Protoporphyrin IX Concentration Research Articles

δ-Aminolevulinic acid-based photodynamic reaction in HaCaT cells is enhanced by a low concentration of dimethyl sulfoxide

Objective To investigate the effect of 0.1% dimethyl sulfoxide (DMSO) on photody-namic reaction in a human keratinocyte cell line, HaCaT, induced by 5-aminolevulinic acid. Methods HaCaT cells were cultured in vitro with or without the presence of 0.1% DMSO at 37 ℃ and stimulated by δ-aminolevulinic acid (ALA) for 3 hours with light flee. Then, cellular protoporphyrin Ⅸ (PpⅨ) concen-tration in, fluorescence intensity in, proliferation and survival rates of HaCaT cells were determined by high performance liquid chromatography with fluorescence detection (HPLC-FLD), laser scanning confocal microscopy (LSCM), MTT coiorimetric method and trypan blue staining, respectively. Also, a portion of HaCaT cells were treated with ALA-based photodynamic therapy (ALA-PDT) and irradiated by 632.8-nm laser, and 12 hours later, cellular apoptosis and necrosis were detected by flow cytometry with annexin V/PI staining. Results Increased concentrations of PpⅨ were found in DMSO-pretreated HaCaT cells compared with untreated cells (0.57 ± 0.05 μg/L vs 0.44 ± 0.04 μg/L, P 0.05 ) or cell survival rate ( (96.18 ± 2.25 )% vs (94.64 ± 2.40)%, x2 = 1.84, P > 0.05 ) between DMSO-pretreated and untreated cells. After ALA-PDT, the apoptosis and necrosis rate were significantly increased in DMSO-treated HaCaT cells compared with untreated HaCaT cells (2.2% vs 1.5%, x2 = 4.05, P < 0.05; 8.9% vs 0.1%, x2 = 8.23, P< 0.05). Conclusion Low concentration (0.1%) of DMSO could enhance the effect of ALA-PDT on HaCaT cells. Key words: Dimethyl sulfoxide; Aminolevulinic acid; Heme; Keratinocytes; Photochemotherapy

Porphyrins Promote the Association of GENOMES UNCOUPLED 4 and a Mg-chelatase Subunit with Chloroplast Membranes

In plants, chlorophylls and other tetrapyrroles are synthesized from a branched pathway that is located within chloroplasts. GUN4 (GENOMES UNCOUPLED 4) stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits porphyrins to the chlorophyll branch. GUN4 stimulates Mg-chelatase by a mechanism that involves binding the ChlH subunit of Mg-chelatase, as well as a substrate (protoporphyrin IX) and product (Mg-protoporphyrin IX) of Mg-chelatase. We chose to test whether GUN4 might also affect interactions between Mg-chelatase and chloroplast membranes, the site of chlorophyll biosynthesis. To test this idea, we induced chlorophyll precursor levels in purified pea chloroplasts by feeding these chloroplasts with 5-aminolevulinic acid, determined the relative levels of GUN4 and Mg-chelatase subunits in soluble and membrane-containing fractions derived from these chloroplasts, and quantitated Mg-chelatase activity in membranes isolated from these chloroplasts. We also monitored GUN4 levels in the soluble and membrane-containing fractions derived from chloroplasts fed with various porphyrins. Our results indicate that 5-aminolevulinic acid feeding stimulates Mg-chelatase activity in chloroplast membranes and that the porphyrin-bound forms of GUN4 and possibly ChlH associate most stably with chloroplast membranes. These findings are consistent with GUN4 stimulating chlorophyll biosynthesis not only by activating Mg-chelatase but also by promoting interactions between ChlH and chloroplast membranes.

Desferrioxamine shows different potentials for enhancing 5-aminolaevulinic acid-based photodynamic therapy in several cutaneous cell lines

Protoporphyrin IX (PpIX) is the photosensitizer in 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT). Its further bioconversion to heme requires iron and can be suppressed by iron chelators such as desferrioxamine (DFO). To investigate the effectiveness of DFO in enhancing PpIX-based PDT in skin tissue, we selected fibroblasts, HaCat cells and Hep-2 cells as targets co-cultured with ALA, that have different biological characteristics for PpIX conversion. Evaluated interventions included: (1) blank control (no ALA, no DFO); (2) DFO alone; (3) ALA alone; and (4) DFO in combination with ALA. Before photodynamic irradiation, cellular PpIX level and fluorescence were measured. After irradiation, cell death ratio was calculated and morphological changes in the cells were observed. The results showed that the content and photodynamic effects of cellular PpIX presented in the order Hep-2 cells > HaCat cells > fibroblasts, either co-cultured with ALA alone or with ALA plus DFO. DFO was found to have increasing effects on both PpIX level and cell death ratio in the same order. It was found that DFO had different potentials for augmenting ALA-PDT in these cutaneous cell lines. The cells proliferating more rapidly might be more susceptible to enhancement of DFO.

Characterization of reactive oxygen species generated by protoporphyrin IX under X-ray irradiation

As a possible radiosensitizer candidate having biological compatibility, oncotropic property, and X-ray activation capability, contribution of protoporphyrin IX (PpIX) to enhanced generation of reactive oxygen species (ROS) under X-ray and UV irradiations were examined. To identify the kinds of ROS, 2-[6-(4-amino)phenoxy-3 H-xanthen-3-on-9-yl] benzoic acid (APF) and dihydroethidium (DHE) were used together with ethanol as a hydroxyl radical (OH ) quencher. All of the three species of our interest (OH , superoxide radical (O 2 − ), and singlet oxygen ( 1O 2)) were enhanced by PpIX under X-ray and UV irradiations in addition to those by radiolysis and photolysis. Its enhancement factors exceeded 1.7 depending on the concentrations of PpIX from 1.5 to 15.0 μg/ml.

Sonodynamic Antitumor Effect of Protoporphyrin IX Disodium Salt on S180 Solid Tumor

Background: The sonodynamically induced antitumor effect of protoporphyrin IX (PPIX) disodium salt was studied in mice bearing sarcoma 180 solid tumors. Methods: In order to determine the optimum timing of ultrasound exposure after administration of PPIX, the PPIX concentrations in plasma, skin, muscle and tumor were estimated by measuring the fluorescence intensity of tissue extractions with a fluorescence photometer based on the standard curve. Antitumor effects were estimated by measuring tumor size and calculating the average survival time of tumor-bearing mice after sonodynamic therapy; additionally, the morphological changes of sarcoma 180 cells were evaluated by transmission electron microscope observation in vivo. Results: Our experiments suggested a time of 24 h after the administration of PPIX to be best for ultrasound exposure. At an ultrasound intensity ≧5 W/cm² and a PPIX dose ≧5 mg/kg, a significant synergistic effect of ultrasound combined with PPIX was observed, reducing tumor volume and increasing average animal survival time; this synergistic effect was obviously stronger than ultrasound treatment alone, while PPIX alone showed no significant effect. Transmission electron microscope observation indicated that changes in cell ultrastructure, such as cell membrane destruction, mitochondria swelling and chromatin condensation, were important factors that inhibited tumor growth and even induced cell death. Conclusion: The results implied that the antitumor effect of ultrasound could be enhanced in the presence of PPIX which might be involved in a sonochemical mechanism.

Visualization of basal cell carcinoma by fluorescence diagnosis and independent component analysis

Photodynamic detection (PDD) of skin tumours is based on the visualization of a fluorophores, with the ability to accumulate in tumour tissue, by the use of fluorescence imaging. Of particular importance is the application of δ-5-aminolaevulinic acid (ALA) that, through the process of biosynthesis causes formation of the protoporphyrin IX (PpIX). The PpIX has the ability of selective fluorescence after basal cell carcinoma (BCC) has been treated with ALA. Higher concentration of PpIX in tumour tissue compared to surrounding normal skin is the basis for PDD. Our contribution in this preliminary study is application of the independent component analysis (ICA) to extract the BCC spatial map, by processing fluorescent RGB image acquired under excitation with 405nm light. Comparative performance analysis with other two widely used image processing methods: ratio imaging and optimal threshold based imaging, reveals that ICA produces BCC spatial map that is most consistent in term of diagnostic quality by both visual assessment and calculation of the BCC demarcation line. We believe this represents a solid basis for the design of a compact and low-cost multi-spectral fluorescence imaging system, capable for real time calculation of the skin tumour demarcation.

Photodynamic Therapy of Vertebral Metastases: Evaluating Tumor‐to‐Neural Tissue Uptake of BPD‐MA and ALA‐PpIX in a Murine Model of Metastatic Human Breast Carcinoma†

Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin-derivative monoacid ring A (BPD-MA) and by 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor-bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD-MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD-MA, ALA-PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor-bearing vertebrae compared to spinal cord. The highest ratio for BPD-MA concentration was found 15 min after injection, which can be recommended for therapy in this model.

Phototransformations of 5-Aminolevulinic Acid-induced Protoporphyrin IX in vitro: A Spectroscopic Study¶

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorin-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blueshifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

Comparison between sonodynamic effect with protoporphyrin IX and hematoporphyrin on sarcoma 180

The comparison between sonodynamic antitumor effect with protoporphyrin IX (PPIX) and hematoporphyrin (Hp) at a concentration of 5 mg/kg on Sarcoma 180 (S180) cells was studied in vivo, and the potential cell damage mechanism was also investigated. The sonodynamically induced anti-tumor effect of PPIX was studied in mice bearing S180 solid tumors. In order to determine the optimum timing of ultrasound exposure after administration of PPIX, the PPIX concentrations in plasma, skin, muscle and tumor were determined by the fluorescence intensity of tissue extractions with a fluorescence spectrophotometer based on the standard curve. Anti-tumor effects were estimated by measuring the tumor size and the tumor weight. Additionally, the morphological changes of S180 cells were evaluated by transmission electron microscope (TEM) observation immediately after sonodynamic therapy (SDT) treatment. A time of 24 h after the intravenous administration of PPIX was chosen as the best time for ultrasound exposure. The antitumor effect induced by PPIX mediated sonodynamic therapy (PPIX-SDT) was in a dose dependent manner when ultrasound intensity was at or above the inertial cavitation threshold (5 W/cm(2)). A significant tumor growth delay was observed both in PPIX mediated sonodynamic therapy and in Hp mediated sonodynamic therapy treatments (Hp-SDT), and the tumor weight inhibition ratios after the synergistic treatments were 42.82 +/- 0.03 and 35.22 +/- 0.03%, respectively, this difference was significant at P < 0.05. While ultrasound alone (5 W/cm(2)) showed a slight tumor growth inhibitory effect compared with the control group, and PPIX or Hp alone showed almost no significant effect. Furthermore, TEM observation indicated cell damage was more serious in PPIX-SDT treatment group than in Hp-SDT treatment group. After sonication, the cell ultra-structure such as cell membrane destruction, mitochondria swelling, chromatin condensation might be important factors that inhibited the tumor growth and even induced cell death. The comparative results suggested that PPIX as a sonosensitizer might have more potential cytotoxicity than Hp when irradiated with ultrasound, and the ultra-structural changes may account for cell destruction induced by sonodynamic therapy in our experiment mode.

5-Aminolevulinic Acid-Mediated Photodynamic Therapy on Hep-2 and MCF-7c3 Cells

The cytotoxic effect of 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) on two human carcinoma cell lines, MCF-7c3 cells and Hep 2 cells, was studied. In both cell lines, PPIX content depends on the ALA concentration and incubation time. The maximal PPIX content was higher in the MCF-7c3 cells, reaching a value of 8 microg/10(6) cells, compared to the Hep-2 cells, which accumulated 3.2 microg/10(6) cells. Treatment of cells with the iron chelator desferrioxamine prior to ALA exposure enhances the amount of PPIX, consequently diminishing enzymatic activity of ferroquelatase. Photo sensitization of the cells was in correlation with the PPIX content; therefore, conditions leading to 80% cell death in the MCF-7c3 cells provoke a 50% cell death in the Hep 2 cells. Using fluorescence microscopy, cell morphology was analyzed after incubation with 1 mM ALA during 5 hr and irradiation with 54 Jcm(-2); 24 hr post-PDT, MCF-7c3 cells revealed the typical morphological changes of necrosis. Under the same conditions, Hep-2 cells produced chromatine fragmentation characteristic of apoptosis. PPIX accumulation was observed to occur in a perinuclear region in the MCF-7c3 cells; while in Hep-2 cells, it was localized in lysosomes. Different mechanisms of cell death were observed in both cell lines, depending on the different intracellular localization of PPIX.

Hexyl‐aminolevulinate‐mediated photodynamic therapy: How to spare normal urothelium. An in vitro approach

Photodynamic therapy (PDT) of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizer concentration, irradiation parameters, and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium invitro. The aim was to determine the threshold conditions for the cell survival. Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm(2) (irradiance 30 and 75 mW/cm(2), respectively) and for white light 0.55 J/cm(2) (irradiance 30 mW/cm(2)). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. We have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.

Enhancement of the photodynamic effects on human oral squamous cell carcinoma cell lines by treatment with calcipotriol

Summary Background 5-Aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) for patients with skin and oral diseases is a highly sophisticated procedure, but the incidence of disease recurrence after treatment with ALA-based PDT is somewhat alarming. Calcipotriol, an analogue of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been reported to regulate the proliferation and differentiation of keratinocytes. Objective In order to obtain even greater efficacy of ALA-based PDT, we investigated the synergistic effects of calcipotriol as an adjunct to ALA-based PDT for human oral squamous cell carcinoma (SCC) cell lines. Methods Intracellular protoporphyrin IX (PpIX) converted from exogenous ALA in SCC cell lines treated with/without calcipotriol was measured by a fluorescencemeter. Then, the in vitro effects of calcipotriol, the cyclooxygenase (COX)-2 selective inhibitor (nimesulide), ALA-based PDT and their combination on two SCC cell lines, HSC-2 (a COX-2 high expresser) and HSC-4 (a COX-2 non-expresser), were determined by MTT assay and double-staining for annexin V and propidium iodide. Results The concentration of intracellular PpIX was increased in four of the eight SCC cell lines (50%) treated with calcipotriol. The greatest alteration of intracellular PpIX was found in HSC-4 (1.9-fold). The combination of calcipotriol and ALA-based PDT remarkably inhibited cellular proliferation and induced cellular death of both HSC-2 and HSC-4. Whereas, this morphological damage was more serious in HSC-4 than in HSC-2. Furthermore, these effects were almost equivalent to the synergistic effect of the combination of nimesulide and ALA-based PDT on HSC-2. Conclusions The present study suggests that treatment with calcipotriol enhances the photodynamic effects on SCC via the accumulation of exogenous ALA-dependent PpIX.

Clinical Pharmacokinetics of the PDT Photosensitizers Porfimer Sodium (Photofrin), 2‐[1‐Hexyloxyethyl]‐2‐Devinyl Pyropheophorbide‐a (Photochlor) and 5‐ALA‐Induced Protoporphyrin IX

Photodynamic therapy (PDT) uses a photosensitizer activated by light, in an oxygen-rich environment, to destroy malignant tumors. Clinical trials of PDT at Roswell Park Cancer Institute (RPCI) use the photosensitizers Photofrin, Photochlor, and 5-ALA-induced protoporphyrin IX (PpIX). In some studies the concentrations of photosensitizer in blood, and occasionally in tumor tissue, were obtained. Pharmacokinetic (PK) data from these individual studies were pooled and analyzed. This is the first published review to compare head-to-head the PK of Photofrin and Photochlor. Blood and tissue specimens were obtained from patients undergoing PDT at RPCI. Concentrations of Photofrin, Photochlor, and PpIX were measured using fluorescence analysis. A non-linear mixed effects modeling approach was used to analyze the PK data for Photochlor (up to 4 days post-infusion; two-compartment model) and a simpler multipatient-data-pooling approach was used to model PK data for both Photofrin and Photochlor (at least 150 days post-infusion; three-compartment models). Physiological parameters were standardized to correspond to a standard (70 kg; 1.818 m2 surface area) man to facilitate comparisons between Photofrin and Photochlor. Serum concentration-time profiles obtained for Photofrin and Photochlor showed long circulating half-lives, where both sensitizers could be found more than 3 months after intravenous infusion; however, estimated plasma clearances (standard man) were markedly smaller for Photofrin (25.8 ml/hour) than for Photochlor (84.2 ml/hour). Volumes of distribution of the central compartment (standard man) for both Photofrin and Photochlor were about the size (3.14 L, 4.29 L, respectively) of plasma volume, implying that both photosensitizers are almost 100% bound to serum components. Circulating levels of PpIX were generally quite low, falling below the level of instrument sensitivity within a few days after topical application of 5-ALA. We have modeled the PK of Photochlor and Photofrin. PK parameter estimates may, in part, explain the relatively long skin photosensitivity attributed to Photofrin but not Photochlor. Due to the potential impact and limited experimental PK data in the PDT field further clinical studies of photosensitizer kinetics in tumor and normal tissues are warranted.

INHIBITION OF HO‐1 INCREASES RESPONSIVENESS OF MELANOMA CELLS TO ALA‐PDT

Based on the observation that 5-aminolevulinic acid (ALA) induced the expression of heme oxygenase-1 (HO-1) in cultured melanoma cells, we examined the role of HO-1 on the effectiveness of ALA-based photodynamic therapy (ALA-PDT). Transcriptional activation of the HO-1 gene is considered as an adaptive response to oxidative and cellular stress and confers a protective capacity to cell and tissue injury, which may affect the responsiveness to ALA-PDT. A time dependent accumulation (0h-16h) of protoporphyrin IX (PPIX) within melanoma cells was seen after ALA supply (0.5 mM ALA). In the same time interval a significant increase (up to 25-fold) in HO-1 protein expression was observed. Thus, production and degradation of PPIX (by HO-1) were simultaneously enabled, leading to a reduced intracellular concentration of PPIX. Diminishing of HO-1 activity by the HO-1 inhibitor tin protoporphyrin IX (SnPPIX) significantly enhanced the formation of PPIX up to 1.8 fold. A further strong increase in HO-1 protein expression (up to 128-fold) was seen after ALA-PDT treatment. The inhibition of HO-1 activity by SnPPIX made tumor cells considerably more sensitive to ALA-PDT treatment. At low radiation doses (0.42 J/cm2) the death rate increased significantly from 9.8 ± 5.0 % to 50.7 ± 19.7 %. Therefore, specific inhibition of HO-1 activity may be used to increase the effectiveness of ALA-PDT.

Probing strong optical fields in compact aggregates of silver nanoparticles by SERRS of protoporphyrin IX

TEM images and measurements of SERRS (surface-enhanced resonance Raman scattering) spectra as a function of the porphyrin concentrations in systems with unmodified and chloride-modified Ag nanoparticles and protoporphyrin IX (PPIX) are reported. TEM images have shown formation of compact aggregates in systems with chloride modified Ag nanoparticles, as opposed to systems with the unmodified particles constituted by isolated particles. SERRS spectra of PPIX as a function of PPIX concentration were measured and subjected to factor analysis. Two spectral components were identified and tentatively attributed to unperturbed PPIX and to Ag+ -PPIX surface species. Concentration value of the SERRS spectral detection limit of the latter species was determined to be nearly three orders of magnitude lower in the system with the compact aggregates than in the system with separated nanoparticles and achieves the value of 1 x 10(-10) M in a macrosampling Raman experiment. TEM images and SERRS-micro-Raman spectra of single compact aggregates of chloride-modified Ag nanoparticles incorporating PPIX molecules were acquired from a sample prepared by attachment of the aggregates to amine groups of derivatized, SiOx/formvar coated copper grids for TEM. The SERRS signal has shown large temporal fluctuations as well as variations from one aggregate to another. Within the signal fluctuations, a SERRS spectrum showing the characteristic bands of both SERRS spectral forms of PPIX and originating most probably from a few PPIX molecules located in hot spots in the interstices between the Ag nanoparticles, was obtained.

Correlation Between Macroscopic Fluorescence and Protoporphyrin IX Content in Psoriasis and Actinic Keratosis Following Application of Aminolevulinic Acid

In fluorescence diagnosis with 5-aminolevulinic acid (ALA)-induced porphyrins (FDAP), protoporphyrin IX (PpIX) accumulation can be macroscopically visualized. Interpretation of these data is still problematic because of the low reproducibility of the procedure and poor understanding of the mechanisms involved in PpIX tumor selectivity. In this study, PpIX accumulation is investigated in patients with psoriasis and actinic keratosis (AK) following FDAP. For this purpose, desquamated lesional and non-lesional skin were incubated with 20% ALA ointment for 3 h, FDAP was performed, and highly fluorescing lesional skin and non-lesional skin were biopsied. In extracts from these biopsies, PpIX, protein, and dsDNA were quantified by spectrofluorometry. Digital images acquired with FDAP were analyzed using image analysis software. PpIX per biopsy in lesional skin in both psoriasis and AK was significantly higher than in non-lesional skin (p < 0.05). When corrected for epidermal involvement, only lesional psoriatic skin showed significantly higher PpIX levels than non-lesional skin. The PpIX-ratio lesional:non-lesional skin (mean(pmol per mL)+/-SEM) was 4.12+/-0.91 in psoriasis and 1.96+/-0.24 in AK. In FDAP, the ratio of lesional:non-lesional skin was 1.77+/-0.06 in psoriasis and 1.37+/-0.07 in AK. Macroscopic fluorescence and PpIX content appeared to be well correlated (r = 0.73), thus making FDAP a good predictor of PpIX content.

In Vivo Study of Biological Effects of Photodynamic Therapy on Cervical Cancer

In 1999, Ramon et al examined the effects of δ-aminolevulinic acid (δ-ALA) on two cervical cancer cell lines (HeLa and CaLo) and normal cervical cells. The results reported additionally support the proposal that Photodynamic Therapy (PDT) could be an effective tool for treatment of cervical cancer (CeCa) by protoporphyrin IX (PpIX) accumulation. This cancer is in México considered a problem of public health.In this project PDT was applied in a CeCa mouse model by implantation of HeLa cells in nu/nu mice, exposing it to δ-ALA to a dose that induces the greater concentration of PpIX by intratumoral and oral route, as well as the power and the number of optimal irradiations at 630 nm with a diode laser; verifying which of the used doses does not produce significant damage to the organs by means of histopatologic techniques: liver, spleen, brain and kidney, the determination of reactive substances to tiobarbituric acid (TBAR'S) and measured the amount of residual δ-ALA, the size of the tumor at the beginning and at the end of the application of the PDT.Results. The cervical cancer mouse model is developed by implantation of 1,5 million HeLa cells, appearing at 15 days. The dose of δ-ALA that induces the greater concentration of PpIX in the tumor by intratumoral route was 40 mg of δ-ALA/kg body weight and for oral route it was 20 mg of δ-ALA/kg body weight, both routes did not present significant damage in the organs and according to the Analysis of Bifactorial Variance for the doses of δ-ALA and the organs no difference exists (p > 0.05).The power of the used laser was 150 J/cm2 and 250 J/cm2, both applied by intratumoral and oral route after the δ-ALA administration. The greater diminution in the size of the tumor (53%) was by the δ-ALA oral route administration to a power of 150 J/cm2 with 5 irradiations in intervals of 48 h.

Phototoxicity of Protoporphhyrin IX, Diarginine Diprotoporphyrinate and N,N‐Deiphenylalanyl Protoporphyrin Toward Human Fibroblasts and ketratinocytes In vitro: Effect of 5‐Methoxypsoralen†;

AbstractThe phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N‐diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5‐methaoxyposralen (5‐MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet A (UV‐A) irradiation (λ= 365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratino‐cytes decerases in the order: PPIX &gt; PP(Arg)2 &gt; PP(Phe)2. A synergistic efect of 5‐MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1–0.5 μM 5‐MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water‐soluble PP(Arg)2 and 5‐MOP concentrations lowere than 0.75 μM. Above this 5‐MOP concnetration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5‐MOP. On the other hand, the intracellular contnet of PP(Arg)2 is decerased in concentration‐dependent manner by the psoralen. Illumination with red light, not absorbed by 5‐MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5‐MOP, suggesting that dark interaction of 5‐MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structure of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5‐MOP on the PP(Arg)2 phototoxicity at high 5‐MOP concentration. This study suggests that PP(Arg)2 in combinatin with 5‐MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.

Metronomic Photodynamic Therapy as a New Paradigm for Photodynamic Therapy: Rationale and Preclinical Evaluation of Technical Feasibility for Treating Malignant Brain Tumors¶

ABSTRACTThe concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low‐dose PDT using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX(PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA‐mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate, sodium salt nick‐end labeling positive) cells as compared with an acute, high‐dose regimen (ALA‐αPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectro‐fluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor‐to‐brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light‐emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.

Phototoxicity of Protoporphyrin IX, Diarginine Diprotoporphyrinate and N,N-Diphenylalanyl Protoporphyrin Toward Human Fibroblasts and Keratinocytes In Vitro: Effect of 5-Methoxypsoralen ¶

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N-diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (lambda=365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)2 > PP(Phe)2. A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1-0.5 microM 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)2 and 5-MOP concentrations lower than 0.75 microM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)2 is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)2 phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)2 in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.