Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

Novel Hydrogen Sulfide (H2S)-Releasing BW-HS-101 and Its Non-H2S Releasing Derivative in Modulation of Microscopic and Molecular Parameters of Gastric Mucosal Barrier.

Hydrogen sulfide (H2S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H2S-prodrug, BW-HS-101 with the ability to release H2S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5-50 µmol/kg) or its analogue without the ability to release H2S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE2 concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 µmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H2S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H2S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H2S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.

Oxygen-producing proenzyme hydrogels for photodynamic-mediated metastasis-inhibiting combinational therapy.

Photodynamic therapy (PDT) has provided a promising approach for the treatment of solid tumors, while the therapeutic efficacy is often limited due to the hypoxic tumor microenvironment, resulting in tumor metastasis. Herein, we report an oxygen-producing proenzyme hydrogel (OPeH) with photoactivatable enzymatic activity for PDT enabled metastasis-inhibiting combinational therapy of breast cancer. This OPeH based on alginate is composed of protoporphyrin IX (PpIX) conjugated manganese oxide (MnO2) nanoparticles, which act as both the photosensitizer and oxygen-producing agent, and singlet oxygen (1O2)-responsive proenzyme nanoparticles. In the hypoxic and acidic tumor microenvironment, MnO2 can generate 1O2 to promote PpIX-mediated PDT with an amplified 1O2 generation efficiency, which also triggers the cleavage of 1O2-responsive linkers and cascade activation of proenzymes for cancer cell death. This combinational therapy upon photoactivation not only greatly inhibited the tumor growth, but also suppressed lung metastasis in a mouse xenograft breast tumor model, which is impossible in the case of PDT alone. This study thus provides a proenzyme hydrogel platform with photoactivatable activity for metastasis-inhibiting cancer therapy with high efficacy and safety.

Broaden sources and reduce expenditure: Tumor-specific transformable oxidative stress nanoamplifier enabling economized photodynamic therapy for reinforced oxidation therapy.

Cancer cells immersed in inherent oxidative stress are more vulnerable to exogenous oxidative damages than normal cells. Reactive oxygen species (ROS)-mediated oxidation therapy preferentially aggravating tumor oxidative stress to disrupt redox homeostasis, has emerged as an effective and specific anticancer treatment. Herein, following an ingenious strategy of "broaden sources and reduce expenditure", we designed a versatile tumor-specific oxidative stress nanoamplifier enabling economized photodynamic therapy (PDT), to achieve synergistic oxidative stress explosion for superior oxidation therapy. Methods: Cinnamaldehyde (CA) as a therapeutic ROS generator was first conjugated to hyaluronic acid (HA) through acid-labile hydrazone bond to synthesize tailored amphiphilic HA@CA conjugates, which could surprisingly self-assemble into uniform nanofibers in aqueous media. Photosensitizer protoporphyrin (PpIX) was efficiently encapsulated into HA@CA nanofibers and transformed HA@CA nanofibers to final spherical HA@CAP. Results: With beneficial pH-responsiveness and morphology transformation, improved bioavailability and selective tumor accumulation, HA@CAP combining ROS-based dual chemo/photodynamic treatment modalities could induce cytotoxic ROS generation in a two-pronged approach to amplify tumor oxidative stress, termed "broaden sources". Moreover, utilizing CA-induced H2O2 production and cascaded Fenton reaction in mitochondria to consume intracellular overloaded Fe(II), HA@CAP could skillfully block endogenic heme biosynthesis pathway on site to restrain undesired elimination of PpIX for economized PDT, termed "reduce expenditure". Both in vitro and in vivo results demonstrated the superior antitumor performance of HA@CAP. Conclusion: This study offered an inspiring strategy of "broaden sources and reduce expenditure" to specifically boost tumor oxidative stress for reinforced oxidation therapy.

Heme oxygenase-1 alleviated non-alcoholic fatty liver disease via suppressing ROS-dependent endoplasmic reticulum stress.

AIMS: The endoplasmic reticulum (ER) stress response plays a crucial role in the development of nonalcoholic steatohepatitis (NASH). Heme oxygenase-1 (HO-1) exerts beneficial effects against oxidative injury in NASH. This study is aimed to clarify whether HO-1 is an effective therapeutic strategy for NASH via regulation of ER stress. METHODS: The C57BL/6J mice were fed with methionine-choline deficient (MCD) for 4 weeks and high fat-high carbohydrate-high cholesterol (HFD) diet for 16 weeks, with hemin or zinc protoporphyrin IX (ZnPP-IX), respectively. The LO-2 cells were cultured in palmitic medium, with transfected pEX-HO-1 or sh-HO-1 plasmid for 24 h. Meanwhile, thirty NASH patients and 15 health controls were enrolled. The ER ultrastructure was observed by transmission electron microscopy (TEM) and confocal microscopy. The expressions of mRNAs and proteins of HO-1, ER stress related genes were detected by real time PCR, western blot and immunohistochemical staining, respectively. RESULTS: The swelled and broken rough endoplasmic reticulums were observed in MCD and HFD fed mice. The reactive hepatic expression of HO-1 was related with the increased ROS production and ER stress, companied with upregulation of GRP78, p-IRE1, PERK, ATF6. Through hemin administration, hepatocyte apoptosis was suppressed companied down-regulation of CHOP, caspase12 and up-regulation of BCL2. Conserved results were exhibited in ZnPP-IX administrated mice and HO-1 silent cells. Consistent results were observed in the NASH Patients. CONCLUSIONS: HO-1 could serve as a protective factor in the progression of nutritional steatohepatitis by suppresses hepatocyte excessive ER stress and might be a potential target for therapy of nonalcoholic steatohepatitis.

Optically controlled hybrid metamaterial of plasmonic spiky gold inbuilt graphene sheets for bimodal imaging guided multimodal therapy.

The development of multifunctional molecular diagnostic platforms for the concordant visualization and treatment of diseases with high sensitivity and resolution has recently become a crucial strategy in cancer management. Thus, engineering functional metamaterials with high therapeutic and imaging capabilities to elucidate diseases from their morphological behaviors to physiological mechanisms is an unmet need in the current scenario. Here, we report the design of a unique hybrid plasmonic nanoarchitecture for targeted multiple phototherapies of breast cancer by simultaneous real-time monitoring through fluorescence and surface-enhanced Raman scattering (SERS) techniques. The nanoframework consisted of plasmonic gold-graphene hybrids tethered with folic acid-ligated chitosan-modified photosensitizer (PpIX) to afford target-specific localized photothermal and photodynamic therapy. The hybrid vehicle also served as an excellent nanocarrier for the efficient loading and stimuli-responsive release of the chemotherapeutic drug doxorubicin (DOX) to enhance the therapeutic efficacy, thereby forming a trimodal nanomedicine against cancer. The cytotoxic effects induced by the cumulative action of the triplet therapeutic tools were visualized through both fluorescence and SERS imaging channels. Moreover, it also generated synchronized therapeutic effects resulting in the effective regression of tumor volume without propagating any toxic effects to other organs of the animals. Taken together, by virtue of strong light-matter interactions, the nanoprobe showed enhanced photoadsorption, which facilitated amplified light-reactive therapeutic and imaging efficacies along with targeted and enhanced chemotherapy, both in vitro and in vivo, which may offer promising outcomes in clinical research.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas.

BACKGROUND: The usefulness of 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is intensely discussed. However, data about kinetics of 5-ALA and protoporphyrin (Pp) IX in meningiomas are lacking. METHODS: As the first study so far, we performed longitudinal intraoperative real-time ex situ measurements of fluorescence intensity and PpIX concentrations during FGS of ten benign and two atypical meningiomas. Kinetics were subsequently compared with data from 229 glioblastomas. RESULTS: Spectroscopy revealed fluorescence (median 2945.65 a.u.) and PpIX accumulation (median 18.31 µg/ml) in all 43 analyzed samples. Fluorescence intensity (2961.50 a.u. vs 118.41 a.u.; p < .001) and PpIX concentrations (18.72 µg/ml vs .98 µg/ml; p < .001) were higher in samples with (N = 30) than without (N = 2) visible intraoperative tumor fluorescence. ROC curve analyses revealed a PpIX cut-off concentration of 3.85 µg/ml (AUC = .992, p = .005) and a quantitative fluorescence cut-off intensity of 286.73 a.u. (AUC = .983, p = .006) for intraoperative visible tumor fluorescence. Neither fluorescence intensity (p = .356) nor PpIX (p = .631) differed between atypical and benign meningiomas. Fluorescence and PpIX peaked 7-8 h following administration of 5-ALA. Meningiomas displayed a higher fluorescence intensity (p = .012) and PpIX concentration (p = .005) than glioblastomas 5-6 h after administration of 5-ALA. Although fluorescence was basically maintained, PpIX appeared to be cleared faster in meningiomas than in glioblastomas. CONCLUSIONS: Kinetics of PpIX and fluorescence intensity differ between meningiomas and glioblastomas in the early phase after 5-ALA administration. Modification of the timing of drug administration might impact visibility of intraoperative fluorescence and helpfulness of FGS and should be investigated in future analyses.

Investigation of the emission spectra and cytotoxicity of TiO2 and Ti-MSN/PpIX nanoparticles to induce photodynamic effects using X-ray.

BACKGROUND: Photodynamic therapy (PDT) has been recognized as an effective method for cancer treatment; however, it suffers from limited tissue penetration depth. X-rays are ideal excitation sources for activating self-lighting nanoparticles that can penetrate through deep tumor tissues and convert the X-rays to visible light. In this study, Ti-MSN/PpIX nanoparticles for X-ray induced photodynamic therapy was synthesized. Preparation, characterization, and emission spectrum of Ti-MSN/PpIX nanoparticles as well as PDT activity and toxicity of the nanoparticles on HT-29 cell line were investigated. METHODS: Firstly, mesoporous silica nanoparticles (MSN) were synthesized through sol-gel method. Then, TiO2 and PpIX were loaded in MSN. Next, the emission spectra of TiO2, Ti-MSN, and Ti-MSN/PpIX nanoparticles, while activated by X-ray (6 MVp), were recorded. In addition, viability of cells after treatment by Ti-MSN/PpIX nanoparticles and X-ray irradiation was studied. RESULTS: SEM, TEM and FESEM images of the spherical composite nanoparticles showed that their dimensions were changed by incorporating Ti and organic compound of PpIX. Two-dimensional hexagonal structure with d100-spacing was about 3.5 nm with particle sizes of 70-110 nm. The optical characteristics of TiO2 nanoparticles showed strong emission lines, which effectively overlapped with the absorption wavelengths of protoporphyrin IX (PpIX). Cellular experiments showed Ti-MSN/PpIX nanoparticles have proper biocompatibility, however, after X-ray irradiation, significant decrease of cell viability in the presence of the nanoparticles was observed. CONCLUSION: The presented X-PDT method could enhance RT efficacy and is enable that allows for reducing X-ray dose exposure to healthy tissues to overcome radio-resistant tumors.

A Pharmacologic "Stress Test" for Assessing Select Antioxidant Defenses in Patients with CKD.

BACKGROUND AND OBJECTIVES: Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves. DESIGN: , setting, participants, & measurementsA total of 18 healthy volunteers and 24 participants with stage 3 CKD (n=12; eGFR 30-59 ml/min per 1.73 m2) or stage 4 CKD (n=12; eGFR 15-29 ml/min per 1.73 m2) were injected once with SnPP (9, 27, or 90 mg). Plasma and/or urinary antioxidant proteins were measured at baseline and for up to 4 days post-SnPP dosing. Kidney safety was gauged by serial measurements of BUN, creatinine, eGFR, albuminuria, and four urinary AKI biomarkers (kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, cystatin C, and N-acetyl glucosaminidase). RESULTS: Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (r=-0.85 to -0.95). All four proteins manifested statistically significant dose- and time-dependent elevations after SnPP injection. However, marked intersubject differences were observed. p21 responses to high-dose SnPP and HO-1 responses to low-dose SnPP were significantly suppressed in participants with CKD versus healthy volunteers. SnPP was well tolerated by all participants, and no evidence of nephrotoxicity was observed. CONCLUSIONS: SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.

Protein assembled nano-vehicle entrapping photosensitizer molecules for efficient lung carcinoma therapy.

The efficiency of drug depends not only on its potency but also on its ability to reach the target sites in preference to non-target sites. In this regard, protein assembled nanocarrier is the most promising strategy for intracellular anti-cancer drug delivery. The key motive of this study is to fabricate biocompatible protein assembled nanocarrier conjugated photosensitizer system for stimuli-responsive treatment of lung carcinoma. Here, we have synthesized a unique nanohybrid of protein assembled gold nanoparticles (AuNPs), attaching a model photosensitizer, Protoporphyrin IX (PpIX) to the protein shell of the nanoparticles (NPs) imparting an ideal drug-carrier nature. Photo-induced alteration in hydrodynamic diameter suggests structural perturbation of the nanohybrid which in terms signifies on-demand drug delivery. The drug release profile has been further confirmed by using steady-state fluorescence experiments. AuNP-PpIX showed excellent anti-cancer efficiency upon green light irradiation on lung adenocarcinoma cell line (A549) through intracellular reactive oxygen species (ROS) generation. The cellular morphological changes upon PDT and internalization of nanohybrid were monitored using confocal laser scanning microscope. This anti-cancer effect of nanohybrid was associated with apoptotic pathway which was confirmed in the flow cytometric platform. The developed nanomedicine is expected to find relevance in clinical anti-cancer PDT models in the near future.

Near-infrared light-triggered degradable hyaluronic acid hydrogel for on-demand drug release and combined chemo-photodynamic therapy.

In this study, an injectable and near-infrared (NIR) light-triggered ROS-degradable hyaluronic acid hydrogel platform was developed as localized delivery vehicle for photosensitizer protophorphyrin IX (PpIX) and anticancer drug doxorubicin (DOX), to achieve superior combined chemo-photodynamic therapy with light-tunable on-demand drug release. The in situ-forming hydrogel fabricated readily via the formation of dynamic covalent acylhydrazone bonds could efficiently prevent severe self-quenching effect of water-insoluble PpIX due to the covalent binding, leading to localized enhanced photodynamic therapy (PDT). Moreover, the extensive ROS generated by the hydrogel under NIR light irradiation could not only realize efficient PDT effect, but also cleave the ROS-cleavable small molecule crosslinker, inducing the desirable degradation of hydrogel and subsequent on-demand DOX release for cascaded chemotherapy. The developed versatile hyaluronic acid hydrogels have tunable properties, excellent biocompatibility, biodegradability and exhibit outstanding therapeutic effects in both in vitro cellular experiments and in vivo antitumor studies.

Improving the color of meat products without adding nitrite/nitrate using high zinc protoporphyrin IX-forming microorganisms.

Zinc protoporphyrin IX (ZnPP) mainly contributes to the red color of dry cured ham without nitrites/nitrates. Here, we examined the effects of acids used for pH adjustment, pH, and microorganisms on ZnPP formation. The results showed that ZnPP formation and optimal pH were dependent upon the acid type. In the presence of microorganisms, the optimal pH for ZnPP formation shifted to higher values, with the amount of formed ZnPP markedly increased at the shifted optimal pH. Additionally, two bacterial strains isolated from incubated pork homogenate exhibited an enhanced ability to form ZnPP. Although the two isolated bacteria are not edible, inoculation with one bacterium into minced meat resulted in formation of large amounts of ZnPP and color closer to that of nitrite-added meat. These results suggest that appropriate food-grade bacterial strains can improve the color of various fermented meat products in the absence of nitrites/nitrates.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair.

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Hemin attenuated oxidative stress and inflammation to improve wound healing in diabetic rats.

Oxidative stress and persistent inflammation play crucial role in the progression of diabetic wound complications. Hemeoxgenase-1 (HO-1) by degrading hemin has been shown to display anti-oxidant and anti-inflammatory effects. Further, hemin is a potent HO-1 inducer. Thus, the current study was aimed to evaluate the effect of topical application of hemin on diabetic wound in rats. Four hundred square millimeter open excision wound were created 2 weeks after induction of diabetes with single intraperitoneal injection of streptozotocin (60 mg/kg), and the diabetic rats were divided into three groups namely diabetic control, hemin, and tin protoporphyrin (SnPPIX). Ointment base, hemin (0.5% in ointment base), and SnPPIX (0.5% in ointment base) were applied topically to wounded area in diabetic control, hemin, and SnPPIX group rats, respectively, twice daily for 19 days. Hemin significantly increased the wound contraction in comparison to control and SnPPIX-treated rats. Time-dependent analysis revealed significant increase in anti-oxidants with concomitant decrease in oxidants in hemin-treated rats as compared to diabetic control rats. Further, mRNA expression decreased for inflammatory cytokine and increased for anti-inflammatory cytokine in hemin group as compared to diabetic control rats. Expression of HO-1 also increased in hemin group as compared to diabetic control rats. However, SnPPIX group results were in disagreement with results of hemin which is clearly reflected in histopathology. Results indicate the ability of hemin to accelerate wound healing in diabetic rats by combating inflammation and oxidative stress probably via HO-1.

Synthesis of Pegylated Manganese Protoporphyrin as a Catalase Mimic and Its Therapeutic Application to Acetaminophen-Induced Acute Liver Failure.

Metalloporphyrin derivatives have been investigated for their therapeutic potential for oxidative stress-related diseases because of their scavenging of reactive oxygen species (ROS). Here, we describe the synthesis, physicochemical properties, and ROS-scavenging activities of one such derivative-polyethylene glycol (PEG)-conjugated manganese protoporphyrin (PEG-MnPP). Carboxyl groups of the protoporphyrin ring at the C6 and C7 positions were first conjugated with ethylenediamine to introduce amino groups into the protoporphyrin structure. The amino groups were then reacted with succinimidyl PEG, with an average molecular weight of 2000, to obtain pegylated protoporphyrin (PEG-PP). Manganese was chelated to the protoporphyrin ring by incubating PEG-PP and manganese acetate in methanol. Dynamic light scattering and fluorescent spectrometry analyses revealed that PEG-MnPP self-assembled into nanoparticles in aqueous media with an apparent diameter of 70 nm. PEG-MnPP effectively eliminated hydrogen peroxide from cell culture media and protected cultured mammalian cells from toxic insults induced by hydrogen peroxide exposure or by 6-hydroxydopamine treatment. Intravenous administration of PEG-MnPP to mice significantly suppressed acute liver failure that had been induced by acetaminophen overdose. These data warrant additional investigation to study the therapeutic potential of PEG-MnPP as a water-soluble metalloporphyrin-based catalase mimic for oxidative stress-associated diseases.

Ultraviolet radiation exposure during daylight Photodynamic Therapy.

BACKGROUND: Daylight photodynamic therapy (dPDT) is an effective treatment for field-change actinic keratoses (AK), with similar efficacy to conventional PDT but lower patient pain scores. Whilst AK occur consequent to chronic solar ultraviolet (UV) exposure, paradoxically solar visible radiation is used during PDT. OBJECTIVES: To investigate the nature and levels of UV exposure, both erythemal UV and UVA, occurring during dPDT. METHODS: Four years of solar erythemally effective UV (UVE) irradiance, UVA irradiance and illuminance data were obtained from Pubic Health England for 12 locations. For a standard 2 h treatment period, the data were converted into standard erythemal doses (SEDs), UVA dose and protoporphyrin-IX (PpIX)-weighted dose from UVE irradiance, UVA irradiance and illuminance respectively. These three parameters were compared ascertaining the UV exposure received during dPDT. RESULTS: Analysis of UV exposure during dPDT showed a UK maximum average UVE exposure of 8.2 SED at Camborne (PpIX dose 23.4 J cm-2). Treatment earlier in the day reduces average UV exposure (Camborne 5.2 SED, PpIX dose 18.2 J cm-2), whilst PpIX dose achieves threshold during winter months (Camborne, November, 0.8 SED, PpIX dose 7.1 J cm-2). Cyprus and Gibraltar (with high UV exposure during dPDT) experience a maximum of 14.3 SED and 12.9 SED, with respective PpIX doses of 36.1 J cm-2 and 35.1 J cm-2, in June. UVA exposure is also presented for comparison. CONCLUSION: Therapeutically effective dPDT doses can be achieved at times of the day and year when UV exposure is minimal.

Topical and intradermal delivery of PpIX precursors for photodynamic therapy with intense pulsed light on porcine skin model.

In order to purposely decrease the time of the photodynamic therapy (PDT) sessions, this study evaluated the effects of PDT using topical and intradermal delivery of two protoporphyrin (PpIX) precursors with intense pulsed light (IPL) as irradiation source. This study was performed on porcine skin model, using an IPL commercial device (Intense Pulse Light, HKS801). IPL effect on different administration methods of two PpIX precursors (ALA and MAL) was investigated: a topical cream application and an intradermal application using a needle-free, high-pressure injection system. Fluorescence investigation showed that PpIX distribution by needle-free injection was more homogeneous than that by cream, suggesting that a shorter drug-light interval in PDT protocols is possible. The damage induced by IPL-PDT assessed by histological analysis mostly shows modifications in collagens fibers and inflammation signals, both expected for PDT. This study suggested an alternative protocol for the PDT treatment, possibility half of the incubation time and with just 3 min of irradiation, making the IPL-PDT, even more, promising for the clinical treatment.

Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid-mediated Protoporphyrin IX by Iron Chelator Deferoxamine.

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron-dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA-PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA-PpIX fluorescence in tumor cells with lower FECH activity (MDA-MB-231, Hs 578T) than in tumor cells with higher FECH activity (MDA-MB-453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.

Photodynamic therapy for actinic keratosis of the forehead and scalp with the Aktilite CL 128: Is there a cut-off value for PpIX-weighted irradiance for effective treatment?

BACKGROUND/PURPOSE: Photodynamic therapy (PDT) is an established treatment for actinic keratosis (AK). Among the approved protocols in Europe, the most widely used involves irradiation with the Aktilite CL 128 (C-PDT). We aimed to assess the heterogeneity of irradiance over the treatment area when using C-PDT. We also investigated whether there is a cut-off value for protoporphyrin IX (PpIX)-weighted irradiance that may predict the treatment outcome of C-PDT. METHODS: An Ophir PD300 photodiode sensor connected to an Ophir Laser Star power meter was used to measure the irradiance delivered to 114 AKs of the scalp and forehead of 19 patients treated with C-PDT. The PpIX-weighted irradiances were deduced and cross-referenced with the complete responses at 3 months. RESULTS: From the measured irradiances ranging from 0.25 to 60 mW/cm2 (average: 31.94 mW/cm2 ), a standard deviation of 17.17 mW/cm2 was computed. Irradiance heterogeneity over the treatment area during C-PDT was demonstrated. The 66/114 AKs with a complete response at 3 months (57.89%) received a mean PpIX-weighted irradiance of 0.52 mW/cm2 vs 0.56 mW/cm2 for the resistant 48/114 AKs (42.11%). No significant effect of PpIX-weighted irradiance on the complete response at 3 months was found (odds ratio for a 0.1-unit change, 0.96; 95% confidence interval, 0.83 to 1.10; P = 0.53). Therefore, no cut-off value for PpIX-weighted irradiance that predicts treatment outcome could be identified. CONCLUSIONS: A device enabling homogeneous irradiation at a lower irradiance than the Aktilite CL 128 may therefore be suitable. This lower irradiance may further increase the treatment tolerance by patients.