ABSTRACT

1

• ABSTRACT
• Photodynamic Therapy (PDT) is a cancer treatment
  that utilizes a photosensitizer, light and
  oxygen.
• Photosensitizers (PS) should have intense
  visible absorptions high intersystem crossing
  efficiencies ease of modification
  biodegradability etc., Phenothiazine (PHT)
  derived and halogenated Squarine (SQ) dyes should
  be ideal sensitizers.
• Treatment efficacy is enhanced by linking the PS
  to a cell recognition probe. Synthesis, targeting
  spectroscopic characterization of some of
  these promising PDT agents will be discussed.

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Synthesis, Characterization, and Targeting of
Phenothiazine- and Squaraine-based
Photosensitizers for PhotodynamicTherapy Dyeing
for a Cure
Prakash B Sanjeevaiah, Ping Hu, Basel Zaitoun,
Kenneth Olsen, and David S. Crumrine, Department
of Chemistry, Loyola University Chicago, IL
60626 dcrumri_at_luc.edu
3
Introduction to PDT

• Photodynamic therapy relies on biological
  reactions initiated by oxidative species produced
  in photochemical reactions. The key elements
  are
• A photosensitizer (PS) that, after excitation by
  light, rapidly transforms from the excited
  singlet to the excited triplet state.
• Oxygen in the triplet ground state accepts
  triplet energy from the sensitizer and is
  transformed into the very reactive excited
  singlet state.
• Singlet oxygen is believed to be the primary
  cytotoxic agent responsible for photo-biological
  activity (Type II Mechanism), however various
  radical reactions could play a supporting role
  (Type I Mechanism).

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Advantages of Current PDT

• A relatively nontoxic PS is administered (orally,
  topically, or intravenously) and equilibrated for
  a period of time, allowing for maximum
  tumor/normal tissue differentiation.
• The tumor is then irradiated by light of a
  wavelength absorbed by the PS.
• The cytotoxic products generated by the excited
  PS cause destruction of the tumor, preferably
  without damaging normal tissue.

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An Ideal Photosensitizer

• Should be non-toxic and stable in the dark.
• Should have absorption between 600 and 800 nm,
  where tissue penetration is greater with
  sufficient energy to generate singlet oxygen.
• A molar absorptivity of 20,000 30,000 M-1 cm-1
  would minimize the dose needed.
• A relatively easy synthesis from readily
  available starting materials with feasible large
  scale production.
• Good solublity in aqueous solvent mixtures.

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Promising PDT Photosensitizers
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Specific Aims

• The specific aims for this project are to
• Develop a group of phenothiazine and squaric acid
  based photo-sensitizers
• Attach them to targeting agents
• Test their quantum efficiencies
• Test their PDT efficacies

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First System Azure B dye-linker synthesis
Milind Choubal Carla Edwards
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HPLC Data for Compounds of System 2

• C18 column with CH3CN/H2O/TFA as eluent and UV
  detection.
• Compound Retention time (min)
• Azure B 33
• Az-CFMRFa 21
• Az-CLRFa 20.3
• Az-CFMRF 19.8
• Az-linker-Cl 17.5
• CLRFa 6.0
• CFMRF 5.5
• CFMRFa 5.3 (M. Choubal)
• Peptides, 1996, 17, 991-994.

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Singlet Oxygen Quantum Yields

• Conditions MethB Azure B Az-CFMRFa
  Az-CFMRF Az-CLRFa
• pD9 100mM borate 0.54 0.47 0.72
  0.71 0.75
• pD8 100mMphosph 0.46 0.40 0.65
  0.63 0.66
• pD7 100mMphosph 0.22 0.21 0.50 0.45
  0.50
• pD6 100mMphosph 0.09 0.11 0.25 0.25
  0.27
• pD7 10mMphosph 0.31 0.29 0.47 0.48
  0.46
• pD7 100mMphosph 0.44 0.33 0.45 0.45
  0.43
• 35 mM Sodium lauryl sulfate added. (J.
  Kanofsky)
• Determined from emission at 1270 nm measured 90o
  to laser excitation.
• Small peptide PROBABLY wraps around dye to avoid
  stacking
• Peptides, 1996, 17, 1213-1217.

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Selectivity for Binding Sites

• Neuropeptides and antibodies have very specific
  binding sites.
• Need a derivative that doesnt affect binding
  specificity
• A simple test involves attempting to displace a
  radio iodine labeled derivative from the
  receptor.
• Level of radioactivity in solution is a measure
  of specificity.
• Receptor Binding Assay Summary for Helix Brain
• Ligand IC50 (mM) Ligand IC50 (mM)
• FMRFa 2.8 CLRFa 10
• CFMRFa 0.8 Az-CLRFa 4.2
• AzCFMRFa 1.6 Azure B gt100
• CFMRF 8.4 Methylene Blue gt100
• AzCFMRF 20 (K. Payza)
• Peptides, 1996, 17, 1279-1284

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Synthesis of X-Linked Hb-Dye Conjugate(a
possible Oxygen donor/scavenger)
Claudia Davila Ping Hu
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System 3 Folic Acid for Targeting
MW 441

• non-destructive delivery of macromolecules into
  living cell
• (antibodies, genes, enzymes and peptides) P.
  Low, Purdue U.
• Folate receptors are over-expressed in many
  cancer cells

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Folate Endocytosis for Targeting
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System 3 Targeting Outline

• Dye
  Folate Conjugate

C.P. Leamon P. Low, Purdue Univ, JBC, 1992,
267, 24966-24971
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Human Neuroblastoma Cells Uptake Protocol
Human neuroblastoma cells (SH-SY5Y) from
Dr. Freitas Lab Folate receptor
overexpression has not been reported.
Cell film

• Hb-FITC
• Control sample 30
  min 60 min

(b) Folate-Hb-FITC 30 min
60 min PDT model sample

• Folate-Hb-FITC 30 min
  60 min
• 0.1 M folic acid
• Folate inhibition

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Fluorescence Images of Nerve Cell Uptake
Experiment
A
C
(a)

(b)
(c)
(a) Hb-FITC (b) Folate-Hb-FITC
(c) Folate-Hb-FITC

1 mM
folic acid
Ping Hu Susan McGuire
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Microscope Images of SHSY5Y Cells Before and
After Applying PDT Agent
White Light Control No Hb-Folate-FITC
Fluorescence filter 520 nm Applied
Hb-Folate-FITC after 30 mins
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Western Blot for Folate Specific Binding on
SHSY5Y Cells

• Results
• Folate receptor on the SHSY5Y cells
• Folate receptor not affected by Folate in the
  media

SHSY5Y whole cell lysate, anti-mouse IgG-HRP,
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System 4Proof of Concept Phenothiazine
Monoester of Folic Acid FA-PHT
UV, MS, NMR characterized, used for a cellular
intake study
M peak for FA-PHT ester
UV shows modification of both FA PHT
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System 5 Phenothiazine Trimer and Monoester of
Folic Acid
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Spectra for Phenothiazine Trimer
l max at 680 840 nm
M Peak of Trimer
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Spectroanalytical Studies

• The PHT trimer was subjected to cyclic
  voltammetry to study its redox potential and the
  effect of light.
• Upon irradiation with a Xenon lamp, an
  additional oxidation peak at 0.45 mV appeared
  and a black layer formed on
• surface of the electrolytic cell indicating
  photooxidation

Augustine Agyeman
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Spectroanalytical Studies
Additional peak due to photo oxidation with
Xenon light source
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PhotosensitizersSome of the phenothiazine
derivatives currently being synthesized are given
below to study substituent effects on generation
of singlet oxygen. Trimer derivatives will also
being synthesized.
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Another Synthetic Route for PHTsSubsequent
reactions are being carried out to link PHT
trimer to a maleimide derivative. This derivative
could be effectively linked to an oxygen source
such as Hemoglobin via the sulfhydryl group of
cysteines
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System 5 Squaric Acid-Linker-Folate Complex
B. Zaitoun
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Conclusions and Future Work

• Some Phenothiazine based photosensitizers have
  been synthesized and characterized. They show
  promise as PDT agents. The PHT backbone can be
  readily modified.
• Preliminary data shows the feasibility of
  inserting a PS into the cell with Folic acid
  targeting. Cell tests on the trimer are underway.
• Different side chains or functional groups on the
  phenothiazine backbone might help increase
  quantum efficiencies.
• Some Squaric Acid derivatives show promise in
  PDT.
• Mouse bone-marrow derived cancer stem cells will
  be tested for reaction selectivity.

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Acknowledgements

• LU for Arthur. J. Schmitt Dissertation
  Scholarship and Teaching Fellowship to P.
  Sanjeevaiah and for Research Leave for DC
• NIH, NSF-REU, Am. Inst. of Biochemistry
• Matthew Wlodarski, Alan Barlan.
• Dr. Jeff Kanofsky, Hines Veterans Hospital
• Dr. Kemal Payza, NIH
• Dr. Susan McGuire, LUMC
• Dr. Paul Chiarelli, LC-MS support
• Dr. David French, Manager, NMR Facility
• Augustine Agyeman, Godfred Boateng
• Dr. Jeff Feigenbaum

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References

• Phenazathionium Cations II. Revue Roumaine de
  Chimie, 1980,25, 8, 1239-1244, I. A. Silberg
  A. U. Diudea
• Design and Synthesis of 111-InDTPA-Folate for
  use as tumor-targeted radiopharmaceutical.
  Bioconjugate Chem, 1997, 8, 673-679, Philip. S.
  Low et al
• Preparation of 1,3,7,9-tetrabromophenothiazine.
  Nippon Kagaku Kaishi, 1997, 3, 437-8, Ikeda,
  Yoshikazu
• Remarkable structure deformation in phenothiazine
  trimer radical cation. Org. Letts, 2004, 6,
  3493-96, Keiji Okada et al . Background pic www.
  photochembgsu.edu
• Photochemistry of Squarine Dyes I, J. Phys.
  Chem., 1992, 96, 195-199, P. V. Kamat et. al.
• Chemical Aspects of Photodynamic Therapy, Raymond
  Bonnett, CRC Press, 2000.

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Initial HeLa Cell work with FluoresceinITC-FA
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Cell Accumulation Assay

• SH-SY5Y cells were grown in a 75 cm2 plastic
  tissue culture flask in DMEM medium and incubated
  for 72 h at 37?C for proper attachment. The cells
  were then exposed to PHT and the FA-PHT ester
  (21.0 ?M) and incubated for 24 h, washed with HBS
  buffer, and harvested with Pucks D1. Cell
  suspension was centrifuged and washed 3X with HBS
  buffer. The fluorescence emission from the loaded
  cells was measured using the fluorometer with an
  excitation/emission wavelength of 300/362nm. The
  cellular uptake of FA-PHT was on average about
  same as that of untreated cells, this indicates
  that folic acid may be aiding in endocytotic
  uptake of the dye. It was noted that to observe a
  clear difference between the cellular intake and
  the background noise in fluorescence, the
  concentration of the FA-PHT ester should be at
  least 50mM. Studies are continuing.

Susan McGuire G. Boateng