e = Molar absorptivity, or molar extinction coefficient, M1 cm1
c = Concentration of the solute, M or pg/ml since the molecular weight of
Deuteporfin is not known
l = path length, cm, in this case 1 cm for the 3 ml quartz cuvette used
Therefore there is a relationship between Absorbance and Transmittance, where:
100
A = Logw and%Absorption = 100 %T,Absorbance =£ Absorption
%T
The last note is of importance since Absorbance is defined as the measured value or
magnitude of incident light absorbed by the sample, whereas Absorption is the process by
which an electron from a lower energy level is promoted to a higher energy level.
Absorption Spectra. Deuteporfin was diluted in DMSO, NPH2O, and
1 xPhosphate Buffered Saline (PBS). Standard one-photon absorption curves for each
solvent were obtained by successively diluting a 100 pg/ml sample in 10 pg/ml steps for
the Soret band and subsequently a 10 pg/ml sample in 1 pg/ml step dilutions for Q-bands
at room temperature (22 2C). These wavelength bands were chosen to appropriately
capture the range of the Soret band and four Q-band peaks as well as beyond the range to
check for any spurious anomalies. Three series dilution sets were taken and each dilution
point was averaged. All absorption readings were taken using the same quartz cuvette to
eliminate differences in readings caused by any cuvette-specific anomalies.
Measurements were recorded by the Cary 100 WinUV software (Agilent Technologies,
Inc., Santa Clara CA, USA).
29


One-Photon Fluorescence Measurements
Fluorescence Measurement Instrumentation and Theory. Fluorescence
emission spectra generated from one-photon excitation were obtained using an ISS PCI
steady-state photon counting spectrofluorimeter (ISS Inc., Champaign, IL, USA).
Figures II.3 and II.4 shows the ISS PCI and its associated light path schematic.
Figure II.3 ISS PCI Steady-State Photon Counting Spectrofluorimeter.
30


Unrip
Figure II.4 ISS PCI Top Level Schematic Diagram.
A spectrofluorimeter operates by light excitation via a xenon arc lamp through a
monochromator which accepts the incoming light and provides color selection by rotation
of a grating which determines the excitation wavelength for the sample measurement.
Next the light path encounters a beam splitter which diverts part of the light through a
quantum counter, which is to correct for fluctuations in lamp intensity as well as to
provide a reference wavelength of light for the reference PMT. Usually a highly
concentrated solution of Rhodamine B in ethanol or ethylene glycol is used for the
quantum counter [21], The light that is not diverted continues through an optional
excitation polarizer used for anisotropic measurements, which can adjust for vertical or
horizontal polarization for optimizing light coming from the excitation monochromator.
31


The light enters the sample and for the case of this study goes to the right though another
polarizer (optional for antistrophic measurements) to adjust emission polarity for the light
path onward, going through an optional long-pass filter used to block undesired higher
order scattered excitation light where it arrives to an emission monochromator, for
selecting the wavelength of light to be received by the emission PMT for measurement.
The ISS PCI measures fluorescence intensity in photons/second. In the case of
this study, this can be considered an arbitrary number.
Fluorescence Emission Spectra. Readings were taken at room temperature
(22C 5C) and recorded via the spectrofluorimeter software (Vinci version 1.6.SP7,
ISS Inc., Champaign, IL, USA). Excitation wavelength was 400 nm. Fluorescence
emission spectra in the 400800 nm range revealed no emission except two peaks at
approximately 615 nm and 678 nm depending on solvent, respectively. Therefore most
subsequent readings were taken in the 610710 nm range except where noted.
In addition, for each Q-band excitation wavelength from 450 to 585 nm at 5 nm
increments, synchronized emission spectra were obtained by using the ISS
spectrofluorometer to measure fluorescence intensity at 5 nm increments in the 600700
nm range. For these experiments, Soret band excitation wavelengths were not considered
as this band is not typically utilized in PDT treatment. All emission readings were taken
using the same quartz cuvette to ensure measurement consistency.
32


Photobleaching Experiment
Laser Photobleaching. To measure the effects of photobleaching on
Deuteporfin, a 630 nm laser was used with a set 1W output connected to a microlens
fiber (Medlight S.A. Ecubens, Switzerland), delivering light to a glass bottom petri dish
(MatTek Corp, Ashland, MA, USA) at 150mW/cm2. 3 ml of Deuteporfin diluted in
solvent was photobleached in the petri dish for a set period of time. After irradiation, the
solution was transferred to a 3ml quartz cuvette and measurements taken using the ISS
PCI spectrofluorometer. This process was repeated at exposure times of 0, 10, 30, 60,
120, 300, 600 seconds and results plotted.
Solar Photobleaching. An experiment utilizing sunlight was performed to
determine if broad spectrum light effects on photobleaching. 75 cm2 culture flasks
containing 100 and 10 pg/ml dilutions of Deuteporfin in DMSO. To prevent solvent
evaporation, the flasks were sealed prior to solar irradiation. Also three initial absorption
measurements were taken prior to photobleaching using the Cary 100 Spectrophotometer
at room temperature (22 2C) in the range of 350 450 nm for Soret, and 450 700nm
for Q-band observations and averaged. The samples were then exposed to bright sunlight
with an average 162 mW/cm2 intensity measured on the surface of the sample for 10
minutes. The irradiated samples absorption levels were measured again using the same
measurement process as before and graphed for comparison against the unbleached
measurements.
33


Experiments to Compare Properties of Deuteporfin to HMME
One Photon Absorption Spectra. A fortunate opportunity arose when exploring
the possibility of comparing Deuteporfin Q-band absorption spectra measurements
against equivalent HMME data, as the same concentrations and absorption spectra
measurements were performed as part of a previous study at the University of Colorado
Denver in 2010 using the same laboratory dilution preparation processes and
measurement procedures with the same Cary 100 Spectrophotometer [22], To confirm
that the equipment calibration matched previous calibration measurements taken at the
approximate time of the HMME data collection, a measurement was performed with a
reference 020 glass filter placed in the light path and compared against archived
measurement data which matched the original baseline measurement. With that in mind
there is high confidence in previous absorption peak wavelength data, although there is
still albeit minimal concern that there could be slight deviations in dilution practices that
could affect absorption intensity outcomes. As a result, Deuteporfin data obtained from
the previous one photon Q-band absorption spectra measurements in various solvents
were compared against the HMME absorption data to inspect the maximum Q-band peak
intensity and the phase shift of the peaks for both photosensitizers.
Fluorescence Emission Spectra. An experiment comparing Deuteporfin and
HMME was devised to see if there were significant differences in emission intensity and
peak emission frequency. The photosensitizers were diluted with DMSO to 1 pg/ml and
measurements taken with the ISS PCI Spectrofluorometer at room temperature
(22 2C) and measured in the 610-640 nm range. The resultant data was normalized
34


first as a group and then setting the range from 0 to 1 a.u. in order to compare the
maximum peak intensity as well as the phase shift of the peaks of the sensitizers.
35


CHAPTER III
RESULTS
One-Photon Absorption Profile of Deuteporfin
As noted earlier, Deuteporfin exhibits a very strong Soret absorption Soret peak
when diluted at 8 pg/ml. As seen in Figure III. la, The strongest absorption was obtained
in DMSO at 401 nm, followed by PBS and H2O at 393 and 391 nm. The solvents also
presented four distinct Q-band peak readings as shown in Figure III. lb in the range of
499 621 nm, with H2O and PBS wavelengths in family at the three higher intensity
peaks at 503 504 nm, 538 nm, 564 and 566 nm, with the greatest difference at 610 and
618 nm for H2O and PBS respectively. The Q-band DMSO peaks were more spread out
in comparison to the water based solvents, with peaks at 499, 533, 568 and 621 nm, and
showed a significantly higher absorption compared to the other water based solvents as
well.
36


Wavelength (nm)
Wavelength (nm)
H20
PBS
DMSO
H20
PBS
DMSO
Figure III.1 Absorption Spectra of Deuteporfin in Various Solvents at 22 +2C.
(a) Soret band obtained at Deuteporfin concentration of 8 pg/ml.
(b) Q-band obtained at Deuteporfin concentration of 100 pg/ml.
37


Fluorescence Properties of Deuteporfin
Due to the close proximity the lowest Q-band absorption peaks, low
concentrations were used in order to minimize reabsorption which can affect fluorescence
measurements. The strongest fluorescence peak range for Deuteporfin solutes was
between 618 and 628 nm. Deuteporfin dissolved in DMSO showed the strongest
fluorescence peak at 628 nm, followed by PBS at 618 nm, PBS/FBS at 626 nm and H2O
at 618 nm. A second peak was observed with water based solvents in the range of 678 -
686 nm, with a stronger DMSO peak further out at 694 nm. Interestingly the PBS/FBS
dilution peaks were more in family with DMSO with a left shift in the strongest peak of
only 2 nm, and an 8 nm left shift for the lower secondary peak.
Wavelength (nm)
------H20
...... PBS
PBS/FBS
-DMSO
Figure III.2 Deuteporfin Emission Spectra in the Range 610710 nm at 22 +2C.
Concentration of Deuteporfin in solution was 1 pg/ml. Excitation was at 400 nm.
38


A standard curve was generated for Deuteporfin in DMSO for the dilution range
1.0 O.lpg/ml, and trend lines added for inspection. The plots for both 628 and 694nm
emission peaks were almost perfectly linear throughout the dilution steps as seen in
Figure in.3.
628
694
Linear (628)
Linear (694)
Concentration (|ig/ml)
Figure 111.3 Deuteporfin in DMSO standard dilution curves.
Concentration range of Deuteporfin in solution was 1.0 0.1 pg/ml. Excitation was at
400 nm.
Q-band synchronized emission spectra from 610 710 nm versus 350 600 nm
excitation wavelength measurements taken incrementally in 5 nm steps were also
obtained using the ISS PCI spectrofluorometer. The resulting measurements were then
plotted by generating a topographic chart as shown in Figure III.4.
39


Figure III.4 Emission chart of Deuteporfin lpg/ml in DMSO at 22 +2C.
Contour lines represent an increase of 1 a.u. and 10 a.u. as labeled, normalized for the for
the Q and Soret band regions respectively.
40


Photobleaching Kinetics of Deuteporfin
Prior to the photobleaching experiment it was noted that for the for previous
Deuteporfin absorption and fluorescence standard curve trials that there was no self-
bleaching characteristics in the resulting measurements (emission and absorbance level
consistency with no phase shift) as this could taint the outcomes of the procedure. As
such it was determined that a series of trial timed instrumentation runs with samples not
exposed to light were not required. Photobleaching of Deuteporfin in DMSO diluted to
1 pg/ml was performed in an open petri dish at 630 nm and 150mW/cm2 which produced
an initial rapid reduction in fluorescence intensity at a first order rate, however this
changed at approximately 60 seconds exposure to a second order rate as seen in Figure
III.4a. Figure III.4b shows the overall photobleaching fluorescence spectra for the times
measured.
41


180000
0 100 200 300 400 500 600
Photobleaching Time (sec)
-----10
-----30
60
-----120
-----300
-----600
Emission Wavelength (nm)
Figure III.5 Photobleaching kinetics of Deuteporfin in DMSO.
(a) Exposure time curve: 1 pg/ml, 400nm excitation, 628 nm emission at 150mW/cm2.
(b) Fluorescence spectra from 600 700 nm. Exposure times: 0, 10, 30, 60, 120, 300,
600 from top to bottom.
Solar photobleaching was also examined, with Deuteporfin in 100 pg/ml and 10
pg/ml dilutions of DMSO exposed in direct sunlight to a measured average power of 168
mW/cm2 for a period of 10 minutes. No Soret peak shift was detected and the absorption
intensity decreased by just under one half, whereas the Q-band showed minimal shifts in
42


the 3 major peaks with expected reduction in intensity for each of the band peaks. An
unexpected minor peak emerged at 643 nm which may be a potential anomaly that could
be investigated in future research.
Wavelength (nm)
Wavelength (nm)
Figure III.6 Solar bleaching of Deuteporfin in DMSO.
(a) lOOpg/ml concentration Soret peak absorption decreased by. 54% at 401 nm with no
peak shift, (b) lOpg/ml concentration Q-band absorption peaks decreased by 24% at 499
nm, 23% at 535 nm, 20% at 568 nm and 15% at 621 nm respectively. Note there the low
level points of the solar bleached curve came to rest at a higher level than the unbleached
curve, and a red arrow points to a new peak at 643 nm, possibly indicating the presence
of photoproducts as a result of photobleaching.
43


Comparison of Deuteporfin to HMME
Some key Deuteporfin data was compared to current and previously measured
HMME data to compare each of the PS qualities and to determine what characteristics
could support the claims on Deuteporfin effectiveness for tumor PDT stated in the
manufacturers literature can be verified. Some HMME data was able to be obtained
from research that led to a journal paper that was published in 2012, of which I
contributed in laboratory test procedures and compiling results, and one experiment was
performed post HMME study as part of this research [22], A wealth of the HMME study
data could not be used as many of the laboratory process parameters followed in the
initial HMME investigation did not conform to laboratory procedure parameters for this
study, such as solute concentration for Soret band absorption spectra measurements,
photobleaching procedure process/concentration differences and temperature differences
during measurement procedures to name a few. As such the ability to objectively
compare the majority of the two sets of research data is limited. However, the key data
and results presented here give a good insight to the characteristic qualities and
differences of these two photosensitizers.
HMME, like Deuteporfin is also a HpD preparation which consists of a mixture of
two positional isomers of 7(12)-(l-methoxyethyl)-12(7)-(l-hydroxyethyl)-3,8,13,17-
tetramethyl-21H, 23H-porphrin-2, 18-dipropionic acid, as seen in Figure III.6 for the
treatment of port wine stain birthmarks. The drug has been indicated for use as a
vascular targeted PDT, and it has also been suggested that HMME could also be utilized
in in ocular and anti-tumor PDT [22],
44


(OCH3)
OH
I
(OH)
OCH3
I
ch-ch3
ch3
Figure III.7 Chemical structure of HMME.
An observation was initially made between previously obtained HMME data and
the Deuteporfin absorption data Q-band results. As shown in Figure III.8 there appears to
be a close similarity between both photosensitizers in not only their peak wavelengths but
absorption levels as well. As shown in Figure III.8 there is a strong correlation between
the two PS Q-band absorption peak levels for DMSO and PBS solutes, however there is a
noted downward shift in Deuteporfin peak levels in comparison to HMME in H2O. This
was not surprising as it was found in other HMME absorption data that solutions in water
appeared to be notably higher than in Deuteporfin H2O absorption spectra measurements.
45


DP DMSO .... HMMEDMSO
Wavelength (nm)
b DP PBS ... HMMEPBS
C ----DP NP H20 .... HMMEDIH20
Figure III.8 Absorption Q-band comparison of Deuteporfin and HMME in various
solutes at 100 pg/ml.
(a) DP in DMSO, (b) PBS, and (c) H2O, noting that in Deuteporfin NP H2O was used
compared to DIH2O which should have a negligible impact on the resulting
measurements.
46


An experiment was performed to compare the fluorescence characteristics of
Deuteporfin and HMME, with both PS samples diluted in lpg/ml DMSO and the highest
emission peak was measured from 610-640 nm. Although the two compounds shared
similar emission peak wavelengths, the intensity of fluorescence of Deuteporfin was
substantially higher by a factor of 19.6. This evidence shows favorably that Deuteporfin
is better suited to activation in deep tissue due to its increased emission levels at the same
excitation wavelength compared to HMME, which in turn suggests that more released
energy could be available for 'CE production.
47


DP 400 nm
HMME 400 nm
DP 400 nm
HMME 400 nm
Figure III.9 Emission comparison of Deuteporfin and HMME.
Photosensitizers diluted in 1 jug/ml in DMSO and excited at 400 nm.
(a) Fluorescence in Photons/sec
(b) Normalized Fluorescence intensity. Note the similarity in emission and the
significant difference in Fluorescence intensity of the two compounds.
48


CHAPTER IV
DISCUSSION
Measurement Observations
One Photon Absorption. Not surprisingly, Deuteporfin exhibits characteristics
similar to other hematoporphyrin-based sensitizers with Q-band absorption peaks which
reside within 500 700 nm and which is mostly within the practical PDT treatment range
of 600- 800 nm for tumor treatment, although a longer wavelength is preferred for deeper
tissue penetration. For current clinically used sensitizers the PS activation wavelength
varies from 420 nm (blue) to 780 nm (deep red). Light waves of greater length penetrate
tissue further; blue light attenuates greatly within 1-2 mm whereas red light can penetrate
more than 5 mm [23], For example, the average effective PDT penetration depth for the
PS Photofrin (intensity reduced to 37%) is about 1-3 mm at 630 nm, the wavelength used
for clinical treatment, while penetration is approximately twice that at 700-850 nm [24],
The strong Soret peak absorbance peak observed between 390 405 nm could also be
effective for PDT although the shorter wavelength is only suited for surface treatment or
for superficial lesions via intraluminal treatment since light penetration into the tissue is
minimal. These peaks are useful in determining which wavelengths to use for PDT based
on the target tissue type and thickness in combination with the location of treatment. The
manufacturers recommendation is to use longer wavelengths to enable deeper
penetration into tissue which is the intended application of Deuteporfin, whereas for
HMME it is more suited for shorter wavelength emission for safely treating vascular
conditions such as Port Wine Stain or surface lesions [25],
49


Fluorescence Properties and Photobleaching Kinetics. Examination of
fluorescence emission showed two peaks at approximately 623 5 nm and 686 8nm
depending on solvent used. Also, when a series dilution was performed the result was a
near perfect linear decrease in intensity from 1.0 0.1 pg/ml. Although not shown in this
paper, absorbance series dilutions were performed from 100 10 pg/ml for Soret and
10-1 pg/ml for Q-bands, and the results exhibited a similar peak height linear step
decrease for absorbance versus dilution. The high count for fluorescence (13519
photons/sec at lpg/ml) suggests a potential for good 'Cb production, however further
investigation through singlet oxygen phosphorescence measurements are needed to verify
this assumption. A comparison to fluorescence intensity of HMME in DMSO is explored
in the following section that adds support to this hypothesis.
Photobleaching observations are important to determine the amount of
photodegradation of the PS to understand whether the photobleaching is an advantage or
disadvantage to treatment. Too rapid and malignant tumor/tissue destruction may not be
complete, and depending on the PS damage or irritation to surrounding tissue could occur
if proper PS dosimetry and photobleaching rates were not taken into account to control
the PS reaction [26], Figure III. 5 shows how photobleaching initially impacted
Deuteporfin in the first 60 seconds of exposure, decreasing 20 percent from its initial
intensity. However from 60 to 600 seconds the degradation dropped by only 10 percent
with an intensity of 123850 photons/sec, hinting that 'CE production although decreased
should continue at an effective rate for treatment, although further evidence would be
preferred to verify this.
50


There is a strong interest if photoproducts can be produced by intense multi
wavelength irradiation such as by photobleaching which then can be potentially exploited
for PDT [27], It has been observed that there have multiple instances where porphyrins
have exhibited stable photoproducts in the 640-660 nm range that can be used in tumor
therapy due to increased absorbance in this region and increased light penetration depth
into tissue [28], Solar photobleaching Deuteporfin in DMSO at 100 pg/ml for 10 minutes
did indeed result in a new peak at 643 nm which is consistent with other porphine studies.
This result is interesting since when a similar 1 hour experiment was performed with
HMME in PBS at the same concentration for a 1 hour solar irradiation exposure time no
additional peaks were detected, only minor absorbance peak shifts [22], Further
investigation would be required to fully understand the extent of this new peak and
present confirmation of photoproduct formation with different solutes and dilutions along
with a set of time incremented solar photobleaching measurements.
Deuteporfin and HMME Comparison Observations. The similarities and
differences between Deuteporfin and HMME helped to verify the manufacturers
assertions as to the dosimetry methodologies for these medications. The Absorption
peaks were strikingly similar for both compounds, with minor fluctuations in absorbance
and similar Q-band peak wavelengths. As for the fluorescence measurements, it was a
pleasant surprise to see the dramatic increase by a factor of nearly 20 for the intensity of
Deuteporfin in contrast to HMME levels. Even though these measurements were for a
single solute (DMSO), it can be assumed that similar results would be observed for other
solutes. This adds further weight to the assumption that 'Oi production should be
significantly higher than that of HMME, and therefore should support the claim that this
51


PS would be useful in for tumor PDT. Again, to confirm this assumption further
characterization analysis is needed for confirmation as well as to determine the proper
dosimetry regimen for treatment.
Considerations for Future Analysis
There are still areas which need to be investigated further in order to have a
complete characterization worthy of submittal for a journal article, as this is the ultimate
goal when this research was initiated in 2011 when sample Deuteporfin was obtained for
characterization. Since there are multiple factors involved in effective PS activation, 102
production and biological uptake to effectively eradicate malignant tissue, multiple
studies will have to be ultimately be performed in order for determining proper dosimetry
parameters needed to proceed to a formal medical trial. Only when this is accomplished
can Deuteporfin be considered by the FDA as an acceptable PDT treatment.
Photobleaching Quantum Yield. One item that was overlooked was fulfilling
measurements to obtain photobleaching quantum yield data, which was successfully
accomplished as part of the study involving HMME characterization as noted earlier, and
is described in the process below. To measure initial photobleaching quantum yield, the
first steps that should have been performed in the photobleaching experiment were to
measure the fluence rate emitted from the microfiber through the solvent containing
Deuteporfin in the petri dish, using a an optical Si-detector with wattmeter with the
sensor directly below the petri dish to measure the transmitted power though the solution
before and after photobleaching. Converting the power measurements from watts to
photons per second, the quantum yield can be calculated.
52


The calculation process is as follows [22]:
after Y^before) ^
Where: i?i = initial rate of photon absorption by the reaction mixture
Ysafter = transmitted power difference in photons/s after
photobleaching
Ysbefore = transmitted power difference in photons/s before
photobleaching
t = bleaching time in seconds
and:
N ( fafter \ 6-0 2 2 X 1023 / fafter\
2 MWC\ fbefore) MW C{ fbefore)
Where: Ri = initial rate of reduction of Deuteporfin molecules
fafter = fluorescence intensity after photobleaching
fbefore = fluorescence intensity before photobleaching
cMg = initial concentration
N = Avogadros number
MW = is the molecular weight of Deuteporfin (unknown)
The initial quantum yield of photobleaching (P can now be calculated:
A factor that makes calculating the quantum yield not possible is that the molecular
weight of Deuteporfin is not known due to the proportionalities of the formulation is
estimated as the drug composition is deemed as proprietary information by the
53


manufacturer. In order to properly accomplish this characterization special permission to
obtain the molecular weight from the manufacturer would need to be obtained or if
possible a series of experiments would need to be performed to elaborately reverse
engineer the formulation characteristics individually to obtain a close approximation.
Only one article of research on Deuteporfin characterization has been found in regards to
determining the Deuteporfin components DMD and MHD concentration levels in dog
plasma, but the molarity of the combined reactive compounds or study into the remaining
(<15%) constituent formulation was not analyzed [29], This may be the reason why no
formal characterization journal papers have been published to date.
Fluorescence Quantum Yield. Another area to be worked include fluorescence
lifetime measurement, the time which a fluorophore remains in the excited state. The
purpose is to determine the rate of decay of the PS in order to prescribe the proper dose
and number of treatments for the proper PDT regimen. This would require in vitro and
in vivo studies since fluorescence lifetime measurements have the potential to
significantly decrease in in a biological environment in comparison to the PS in pure
solvent. The decay of the intensity as a function of time is defined as [30]:
11 _tA
~ lo
Where: It = intensity at time t
I0 = intensity at time 0
t = time
r = fluorescence lifetime
54


On a side note, x is the inverse of the of the total decay rate such that [30]:
T = (r + KrY1
Where: Y = radiative decay rate
knr = non-radiative decay rate
Studies have been performed in determining fluorescence lifetimes in the biological
environment through the use of Time-Resolved Fluorescence (TRF), which takes into
account biological phenomena which introduces a normalization term A into the intensity
decay equation above such that [31]:
It = Ae~t/T
Note that this is a simplification that addresses multiple biological conditions that are
beyond the scope of this research. Addressing lifetime measurements in a more natural
environment could further refine proper dosimetry requirements based on particular
treatment conditions and regimen. As such, this area of evaluation could be researched as
its own subset of PDT characterization analysis.
Photodynamic Diagnosis. As mentioned earlier in this study, the utilization of a
PS as an indicator of a malignancy in a process called Photodynamic Diagnosis (PDD)
has been explored and has promise in detecting cancers as well as infections and diseases.
A study with Deuteporfin was conducted to visually detect lymphatic metastases in rats
by visual fluorescence observation using a Woods lamp, which is essentially an
ultraviolet light in the range of 320 400 nm that is primarily used in dermatological
examinations to observe skin disorders and bacterial/fungal infections [32], It was
determined that there was an increase in concentration in Deuteporfin in cancerous tissue
in comparison to normal healthy tissue, which resulted in the observation of malignancies
55


increased fluorescence when irradiated by a Woods lamp [4], Research performed on
other PDT PSs has shown that their application as PDD is viable by procedural
adjustments such as modifying the excitation light dose [33],
In Vivo Characterization Utilizing Various Light Sources/Configurations.
This area of PDT characterization research has not been addressed, although it appears to
be utilized more in line with clinical trials. To maximize PDT results while minimizing
test subject complications during treatment, some of this analysis can be brought into the
characterization procedures to explore light source placement in tumor treatment.
Two Photon Excitation. A potential experiment under consideration would be
two-photon excitation (TPE). In PDT this process has been investigated to exploit the
ability to excite many PS formulations at approximately twice the optical wavelength
compared to one-photon excitation, which in turn allows deeper tissue penetration. This
combined with minimizing collateral tissue damage as the probability of absorption
increases with the square of the light intensity, enabling spatial confinement of PS
activation as well as femtosecond lasers can be utilized minimizing photothermal damage
makes TPE an attractive candidate for PDT treatment [22, 34], For HMME a two-photon
experiment was performed achieving favorable results including obtaining an absorption
peak at 740 nm, that given the absorption comparison with Deuteporfin would suggest
that the PS would also exhibit a similar response to TPE [22],
Another novel process in development is to use secondary sensitizers in
combination with a PS for TPE PDT treatment. A secondary sensitizer such as a Carbon
Quantum Dot (CQD) would be activated at the TPE wavelength range and the sensitizer
can be then excited indirectly with two near-IR photons through an energy transfer
56


mechanism known as either Forster Resonance Energy Transfer or Fluorescence
Resonance Energy Transfer (FRET) in the activation band for the PDT PS, thereby
providing excellent light penetration characteristics up to 2 cm for tumor treatment
efficacy [35, 36], This process could be performed in vitro prior to clinical trials to
investigate the biological environment impacts to TPE to add fidelity to characterization
measurements.
Figure IV.l FRET process as applied to secondary sensitizer use in PDT.
The concerted transitions of donor and acceptor require energy matching of fluorescence
of the donor (dashed green arrows and spectrum) and absorption of the acceptor (dashed
orange arrows and spectrum). However, they occur without emission of the donor. After
the energy transfer to the acceptor, its specific fluorescence spectrum can be observed
(red arrows, spectrum), or in the case of having protoporphyrin IX PS the acceptor
molecules will go through the ISC into the Ti state and 'CE production would occur [35],
57


0

2x800nmi
Figure IV.2 Schematic representation of the CQD-P conjugate illustrating indirect
excitation of the protoporphyrin IX upon two-photon excitation of the CQD.
CQD is excited at 400nm, and results in FRET energy transfer to the protoporphyrin IX
PS, resulting in 'CF at a tissue penetration depth not possible utilizing the PS alone [35],
Light Source Delivery. Utilizing the correct medically approved lasers and
optical fibers to administer the required light dosimetry to the affected site can
significantly affect dosimetry requirements and treatment outcomes of PDT. Recent
developments in research and the manufacturing of FDA approved medical grade laser
equipment has produced systems specifically designed for the field of PDT in
administering multiple light sources of varying wavelengths and intensities for various
site and PS applications. This type of system could be enhanced with the ability to
provide a reactionary light source in multiple regions of the treatment site for interstitial
light delivery, by providing customized irradiation to surrounding the tissue using
multiple optical fibers that dosimetry parameters can be individually measured and
adjusted for each fiber channel, minimizing multiple fiber placement and irradiation
58


procedures. One application that shows promise for both research and application of
PDT is a system that uses multiple fibers for administrating laser light to the site as well
as for monitoring levels of irradiation to monitor treatment progression by the ability of
switching the fibers from the output laser via a switching interface to a spectrometer for
measurement. [37], An approved laser example is the Modulight ML7710-PDT medical
laser system (Modulight, Inc. Finland, www.modulight.com) is capable of delivering up
to 8 individually controlled fiber output channels of wavelengths of 630, 635, 652, 660-
690 and 753nm at a power of 1-15 W. This could be coupled to a similar switching
interface apparatus to alternate between the various emission/detection sources, or
additional fibers can be inserted to the site for direct coupling to spectrometer(s) for
active PDT monitoring. This method could be of interest in the pre-clinical phase of PS
characterization by monitoring tissue PDT process from multiple regions to monitor
biological variations in fluorescence and photobleaching quantum yields as well as
developing processes to accurately determine 'CF quantum yield under different
biological conditions.
Other Concerns. The website ClinicalTrials.gov is maintained by the US
National Library of Medicine and National Institutes for Health as a resource for
accessing clinical studies process and their outcomes by researchers and medical
professionals as well as for the general public. This is a useful tool in determining a drug
or medical device status for acceptance for further study or for proceeding toward the
next steps for FDA approval. A search on Deuteporfin was performed to see if any
preliminary clinical trials were performed and if so the corresponding study findings.
The search revealed that there was a record of a clinical trial performed under record
59


number NCT01481597 initiated October 2011 and completed May 2012. The study was
to investigate on the bodys tolerance to the drug and on pharmacokinetics, which is the
time course of absorption, bioavailability, distribution and metabolism, of single-dose of
Deuteporfin administered intravenously in 32 healthy volunteers to determine the
potential for adverse events that could be a health concern. Although the study was
completed, the findings have not been entered into the database, nor upon searching for
journal papers or medical publications has there been found any published outcomes to
this study. This is a potential concern since if there are any results suggesting that
Deuteporfin is not a safe and viable PDT treatment then further research into its
properties may have to be reconsidered.
60


CHAPTER V
CONCLUSION
Deuteporfin in the PDT Arsenal
There are many observations that have shown that Deuteporfin is similar to other
HpD PS drugs. There is a very strong Soret peak followed by four weaker Q-band peaks
in the 499 621 nm range which is within the common spectrum for performing PDT
and very similar to HMME Q-band peak spectra. There are also two strong fluorescence
peaks at approximately 623 and 686 nm, with the strongest fluorescence peak of the
various solvent dilutions having some similar qualities with the Soret absorption peak.
There may be a possibility of photoproducts being generated when exposed to broad
spectrum light which needs to be further investigated, although no preliminary evidence
of photoproducts appeared after laser (630 nm) irradiation. The initial characterization
data in this report supports that Deuteporfin is a promising candidate for tumor PDT
treatment. The investigation into photobleaching quantum yield needs to be performed to
determine the extent of singlet oxygen production in order to perform further research
into dosimetry protocols. Comparison of DP against other FDA approved PSs such as
Photofrin and Levulan needs to be performed in order to verify that this drug at a
minimum can be utilized as a substitute for PDT treatment, and to verify that the potential
for enhanced treatment regimen is possible. Safety into its utilization during treatment
and post procedure needs to be verified as well, and if confirmed this could lead the way
toward further clinical research toward ultimately being considered for FDA approval
61


Final Thoughts
This analysis has been a valuable insight to the field of Photodynamic Therapy
and its contributing components that make this medical treatment possible. Although this
therapeutic method has been historically recorded as being utilized in some form for
centuries, only recently has PDT been rediscovered and applied in modern medical
practice for almost two decades, with the full implications of this treatment just now
being discovered [38], As the fields of engineering and medicine converge, new
approaches and methodologies are being discovered and put into practice. Photodynamic
Therapy is one of those areas where this medical/engineering relationship will be tested,
and ultimately patients will benefit from the resulting outcomes of this development.
Deuteporfin characterization is just one small step in the process of expanding
potential treatments to aid in eradicating cancerous lesions and tumors, port wine stains
and other epidermal cosmetic treatments, bacterial infections, and even aid in the
detection of malignancies and diseases. The opportunity to expand this field has arrived,
and it is up to engineers and medical researchers to combine their talents to further
develop this field for future generations to benefit from its medical capabilities.
62


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Full Text

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OPTICAL CHARACTERISTICS OF DEUTEPORFIN (DEUXEMETHER ), A PHOTODYNAMIC THERAPY SENSITIZER By MICHAEL CHARLES DUFFY B.S., University of Colorado Denver, 2004 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Electrical Engineering 2016

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ii This thesis for the Master of Science degree by Michael Charles Duffy has been approved for the Electrical Engineering Program by Tim Lei, Chair Yiming Deng Hamid Fardi April 15, 2016

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iii Duffy, Michael Charles (M.S., Electrical Engineerin g) Optical Characteristics of Deuteporfin (Deuxemether ), a Photodynamic Therapy Sensitizer Thesis directed by Associate Professor Tim C. Lei ABSTRACT Laboratory results on some of the optical propertie s of Deuteporfin, a relatively new photosensitizing drug that has been in clinical tri als in China since around 2009, is discussed. The drug was characterized on the basis of one photon absorption and fluorescence emission for providing data for proper drug applications and dosimetry. In addition, the effects of photobleaching were invest igated to characterize decay kinetics. The results of this research on this photosensitize r were also compared against HMME Hematoporphyrin monomethyl ether (HMME) (Hemoporfin) key characterization data which includes Q-band absorption to compare peak wa velengths and fluorescence intensity to show that Deuteporfin has similar abso rption profile to HMME while it has superior fluorescence characteristics. The finding s help to support the manufacturerÂ’s claim that Deuteporfin can be an effective photosen sitizer for tumor treatment. The form and content of this abstract were approved I recommend its publication. Approved: Tim Lei

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iv DEDICATION I dedicate this work to Raquel Duffy, whose infinit e love and support helped made this possible. To all who have the desire to succeed in their goal s, it is truly possible with hard work and determination. “The three great essentials to achieve anything wor th while are: Hard work, Stick-to-itiveness, and Common sense.” – Thomas A. Edison

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v ACKNOWLEDGMENTS I would like to thank Dr. Tim Lei, for his support and encouragement has guided me beyond what I thought possible in making this th esis a reality. Zheng Huang MD PhD for supplying the research materials and advisi ng me through the laboratory process. Gregory F. Glazner, without whose inspiration, foun dation research and laboratory support at the University of Colorado Anschutz Medi cal Campus this work would not be possible. A big thank you to Brian Atkinson, for your classes advisement and camaraderie; keeping me focused on my undergraduate career while making it enjoyable no matter the challenges that the outside world at the time had t o offer. I would also like to thank posthumously Dr. Carl T. A. Johnk, whose instruction, enthusiasm, advice and friendship enabled me to ste p outside my comfort zone to consider this research. His Electromagnetic Fields classes and TA experience he requested on me were instrumental in my path leadin g to this point in my academic career. Last, and certainly not least, I thank my family. My amazing wife Raquel, who never complained about the countless hours and slee pless nights of work, along with her steadfast support helped me persevere to fulfill th is research. My wonderful daughters Gwyneth and Breanna, who keep the child in me alive with enthusiasm in everything I do no matter the tasks I have in front of me. Thank y ou.

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vi TABLE OF CONTENTS CHAPTER I. INTRODUCTION ...................................... ................................................... .............. 1 Deuteporfin Drug Description ...................... ................................................... ....... 1 PDT Concept and Treatment ......................... ................................................... ...... 3 Light Administration used in PDT .................. ................................................... ..... 7 Surface Illumination. ............................. ................................................... ......... 8 Intraluminal Illumination. ........................ ................................................... .... 10 Interstitial Illumination. ........................ ................................................... ....... 15 Bronchial Tumor Interstitial PDT. ................. ........................................... 16 Prostate Cancer Interstitial PDT. ................. ............................................. 19 Objective ......................................... ................................................... ................... 24 II. MATERIALS AND METHODS ............................. ................................................. 2 5 Process ........................................... ................................................... .................... 25 Preparation of Deuteporfin Solutions .............. ................................................... .. 25 One-Photon Absorption Measurements ................ ................................................ 26 Absorption Measurement Instrumentation and Theory. ................................. 26 Absorption Spectra................................. ................................................... ...... 29 One-Photon Fluorescence Measurements .............. ............................................... 30 Fluorescence Measurement Instrumentation and Theory .............................. 30 Fluorescence Emission Spectra. .................... .................................................. 32 Photobleaching Experiment ......................... ................................................... ...... 33 Laser Photobleaching. ............................. ................................................... ..... 33 Solar Photobleaching. ............................. ................................................... ..... 33 Experiments to Compare Properties of Deuteporfin to HMME ........................... 34

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vii One Photon Absorption Spectra. .................... ................................................. 3 4 Fluorescence Emission Spectra. .................... .................................................. 34 III. RESULTS ........................................... ................................................... .................... 36 One-Photon Absorption Profile of Deuteporfin ...... .............................................. 36 Fluorescence Properties of Deuteporfin ............ ................................................... 38 Photobleaching Kinetics of Deuteporfin ............ ................................................... 41 Comparison of Deuteporfin to HMME ................. ................................................ 44 IV. DISCUSSION ........................................ ................................................... ................. 49 Measurement Observations .......................... ................................................... ...... 49 One Photon Absorption.............................. ................................................... .. 49 Fluorescence Properties and Photobleaching Kinetics .................................. 50 Deuteporfin and HMME Comparison Observations. ..... ................................ 51 Considerations for Future Analysis ................ ................................................... ... 52 Photobleaching Quantum Yield. ..................... ................................................ 52 Fluorescence Quantum Yield. ....................... .................................................. 54 Photodynamic Diagnosis. ........................... ................................................... 55 In Vivo Characterization Utilizing Various Light Sources/C onfigurations. ... 56 Two Photon Excitation. ............................ ................................................ 56 Light Source Delivery. ............................ .................................................. 58 Other Concerns. ................................... ................................................... ........ 59 V. CONCLUSION ........................................ ................................................... ............... 61 Deuteporfin in the PDT Arsenal .................... ................................................... .... 61 Final Thoughts .................................... ................................................... ............... 62 REFERENCES ....................................... ................................................... ................ 63

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viii LIST OF TABLES Table I.1 Photofrin Treatment Regimen for High-Grade Dys plasia in Barrett's Esophagus from the Prescribing Information ....................... ................................................... .................... 13 I.2 Fiber Optic Power Outputs and Treatment Times Required to Deliver 130 J/cm of Diffuser Length Using the Centering Balloon per Len gth of BarrettÂ’s Mucosa to be treated. .......................................... ................................................... .................................. 13 I.3 Short Fiber Optic Diffusers to be Used Without a Centering Balloon to Deliver 50 J/cm of Diffuser Length at a Light Intensity of 400 mW/cm ........................................... 14

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ix LIST OF FIGURES Figure I.1 Chemical structure of MHD and DMD, the main co mponents of Deuteporfin. .......... 2 I.2 Simplified Jablonski Energy Level diagram for photosensitizer excitation leading to singlet oxygen production. ........................ ................................................... ....................... 4 I.3 BLU-U Model 4170 Blue Light Photodynamic Therapy Illumin ator (DUSA Pharmaceuticals, Inc.). ................................................. ................................................... .. 9 I.4 Diomed™ PDT 630 laser system and close-up view of an illuminated OPTIGUIDE™ cylindrical fiber optic diffuser (Pinnac le Biologics, Inc.). ..................... 11 I.5 PDT to treat precancerous changes in Barrett’s esophagus (the dark red, smooth areas), process descriptions below. ............... ................................................... ................. 15 I.6 Intrinsic airway compression following photody namic therapy. ............................... 17 I.7 Combined rigid and flexible bronchoscope used in interstitial bronchoscopic photodynamic therapy. ............................. ................................................... ...................... 18 I.8 Example of interstitial procedure of tumor mas s in the right upper lobe. .................. 18 I.9 Illustration of prostate with interstitial fib er placement in PDT treatment. ................ 19 I.10 Illustration of calculated treatment light fl uence being delivered through multiple optical fibers positioned inside the prostate gland ................................................. .......... 20 I.11 Geometry description of prostate relative to the urethra and rectum. ...................... 20 I.12 Example of an interstitial procedure of a pro state with a tumor mass in the right upper lobe. ....................................... ................................................... ............................... 23 II.1 Cary 100 UV/Vis Spectrophotometer. .......... ................................................... ......... 26 II.2 Cary 100 Top Level Schematic Diagram. ....... ................................................... ....... 27 II.3 ISS PC1 Steady-State Photon Counting Spectrof luorimeter. .................................... 30 Figure II.4 ISS PC1 Top Level Schematic Diagram. ................................................... ... 31 III.1 Absorption Spectra of Deuteporfin in Various Solvents at 22 2C. ...................... 37 III.2 Deuteporfin Emission Spectra in the Range 61 0—710 nm at 22 2C. .................. 38 III.3 Deuteporfin in DMSO standard dilution curves ................................................. .... 39

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x III.4 Emission chart of Deuteporfin 1g/ml in DMSO at 22 2C. ................................ 40 III.5 Photobleaching kinetics of Deuteporfin in DM SO. ............................................... .. 42 III.6 Solar bleaching of Deuteporfin in DMSO. .... ................................................... ....... 43 III.7 Chemical structure of HMME. ................ ................................................... ............. 45 III.8 Absorption Q-band comparison of Deuteporfin and HMME in various solutes at 100 g/ml. ........................................ ................................................... .............................. 46 III.9 Emission comparison of Deuteporfin and HMME. ................................................. 48 IV.1 FRET process as applied to secondary sensitiz er use in PDT. ................................ 57 IV.2 Schematic representation of the CQD–P conjuga te illustrating indirect excitation of the protoporphyrin IX upon two-photon excitation of the CQD. ..................................... 58

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xi LIST OF ABBREVIATIONS DI H2O Deionized water (H2O) DMSO Dimethyl Sulfoxide DP Deuteporfin, also known as Deuxemether DXM Deuxemether, also known as Deuteporfin FDA Food and Drug Administration HMME Hematoporphyrin Monomethyl Ether, also known a s Hemoporfin HpD Hematoporphyrin Derivative MRI Magnetic Resonance Imaging NP H2O Nanopure (18.3M /cm) water (H2O) O2 Oxygen PMT Photomultiplier Tube PDD Photodynamic Diagnosis PDT Photodynamic Therapy PBS Phosphate Buffered Saline PBS/FBS Phosphate Buffered Saline/ Fetal Bovine Ser um PS Photosensitizer ROS Reactive Oxygen Species 1O2 Singlet oxygen TPE Two-photon Excitation TRF Time-Resolved Fluorescence US United States UV Ultraviolet

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1 CHAPTER I INTRODUCTION Deuteporfin Drug Description Deuteporfin (DP), also known as Deuxemether (DXM), is a relatively new photosensitizing drug used in Photodynamic therapy (PDT), which has been in clinical trials in China since around 2009. The photosensit izer (PS) is manufactured by Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd., whic h also manufactures Hematoporphyrin monomethyl ether (HMME) (Hemoporfin), which has been also been researched for its optical properties at the Univer sity of Colorado Denver. The primary indication of Deuteporfin is for oncological (tumor ) reduction therapy, where PDT treatment can be administered either externally or internally to the site with delivery of the light source to the affected area for activatio n of the drug being the only limitation to treatment. Deuteporfin is a Hematoporphyrin Deriv ative (HpD) preparation which mainly consists of 3(or 8)-(1-methoxyethyl)-8(or 3) -(1-hydroxyethyl)-deuteroporphyrin IX (MHD), 3,8-di(1-methoxyethyl)-deuteroporphyrin I X (DMD), 3(or 8)-(1methoxyethyl)-8(or 3)-vinyl-deuteroporphyrin IX (MV D), 3(or 8)-(1-hydroxyethyl)-8(or 3)-vinyldeuteroporphyrin IX (HVD) and small portion of protoporphyrin IX [1]. The quantities of the components are proprietary, and b ecause of this the molecular weight is not available. Estimations on the two primary acti ve components of the drug have been given to be that MHD and DMD comprise of greater th an 85% of the composition of Deuteporfin, with the proportion of photosensitive porphyrin that has poor tumor selectivity is much lower than that in other HpDs s uch as Photofrin, and a preclinical

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2 study on rats has shown good tolerance to the as we ll as favorable elimination characteristics that support the prospects of being a favorable PDT drug [2]. The core structure, a porphyrin ring (porphine), de fined as a heteroaromatic compound characterized by a tetrapyrrolic structure consisting of four pentagonal pyrroles linked by four methylene bridges [3]. The chemical structure for MHD and DMD is shown in Figure I.1. Figure I.1 Chemical structure of MHD and DMD, the main components of Deuteporfin. MHD: R1=CH3CH (OCH3) and R2= CH3CHOH or R1=CH3CHOH and DMD: R2=CH3CH (OCH3) or R1=CH3CH (OCH3) and R2=CH3C H (OCH3).

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3 Because the molecular weight of Deuteporfin is not known, various analyses involving molecular quantities cannot be performed without either having the weight be supplied by the manufacturer or by extrapolation wh ich is a time consuming process. Further details into the analyses involved will be explained the discussion of this study. Research has also been recently performed to test i ts effectiveness for malignant tumor detection by Photodynamic Diagnosis (PDD). T his procedure is common practice but is currently in the early stages of research fo r Deuteporfin and has not been performed in vivo to date [4]. PDT Concept and Treatment Although this not a study on photochemistry or phot obiology, a brief explanation into PDT reaction principles and cell response are required to gain an understanding into the need for optical characterization of photosensi tizers. PDT occurs through a photochemical reaction initiated by three non-toxic components; a photosensitizer (PS), oxygen and light. However when the photosensitizer is excited by the appropriate wavelength of light combined with oxygen supplied a t the site the resulting reaction generates Reactive Oxygen Species (ROS), which incl ude singlet oxygen (1O2), which is cytotoxic meaning toxic to cells. Singlet oxygen i s more toxic to malignant cells but is less harmful to healthy tissue. This reaction occurs when a light source typically in the range of 400 – 700 nm, irradiates the PS where a molecule in turn absorbs a photon which in turn excites the molecule from the singlet S0 to S1 state through a process called internal conversion (IC). S1 has a short lifetime in the nanoseconds; molecules can then either relax back to the S0 ground state via IC generating heat or by fluoresce nce. However, there are some

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4 molecules that cross over to the triplet T1 state through an intersystem crossing (ISC); a state that has a significantly longer lifetime the micro to millisecond range. The time advantage for molecules in this triplet state is th e ability to efficiently transfer energy, and when this transfer to naturally occurring tripl et oxygen (3O2) occurs the result is the generation of 1O2. This process is shown in the energy level diagra m in Figure I.2 [5, 6]. Figure I.2 Simplified Jablonski Energy Level diagr am for photosensitizer excitation leading to singlet oxygen production. A light source irradiating the photosensitizer, or PS at the singlet S0 state which in turn a molecule of the PS absorbs a photon promoting it to the excited singlet state S1 by internal conversion (IC). While many molecules con vert the light to heat through IC or by fluorescence when returning to S0, some molecules through the intersystem crossing (ISC) enter the triplet T1 state which in turn an energy transfer between the exited PS molecules and naturally occurring triplet oxygen pr oduces reactive singlet oxygen.

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5 For PDT to be effective, the PS ideally has to be p ossess the following characteristics: be chemically pure and of known composition, have minimal dark toxicity and only be cytotoxic in the presence of light, be preferentially retained by the target tissue, wi th high contrast to the neighboring normal tissue, be able to be rapidly excreted from the body to pro vide low systemic toxicity, have a high quantum yield for the photochemical eve nt, such as the generation of singlet oxygen (1O2) to effectively kill cancerous tissue, high photostability (low photobleaching) by prevent ion of degradation of the PS, again for optimal 1O2 production to promote consistent and thorough destruction of malignant cells, high quantum yield of natural fluorescence for PDD by fluorescence spectroscopy and for optical dosimetry measurements and have strong absorbance with a high extinction coeff icient in the 600-800 nm range where tissue penetration of light is at a maximum and where the wavelengths of light are still energetic enough to produce singlet oxygen [7, 8]. The PS is typically administered by intravenous inj ection and therefore is dispersed throughout the body; however the ideal sc enario is to have PS localization at the treatment site. Many photosensitizers have the property of being retained for longer periods in malignant or target tissue versus health y tissue, so this can be used to an

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6 advantage for treatment [8]. Furthermore, PDT acti vation occurs only where light is applied, thereby it is possible to minimize damage to healthy tissue, but at the same time to have the ability to properly illuminate the volu me of target tissue in order to effectively damage and kill the population of target cells is t he goal. This delicate balance of drug and light administration in combination with an ade quate oxygen source to for the energy transformation to 1O2 at a level appropriate for successful PDT treatmen t requires analysis of the PS characteristic properties. What is the minimal concentration of PS required to generate the proper quantity of 1O2 to effectively treat the site? At what time after delivery of the PS should PDT be administered for vascular or tumor treatment? At what wavelength(s) of light can the PS be activated ? How far can the light penetrate into tissue before being absorbed to the point where it does not effectively activate the PS? Is density of tissue a factor on light administration? Determining the optimal wavelength and intensity for a specific PS activation, examini ng photobleaching characteristics and PS concentration effects are just some of the facto rs in prescribing the proper PDT drug dosage and light administration techniques [9]. Th is can be a complicated evaluation that could be researched to a level of extensive chemica l, optical en vitro and en vivo experiments that could easily be the subject of a d octoral dissertation. While the experiments performed in this paper are but a subse t of which can and should be explored, the research that has been completed to d ate is a substantial step in fully understanding DeuteporfinÂ’s optical properties.

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7 Light Administration used in PDT Taking from a phrase, not all PSs and treatment reg imens are alike, and because of this different methods of light administration a re used depending on the medical condition and drug being utilized for treatment. L ight regimen is as important as drug regiment in order for a treatment to be successful. This is due to the PSÂ’s particular characteristics such as absorption spectra and sing let oxygen quantum yield, and therefore have different PDT treatment methodologie s to best treat a particular medical condition [10]. This section will address three me thodologies in use that could ultimately be utilized in Deutepofrin PDT administration. Three examples of PS drugs which are approved by th e FDA and actively used for PDT treatment in the United States (US) include: porfimer sodium injection (Photofrin), for treatment of esophageal cancer, endobronchial cancer and high-grade dysplas ia in BarrettÂ’s esophagus, aminolevulinic acid HCl for Topical Solution, 20% ( Levulan) to treat actinic keratosis, and, vertiporfin injection (Visudyne) for treatment of macular degeneration; to seal leaks at the center of the macula without dama ging the central vision [10]. For purposes of describing treatment methodologies that Deuteporfin would be considered, Photofrin and Levulan treatment will be discussed but Visudyne will not be explored as Deuteporfin has not been indicated in t his type of macular degeneration surface treatment. What follows is an introduction to the three types of light

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8 administration (surface, intraluminal and interstit ial) and how it could ultimately be utilized in DP drug utilization. These examples ar e intended for describing light dosage techniques only and not the chemical mechanisms of these PDT drugs. Surface Illumination. This is a superficial methodology primarily used for treatment of epidermal conditions such as acne, pso riasis, actinic keratosis or melanoma [11]. Since these are surface related conditions P DT can easily administered by using a topical solution ointment drug formulation and a li ght of either broad spectrum or particular wavelength be applied based on the Q-ban d absorption spectra. An FDA approved PS for this application used in the US is aminolevulinic acid HCl for Topical Solution, 20% (Levulan, DUSA Pharmaceuticals, Inc.) to treat actinic keratosis which is a patch of moderate to thick scaly or crusty UV damaged skin (usually facial) that is potentially 10% pre-cancerous leading to squamous c ell carcinoma [12]. As noted in the manufacturer prescribing information, the drug is f irst applied topically to the affected area and surrounding tissue to ensure all pre-cance rous subject tissue is treated and then left to be absorbed by the tissue for 14 hours, whi ch during that time the patient has to be careful to not be exposed to bright indoor light or sunlight. After the time period has elapsed there is a two hour window of opportunity t o perform PDT treatment. Surface illumination via blue light (400-450 nm) lamp expos ure is administered for a period of 1,000 seconds (16 minutes, 40 seconds) in order to completely activate the PS with a total light dose of 10 J/cm2. After treatment the treated lesions will become red and inflamed with possibly some scaling, and hypersensitivity to light with pain and tingling to the treated area may occur. The lesions will typically heal after approximately four weeks

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9 after treatment. A recommended light source which is also manufactured by the drug manufacturer that is shown in Figure I.3. Figure I. 3 BLU-U Model 4170 Blue Light Photodynamic Therapy Illumin ator (DUSA Pharmaceuticals, Inc.).

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10 Levulan PDT treatment can be repeated twice in an e ight week period per the drug manufacturer to optimize results. This method of light delivery is the simplest to administer compared to using laser light to accompl ish PDT from a procedural standpoint, but obviously this is limited to extern al applications. Deuteporfin is not indicated for epidermal PDT, but this procedure cou ld possibly be explored if a topical formulation of the PS is developed in a dosimetry f avorable for this application in the future. Intraluminal Illumination. The term intraluminal seems obvious grammatically but needs to be properly explained in order to unde rstand the full connotation of the word. Oxford English Dictionary (2015) defines Lumen in physics as The SI unit of luminous flux, equal to the amount of light emitted per second in a unit solid angle of one steradian from a uniform source of one candela; how ever in anatomical terms it is the central cavity of a tubular or other hollow structu re in an organism or cell. The esophagus, trachea, bronchial passages, arteries an d veins, and intestines would be considered to have luminous cavities. In the field of PDT, these areas if affected by a malignancy could conceivably be treated by PDT via use of superficial intraluminal light administration. The colon and lower gastrointestin al tract, esophagus and bronchial passages are good candidates for this methodology. An FDA approved drug with a proven success rate in intraluminal applications in PDT is Porfimer sodium (Photofrin, Pinnacle Biologics, Inc.), which has been prescribe d for treatment of endobronchial and esophageal cancers as well as High-Grade Dysplasia (HGD) of BarrettÂ’s Esophagus (BE) [10]. BE is a precancerous condition that if left untreated has a 59% risk of developing into adenocarcinoma of the esophagus which has a 5year survival rate of less than 10%,

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11 thus early detection and treatment of this conditio n can be the difference between life and death [13]. The following scenario describes the p rocess utilizing intraluminal 630 nm laser light delivery to activate Photofrin to treat HGD in BE As with DUSA who manufactures Levulan and the BLU-U illuminator, Pinnacle Biologics also manufactures light delivery equipmen t for intraluminal PDT treatment. The Diomed™ PDT 630 laser system is a recommended l ight source, and per the manufacturer’s prescribing information alternate la ser systems must be approved for delivery of a stable power output at a wavelength o f 630 3 nm for use; however the OPTIGUIDE™ cylindrical fiber optic diffusers for us e in intraluminal therapy are required by the manufacturer to ensure required tis sue illumination to activate the PS [14]. The laser system and associated diffuser is shown in Figure 1.4. Figure I.4 Diomed™ PDT 630 laser system and close-up view of an illum inated OPTIGUIDE™ cylindrical fiber optic diffuser (Pinnac le Biologics, Inc.).

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12 According to the manufacturerÂ’s prescribing informa tion, the first step is to administer the PS drug intravenously, in a dosage o f 2 mg/kg of body weight in 3 to 5 minute period 40-50 hours prior to light treatment [14]. This will enable the Photofrin to be absorbed and selectively retained in the affecte d lumen tissue to be treated, while at the same time healthy tissue will have the ability to excrete the drug clear of the PS in the allotted wait time prior to treatment. At the time of treatment, an endoscope aides in placement of a clear windowed centering balloon tha t is of a predetermined length of the BarretÂ’s mucosa (tissue) to be treated through the lumen and to the treatment site, followed by a complementary length cylindrical fibe r optic diffuser which is placed into the center channel of the balloon. The balloon is used to surround the fiber tip as an aid in centering the light source in the area to be tre ated. A 630 nm laser light source is then applied to deliver a required light dose of 130 J/c m of diffuser length where an acceptable intensity of 200-270 mW/cm of diffuser length is re commended. To calculate the proper dose the following equation is used: Tables are provided in the prescribing information for treatment regimen and to account for different diffuser lengths and power output com binations to successfully treat an area for a required minimal time, as represented below. Table 1 is the procedure regimen in the prescribing information HGD of BE treatment, an d Table 2 lists the light dosimetry at 480 seconds at a light intensity of 270 mW/cm, with an option at a light intensity of 200mW/cm for lower power lasers with a total output up to 2.5W. Table 3 applies to the optional short fiber 50 J/cm skip area treatment at a light intensity of 400 mW/cm.

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13 Table I.1 Photofrin Treatment Regimen for High-Gra de Dysplasia in Barrett's Esophagus from the Prescribing Information Procedure Study Day Light Delivery Devices Treatment Intent PHOTOFRIN Injection Day 1 NA Laser Light Application Day 3a 3, 5 or 7 cm balloon (130 J/cm) Photoactivation Laser Light Application (Optional) Day 5 Short ( 2.5 cm) fiber optic diffuser (50 J/cm) Treatment of "skip" areas only a Discrete nodules will receive an initial light ap plication of 50 J/cm (using a short fiber optic dif fuser without balloon) before the balloon light applicati on. NA: Not Applicable. Table I.2 Fiber Optic Power Outputs and Treatment Times Required to Deliver 130 J/cm of Diffuser Length Using the Centering Balloon per Length of BarrettÂ’s Mucosa to be treated. Treated BarrettÂ’s Mucosa Length (cm) Balloon Window Length (cm) Fiber Optic Diffuser Length (cm) Light Intensity (mW/cm) Required Power Output from Diffusera (mW) Treatment Time (sec) (min:sec) 1-3 3 5 270 1350 480 8:00 4-5 5 7 270 1900 480 8:00 6-7 7 9 270 2440 480 8:00 200 1800 480 10:50 a As measured by immersing the diffuser into the cu vette in the power meter and slowly increasing the laser power. Note: No more than 1.5 times the required diffuser power output should be needed from the laser. If mo re than this is required, the system should be checked

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14 Table I.3 Short Fiber Optic Diffusers to be Used W ithout a Centering Balloon to Deliver 50 J/cm of Diffuser Length at a Light Inten sity of 400 mW/cm Fiber Optic Diffuser Length (cm) Required Power Output from Diffusera (mW) Treatment Time (sec) (min:sec) 1.0 400 125 2:05 1.5 600 125 2:05 2.0 800 125 2:05 2.5 1000 125 2:05 a As measured by immersing the diffuser into the cu vette in the power meter and slowly increasing the laser power. Note: No more than 1.5 times the required diffuser power output should be needed from the laser. If more than this is require d, the system should be checked. An initial BE treatment length of 7 cm is performed per the dosimetry. A secondary light treatment may be applied immediately after up to fi ve days from the PS injection to address areas that visually appear as “skipped” or did not properly respond to treatment in the initial procedure. A follow up full dose treat ment can be performed to address additional lengths of BE greater than 7 cm or to re treat the original area 90 days after the initial PDT. For HGD of BE no debridement, or remo val of dead tissue, is necessary after treatment as compared to lung cancer procedur es where residual dead tumor tissue can potentially block the airways and does not natu rally dissipate which in turn impedes the healing process.

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15 Figure I.5 PDT to treat precancerous changes in Ba rrettÂ’s esophagus (the dark red, smooth areas), process descriptions below. (a) View before therapy. (b) View one day after PDT showing destruction of t he esophageal mucosa (the innermost layer of the wall of the esophagus, where the disease arises). (c) View one month after PDT showing regeneration o f the normal lining of the esophagus. (d) Shows the balloon encapsulating a cylindrical f iber for light delivery to the esophagus. The balloon is first inserted over a gu ide wire positioned endoscopically at the treatment site. The wire is then removed and re placed by a diffuser laser fiber, which can be seen as a thin red line in the center of the balloon [15]. In the case of Deuteporfin, this would be one of th e preferred methods of light delivery and treatment of precancerous and malignan t tissues, and could be a potential alternative to Photofrin in PDT. Clinical trials w ould have to be completed in order to determine its efficacy. Interstitial Illumination. Interstitial implies that the optical fibers are directly inserted into the tissue being treated such as a ma lignant tumor, which is normally dense to where superficial light would not be able to pen etrate to the center of the tumor. This is the most invasive process used in PDT, and great care has to be used in this process of treatment. Interstitial light treatment is approve d in the US for lung cancer on a b c d

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16 noncircumferential endobronchial tumors that are so ft enough to penetrate the tissue, as detailed in the Photofrin manufacturerÂ’s prescribin g information, but no evidence of pre or post clinical trial results or use in this appli cation could be found in the prescribing information or elsewhere for reporting in this stud y [14]. However, international publications have documented this procedure as well as for other malignant tumor interstitial techniques [16, 17]. There are also o ngoing interstitial treatment regimen studies that are in phase II US clinical trials whi ch are expected to complete in 2017, as documented in the ClinicalTrials.gov website databa se, that involve candidates who have recurring malignant tumors of the head and neck. The following presents two examples of the applicat ion of interstitial illumination in PDT cancer treatment. The first which is author ized in the US utilizes the FDA approved photosensitizer Photofrin, prescribed in t he same drug and light dosimetry as intraluminal PDT but the light delivery method is n ow more direct in that a small cylindrical optical diffuser is directly placed in the tumor tissue. Next to be described is a procedure that is being used in trials internationa lly in treating prostate cancer through the same interstitial illumination method using mul tiple illumination fibers which has also evidence of promising outcomes. Bronchial Tumor Interstitial PDT. In the treatment of endobronchial cancer, Photofrin is administered per the same manufacturer Â’s prescribed dosimetry regimen used in the BE intraluminal procedure [14]. After 40-50 hours the interstitial procedure is performed as determined by the size and geometry of the tumor. Two to three days after treatment, debridement, which is the process of rem oving necrotic tissue which can obstruct the airways, inhibit lesion healing, and p otentially contribute to secondary

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17 infection needs to be performed. The added benefit of debridement from a secondary PDT treatment standpoint is the remaining malignant live tissue is now exposed surface area making light treatment more effective should a dditional intraluminal treatment be prescribed. Secondary spot treatment can be admini stered between 96-120 hours after the initial Photofrin injection. Additional PDT tr eatment can be administered a minimum of 30 days after the initial therapy, and up to thr ee courses (each separated by a minimum of 30 days) can be given as needed. Figure I.6 Intrinsic airway compression following photodynamic therapy. Endoscopic view of left lower lobe of a patient wit h intrinsic compression related to endobronchial carcinoid upon completion of photodyn amic therapy (PDT). (a) View during therapy, using a rigid bronchoscope containing thin laser fiber to deliver 633nm interstitial illumination. Fiber is placed in the center of the tumor in order to evenly illuminate throughout the tumor to ensure ev en PS activation. (b) Debridement of the tissue would occur 2-3 days after light administration to prevent intrinsic airway compression (obstruction) due to n ecrotic debris. (c) Post debridement, laser cauterization of the tu mor base (Hemostasis) may be required to minimize bleeding [18].

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18 Figure I.7 Combined rigid and flexible bronchoscop e used in interstitial bronchoscopic photodynamic therapy. Note the cylindrical optical fiber with its end dif fuser protruding through the flexible biopsy channel of the fiber optic bronchoscope for illumination [16]. Figure I.8 Example of interstitial procedure of tu mor mass in the right upper lobe. (a) Cylindrical diffuser in the tumor and (b) with illumination in progress [16].

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19 Prostate Cancer Interstitial PDT. A more complex treatment regimen is required for prostate cancer due to the physiology of the tumor and surrounding tissues. The prostate gland surrounds the urethra and is in close proximity to the rectum. This makes any treatment processes susceptible to compli cations from collateral tissue necrosis that can effect quality of life, such as E rectile Dysfunction (ED) or incontinence because isolating radiation treatment to the gland is very difficult given the close proximity to the urethra and rectum [19]. This is where interstitial PDT shows promise in that light levels can be adjusted via fiber placeme nt in the organ along with tailored light dosimetry as shown in Figures I.9 through I.11 belo w. Figure I.9 Illustration of prostate with interstit ial fiber placement in PDT treatment. For localized prostate cancer interstitial treatmen t, cylindrical fiber optic diffusers can be positioned and quantity adjusted to deliver PDT to either a portion of or to the entire gland determining on the nature of the treatment [1 9].

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20 Figure I.10 Illustration of calculated treatment l ight fluence being delivered through multiple optical fibers positioned inside t he prostate gland. Two different instances during the same treatment s ession, where the isosurfaces indicate tissue that has received at least the threshold dos e. Note the urethra in the central region of the gland and the rectum just below the prostate [17]. Figure I.11 Geometry description of prostate relat ive to the urethra and rectum. 3 dimensional rendition of the geometry of the pros tate, urethra and rectum are indicated (in red, green and blue, respectively). The templat e grid is shown on the x, y-plane. The maximum projection of the prostate and cross sectio ns of the urethra and rectum at the same height are superimposed on the template, with rectal surface up to y > -1 observed for determine the correct PDT exposure [20].

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21 As seen in the three figures above, to effectively treat the cancerous tissue without impacting the urethra and rectum is a difficult pro blem to solve, since both have also been exposed to the photosensitizer meant to treat solely the prostate and all associated cells are highly susceptible to PDT damage upon exp osure to the threshold light activation dosage. Therefore a threshold light dos e has to be determined in each individual case that ensures the minimum exposure r equired in order to activate the PS in the treatment region only in order to prevent unint ended cell death is paramount in preventing post procedure medical complications and retaining optimal quality of life for the patient post treatment. A simplified example in three steps can be describe d for determining light dose [17]. First using the light distribution, or fluen ce rate, and taking the PS concentration into account as shown in the process below: Where: DPDT = PDT dose T = treatment time = extinction coefficient of the PS [ PS ] = PS concentration = fluence rate, “light distribution”

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22 Next, interstitial treatment [ PS ] and needs to be addressed three dimensionally. Assuming for simplicity that the PS is homogeneousl y distributed in the target volume a fluence dose can be determined: n Where: DFluence = fluence dose T = treatment time = fluence rate Using calibrated fiber optic probes can aid in dete rmining the fluence rate. Through integration of the signal for a specified treatment time a fluence dose can be found for a particular fiber location. Third, to develop a spa tial map for the entire treatment volume, the photon propagation is theoretically calculated and optical properties of the tissue undergoing treatment are determined, which is aided by prior ultrasound and/or Magnetic Resonance Imaging (MRI) procedures taken from prior examinations, and an imaging procedure with the fibers in place before treatment A photon propagation model is then used, such as the theoretical model for the analyti cal solution to the diffusion equation: Where: ( r ) = radial fluence rate P = power (watts) D = diffusion coefficient (cm) eff = effective attenuation coefficient of the light i n the treated tissue (cm-1) r = radial distance from the point source (cm)

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23 Figure I.12 Example of an interstitial procedure o f a prostate with a tumor mass in the right upper lobe. Fluence rate map for 7 diffusers, with eff = 2.5/cm. The red line corresponds to the prostate, the green to the urethra, and the blue to the rectum. The black lines are contour levels at 100 (inner curve) and 62.5 (outer curve) arbitrary dose units [20]. As observed from this interstitial procedure, there are many variables that have to be accounted for in order for this form of PDT trea tment to be successful, but at the same time the benefit can far outweigh the potential ris ks when you take into consideration other treatment options. Modalities such a radiati on therapy or brachytherapy have a high incidence of side effects (incontinence, ED) w hich is why interstitial PDT is being explored in preventing these outcomes. Photofrin i s one PS that has been explored in this PDT procedure, and Deuteporfin could be a potential candidate as well [17].

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24 Objective Little has been published to date in regards to Deu teporfin optical properties, which is the purpose behind this research. The res ults from this initial optical characterization of this compound can aid in develo ping treatment regimen and dosimetry recommendations in regards to excitation methodolog y. The goal of this thesis is to document preliminary laboratory analysis data and e valuate the characterization of Deuteporfin using techniques including one-photon a bsorption and fluorescence series dilution and photobleaching measurements in various solvents. Additionally, a comparison of key Deuteporfin characterization resu lts will be compared against HMME data to reinforce previous published statements not ing the potential effectiveness of this photosensitizer for use in tumor PDT treatment.

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25 CHAPTER II MATERIALS AND METHODS Process Preparation and experiments with Deuteporfin requir e careful processes to ensure that drug light exposure be eliminated/minimized wh erever possible to ensure not to skew laboratory results. In preliminary measurements to determine the correct concentration for absorption and fluorescence it was noticed that the slightest variation can have drastic changes in experiment outcomes, and experiments rer un to address anomalies confirmed this scenario was usually the case. Processes to e nsure accuracy and repeatable outcomes were developed and adhered to ensure that experimen ts were repeatable and verifiable. These processes are detailed in the following secti ons. Preparation of Deuteporfin Solutions Sample Deuteporfin was obtained in drug grade powde r form and kept refrigerated at 4C. Deuteporfin powder is similar to HMME in appearance however the powder is lighter in consistency and tends to dispe rse in a fine film shortly after transferring a sample to a plastic test tube. As s uch a base stock solution is required to have readily available liquid form of the drug for experiments. The base stock solution was made at 10mg/ml Deuteporfin with Dimethyl Sulfo xide (DMSO) for later use in target dilutions for experiments. The stock soluti on was diluted with the solvents to be evaluated prior to measurement to obtain 100g/ml c oncentration for Q-band absorption measurements, 8 g/ml for Soret absorption/emission measurements and 1 g/ml for fluorescence measurements unless otherwise noted. These dilutions were based on the

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26 sensitivity range of the instruments used for measu rement. All Deuteporfin solutions were kept in light safe containers at all times up to measurement. One-Photon Absorption Measurements Absorption Measurement Instrumentation and Theory. One-photon absorption spectra measurements were obtained to de termine absorption peaks in the Soret and Q-bands for the purpose of checking for o ptimal conditions for tissue light absorption as higher wavelength light is more prefe rable for tissue absorption. A Cary 100 UV/Vis spectrophotometer (Agilent Technologies, Inc., Santa Clara CA, USA) was used to measure absorbance, which is shown in Figur e II.1. Figure II. 1 Cary 100 UV/Vis Spectrophotometer.

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27 Figure II.2 Cary 100 Top Level Schematic Diagram. The step by step process which a spectrophotometer operates is described as follows. A light source comprised of UV and visibl e light lamps are used to provide the 190 – 900nm instrument range which the bounds can b e adjusted to a specific measurement. The light enters a monochromator to s elect a particular wavelength of light for evaluation, which then is sent through a series of mirrors to a beam diverting chopper which either blocks, reflects or allows lig ht to pass straight through depending on the cell absorbance to be measured, ensuring tha t either the reference or sample beam is aligned for accurate reproducible measurement. In the case of this example the light will pass through to the path leading to the sample cell and continue until it is reflected at a second chopper where the absorbance light is rece ived by the Photomultiplier Tube detector (PMT) for measurement.

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28 In the case of the Cary 100, there are two ways to compensate for light absorption discrepancies from the cuvette and solvent properti es that can be subtracted from the sample substance measurement. A reference cell of the same material composition can be filled with the solute used for dilution and mea sured in the reference path along with the sample cuvette and systematically subtracted ou t to produce the final measurement, or the reference cuvette with solute can be measured f irst in a process called zero/baseline, where the system software can record the reference measurements through a range and calculations to compensate for the cuvette and solu te can be performed during the final measurement. The advantage of the second process i s that the same cuvette can be used for the reference and sample measurements removing any minimal discrepancies of cuvettes composed of the same material that may hav e slightly different optical properties, and also this process keeps the chopper s in the same position which further removes any variances that could occur due to chopp er position anomalies. The purpose behind performing absorption measuremen ts is to confirm linearity of concentration versus absorbance levels of a solu tion, to determine absorption intensity peak wavelengths for the Soret and Q-band, to compa re absorbance levels of the PS against various solutes and compare levels against other compound/solute measurements, in this case HMME. This measurement is based on th e Beer-Lambert law, which states that the concentration of a solute is directly prop ortional to the absorbance of a solution [6]. First by defining Transmittance as: r r Where: IS = Sample Intensity IR = Reference Intensity of cuvette with solvent

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29 The Beer-Lambert law then defines Absorbance A as: r Where: = Molar absorptivity, or molar extinction coeffici ent, M-1cm-1 c = Concentration of the solute, M or g/ml since the molecular weight of Deuteporfin is not known l = path length, cm, in this case 1 cm for the 3 ml quartz cuvette used Therefore there is a relationship between Absorbanc e and Transmittance, where: The last note is of importance since Absorbance is defined as the measured value or magnitude of incident light absorbed by the sample, whereas Absorption is the process by which an electron from a lower energy level is prom oted to a higher energy level. Absorption Spectra. Deuteporfin was diluted in DMSO, NPH2O, and 1 Phosphate Buffered Saline (PBS). Standard one-ph oton absorption curves for each solvent were obtained by successively diluting a 10 0 g/ml sample in 10 g/ml steps for the Soret band and subsequently a 10 g/ml sample in 1 g/ml step dilutions for Q-bands at room temperature (22 2C). These wavelength b ands were chosen to appropriately capture the range of the Soret band and four Q-band peaks as well as beyond the range to check for any spurious anomalies. Three series dilu tion sets were taken and each dilution point was averaged. All absorption readings were t aken using the same quartz cuvette to eliminate differences in readings caused by any cuv ette-specific anomalies. Measurements were recorded by the Cary 100 WinUV so ftware (Agilent Technologies, Inc., Santa Clara CA, USA).

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30 One-Photon Fluorescence Measurements Fluorescence Measurement Instrumentation and Theory Fluorescence emission spectra generated from one-photon excitati on were obtained using an ISS PC1 steady-state photon counting spectrofluorimeter (IS S Inc., Champaign, IL, USA). Figures II.3 and II.4 shows the ISS PC1 and its ass ociated light path schematic. Figure II.3 ISS PC1 Steady-State Photon Counting S pectrofluorimeter.

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31 Figure II.4 ISS PC1 Top Level Schematic Diagram. A spectrofluorimeter operates by light excitation v ia a xenon arc lamp through a monochromator which accepts the incoming light and provides color selection by rotation of a grating which determines the excitation wavele ngth for the sample measurement. Next the light path encounters a beam splitter whic h diverts part of the light through a quantum counter, which is to correct for fluctuatio ns in lamp intensity as well as to provide a reference wavelength of light for the ref erence PMT. Usually a highly concentrated solution of Rhodamine B in ethanol or ethylene glycol is used for the quantum counter [21]. The light that is not divert ed continues through an optional excitation polarizer used for anisotropic measureme nts, which can adjust for vertical or horizontal polarization for optimizing light coming from the excitation monochromator.

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32 The light enters the sample and for the case of thi s study goes to the right though another polarizer (optional for antistrophic measurements) to adjust emission polarity for the light path onward, going through an optional long-pass fi lter used to block undesired higher order scattered excitation light where it arrives t o an emission monochromator, for selecting the wavelength of light to be received by the emission PMT for measurement. The ISS PC1 measures fluorescence intensity in phot ons/second. In the case of this study, this can be considered an arbitrary num ber. Fluorescence Emission Spectra. Readings were taken at room temperature (22C 5C) and recorded via the spectrofluorimeter software (Vinci version 1.6.SP7, ISS Inc., Champaign, IL, USA). Excitation waveleng th was 400 nm. Fluorescence emission spectra in the 400—800 nm range revealed n o emission except two peaks at approximately 615 nm and 678 nm depending on solven t, respectively. Therefore most subsequent readings were taken in the 610—710 nm ra nge except where noted. In addition, for each Q-band excitation wavelength from 450 to 585 nm at 5 nm increments, synchronized emission spectra were obta ined by using the ISS spectrofluorometer to measure fluorescence intensit y at 5 nm increments in the 600—700 nm range. For these experiments, Soret band excitat ion wavelengths were not considered as this band is not typically utilized in PDT treat ment. All emission readings were taken using the same quartz cuvette to ensure measurement consistency.

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33 Photobleaching Experiment Laser Photobleaching. To measure the effects of photobleaching on Deuteporfin, a 630 nm laser was used with a set 1W output connected to a microlens fiber (Medlight S.A. Ecubens, Switzerland), deliver ing light to a glass bottom petri dish (MatTek Corp, Ashland, MA, USA) at 150mW/cm2. 3 ml of Deuteporfin diluted in solvent was photobleached in the petri dish for a s et period of time. After irradiation, the solution was transferred to a 3ml quartz cuvette an d measurements taken using the ISS PC1 spectrofluorometer. This process was repeated at exposure times of 0, 10, 30, 60, 120, 300, 600 seconds and results plotted. Solar Photobleaching. An experiment utilizing sunlight was performed to determine if broad spectrum light effects on photob leaching. 75 cm2 culture flasks containing 100 and 10 g/ml dilutions of Deuteporfi n in DMSO. To prevent solvent evaporation, the flasks were sealed prior to solar i rradiation. Also three initial absorption measurements were taken prior to photobleaching usi ng the Cary 100 Spectrophotometer at room temperature (22 2C) in the range of 350 – 450 nm for Soret, and 450 – 700nm for Q-band observations and averaged. The samples w ere then exposed to bright sunlight with an average 162 mW/cm2 intensity measured on the surface of the sample fo r 10 minutes. The irradiated samples absorption levels were measured again using the same measurement process as before and graphed for compa rison against the unbleached measurements.

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34 Experiments to Compare Properties of Deuteporfin to HMME One Photon Absorption Spectra. A fortunate opportunity arose when exploring the possibility of comparing Deuteporfin Q-band abs orption spectra measurements against equivalent HMME data, as the same concentra tions and absorption spectra measurements were performed as part of a previous s tudy at the University of Colorado Denver in 2010 using the same laboratory dilution p reparation processes and measurement procedures with the same Cary 100 Spect rophotometer [22]. To confirm that the equipment calibration matched previous cal ibration measurements taken at the approximate time of the HMME data collection, a mea surement was performed with a reference 020 glass filter placed in the light path and compared against archived measurement data which matched the original baselin e measurement. With that in mind there is high confidence in previous absorption pea k wavelength data, although there is still albeit minimal concern that there could be sl ight deviations in dilution practices that could affect absorption intensity outcomes. As a r esult, Deuteporfin data obtained from the previous one photon Q-band absorption spectra m easurements in various solvents were compared against the HMME absorption data to i nspect the maximum Q-band peak intensity and the phase shift of the peaks for both photosensitizers. Fluorescence Emission Spectra. An experiment comparing Deuteporfin and HMME was devised to see if there were significant d ifferences in emission intensity and peak emission frequency. The photosensitizers were diluted with DMSO to 1 g/ml and measurements taken with the ISS PC1 Spectrofluorome ter at room temperature (22 2C) and measured in the 610-640 nm range. T he resultant data was normalized

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35 first as a group and then setting the range from 0 to 1 a.u. in order to compare the maximum peak intensity as well as the phase shift o f the peaks of the sensitizers.

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36 CHAPTER III RESULTS One-Photon Absorption Profile of Deuteporfin As noted earlier, Deuteporfin exhibits a very stron g Soret absorption Soret peak when diluted at 8 g/ml. As seen in Figure III.1a, The strongest absorption was obtained in DMSO at 401 nm, followed by PBS and H20 at 393 and 391 nm. The solvents also presented four distinct Q-band peak readings as sho wn in Figure III.1b in the range of 499 – 621 nm, with H20 and PBS wavelengths in family at the three higher intensity peaks at 503 – 504 nm, 538 nm, 564 and 566 nm, with the greatest difference at 610 and 618 nm for H20 and PBS respectively. The Q-band DMSO peaks were more spread out in comparison to the water based solvents, with pea ks at 499, 533, 568 and 621 nm, and showed a significantly higher absorption compared t o the other water based solvents as well.

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37 Figure III.1 Absorption Spectra of Deuteporfin in Various Solvents at 22 2C. (a) Soret band obtained at Deuteporfin concentratio n of 8 g/ml. (b) Q-band obtained at Deuteporfin concentration of 100 g/ml. n nrr !"# $%# n rr !"# $%# r n n n n

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38 Fluorescence Properties of Deuteporfin Due to the close proximity the lowest Q-band absorp tion peaks, low concentrations were used in order to minimize reabs orption which can affect fluorescence measurements. The strongest fluorescence peak range for Deuteporfin solutes was between 618 and 628 nm. Deuteporfin dissolved in D MSO showed the strongest fluorescence peak at 628 nm, followed by PBS at 618 nm, PBS/FBS at 626 nm and H20 at 618 nm. A second peak was observed with water ba sed solvents in the range of 678 – 686 nm, with a stronger DMSO peak further out at 69 4 nm. Interestingly the PBS/FBS dilution peaks were more in family with DMSO with a left shift in the strongest peak of only 2 nm, and an 8 nm left shift for the lower sec ondary peak. Figure III.2 Deuteporfin Emission Spectra in the R ange 610—710 nm at 22 2C. Concentration of Deuteporfin in solution was 1 g/m l. Excitation was at 400 nm. n n&'!( !"# !"#(&"# $%# n n n n n n

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39 A standard curve was generated for Deuteporfin in D MSO for the dilution range 1.0 – 0.1g/ml, and trend lines added for inspectio n. The plots for both 628 and 694nm emission peaks were almost perfectly linear through out the dilution steps as seen in Figure III.3. Figure III.3 Deuteporfin in DMSO standard dilution curves. Concentration range of Deuteporfin in solution was 1.0 – 0.1 g/ml. Excitation was at 400 nm. Q-band synchronized emission spectra from 610 – 710 nm versus 350 – 600 nm excitation wavelength measurements taken incrementa lly in 5 nm steps were also obtained using the ISS PC1 spectrofluorometer. Th e resulting measurements were then plotted by generating a topographic chart as shown in Figure III.4. )*+, )*+, n n&'!(-./( n 0.n 0.

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40 Figure III.4 Emission chart of Deuteporfin 1g/ml in DMSO at 22 2C. Contour lines represent an increase of 1 a.u. and 1 0 a.u. as labeled, normalized for the for the Q and Soret band regions respectively.

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41 Photobleaching Kinetics of Deuteporfin Prior to the photobleaching experiment it was noted that for the for previous Deuteporfin absorption and fluorescence standard cu rve trials that there was no self– bleaching characteristics in the resulting measurem ents (emission and absorbance level consistency with no phase shift) as this could tain t the outcomes of the procedure. As such it was determined that a series of trial timed instrumentation runs with samples not exposed to light were not required. Photobleaching of Deuteporfin in DMSO diluted to 1 g/ml was performed in an open petri dish at 630 nm and 150mW/cm2 which produced an initial rapid reduction in fluorescence intensit y at a first order rate, however this changed at approximately 60 seconds exposure to a s econd order rate as seen in Figure III.4a. Figure III.4b shows the overall photobleac hing fluorescence spectra for the times measured.

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42 Figure III.5 Photobleaching kinetics of Deuteporfi n in DMSO. (a) Exposure time curve: 1 g/ml, 400nm excitation, 628 nm emission at 150mW/cm2. (b) Fluorescence spectra from 600 – 700 nm. Exposu re times: 0, 10, 30, 60, 120, 300, 600 from top to bottom. Solar photobleaching was also examined, with Deutep orfin in 100 g/ml and 10 g/ml dilutions of DMSO exposed in direct sunlight t o a measured average power of 168 mW/cm2 for a period of 10 minutes. No Soret peak shift w as detected and the absorption intensity decreased by just under one half, whereas the Q-band showed minimal shifts in n &'!(!r.1. n n n &'!(2.. r

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43 the 3 major peaks with expected reduction in intens ity for each of the band peaks. An unexpected minor peak emerged at 643 nm which may b e a potential anomaly that could be investigated in future research. Figure III.6 Solar bleaching of Deuteporfin in DMS O. (a) 100g/ml concentration Soret peak absorption de creased by. 54% at 401 nm with no peak shift. (b) 10g/ml concentration Q-band absor ption peaks decreased by 24% at 499 nm, 23% at 535 nm, 20% at 568 nm and 15% at 621 nm respectively. Note there the low level points of the solar bleached curve came to re st at a higher level than the unbleached curve, and a red arrow points to a new peak at 643 nm, possibly indicating the presence of photoproducts as a result of photobleaching. n nrr 3r4 "4.'5. n nnnrr 3r4 "4.'5. n n r

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44 Comparison of Deuteporfin to HMME Some key Deuteporfin data was compared to current a nd previously measured HMME data to compare each of the PS qualities and t o determine what characteristics could support the claims on Deuteporfin effectivene ss for tumor PDT stated in the manufacturerÂ’s literature can be verified. Some HM ME data was able to be obtained from research that led to a journal paper that was published in 2012, of which I contributed in laboratory test procedures and compi ling results, and one experiment was performed post HMME study as part of this research [22]. A wealth of the HMME study data could not be used as many of the laboratory pr ocess parameters followed in the initial HMME investigation did not conform to labor atory procedure parameters for this study, such as solute concentration for Soret band absorption spectra measurements, photobleaching procedure process/concentration diff erences and temperature differences during measurement procedures to name a few. As su ch the ability to objectively compare the majority of the two sets of research da ta is limited. However, the key data and results presented here give a good insight to t he characteristic qualities and differences of these two photosensitizers. HMME, like Deuteporfin is also a HpD preparation wh ich consists of a mixture of two positional isomers of 7(12)-(1-methoxyethyl)-12 (7)-(1-hydroxyethyl)-3,8,13,17tetramethyl-21H, 23H-porphrin-2, 18-dipropionic aci d, as seen in Figure III.6 for the treatment of port wine stain birthmarks The drug has been indicated for use as a vascular targeted PDT, and it has also been suggest ed that HMME could also be utilized in in ocular and anti-tumor PDT [22].

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45 Figure III.7 Chemical structure of HMME. An observation was initially made between previousl y obtained HMME data and the Deuteporfin absorption data Q-band results. As shown in Figure III.8 there appears to be a close similarity between both photosensitizers in not only their peak wavelengths but absorption levels as well. As shown in Figure III. 8 there is a strong correlation between the two PS Q-band absorption peak levels for DMSO a nd PBS solutes, however there is a noted downward shift in Deuteporfin peak levels in comparison to HMME in H20. This was not surprising as it was found in other HMME ab sorption data that solutions in water appeared to be notably higher than in Deuteporfin H20 absorption spectra measurements.

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46 Figure III.8 Absorption Q-band comparison of Deute porfin and HMME in various solutes at 100 g/ml. (a) DP in DMSO, (b) PBS, and (c) H2O, noting that in Deuteporfin NP H2O was used compared to DI H2O which should have a negligible impact on the resu lting measurements. n nnnrr $!$%# %%2$%# n n n nnnrr $!!"# %%2!"# rn n n nnnrr $!6! %%2$7 n n

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47 An experiment was performed to compare the fluoresc ence characteristics of Deuteporfin and HMME, with both PS samples diluted in 1g/ml DMSO and the highest emission peak was measured from 610-640 nm. Althoug h the two compounds shared similar emission peak wavelengths, the intensity of fluorescence of Deuteporfin was substantially higher by a factor of 19.6. This evi dence shows favorably that Deuteporfin is better suited to activation in deep tissue due t o its increased emission levels at the same excitation wavelength compared to HMME, which in tu rn suggests that more released energy could be available for 1O2 production.

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48 Figure III.9 Emission comparison of Deuteporfin an d HMME. Photosensitizers diluted in1g/ml in DMSO and excit ed at 400 nm. (a) Fluorescence in Photons/sec (b) Normalized Fluorescence intensity. Note the si milarity in emission and the significant difference in Fluorescence intensity of the two compounds. n nnnn&'!(2.. $! %%2 nnn n nnn6.84&'7.)2.. $! %%2 r n

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49 CHAPTER IV DISCUSSION Measurement Observations One Photon Absorption. Not surprisingly, Deuteporfin exhibits characteri stics similar to other hematoporphyrin-based sensitizers with Q-band absorption peaks which reside within 500 – 700 nm and which is mostly with in the practical PDT treatment range of 600800 nm for tumor treatment, although a long er wavelength is preferred for deeper tissue penetration. For current clinically used se nsitizers the PS activation wavelength varies from 420 nm (blue) to 780 nm (deep red). Lig ht waves of greater length penetrate tissue further; blue light attenuates greatly withi n 1-2 mm whereas red light can penetrate more than 5 mm [23]. For example, the average effec tive PDT penetration depth for the PS Photofrin (intensity reduced to 37%) is about 1– 3 mm at 630 nm, the wavelength used for clinical treatment, while penetration is approx imately twice that at 700–850 nm [24]. The strong Soret peak absorbance peak observed betw een 390 – 405 nm could also be effective for PDT although the shorter wavelength i s only suited for surface treatment or for superficial lesions via intraluminal treatment since light penetration into the tissue is minimal. These peaks are useful in determining whi ch wavelengths to use for PDT based on the target tissue type and thickness in combinat ion with the location of treatment. The manufacturer’s recommendation is to use longer wave lengths to enable deeper penetration into tissue which is the intended appli cation of Deuteporfin, whereas for HMME it is more suited for shorter wavelength emiss ion for safely treating vascular conditions such as Port Wine Stain or surface lesio ns [25].

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50 Fluorescence Properties and Photobleaching Kinetics Examination of fluorescence emission showed two peaks at approxima tely 623 5 nm and 686 8 nm depending on solvent used. Also, when a series dil ution was performed the result was a near perfect linear decrease in intensity from 1.0 – 0.1 g/ml. Although not shown in this paper, absorbance series dilutions were performed f rom 100 – 10 g/ml for Soret and 10 – 1 g/ml for Q-bands, and the results exhibited a similar peak height linear step decrease for absorbance versus dilution. The high count for fluorescence (13519 photons/sec at 1g/ml) suggests a potential for goo d 1O2 production, however further investigation through singlet oxygen phosphorescenc e measurements are needed to verify this assumption. A comparison to fluorescence inte nsity of HMME in DMSO is explored in the following section that adds support to this hypothesis. Photobleaching observations are important to determ ine the amount of photodegradation of the PS to understand whether th e photobleaching is an advantage or disadvantage to treatment. Too rapid and malignant tumor/tissue destruction may not be complete, and depending on the PS damage or irritat ion to surrounding tissue could occur if proper PS dosimetry and photobleaching rates wer e not taken into account to control the PS reaction [26]. Figure III.5 shows how photo bleaching initially impacted Deuteporfin in the first 60 seconds of exposure, de creasing 20 percent from its initial intensity. However from 60 to 600 seconds the degra dation dropped by only 10 percent with an intensity of 123850 photons/sec, hinting th at 1O2 production although decreased should continue at an effective rate for treatment, although further evidence would be preferred to verify this.

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51 There is a strong interest if photoproducts can be produced by intense multi wavelength irradiation such as by photobleaching wh ich then can be potentially exploited for PDT [27]. It has been observed that there have multiple instances where porphyrins have exhibited stable photoproducts in the 640-660 nm range that can be used in tumor therapy due to increased absorbance in this region and increased light penetration depth into tissue [28]. Solar photobleaching Deuteporfin in DMSO at 100 g/ml for 10 minutes did indeed result in a new peak at 643 nm which is consistent with other porphine studies. This result is interesting since when a similar 1 h our experiment was performed with HMME in PBS at the same concentration for a 1 hour solar irradiation exposure time no additional peaks were detected, only minor absorban ce peak shifts [22]. Further investigation would be required to fully understand the extent of this new peak and present confirmation of photoproduct formation with different solutes and dilutions along with a set of time incremented solar photobleaching measurements. Deuteporfin and HMME Comparison Observations. The similarities and differences between Deuteporfin and HMME helped to verify the manufacturerÂ’s assertions as to the dosimetry methodologies for th ese medications. The Absorption peaks were strikingly similar for both compounds, w ith minor fluctuations in absorbance and similar Q-band peak wavelengths. As for the fl uorescence measurements, it was a pleasant surprise to see the dramatic increase by a factor of nearly 20 for the intensity of Deuteporfin in contrast to HMME levels. Even thoug h these measurements were for a single solute (DMSO), it can be assumed that simila r results would be observed for other solutes. This adds further weight to the assumptio n that 1O2 production should be significantly higher than that of HMME, and therefo re should support the claim that this

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52 PS would be useful in for tumor PDT. Again, to con firm this assumption further characterization analysis is needed for confirmatio n as well as to determine the proper dosimetry regimen for treatment. Considerations for Future Analysis There are still areas which need to be investigated further in order to have a complete characterization worthy of submittal for a journal article, as this is the ultimate goal when this research was initiated in 2011 when sample Deuteporfin was obtained for characterization. Since there are multiple factors involved in effective PS activation, 1O2 production and biological uptake to effectively era dicate malignant tissue, multiple studies will have to be ultimately be performed in order for determining proper dosimetry parameters needed to proceed to a formal medical tr ial. Only when this is accomplished can Deuteporfin be considered by the FDA as an acce ptable PDT treatment. Photobleaching Quantum Yield. One item that was overlooked was fulfilling measurements to obtain photobleaching quantum yield data, which was successfully accomplished as part of the study involving HMME ch aracterization as noted earlier, and is described in the process below. To measure init ial photobleaching quantum yield, the first steps that should have been performed in the p hotobleaching experiment were to measure the fluence rate emitted from the microfibe r through the solvent containing Deuteporfin in the petri dish, using a an optical S i-detector with wattmeter with the sensor directly below the petri dish to measure the transmitted power though the solution before and after photobleaching. Converting the po wer measurements from watts to photons per second, the quantum yield can be calcul ated.

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53 The calculation process is as follows [22]: Where: R1 = initial rate of photon absorption by the reactio n mixture transmitted power difference in photons/s after photobleaching transmitted power difference in photons/s before photobleaching t = bleaching time in seconds and: Where: R2 = initial rate of reduction of Deuteporfin molecul es fluorescence intensity after photobleaching fluorescence intensity before photobleaching cg = initial concentration N = AvogadroÂ’s number MW = is the molecular weight of Deuteporfin (unknown) The initial quantum yield of photobleaching can now be calculated: A factor that makes calculating the quantum yield n ot possible is that the molecular weight of Deuteporfin is not known due to the propo rtionalities of the formulation is estimated as the drug composition is deemed as prop rietary information by the

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54 manufacturer. In order to properly accomplish this characterization special permission to obtain the molecular weight from the manufacturer w ould need to be obtained or if possible a series of experiments would need to be p erformed to elaborately reverse engineer the formulation characteristics individual ly to obtain a close approximation. Only one article of research on Deuteporfin charact erization has been found in regards to determining the Deuteporfin components DMD and MHD concentration levels in dog plasma, but the molarity of the combined reactive c ompounds or study into the remaining (<15%) constituent formulation was not analyzed [29 ]. This may be the reason why no formal characterization journal papers have been pu blished to date. Fluorescence Quantum Yield. Another area to be worked include fluorescence lifetime measurement, the time which a fluorophore remains in the excited state. The purpose is to determine the rate of decay of the PS in order to prescribe the proper dose and number of treatments for the proper PDT regimen This would require in vitro and in vivo studies since fluorescence lifetime measurements h ave the potential to significantly decrease in in a biological environme nt in comparison to the PS in pure solvent. The decay of the intensity as a function of time is defined as [30]: # $%& Where: intensity at time t # intensity at time 0 time fluorescence lifetime

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55 On a side note, is the inverse of the of the total decay rate such that [30]: $ Where: radiative decay rate non-radiative decay rate Studies have been performed in determining fluoresc ence lifetimes in the biological environment through the use of Time-Resolved Fluore scence (TRF), which takes into account biological phenomena which introduces a nor malization term A into the intensity decay equation above such that [31]: $%& Note that this is a simplification that addresses m ultiple biological conditions that are beyond the scope of this research. Addressing life time measurements in a more natural environment could further refine proper dosimetry r equirements based on particular treatment conditions and regimen. As such, this ar ea of evaluation could be researched as its own subset of PDT characterization analysis. Photodynamic Diagnosis. As mentioned earlier in this study, the utilizati on of a PS as an indicator of a malignancy in a process cal led Photodynamic Diagnosis (PDD) has been explored and has promise in detecting canc ers as well as infections and diseases. A study with Deuteporfin was conducted to visually detect lymphatic metastases in rats by visual fluorescence observation using a Wood’s l amp, which is essentially an ultraviolet light in the range of 320 – 400 nm that is primarily used in dermatological examinations to observe skin disorders and bacteria l/fungal infections [32]. It was determined that there was an increase in concentrat ion in Deuteporfin in cancerous tissue in comparison to normal healthy tissue, which resul ted in the observation of malignancies

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56 increased fluorescence when irradiated by a WoodÂ’s lamp [4]. Research performed on other PDT PSs has shown that their application as P DD is viable by procedural adjustments such as modifying the excitation light dose [33]. In Vivo Characterization Utilizing Various Light Sources/C onfigurations. This area of PDT characterization research has not been addressed, although it appears to be utilized more in line with clinical trials. To maximize PDT results while minimizing test subject complications during treatment, some o f this analysis can be brought into the characterization procedures to explore light source placement in tumor treatment. Two Photon Excitation. A potential experiment under consideration would be two-photon excitation (TPE). In PDT this process h as been investigated to exploit the ability to excite many PS formulations at approxima tely twice the optical wavelength compared to one-photon excitation, which in turn al lows deeper tissue penetration. This combined with minimizing collateral tissue damage a s the probability of absorption increases with the square of the light intensity, e nabling spatial confinement of PS activation as well as femtosecond lasers can be uti lized minimizing photothermal damage makes TPE an attractive candidate for PDT treatment [22, 34]. For HMME a two-photon experiment was performed achieving favorable result s including obtaining an absorption peak at 740 nm, that given the absorption compariso n with Deuteporfin would suggest that the PS would also exhibit a similar response t o TPE [22]. Another novel process in development is to use seco ndary sensitizers in combination with a PS for TPE PDT treatment. A sec ondary sensitizer such as a Carbon Quantum Dot (CQD) would be activated at the TPE wav elength range and the sensitizer can be then excited indirectly with two near-IR pho tons through an energy transfer

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57 mechanism known as either Frster Resonance Energy Transfer or Fluorescence Resonance Energy Transfer (FRET) in the activation band for the PDT PS, thereby providing excellent light penetration characteristi cs up to 2 cm for tumor treatment efficacy [35, 36]. This process could be performed in vitro prior to clinical trials to investigate the biological environment impacts to T PE to add fidelity to characterization measurements. Figure IV.1 FRET process as applied to secondary s ensitizer use in PDT. The concerted transitions of donor and acceptor req uire energy matching of fluorescence of the donor (dashed green arrows and spectrum) and absorption of the acceptor (dashed orange arrows and spectrum). However, they occur wi thout emission of the donor. After the energy transfer to the acceptor, its specific f luorescence spectrum can be observed (red arrows, spectrum), or in the case of having pr otoporphyrin IX PS the acceptor molecules will go through the ISC into the T1 state and 1O2 production would occur [35].

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58 Figure IV.2 Schematic representation of the CQD–P conjugate illustrating indirect excitation of the protoporphyrin IX upon two-photon excitation of the CQD. CQD is excited at 400nm, and results in FRET energy transfer to the protoporphyrin IX PS, resulting in 1O2 at a tissue penetration depth not possible utilizi ng the PS alone [35]. Light Source Delivery. Utilizing the correct medically approved lasers a nd optical fibers to administer the required light dos imetry to the affected site can significantly affect dosimetry requirements and tre atment outcomes of PDT. Recent developments in research and the manufacturing of F DA approved medical grade laser equipment has produced systems specifically designe d for the field of PDT in administering multiple light sources of varying wav elengths and intensities for various site and PS applications. This type of system coul d be enhanced with the ability to provide a reactionary light source in multiple regi ons of the treatment site for interstitial light delivery, by providing customized irradiation to surrounding the tissue using multiple optical fibers that dosimetry parameters c an be individually measured and adjusted for each fiber channel, minimizing multipl e fiber placement and irradiation

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59 procedures. One application that shows promise for both research and application of PDT is a system that uses multiple fibers for admin istrating laser light to the site as well as for monitoring levels of irradiation to monitor treatment progression by the ability of switching the fibers from the output laser via a sw itching interface to a spectrometer for measurement. [37]. An approved laser example is th e Modulight ML7710-PDT medical laser system (Modulight, Inc. Finland, www.moduligh t.com) is capable of delivering up to 8 individually controlled fiber output channels of wavelengths of 630, 635, 652, 660690 and 753nm at a power of 1-15 W. This could be coupled to a similar switching interface apparatus to alternate between the variou s emission/detection sources, or additional fibers can be inserted to the site for d irect coupling to spectrometer(s) for active PDT monitoring. This method could be of int erest in the pre-clinical phase of PS characterization by monitoring tissue PDT process f rom multiple regions to monitor biological variations in fluorescence and photoblea ching quantum yields as well as developing processes to accurately determine 1O2 quantum yield under different biological conditions. Other Concerns. The website ClinicalTrials.gov is maintained by t he US National Library of Medicine and National Institute s for Health as a resource for accessing clinical studies process and their outcom es by researchers and medical professionals as well as for the general public. T his is a useful tool in determining a drug or medical device status for acceptance for further study or for proceeding toward the next steps for FDA approval. A search on Deuteporf in was performed to see if any preliminary clinical trials were performed and if s o the corresponding study findings. The search revealed that there was a record of a cl inical trial performed under record

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60 number NCT01481597 initiated October 2011 and compl eted May 2012. The study was to investigate on the bodyÂ’s tolerance to the drug and on pharmacokinetics, which is the time course of absorption, bioavailability, distrib ution and metabolism, of single-dose of Deuteporfin administered intravenously in 32 health y volunteers to determine the potential for adverse events that could be a health concern. Although the study was completed, the findings have not been entered into the database, nor upon searching for journal papers or medical publications has there be en found any published outcomes to this study. This is a potential concern since if t here are any results suggesting that Deuteporfin is not a safe and viable PDT treatment then further research into its properties may have to be reconsidered.

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61 CHAPTER V CONCLUSION Deuteporfin in the PDT Arsenal There are many observations that have shown that De uteporfin is similar to other HpD PS drugs. There is a very strong Soret peak fo llowed by four weaker Q-band peaks in the 499 – 621 nm range which is within the commo n spectrum for performing PDT and very similar to HMME Q-band peak spectra. Ther e are also two strong fluorescence peaks at approximately 623 and 686 nm, with the str ongest fluorescence peak of the various solvent dilutions having some similar quali ties with the Soret absorption peak. There may be a possibility of photoproducts being g enerated when exposed to broad spectrum light which needs to be further investigat ed, although no preliminary evidence of photoproducts appeared after laser (630 nm) irra diation. The initial characterization data in this report supports that Deuteporfin is a promising candidate for tumor PDT treatment. The investigation into photobleaching q uantum yield needs to be performed to determine the extent of singlet oxygen production i n order to perform further research into dosimetry protocols. Comparison of DP against other FDA approved PSs such as Photofrin and Levulan needs to be performed in orde r to verify that this drug at a minimum can be utilized as a substitute for PDT tre atment, and to verify that the potential for enhanced treatment regimen is possible. Safety into its utilization during treatment and post procedure needs to be verified as well, an d if confirmed this could lead the way toward further clinical research toward ultimately being considered for FDA approval

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62 Final Thoughts This analysis has been a valuable insight to the fi eld of Photodynamic Therapy and its contributing components that make this medi cal treatment possible. Although this therapeutic method has been historically recorded a s being utilized in some form for centuries, only recently has PDT been rediscovered and applied in modern medical practice for almost two decades, with the full impl ications of this treatment just now being discovered [38]. As the fields of engineerin g and medicine converge, new approaches and methodologies are being discovered a nd put into practice. Photodynamic Therapy is one of those areas where this medical/en gineering relationship will be tested, and ultimately patients will benefit from the resul ting outcomes of this development. Deuteporfin characterization is just one small step in the process of expanding potential treatments to aid in eradicating cancerou s lesions and tumors, port wine stains and other epidermal cosmetic treatments, bacterial infections, and even aid in the detection of malignancies and diseases. The opport unity to expand this field has arrived, and it is up to engineers and medical researchers t o combine their talents to further develop this field for future generations to benefi t from its medical capabilities.

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