KEYWORDS: Tumors, Photodynamic therapy, Luminescence, Magnetic resonance imaging, Cancer, In vivo imaging, Absorption, Oncology, Breast cancer, Positron emission tomography
Purpurinimides, derived from chlorophyll-a, are tumor avid, stable in vivo and show a strong absorption in the near IR
region (700 nm) with a high singlet oxygen (>50%) producing efficiency. Such a characteristic could be useful in
treating large and deeply seated tumors by photodynamic therapy (PDT). These long wavelength photosensitizers can be
used as vehicles to deliver the imaging agents to tumors. Therefore, by linking suitable tumor imaging moiety with
purpurinimide could produce a novel bifunctional agent, which could possess the desired photo physical properties for
both tumor detection as well as photodynamic therapy. Such image guided therapy would represent an archetype for
cancer treatment.
Various problems arising during molecular imaging of different fluoroprobes and metabolites used in PDT can be
circumvented by focusing on multifunctional therapy agents. Thus an effective photo sensitizer coupled with other
useful roles to play in PDT treatment make nanoparticles as a good vehicle for different delivery assuming
multifunctional roles not only in PDT but also as therapeutic agents for targeted delivery. A new approach is the
involving use of 100 nm NPs as photo sensitizers and/or imaging agents. In our Lab., we employ two such NPs and are
ORMOSIL (organically Modified Silica) and PAA (Polyacrylamide) which are found to be biologically very safe
without disturbing the therapeutic value. The size of the nanoparticles determined by TEM and Dynamic Light
Scattering are ~30 nm. These NPs are taken up in conjunction with cyanine dye at near infra red as it has been reported
in literature that encapsulated NPs shows very low singlet oxygen production compared with the post-loaded NPs though
the reasons are not yet clear. Therefore, we investigated the idea of post-loading or adsorbing vis-a-vis encapsulation.
Photodynamic Therapy (PDT) is emerging as a successful tool to treat both malignant and benign tumors. It involves the
interaction of a photosensitizer which upon activation by the appropriate light dose, leads to a cytotoxic and vasculotoxic
photodynamic reaction. Improvements in PDT in areas such as the delivery and selectivity of photosensitizers,
light-delivery and overall efficacy have helped to increase its attractiveness as an option for therapy. For optimizing the
PDT treatment by a "see and treat approach," we have developed a number of tumor avid photosensitizers (PS) namely
HPPH-Cyanine dye conjugates or other compounds (Iodinated photosensitizers) which have the ability for Optical and/or
PET imaging as well as being effective photosensitizers for treatment. Hyperthermia refers to various techniques of heat
application which may be delivered as a single modality or as part of an adjunct treatment option to the existing cancer
therapies. Depending upon the temperature range used, hyperthermia might either directly induce cell kill or enhance the
efficacy of other treatment modalities. Hyperthermia increases blood flow within the body, which may allow for higher
dose delivery of photosensitizers with subsequent increased therapeutic efficacy of PDT. Hyperthermia could also
increase the sensitivity of molecular imaging. The use of multifunctional photosensitizers for imaging and PDT is an
emerging area and we have developed a few such agents in our lab. We wish to explore the use of hyperthermia to
improve the use of such multifunctional photosensitizers from the point of view of imaging and/or therapy.
Hyperthermia can be performed either as a whole-body mode or as localized mode. Our goal is to see which of the two
heating modalities offers us better outcome.
We constructed a whole-body fluorescence tomography instrument to monitor novel bifunctional phototherapeutic drugs (e.g., HPPH-Cyanine dye conjugate) in small animals. The instrument allows dense source and detector sampling with a fast galvo scanner and a CCD detector for improved resolution and sensitivity (Patwardhan et al., 2005). Here we report tissue phantom measurements to evaluate the imaging performance with a newly constructed tomography instrument. Phantom measurements showed that strong fluorescence generated by HPPH-Cyanine dye (HPPH-CD), having high fluorescence quantum yield and long wavelength fluorescence emission, allowed deep tissue imaging. We also report in vivo fluorescence measurements of the conjugate in Nude mice bearing A549 human non-small cell lung carcinoma (NSCLC) tumors at 24 hr post injection to evaluate tumor detection ability of the conjugate. Our results indicate that the HPPH-CD shows preferential uptake in tumors compared to surrounding normal tissue at 24 hr post injection. This study demonstrates a potential use of HPPH-CD in detection (fluorescence imaging) and treatment (PDT) of deeply seated tumors.
HPPH (a chlorophyll-a analog) was linked with a cyanine dye and the resulting conjugate was found to be an efficient tumor imaging (fluorescence imaging) and photosensitizing agent (PDT). Our preliminary results suggest that tumor-avid porphyrin-based compounds can be used as vehicles for delivering the desired fluorophores to tumor for fluorescence imaging. In an early diagnosis of microscopic lesions in pre-clinical studies (C3H mice implanted with RIF tumors) the HPPH-cyanine dye conjugate showed tumor-imaging capability (λex: 780 nm, λem: 860 nm) at the non- therapeutic doses that are 100 fold lower than those used therapeutically. Compared to the cyanine dye, the corresponding HPPH-conjugate showed enhanced long-term tumor imaging ability.
Photodynamic therapy (PDT) is a novel cancer treatment modality where the therapeutic action is controlled by light and the potency of the photosensitizer used. Development of new potent photosensitizers (PS) for clinical applications requires that the PDT effects are maximized while minimizing dark cytotoxicity. The dark toxicity of photosensitizers is generally confirmed using cell lines. Photososensitizers that appear promising from in vitro assays need further investigations under in vivo conditions. As in vivo MR methods have the potential to provide information on the tumor status, they can be very effective tools to study dark toxicity of tumors. The tumor produced on the mouse foot dorsum was tested on two newly synthesized photosensitizers along with Photofrin as a control. The MR studies consisted of serial 31P spectral measurements both before and after PS injection. The results show significant changes in the tumor metabolism with increased inorganic phosphate while using new photosensitizers. However these changes slowly approached control levels several hours later. The studies performed while using Photofrin did not show any significant changes indicating minimal or no dark cytotoxicity. Similar studies performed on normal tissue such as the muscle indicated that the energy metabolism was minimally compromised. Our studies demonstrate that the effects of dark cytotoxicity can be observed by 31P MR. The growth profiles of tumors treated with PS alone indicate that the metabolic changes are temporary and do not interfere with the tumor growth. The studies suggest that MR is a new method of monitoring the effect of PS administered toxicity in an in vivo model.
The changes in the tumor that occur following photodynamic therapy (PDT) were studied using a small animal MR imager operating at 7Tesla. The animal model used in these studies was mice bearing radiation induced fibrosarcoma (RIF) tumor on the foot dorsum. The mice were injected with 10μM/kg of one of the photosensitizers: (1) Photofrin, (2) Non-fluorinated porphyrin photosensitizer (DOD-1), (3) Fluorinated porphyrin photosensitizer (DOD-2) and, (4) Fluorinated chlorin photosensitizer (DOD-6). Laser light at 630 or 650 nm (150 mW/cm2, 270 joules/cm2) was delivered to the tumor at 2-24 hours of photosensitizer administration. The MR spectroscopic and imaging examination of the tumors involved both the 1H and 31P nuclei. The tumor bioenergetics was measured by 31P spectroscopy. The water proton relaxivity and diffusion measurements were used to obtain local changes in different regions of the tumor. Changes in 31P MR spectra were observed following PDT using Photofrin and fluorinated chlorin sensitizer (DOD-6). However, no significant changes were observed when the fluorinated porphyrin and its nonfluorinated analog were used. The PDT induced changes in tumor volumes showed significant tumor regression with Photofrin, fluorinated porphyrin and chlorin sensitizers. No tumor regression was observed with the non labeled porphyrin sensitizer and the growth profile followed the general pattern of unperturbed tumors. Serial noninvasive measurements of tumor response to PDT are measurable by both MRI and MRS. The MR derived parameters that are characteristic of the tumor status before and after the therapy are discussed here.
Limited therapeutic options exist when chest wafl recurrence from breast cancer progresses despite standard salvage treatment. As photodynamic therapy offers excellent response for cutaneous lesions this may be a possible indication for PDT. A total of 1 02 treatment fields were illuminated on 9 women with biopsy proven chest wall recurrence of breast cancer which was progressing despite salvage surgery, radiation, and chemo-hormonal therapy. PDT consisted of outpatient IV infusion ofPhotofrmn at 0.8 mg/kg followed 48 hours later by illumination at 140-170 J/cm2 via a KTP Yag laser coupled to a dye unit. No patient was lost to follow up. At 6 months post PDT; complete response, defined as total lesion elimination was 89%, partial response 8%, and no response 3%. No photosensitivity was seen and no patient developed scarring, fibrosis, or healing difficulties. Low dose Photofrmn induced PDT is very active against chest wall lesions. Despite fragile and heavily pre-treated tissues, excellent clinical and cosmetic outcome was obtained. PDT is an underutilized modality for this indication.
In preliminary studies, the efficacy of a new photosensitizer, pyropheophorbide-(alpha) - hexyl ether (HPPH #23), for use in photodynamic therapy (PDT) was assessed using the rat 9L gliosarcoma tumor model in subcutaneous flank tumors, intracranial tumors, and in vivo. Flank and intracranial tumors were irradiated with 75 - 203 J/cm2 24 hours after 0.3 - 0.6 mg/kg IV injection of HPPH #23. At 24 hours post-PDT, and flank tumors showed a range of necrosis at the highest laser dose from 50 - 100%. The overlying skin and underlying muscle were spared. Intracranial tumors exhibited moderate to severe hemorrhagic necrosis. Areas of brain adjacent to tumor within the irradiated field also showed some damage. In vitro phototoxicity of HPPH #23 was compared to that of Photofrin II (PhII). Cells growing in culture dishes were exposed to HPPH #23 or PhII for 20 hours, washed free of unbound drug, then irradiated at 2.5 J/cm2, 17 mW/cm2 at 665 nm (HPPH #23) or 630 nm (PhII). Irradiated cultures were maintained in dark incubators for an additional 4 - 5 days, and phototoxic inhibition of cell proliferation was quantified by the sulphorhodamine B spectrophotometric assay. Under identical irradiation conditions, the IC50 for HPPH #23 (0.25 (mu) g/ml) was 10-fold lower than that of PhII (2.5 (mu) g/ml). Complete cell kill was achieved at sensitizer doses of 0.5 (mu) g/ml (HPPH #23) and 5.0 (mu) g/ml (PhII).
In the first part of this paper, syntheses of water-soluble photosensitizers for use in photodynamic therapy are described. Vinylporphyrins and vinylchlorins react with N.N- dimethylmethyleneammonium iodide (Eschenmoser's reagent) to give 2- (dimethylaminomethyl)vinyl derivatives which can be readily quaternized with methyl iodide to give highly water-soluble quaternary ammonium salts. Deuteroporphyrin-IX dimethyl ester undergoes direct substitution with dimethylaminomethyl at the nuclear 2- and 4-positions. The second part of this report describes the syntheses of bacteriochlorin analogues of natural chlorophyll derivatives; these are obtained by osmium tetraoxide oxidation of chlorins to give vic-dihydroxybacteriochlorins. Acid catalyzed pinacol-pinacolone rearrangements of a number of vic-dihydroxybacterochlorins to given ketobacteriochlorins are described, and the chemistry of the rearrangement is investigated. Biological studies of all sensitizers described will be reported elsewhere.
In order to evaluate the effect of substituents in photosensitizing activity, a series of long wavelength absorbing photosensitizers related to pyropheophorbides, bacteriopheophorbides, and benzoporphyrin derivatives were synthesized. Pheophorbide dimers, covalently joining two molecules of pyropheophorbide-a and bacteriopheophorbide-a with lysine as a cross-link were also prepared. The syntheses and spectroscopic properties of these compounds are discussed. Some of these compounds were tested for in vivo photosensitizing activity vis-a-vis Photofrin IITM, using the standard screening system of DBA/2 mice bearing transplanted SMT/F tumors. The preliminary in vivo results suggest that replacement of substituents at peripheral positions of the macrocycles causes a significant difference in photosensitizing efficacy.
Efficient methods for the syntheses of porphyrin and chlorin dimers joined with variable length carbon units, either at meso positions or at (beta) -pyrrolic positions, are described. The in vivo testing results for some of the dimers are also discussed. The main objective of this work is to understand the structure/activity relationships among carbon-carbon linked dimers of porphyrins and chlorins. It is anticipated that this study will assist the authors in designing photosensitizers with required chemical and photochemical properties.
Syntheses and chemical characterization of a number of new porphyrin-derived sensitizers related to ''benzoporphyrin derivative'' (BPD) are described. The work discussed centers in solving the isomer problem inherent in BPD synthesis from protoporphyrin IX, with regard to the 2- and 4-vinyls and the 6- and 7-propionic acid groups (Fischer nomenclature).
In continuation of the effort to search for an ideal
photosensitizer, two groups of potential new photosensitizers
were synthesized and investigated for their photodynamic actions
against tumors in mice. These were derivatives of methyl
pheophorbide-a and of silicon naphthalocyanine. Of the former
group, the 2 (1-0--hexyl) ethyl-desvinyl--methyl pheophorbide-a, or
}IEDP, was the most active sensitizer. HEDP could be readily
produced in large quantities and showed an optimum photodynamic
action at 665 mu where it absorbs strongly. Also HEDP was
cleared from the mouse skin within 4 days after administration,
thus possibly alleviating the long-term phototoxic side-effects
observed in Photofrin-based therapy. Of the second group of
photosensitizers, the bis (dimethyl hydroxypropylsiloxy) silicon
naphthalocyanine (HPSiNc) , and the corresponding acetoxy
derivative (APSiNc) were of particular interest. At a drug-light
dose of 1.0 mg/kg-135 J/cm2 (delivered by a laser at 772 nm),
they showed antitumor activities comparable to that of
PhotofrinTM. Further studies on these photosensitizers are
warranted.
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