Evaluation of an Indiumlabeled Porphyrin for Use in Photon Activation Therapy with Synchrotron Radia

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Evaluation of an Indium-labeled Porphyrin for Use
in Photon Activation Therapy with Synchrotron
Radiation

• Veronique Seror, Itzhak Orion, Sara Novick,
  Dabney Dixon, Peter Hambright and Brenda Laster

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In addition to the need for monochromatic photons
to induce a photoelectric effect in a high Z
atom, another major requirement for successful
PAT studies is the assurance that the high Z atom
is localized to the DNA of tumor cells and will
remain bound to DNA throughout the radiation.
This presentation will describe the studies
undertaken to identify the site of localization
of indium atoms in preparation for synchrotron
studies.
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Confocal Laser Microscopy
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Notice the ubiquitous fluorescence in cells with
InTMPyP(4) (left) compared to control cells
(right). Nuclei of control cells are black. The
cytoplasmic fluorescence can be attributed to the
cytochromes.
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In vitro Uptake in B-16 Murine Melanoma Cells

• Cells were fractionated by trichloroacetic acid
  (TCA) into two components
• TCA-insoluble contains DNA and high molecular
  weight proteins
• TCA-soluble contains the remaining cellular
  degradation products

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In vitro B-16 InTMPyP(4) ICP-MS
Measurements TCA-insoluble and TCA-soluble
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After showing uptake in the TCA-insoluble
fraction of B-16 cells, an Ethidium Bromide
Displacement Assay (EB) was used to determine the
binding strength of InTMPyP(4) to DNA. EB binds
to sites on DNA and gives off fluorescence. The
fluorescence value was assumed to be 100 in the
EB/DNA complex. Therefore, any decrease in the
percent fluorescence observed, after the addition
of InTMPyP(4), would indicate the displacement of
EB and the binding of InTMPyP(4) to the EB site.

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Ethidium Bromide Displacement Assay
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When a strong binding affinity was shown for
InTMPyP(4) and DNA, experiments to determine
uptake in mice were planned. However, these
required an assessment of the degree of
cytotoxicity that could be expected from
InTMPyP(4). Therefore, the MTT Cytotoxicity
Assay was carried out on B-16 cells.
Proliferating or living cells secrete
dehydrogenase enzymes that can convert MTT to a
purple formazan precipitate that can be
solubilized. Optical absorption can measure the
amount of formazan precipitate present.
Therefore, the more absorption measured, the
greater the number of living cells.
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Optical absorbence was read at 540 nm. The
calculation for percentage of cytotoxicity
was Cytotoxicity 1- (Abs of experimental
wells/ Abs of control wells) 100
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The inability of InTMPyP(4) to reduce cell
proliferation in a malignant tumor line compared
to a transformed line gave an indication that no
correction factors would have to be used in
determining the survival of cells after
irradiation that is, no cytotoxicity factors
would impact upon any results obtained from
radiation studies.
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InTMPyP(4) was injected into mice carrying the
B-16 murine melanoma on their footpads. The mice
were euthanized at various intervals of time,
their organs dissected and the number of In atoms
present in the sample was measured using ICP-MS.
The highest uptake of In atoms in tumor cell DNA
was at 5 hours post injection, measuring 108 In
atoms per cell.
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Synchrotron studies were carried out at the ID17
medical beam line at the ESRF. The B-16 melanoma
on the mouse footpad was irradiated above (28
keV) and below (27.8 keV) the K absorption edge
(27.9 keV) of indium. A total dose of 20 Gy was
delivered to each tumor.
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C-57 mice with B-16 melanoma on foot pad
irradiated at ESRF
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• In conclusion,
• InTMPyP(4) accumulated a therapeutically
  sufficient number of In atoms in tumor cell DNA
  with minimal to no toxicity.
• The In atoms remain bound to tumor cell DNA
  throughout the irradiation period.
• The 20 Gy radiation dose to the B16 melanoma on
  the footpad of the mouse was too low to control
  tumor growth.
• The initial growth rate of the tumor was slower
  after irradiation above the K absorption edge of
  indium compared to all other treatment or no
  treatment groups.
• The early retardation of tumor growth can be
  attributed to damage to the DNA from Auger
  electron emission.

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Thank you for your attention!