Increased Protoporphyrin IX Accumulation Research Articles

Experimental investigation of a combinational iron chelating protoporphyrin IX prodrug for fluorescence detection and photodynamic therapy

Photodynamic therapy (PDT) is an oxygen-dependent, light-activated, and locally destructive drug treatment of cancer. Protoporphyrin IX (PpIX)-induced PDT exploits cancer cells’ own innate heme biosynthesis to hyper-accumulate the naturally fluorescent and photoactive precursor to heme, PpIX. This occurs as a result of administering heme precursors (e.g., aminolevulinic acid; ALA) because the final step of the pathway (the insertion of ferrous iron into PpIX by ferrochelatase to form heme) is relatively slow. Separate administration of an iron chelating agent has previously been demonstrated to significantly improve dermatological PpIX-PDT by further limiting heme production. A newly synthesized combinational iron chelating PpIX prodrug (AP2-18) has been assessed experimentally in cultured primary human cells of bladder and dermatological origin, as an alternative photosensitizing agent to ALA or its methyl or hexyl esters (MAL and HAL respectively) for photodetection/PDT. Findings indicated that the technique of iron chelation (either through the separate administration of the established hydroxypyridinone iron chelator CP94 or the just as effective combined AP2-18) did not enhance either PpIX fluorescence or PDT-induced (neutral red assessed) cell death in human primary normal and malignant bladder cells. However, 500 µM AP2-18 significantly increased PpIX accumulation and produced a trend of increased cell death within epithelial squamous carcinoma cells. PpIX accumulation destabilized the actin cytoskeleton in bladder cancer cells prior to PDT and resulted in caspase-3 cleavage/early apoptosis afterwards. AP2-18 iron chelation should continue to be investigated for the enhancement of dermatological PpIX-PDT applications but not bladder photodetection/PDT.

MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1\u03b1-FECH axes

The efficacy of aminolevulinic acid (5-ALA)-based photodynamic diagnosis (5-ALA-PDD) and photodynamic therapy (5-ALA-PDT) is dependent on 5-ALA-induced cancer-specific accumulation of protoporphyrin IX (PpIX). We previously reported that inhibition of oncogenic Ras/MEK increases PpIX accumulation in cancer cells by reducing PpIX efflux through ATP-binding cassette sub-family B member 1 (ABCB1) and ferrochelatase (FECH)-catalysed PpIX conversion to haem. Here, we sought to identify the downstream pathways of Ras/MEK involved in the regulation of PpIX accumulation via ABCB1 and FECH. First, we demonstrated that Ras/MEK activation reduced PpIX accumulation in RasV12-transformed NIH3T3 cells and HRAS transgenic mice. Knockdown of p90 ribosomal S6 kinases (RSK) 2, 3, or 4 increased PpIX accumulation in RasV12-transformed NIH3T3 cells. Further, treatment with an RSK inhibitor reduced ABCB1 expression and increased PpIX accumulation. Moreover, HIF-1α expression was reduced when RasV12-transformed NIH3T3 cells were treated with a MEK inhibitor, demonstrating that HIF-1α is a downstream element of MEK. HIF-1α inhibition decreased FECH activity and increased PpIX accumulation. Finally, we demonstrated the involvement of RSKs and HIF-1α in the regulation of PpIX accumulation in human cancer cell lines. These results demonstrate that the RSK-ABCB1 and HIF-1α-FECH axes are the downstream pathways of Ras/MEK involved in the regulation of PpIX accumulation.

Improving in vitro photodynamic therapy through the development of a novel iron chelating aminolaevulinic acid prodrug.

Photodynamic therapy (PDT) is a light activated drug therapy that can be used to treat a number of cancers and precancers. It is particularly useful in its topical form in dermatology but improvement of efficacy is required to widen its application. An ester between aminolaevulinic acid (ALA) and CP94 was synthesised (AP2-18) and experimentally evaluated to determine whether protoporphyrin IX (PpIX)-induced PDT effectiveness could be improved. A biological evaluation of AP2-18 was conducted in cultured human primary cells with both PpIX fluorescence and cell viability (as determined via the neutral red assay) being assessed in comparison to the PpIX prodrugs normally utilised in clinical practice (aminolaevulinic acid (ALA) or its methyl ester (MAL)) either administered alone or with the comparator iron chelator, CP94. No significant dark toxicity was observed in human lung fibroblasts but AP2-18 significantly increased PpIX accumulation above and beyond that achieved with ALA or MAL administration +/- CP94 in both human dermal fibroblasts and epithelial squamous carcinoma cells. On light exposure, the combined hydroxypyridinone iron chelating ALA prodrug AP2-18 generated significantly greater cytotoxicity than any of the other treatment parameters investigated when the lowest concentration (250 μM) was employed. Newly synthesised AP2-18 is therefore concluded to be an efficacious prodrug for PpIX-induced PDT in these dermatologically relevant human cells, achieving enhanced effects at lower concentrations than currently possible with existing pharmaceuticals.

An experimental investigation of a novel iron chelating protoporphyrin IX prodrug for the enhancement of photodynamic therapy.

ObjectivesNon‐melanoma skin cancers are the most frequently occurring type of cancer worldwide. They can be effectively treated using topical dermatological photodynamic therapy (PDT) employing protoporphyrin IX (PpIX) as the active photosensitising agent as long as the disease remains superficial. Novel iron chelating agents are being investigated to enhance the effectiveness and extend the applications of this treatment modality, as limiting free iron increases the accumulation of PpIX available for light activation and thus cell kill.MethodsHuman lung fibroblasts (MRC‐5) and epithelial squamous carcinoma (A431) cells were treated with PpIX precursors (aminolaevulinic acid [ALA] or methyl‐aminolevulinate [MAL]) with or without the separate hydroxypyridinone iron chelating agent (CP94) or alternatively, the new combined iron chelator and PpIX producing agent, AP2‐18. PpIX fluorescence was monitored hourly for 6 hours prior to irradiation. PDT effectiveness was then assessed the following day using the lactate dehydrogenase and neutral red assays.ResultsGenerally, iron chelation achieved via CP94 or AP2‐18 administration significantly increased PpIX fluorescence. ALA was more effective as a PpIX‐prodrug than MAL in A431 cells, corresponding with the lower PpIX accumulation observed with the latter congener in this cell type. Addition of either iron chelating agent consistently increased PpIX accumulation but did not always convey an extra beneficial effect on PpIX‐PDT cell kill when using the already highly effective higher dose of ALA. However, these adjuvants were highly beneficial in the skin cancer cells when compared with MAL administration alone. AP2‐18 was also at least as effective as CP94 + ALA/MAL co‐administration throughout and significantly better than CP94 supplementation at increasing PpIX fluorescence in MRC5 cells as well as at lower doses where PpIX accumulation was observed to be more limited.ConclusionsPpIX fluorescence levels, as well as PDT cell kill effects on irradiation can be significantly increased by pyridinone iron chelation, either via the addition of CP94 to the administration of a PpIX precursor or alternatively via the newly synthesized combined PpIX prodrug and siderophore, AP2‐18. The effect of the latter compound appears to be at least equivalent to, if not better than, the separate administration of its constituent parts, particularly when employing MAL to destroy skin cancer cells. AP2‐18 therefore warrants further detailed analysis, as it may have the potential to improve dermatological PDT outcomes in applications currently requiring enhancement. Lasers Surg. Med. 50:552–565, 2018. © 2018 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells.Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted.Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model.Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Increased protoporphyrin IX accumulation does not improve the effect of photodynamic therapy for actinic keratosis: a randomized controlled trial

Photodynamic therapy (PDT) with methyl aminolaevulinate (MAL) is highly effective for treating actinic keratosis (AK) on the face/scalp, but less effective on the extremities. Insufficient accumulation of protoporphyrin IX (PpIX) may cause these inferior efficacy rates. However, it is possible to increase PpIX accumulation by extending the MAL application time and/or pretreating the skin with curettage. To investigate whether increased PpIX accumulation improves the effect of MAL-PDT for AKs in a randomized intra-individual study. Twenty-two patients with 533 AKs on both hands were treated with MAL-PDT. To obtain different concentrations of PpIX, four symmetrical areas on each patient were randomly allocated to different regimens: (i) 3-h MAL application without prior curettage (3hC-); (ii) 3 h with curettage (3hC+); (iii) 21 h without curettage (21hC-); and (iv) 21 h with curettage (21hC+). Treatment efficacy was evaluated after 3 months, whereas PpIX fluorescence, pain and erythema were assessed during and after PDT. Extended MAL application with and without curettage increased PpIX accumulation significantly compared with the standard 3hC+ regimen (P = 0·001 and P = 0·002, respectively). However, the median total clearance rates did not improve accordingly: 3hC+ (55·0%), 21hC- (55·0%) and 21hC+ (53·6%). Conversely, insufficient PpIX accumulation in the 3hC- regimen led to a significantly lower clearance rate (33·3%) than the other regimens (P < 0·045). Furthermore, pain and erythema were correlated to PpIX accumulation. Increased PpIX accumulation does not improve the effect of MAL-PDT for AKs on the hands, but leads to worse adverse events. Different strategies are needed to improve PDT on the extremities.

The importance of iron chelation and iron availability during PpIX-induced photodynamic therapy

AbstractBackground:Protoporphyrin IX (PpIX)-induced photodynamic therapy (PDT) is being utilised as a topical method of localised ablation of certain non-melanoma skin cancers and precancers. Standardised protocols have been implemented to good effect when the disease remains superficial but improvement is required to treat thicker or acrally located conditions. Concurrent administration of an iron chelator during PpIX-PDT has been demonstrated to increase cellular accumulation of PpIX by reducing its bioconversion to haem (an iron dependent process) thus increasing cell kill on subsequent irradiation. Iron however, can also play a role in reactive oxygen species (ROS) generation and limiting its availability via chemical chelation could theoretically reduce the efficacy of PpIX-PDT, so that a response less than that maximally feasible is produced.Materials and methods:The effects of iron availability and chelation on PpIX-PDT have therefore been investigated via fluorescence quantification of PpIX accumulation, single-cell gel electrophoresis (comet assay) measurement of ROS-induced DNA damage and trypan blue exclusion assessment of cell viability. Cultured human cells were utilised and incubated in standardised iron conditions with the PpIX precursor’s aminolaevulinic acid (ALA) or its methyl ester (MAL) in the presence or absence of either of the iron chelating agents desferrioxamine (DFO) or hydroxypyridinone (CP94), or alternatively iron sulphate as a source of iron.Results:ALA or MAL incubation was found to significantly increase cellular PpIX accumulation pre-irradiation as anticipated and this observation correlated with both significantly increased DNA damage and reduced cellular viability following irradiation. Co-incubation with either of the iron chelators investigated (DFO or CP94) significantly increased pre-irradiation PpIX accumulation as well as DNA damage and cell death on irradiation indicating the positive effect of iron chelation on the effectiveness of PpIX-induced PDT. The opposite effects were observed however, when the cells were co-incubated with iron sulphate, with significant reductions in pre-irradiation PpIX accumulation (ALA only) and DNA damage (ALA and MAL) being recorded indicating the negative effects excessive iron can have on PpIX-PDT effectiveness. Some dark toxicity produced by iron sulphate administration in non-irradiated control groups was also observed.Conclusion:Iron chelation and availability have therefore been observed to positively and adversely affect the PpIX-PDT process respectively and it is concluded that the effects of increased PpIX accumulation pre-irradiation produced via iron chelation outweigh any limitations reduced iron availability may have on the ability of iron to catalyse ROS generation/cascades following PpIX-induced PDT. Further investigation of iron chelation within dermatological applications where enhanced PpIX-PDT treatment effects would be beneficial is therefore warranted.

Regulation of 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation in Human Urothelial Carcinomas

Purpose: The purpose of this study was to clarify the regulatory mechanism of protoporphyrin IX (PpIX) synthesis mediated by 5-aminolevulinic acid (ALA) in human urothelial carcinoma (UC), leading to improved accuracy in photodynamic diagnosis and therapy using ALA. Experimental Design: PpIX accumulation in cultured UC cells after incubation for 1–5 h with 0.5–5 mM ALA was analyzed by fluorescence analysis using fluorescence microscopy and flow cytometry technique. Results: PpIX fluorescence mediated by ALA was increased, and the intensity of PpIX fluorescence was time-dependently increased in UC cells compared to noncancerous cells. The distribution of endogenous PpIX fluorescence primarily coincided with mitochondria, and then increased at a specific perinuclear region in the cells during the time of incubation. The ALA-mediated PpIX synthesis in UC cells was suppressed by β-alanine, an inhibitor of β-transporters of cell membrane, and carbonylcyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, the ALA-mediated PpIX accumulation was increased by deferoxamine, an iron chelator, manganese and nitric oxide, which is contributed to PpIX metabolism by inhibiting ferrochelatase activity, generated by a nitric oxide-generating reagent NOC-18. As observed above, ALA-mediated PpIX synthesis in human UC cells was regulated by the process of ALA uptake, ALA conversion to PpIX and metabolism of accumulated PpIX to heme. Conclusions: This shows that the suppression of ferrochelatase increased PpIX accumulation in UC cells using small amount of ALA, thus leading to an improved clinical practicability of photodynamic diagnosis and therapy.

Topical glycerol monooleate/propylene glycol formulations enhance 5-aminolevulinic acid in vitro skin delivery and in vivo protophorphyrin IX accumulation in hairless mouse skin

Photodynamic therapy (PDT), a potential therapy for cancer treatment, utilizes exogenously applied or endogenously formed photosensitizers, further activated by light in an appropriate wavelength and dose to induce cell death through free radical formation. 5-Aminolevulinic acid (5-ALA) is a pro-drug which can be converted to the effective photosensitizer, protoporphyrin IX (PpIX). However, the use of 5-ALA in PDT is limited by the low penetration capacity of this highly hydrophilic molecule into appropriate skin layers. In the present study, we propose to increase 5-ALA penetration by using formulations containing glycerol monooleate (GMO), an interesting and useful component of pharmaceutical formulations. Propylene glycol solutions containing different concentrations of GMO significantly increased the in vitro skin permeation/retention of 5-ALA in comparison to control solutions. In vivo studies also showed increased PpIX accumulation in mouse hairless skin, after the use of topical 5-ALA formulations containing GMO in a concentration-dependent manner. The results show that skin 5-ALA penetration and PpIX accumulation, important factors for the success of topical 5-ALA-PDT in skin cancer, are optimized by GMO/propylene glycol formulations.

Mapping ALA-induced PPIX fluorescence in normal brain and brain tumour using confocal fluorescence microscopy

We have mapped the microscopic distribution of aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in normal rabbit brain and in brain tumour at various time intervals after i.v. injection of ALA. In normal brain, regions without a blood-brain barrier (BBB) such as the choroid plexus and meninges and also structures associated with the CSF synthesized the highest amounts of PPIX. We also detected ALA in the CSF within 2 h after ALA administration when the serum concentration was high. Our fluorescence studies have shown that ALA is able to cross an intact BBB and be metabolized in regions with a BBB. This is evident in regions of the brain such as the hippocampus, thalamus, cerebellar folia and brain stem that are remote from the meninges or choroid plexus. However, the relative peak PPIX fluorescence in these regions are much lower than in regions without a BBB. Our imaging studies have shown that neurons in different regions of normal brain with an intact BBB exhibit varying capacities to metabolize ALA. The grey matter regions of the cerebrum and cerebellum also showed higher PPIX fluorescence compared to white matter regions in these same structures. Our study showed that tumour tissue had the highest PPIX fluorescence compared to normal contralateral cerebral cortex. Maximum selectivity was achieved between tumour and normal tissue at 24 h. PPIX fluorescence in normal blood vessels appear to be mainly due to metabolism of internalized ALA from serum and not due to high PPIX levels. In conclusion, ALA does cross the BBB and all regions of the brain with the exception of the hypothalamus, exhibit a dynamic synthesis of PPIX from ALA. Increased PPIX accumulation in tumour and inflamed brain tissue in comparison to normal brain tissue suggest the feasibility of selective photosensitization of such tissue.