A Recap of Heme Metabolism towards Understanding Protoporphyrin IX Selectivity in Cancer Cells.

Mitochondria are essential organelles of mammalian cells, often emphasized for their function in energy production, iron metabolism and apoptosis as well as heme synthesis. The heme is an iron-loaded porphyrin behaving as a prosthetic group by its interactions with a wide variety of proteins. These complexes are termed hemoproteins and are usually vital to the whole cell comportment, such as the proteins hemoglobin, myoglobin or cytochromes, but also enzymes such as catalase and peroxidases. The building block of porphyrins is the 5-aminolevulinic acid, whose exogenous administration is able to stimulate the entire heme biosynthesis route. In neoplastic cells, this methodology repeatedly demonstrated an accumulation of the ultimate heme precursor, the fluorescent protoporphyrin IX photosensitizer, rather than in healthy tissues. While manifold players have been proposed, numerous discrepancies between research studies still dispute the mechanisms underlying this selective phenomenon that yet requires intensive investigations. In particular, we wonder what are the respective involvements of enzymes and transporters in protoporphyrin IX accretion. Is this mainly due to a boost in protoporphyrin IX anabolism along with a drop of its catabolism, or are its transporters deregulated? Additionally, can we truly expect to find a universal model to explain this selectivity? In this report, we aim to provide our peers with an overview of the currently known mitochondrial heme metabolism and approaches that could explain, at least partly, the mechanism of protoporphyrin IX selectivity towards cancer cells.

Enlarging the Scope of 5-Aminolevulinic Acid-Mediated Photodiagnosis towards Breast Cancers.

Today, most research on treating cancers targets one single cancer, often because of the very specific operation principle of the therapy. For instance, immunotherapies require the expression of a particular antigen, which might not be expressed in all cancers or in all patients. What about metastases? Combination therapies are promising but require treatment personalization and are an expensive approach that many health systems are not willing to pay for. Resection of cancerous tissues may be conducted beforehand. However, the precise location and removal of tumors are in most cases, hurdles that require margins to prevent recurrence. Herein, we further demonstrate the wide application of aminolevulinate-based photodynamic diagnosis and therapy toward breast cancers. By selecting four breast cancer cell lines that represent the main breast tumor subtypes, we investigated their ability to accumulate the fluorescent protoporphyrin IX upon treatment with the marketed 5-aminolevulinic acid hexyl ester (ALA-Hex) or our new and more stable derivative PSI-ALA-Hex. We found that all cell lines were able to accumulate PpIX under a few hours independent of their hormonal status with both treatments. Additionally, this accumulation was less dose-dependent with PSI-ALA-Hex and induced similar or higher fluorescence intensity than ALA-Hex in three out of four cell lines. The toxicity of the two molecules was not different up to 0.33 mM. However, PSI-ALA-Hex was more toxic at 1 mM, even though lower concentrations of PSI-ALA-Hex led to the same PpIX accumulation level. Additional illumination with blue light to induce cell death by generating reactive oxygen species was also considered. The treatments led to a dramatic death of the BT-474 cells under all conditions. In SK-BR-3 and MCF-7, ALA-Hex was also very efficient at all concentrations. However, increasing doses of PSI-ALA-Hex (0.33 and 1 mM) surprisingly led to a higher viability rate. In contrast, the triple-negative breast cancer cells MDA-MB-231 showed a higher death induction with higher concentrations of ALA-Hex or PSI-ALA-Hex. Derivatives of ALA seem promising as fluorescence-guided resection tools and may enable subsequent completion of cancer cell destruction by blue light irradiation.

Systematic Approach for Remote Sensing of Historical Conflict Landscapes with UAV-Based Laserscanning.

In order to locate historical traces, drone-based Laserscanning has become increasingly popular in archaeological prospection and historical conflict landscapes research. The low resolution of aircraft-based Laserscanning is not suitable for small-scale detailed analysis so that high-resolution UAV-based LiDAR data are required. However, many of the existing studies lack a systematic approach to UAV-LiDAR data acquisition and point cloud filtering. We use this methodology to detect anthropogenic terrain anomalies. In this study, we systematically investigated different influencing factors on UAV-LiDAR data acquisition. The flight parameters speed and altitude above ground were systematically varied. In addition, different vegetation cover and seasonal acquisition times were compared, and we evaluated three different types of filter algorithms to separate ground from non-ground. It could be seen from our experiments that for the detection of subsurface anomalies in treeless open terrain, higher flight speeds like 6 m/s were feasible. Regarding the flight altitude, we recommend an altitude of 50-75 m above ground. At higher flight altitudes of 100-120 m above ground, there is the risk that terrain characteristics smaller than 50 cm will be missed. Areas covered with deciduous forest should only be surveyed during leaf-off season. In the presence of low-level vegetation (small bushes and shrubs with a height of up to 2 m), it turned out that the morphological filter was the most suitable. In tree-covered areas with total absence of near ground vegetation, however, the choice of filter algorithm plays only a subordinate role, especially during winter where the resulting ground point densities have a percentage deviation of less than 6% from each other.

Double-PEGylated Cyclopeptidic Photosensitizer Prodrug Improves Drug Uptake from In Vitro to Hen's Egg Chorioallantoic Membrane Model.

Cyclopeptidic photosensitizer prodrugs (cPPPs) are compounds designed to specifically target overexpressed hydrolases such as serine proteases, resulting in their specific activation in close proximity to tumor cells. In this study, we explored a series of conjugates that can be selectively activated by the urokinase plasminogen activator (uPA). They differ from each other by their pheophorbide a (Pha) loading, their number of PEG chains and the eventual presence of black hole quenchers (BHQ3). The involvement of a peptidic linker between the drugs and the cyclopeptidic carrier allows specific cleavage by uPA. Restoration of the photophysical activity was observed in vitro on A549 lung and MCF7 breast cancer cells that exhibited an increase in red fluorescence emission up to 5.1-fold and 7.8-fold, respectively for uPA-cPPQ2+2/5. While these cPPP conjugates do not show dark toxicity, they revealed their phototoxic potential in both cell lines at 5 µM of Phaeq and a blue light fluence of 12.7 J/cm2 that resulted in complete cell death with almost all conjugates. This suggests, in addition to the promising use for cancer diagnosis, a use as a PDT agent. Intravenous injection of tetrasubstituted conjugates in fertilized hen eggs bearing a lung cancer nodule (A549) showed that a double PEGylation was favorable for the selective accumulation of the unquenched Pha moieties in the tumor nodules. Indeed, the diPEGylated uPA-cPPP4/52 induced a 5.2-fold increase in fluorescence, while the monoPEGylated uPA-cPPP4/5 or uPA-cPPQ2+2/5 led to a 0.4-fold increase only.

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs.

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free "click" chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Correction: Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher.

Correction for 'Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher' by Jordan Bouilloux et al., Photochem. Photobiol. Sci., 2018, 17, 1739-1748.

Correction: Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs.

Correction for 'Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs' by Jordan Bouilloux et al., Photochem. Photobiol. Sci., 2018, 17, 1728-1738.

Squalene-PEG: Pyropheophorbide-a nanoconstructs for tumor theranostics.

Novel nanoscale drug delivery biomaterials are of great importance for the diagnosis and treatment of different cancers. We have developed a new pegylated squalene (SQ-PEG) derivative with self-assembly properties. Supramolecular assembly with a lipophilic photosensitizer pyropheophorbide-a (Ppa) by nanoprecipitation gave nanoconstructs SQ-PEG:Ppa with an average size of 200 nm in diameter and a drug loading of 18% (w/w). The composite material demonstrates nanoscale optical properties by tight packing of Ppa within Sq-PEG:Ppa resulting in 99.99% fluorescence self-quenching. The biocompatibility of the nanomaterial and cell phototoxicity under light irradiation were investigated on PC3 prostate tumor cells in vitro. SQ-PEG:Ppa showed excellent phototoxic effect at low light dose of 5.0 J/cm2 as a consequence of efficient cell internalization of Ppa by the nanodelivery system. The diagnostic potential of SQ-PEG:Ppa nanoconstructs to deliver Ppa to tumors in vivo was demonstrated in chick embryo model implanted with U87MG glioblastoma micro tumors.

Squalene-PEG-Exendin as High-Affinity Constructs for Pancreatic Beta-Cells.

Novel drug delivery systems targeting native, transplanted, or cancerous beta-cells are of utmost importance. Herein, we present new exendin-4 derivatives with modified unnatural amino acids at strategic positions within the polypeptide sequence. The modified peptides allowed modular orthogonal chemical modifications to attach imaging agents and amphiphilic squalene-PEG groups. The resulting conjugates, SQ-PEG-ExC1-Cy5 and SQ-PEG-ExC40-Cy5 fluorescence probes, display low nanomolar affinity to GLP-1R in fluorescence-based binding assays with EC50 at 1.1 ± 0.2 and 0.8 ± 0.2 nM, respectively. Naturally expressing GLP-1R MIN6 cells and recombinantly transfected CHL-GLP-1R positive cells were specifically targeted by all of the new beta-cell probes in vitro. Specific islet targeting was observed after i.v. injection of SQ-PEG-ExC1-Cy5 with SQ-PEG in normoglycemic mice ex vivo. Semiquantitative biodistribution analysis by epifluorescence indicated prolonged blood half-life (3.8 h) for the amphiphilic Ex conjugate. Liver and pancreas were identified as main biodistribution organs for SQ-PEG-ExC1-Cy5.

Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part I - self-quenched prodrugs.

Herein, we report the synthesis of a new prodrug system consisting of regioselectively addressable functionalized templates bearing multiple pheophorbide A moieties for use in photodynamic therapy. These coupling reactions were achieved using copper-free "click" chemistry, namely a strain-promoted azide-alkyne cycloaddition. This new design was used to obtain well-defined quenched photosensitizer prodrugs with perfect knowledge of the number and position of loaded photosensitizers, providing structures bearing up to six photosentitizers and two PEG chains. These conjugates are ideally quenched in their native state regarding their fluorescence emission (up to 155 ± 28 times less fluorescent for an hexasubstituted conjugate than a monosubstituted non-quenched reference compound) or singlet oxygen production (decreased 8.7-fold in the best case) when excited. After 2 h of proteolytic activation, the fluorescence emission of a tetrasubstituted conjugate was increased 17-fold compared with the initial fluorescence emission.

Cyclopeptidic photosensitizer prodrugs as proteolytically triggered drug delivery systems of pheophorbide A: part II - co-loading of pheophorbide A and black hole quencher.

Previously, we have shown that the use of a cyclopeptidic carrier could be of great interest for the design of fully characterized prodrugs for further use in photodynamic therapy. In order to further optimize the design, we decided to modify the highly quenched conjugate uPA-cPPP4/5 by co-loading a long-distance fluorescence quencher. For this purpose we tethered two black hole quenchers (BHQ3) together with two pheophorbide A moities onto the same PEGylated backbone and assessed the modified photophysical properties. In addition, to prove the reliability of our concept, we designed two analogues, uPA-cPPQ2+2/5 and CathB-cPPQ2+2/5, by using two different peptidic linkers as substrates for uPA and cathepsin B, respectively. These two conjugates proved to be much more water-soluble than their analogues bearing only Phas. These conjugates are not only highly quenched in their native state with regard to their fluorescence emission (up to 850 ± 287 times less fluorescent for CathB-cPPQ2+2/5 as compared to the unquenched monosubstituted reference uPA-cPPP1/5), but also prevent singlet oxygen production (with a total quenching of the emission when the quenchers are co-loaded with photosensitizers) when the photosentistizers are excited. After proteolytic activation, these conjugates recover their photophysical properties in the same way as occurred for uPA-cPPP4/5, with up to a 120-fold increase in fluorescence emission for uPA-cPPQ2+2/5 after two hours of incubation with uPA.

An Activatable Photosensitizer Targeted to γ-Glutamyltranspeptidase.

We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.

Targeting GLP-1 receptors for repeated magnetic resonance imaging differentiates graded losses of pancreatic beta cells in mice.

AIMS/HYPOTHESIS: Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model. METHODS: We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5 T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively. RESULTS: We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells. CONCLUSIONS/INTERPRETATION: The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.

Kondrat'eva ligation: Diels-Alder-based irreversible reaction for bioconjugation.

Diversification of existing chemoselective ligations is required to efficiently access complex and well-defined biomolecular assemblies with unique and valuable properties. The development and bioconjugation applications of a novel Diels-Alder-based irreversible site-specific ligation are reported. The strategy is based on a Kondrat'eva cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles and maleimides that readily react together under mild and easily tunable reaction conditions to afford a fully stable pyridine scaffold. The potential of this novel bioconjugation is demonstrated through the preparation of fluorescent conjugates of biomolecules and a novel Förster resonance energy transfer (FRET)-based probe suitable for the in vivo detection and imaging of urokinase-like plasminogen activator (uPA), which is a key protease involved in cancer invasion and metastasis.

A step ahead to enhancing routine breast cancer resection: Spheroid and hen's egg chorioallantoic membrane models to assess the photodynamic diagnosis efficiency of ALA and PSI-ALA-hex.

Aminolevulinic acid (ALA) and its derivatives have been used in the diagnosis of several diseases through topical, intravesical, and oral administration. However, their intravenous use for the theranostics of cancers has not raised interest despite its potential advantages. In this study, we compared the efficacy of ALA, its hexyl ester ALA-Hex, and our new derivative PSI-ALA-Hex to induce a fluorescent protoporphyrin IX (PpIX) overproduction in breast cancers. First, we tested the drugs on four subtypes of breast cancer spheroids in vitro. Our results demonstrated the capacity of ALA-Hex and PSI-ALA-Hex to produce PpIX in all breast spheroids, although ALA struggled in half of the models. We applied the chick embryo in vivo model to investigate the intravenous administration route of ALA and PSI-ALA-Hex, ALA-Hex being toxic. We engrafted breast cancer nodules having various hormonal profiles onto the chorioallantoic membrane of the eggs. They were all detected by fluorescence imaging with mild efficacy using PSI-ALA-Hex, which displayed a maximum selectivity of 2.2 to 2.9, whereas ALA showed a higher selectivity from 3.2 to 5.1 at 300 µmol/kg. PSI-ALA-Hex was less appropriate for the diagnosis of breast cancer by intravenous administration. We show for the first time, to the best of our knowledge, the photodetection and imaging of a wide range of breast tumors in vivo upon intravenous treatment with ALA.

Unleashing the potential of 5-Aminolevulinic acid: Unveiling a promising target for cancer diagnosis and treatment beyond photodynamic therapy.

The therapeutic properties of 5-aminolevulinic acid (5-ALA) have been extensively studied for cancer detection and treatment using photodynamic therapy (PDT). When administered externally, 5-ALA is converted to protoporphyrin IX (PpIX) in cancer cells, which generates reactive oxygen species (ROS) upon exposure to light. This process enables targeted cell death induction and cancer detection. Given the highly conserved nature of heme biosynthesis over billions of years, we hypothesized that natural mechanisms might exist to prevent excessive accumulation of PpIX or heme resulting from 5-ALA overload. Therefore, we anticipated alterations in protein expression profiles upon exogenous administration of 5-ALA. To understand cellular responses to 5-ALA, we investigated protein expression changes and identified OR1B1 as a promising target in bladder, prostate, lung, and cervical cancer cells. OR1B1 expression was observed only with 5-ALA and ferrous chloride, highlighting the central role of heme in this discovery. Immunofluorescence and electron microscopy confirmed OR1B1's sub-cellular localization. These findings suggest that 5-ALA transformation in cancer cells and OR1B1 expression have potential for enhancing cancer detection and developing alternative treatments, including immunotherapy. This approach overcomes the limitations of PDT and opens new avenues for effective and targeted cancer interventions.

Enhanced prostate cancer targeting by modified protease sensitive photosensitizer prodrugs.

Prodrugs combining macromolecular delivery systems with site-selective drug release represent a powerful strategy to increase selectivity of anticancer agents. We have adapted this strategy to develop new polymeric photosensitizer prodrugs (PPP) sensitive to urokinase-like plasminogen activator (uPA). In these compounds (to be referred to as uPA-PPPs) multiple copies of pheophorbide a are attached to a polymeric carrier via peptide linkers that can be cleaved by uPA, a protease overexpressed in prostate cancer (PCa). uPA-PPPs are non-phototoxic in their native state but become fluorescent and produce singlet oxygen after uPA-mediated activation. In the present work, we studied the influence of side-chain modifications, molecular weight, and overall charge on the photoactivity and pharmacokinetics of uPA-PPPs. An in vitro promising candidate with convertible phototoxicity was then further investigated in vivo. Systemic administration resulted in a selective accumulation and activation of the prodrug in luciferase transfected PC-3 xenografts, resulting in a 4-fold increase in fluorescence emission over time. Irradiation of fluorescent tumors induced immediate tumor cell eradication as shown by whole animal bioluminescence imaging. PDT with uPA-PPP could therefore provide a more selective treatment of localized PCa and reduce side effects associated with current radical treatments.

Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact.

Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.

Pharmaceutical technology at the service of targeted drug delivery.

Research in pharmaceutical technology has drifted from formulation of systems with improved drug absorption and bioavailability to systems targeting molecular sites of diseases. The research unit of Pharmaceutical Technology from the University of Geneva focuses on the development of systems for both diagnostic and therapeutic purposes. Three types of constructs for targeting are reviewed. With a fine-tuning of size and surface composition, polymeric nanoparticles are developed to improve detection of micrometastasis by fluorescence imaging. Furthermore, surface coating with specific antibodies increase the therapeutic efficiency of the encapsulated chemotherapeutic agent for tumor treatment in animal models. Constructs that are activated by remote sources of energy are investigated in the unit. For instance, microbubbles bearing specific antibody fragments at their surface are useful contrast agents for ultrasound molecular imaging. Microbubbles, if combined with a thrombolytic drug and ultrasound, improve clot lysis, which is promising for stroke treatments. Enzymatically activated prodrug scaffolds are also under development. With this approach, intrinsic enzymatic activity of a diseased tissue activates the formulations. This concept led to the development of theranostic agents that can be used for both diagnostic and therapeutic purposes.

Modulation and proteomic changes on the heme pathway following treatment with 5-aminolevulinic acid.

5-ALA-mediated photodynamic therapy (PDT) has been developed around the heme biosynthesis physiological pathway. It is based on the external supplementation of 5 aminolevulinic acid (5-ALA), increasing the activity of the heme pathway and leading to a significant protoporphyrin IX (PpIX) accumulation. Interestingly, this metbolite accumulation is predominant in cancer cells, induced by a highly active metabolism, therefore limiting off-target side effects and increasing therapy specificity. Nevertheless, the intrinsic mechanism responsible of PpIX accumulation on cells following PDT is still unknown, limiting clinical therapy translation. In order to further understand the mechanisms behind 5-ALA-induced PDT, in this study we aimed to evaluate the proteome changes reported on the physiological heme pathway, in response to an external 5-ALA supplementation. We studied two different scenarios following 5-ALA treatment, 5-ALA accumulation (5-ALA metabolization into the heme pathway blocked with inhibitors) and accumulation of PpIX (normal heme pathway with 5-ALA supplementation). Therefore, we were able to characterize enzymatic changes and to describe bottlenecks in the pathway. Following mass spectrometry analysis, we reported significant differences between 5-ALA and PpIX effects on heme biosynthesis and regulation of degradation. 5-ALA accumulation significantly decreased porphobilinogen deaminase (HMBS) expression, while phorphyrins accumulation (PpIX) upregulated heme synthesis, specifically HMBS and uroporphyrinogen decarboxylase (UROD), and enhanced the enzymatic level of the heme degradation pathway, including Heme oxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA). Interestingly, porphyrins induced a significant downregulation effect on oxygen-dependent coproporphyrinogen-III oxidase (CPOX). In conclusion, in this study we demonstrated that porphyrins play the most relevant role in heme biosynthesis modulation, while 5-ALA alone (PDT substrate) is not responsible of the main changes observed in this pathway during PDT treatment. Understanding heme enzyme modulation would help to design a more rational approach for patient treatment in the clinic. AIM: Effect of 5-ALA and porphyrins on the different Heme biosynthesis and degradation enzymes.