Photochemical Activation of Enediyne Warheads: A Potential Tool for Targeted Antitumor Therapy.

Spatial and temporal control over DNA cleavage by photoactivated enediynes can be complemented by additional factors such as the release of internal strain, chelation, pH changes, intramolecular H-bonds, and substituent effects. This review presents design and reactivity of photoactivated enediynes/enynes and analyses the chemical, biological, and photophysical challenges in their applications.

A novel Doxorubicin prodrug with controllable photolysis activation for cancer chemotherapy.

PURPOSE: Doxorubicin (DOX) is a very effective anticancer agent. However, in its pure form, its application is limited by significant cardiotoxic side effects. The purpose of this study was to develop a controllably activatable chemotherapy prodrug of DOX created by blocking its free amine group with a biotinylated photocleavable blocking group (PCB). METHODS: An n-hydroxy succunamide protecting group on the PCB allowed selective binding at the DOX active amine group. The PCB included an ortho-nitrophenyl group for photo cleavability and a water-soluble glycol spacer arm ending in a biotin group for enhanced membrane interaction. RESULTS: This novel DOX-PCB prodrug had a 200-fold decrease in cytotoxicity compared to free DOX and could release active DOX upon exposure to UV light at 350 nm. Unlike DOX, DOX-PCB stayed in the cell cytoplasm, did not enter the nucleus, and did not stain the exposed DNA during mitosis. Human liver microsome incubation with DOX-PCB indicated stability against liver metabolic breakdown. CONCLUSIONS: The development of the DOX-PCB prodrug demonstrates the possibility of using light as a method of prodrug activation in deep internal tissues without relying on inherent physical or biochemical differences between the tumor and healthy tissue for use as the trigger.

Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action of a photodynamic tumor therapy agent.

Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.

Activation of antitumor agent gilvocarcins by visible light.

Gilvocarcins that are antitumor agents are activated by low doses of visible light to induce bacteriophage lambda in Escherichia coli. This result is dependent on interaction with DNA. Gilvocarcin M, an analog without antitumor activity, failed to induce the prophage after light exposure, thus demonstrating a correlation between photosensitizing and antitumor activities. These results raise several possibilities regarding the mode of action of gilvocarcins as antitumor agents in vivo, involving light or enzymatic activating systems, which could be exploited in human cancer therapy.

pH/NIR-responsive semiconducting polymer nanoparticles for highly effective photoacoustic image guided chemo-photothermal synergistic therapy.

Multifunctional drug delivery nanoplatform (PDPP3T@PSNiAA NPs) based on NIR absorbing semiconducting polymer nanoparticles for pH/NIR light-controllably regulated drug release has been successfully prepared. In this strategy, pH/thermal-sensitive multifunctional polymer polystyrene-b-poly(N-isopropylacrylamide-co-acrylic acid) (PSNiAA) was meticulously designed and synthesized using the reversible addition fragmentation chain transfer (RAFT) polymerization method. Furthermore, PSNiAA was used to functionalize diketopyrrolopyrrole-based semiconducting polymer (PDPP3T) to combine photothermal capacity and pH/thermo-responsive drug release in one entity. The prepared PDPP3T@PSNiAA NPs exhibited high photothermal conversion efficiency (η = 34.1%) and excellent photoacoustic (PA) brightness. Meanwhile, benefiting from the photothermal effect of PDPP3T and the pH/thermal-responsive properties of PSNiAA, Dox-loaded PDPP3T@PSNiAA NPs (PDPP3T@PSNiAA-Dox NPs) were able to controllably regulate the release of Dox by pH/NIR light, in which the enhanced drug release at acidic condition upon NIR irradiation phenomenon would minimize unnecessary drug release in normal tissues and was highly beneficial for precise synergistic chemo- and photothermal therapy.

Doxorubicin-loaded PLGA nanoparticles for the chemotherapy of glioblastoma: Towards the pharmaceutical development.

Brain delivery of drugs by nanoparticles is a promising strategy that could open up new possibilities for the chemotherapy of brain tumors. As demonstrated in previous studies, the loading of doxorubicin in poly(lactide-co-glycolide) nanoparticles coated with poloxamer 188 (Dox-PLGA) enabled the brain delivery of this cytostatic that normally cannot penetrate across the blood-brain barrier in free form. The Dox-PLGA nanoparticles produced a very considerable anti-tumor effect against the intracranial 101.8 glioblastoma in rats, thus representing a promising candidate for the chemotherapy of brain tumors that warrants clinical evaluation. The objective of the present study, therefore, was the optimization of the Dox-PLGA formulation and the development of a pilot scale manufacturing process. Optimization of the preparation procedure involved the alteration of the technological parameters such as replacement of the particle stabilizer PVA 30-70 kDa with a presumably safer low molecular mass PVA 9-10 kDa as well as the modification of the external emulsion medium and the homogenization conditions. The optimized procedure enabled an increase of the encapsulation efficiency from 66% to >90% and reduction of the nanoparticle size from 250 nm to 110 nm thus enabling the sterilization by membrane filtration. The pilot scale process was characterized by an excellent reproducibility with very low inter-batch variations. The in vitro hematotoxicity of the nanoparticles was negligible at therapeutically relevant concentrations. The anti-tumor efficacy of the optimized formulation and the ability of the nanoparticles to penetrate into the intracranial tumor and normal brain tissue were confirmed by in vivo experiments.

PEGylated doxorubicin cloaked nano-graphene oxide for dual-responsive photochemical therapy.

Graphene oxide (GO) owns huge surface area and high drug loading capacity for aromatic molecules, such as doxorubicin (DOX). However, its biocompatibility is poor and it might agglomerate in physiological conditions. Chemical modification of GO with hydrophilicpolymer, especially PEGylation, was a common method to improve its biocompatibility. But the chemical modification of GO was complicated, and its drug loading capacity might be reduced because of the occupation of its functional groups. In this study, DOX-PEG polymers with different PEG molecular weights were synthesized to modify nano graphene oxide (NGO) to simultaneously realize the solubilization of NGO and the high loading capacity of DOX. The result showed that the drug release of NGO@DOX-PEG was pH sensitive. NIR irradiation could augment the drug release, cellular uptake, cytotoxicity and nuclear translocation of nanodrugs. Among the three kinds of nanodrugs, NGO@DOX-PEG5K was superior to others. It suggested that after conjugating with PEG, the bond between DOX-PEG and NGO was weakened, which resulted in a better drug release and treatment effect. In summary, the NIR and pH dual-responsive NGO@DOX-PEG nanodrugs were developed by noncovalent modification, and it demonstrated excellent biocompatibility and photochemical therapeutic effect, presenting a promising candidate for antitumor therapy, especially NGO@DOX-PEG5K.

Effect of gamma irradiation on spleen cell function and cytotoxicity of doxorubicin.

The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-gamma and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated doxorubicin (NIRD) was used as a control. The mice injected with NIRD (2mg/kg body weight for 5 days, 24h interval) showed a considerable decrease (P<0.05) in the body, spleen weight, proliferation and cytokine release (IL-2 and IFN-gamma) as compared to control. However, a non-significant variation was observed in IRD treated mice compared with normal. Tumor bearing mice treated with NIRD and IRD (2mg/kg body weight, five doses at 48 h interval) showed diverse results on spleen cell cytokine release, proliferation and metastasis. HPLC results revealed the formation of several trace level degradation (P<0.05) products of IRD. IRD displayed a non-significant variation of cytotoxicity on B16BL6 cells, and low percentage (P<0.01) of cardiotoxicity on H9c2 cells as compared to NIRD. Altogether, this present study emphasis that gamma irradiation altered the property of doxorubicin.

Radiation sterilisation of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles.

Doxorubicin-loaded poly(butyl cyanoacrylate) (PBCA) nanoparticles were prepared by anionic polymerisation under non-aseptic conditions. The feasibility of sterilisation of this formulation using either gamma-irradiation or electron beam irradiation was investigated. The irradiation doses ranged from 10 to 35 kGy. Bacillus pumilus was used as the official test microorganism. The bioburden of the untreated formulation was found to be 100 CFU/g. Microbiological monitoring revealed that at this level of the bioburden the irradiation dose of 15 kGy was sufficient for sterilisation of the nanoparticles. The formulation showed excellent stability with both types of irradiation in the investigated dose range. The irradiation did not influence the physicochemical parameters of the drug-loaded and empty nanoparticles, such as the mean particle size, polydispersity, and aggregation stability. The molecular weights of the PBCA polymer as well as the polydispersity indices (M(w)/M(n)) remained nearly unchanged. The drug substance was stable to radiolysis. Additionally, the presence of irradiation-induced radicals was evaluated by ESR spectroscopy after storage of the particles at ambient temperature. The paramagnetic species found in the formulation were mainly produced by irradiation of mannitol and dextran used as excipients.

Effects of extremely low-frequency electromagnetic fields on delayed chromosomal instability induced by bleomycin in normal human fibroblast cells.

This study was carried out to examine the interaction of extremely low-frequency electromagnetic fields (ELF-EMF) on delayed chromosomal instability by bleomycin (BLM) in human fibroblast cells. A micronucleus-centromere assay using DNA probes for chromosomes 1 and 4 was performed and a 60-Hz ELF-EMF of 0.8 mT field strength was applied either alone or with BLM throughout the culture period. The frequencies of micronuclei (MN) and aneuploidy were analyzed at 28, 88, and 240 h after treatment with BLM. The coexposure of cells to BLM and ELF-EMF led to a significant increase in the frequencies of MN and aneuploidy compared to the cells treated with BLM alone. No difference was observed between field-exposed and sham-exposed control cells. The frequency of MN induced by BLM was increased at 28 h, and further analysis showed a persistent increase up to 240 h, but the new levels were not significantly different from the level at 28 h. BLM increased the frequencies of aneuploidy at 28, 88, and 240 h, and significantly higher frequency of aneuploidy was observed in the cells analyzed at 240 h compared to the cells examined at 28 h. No interaction of ELF-EMF on delayed chromosomal instability by BLM was observed. Our results suggest that ELF-EMF enhances the cytotoxicity of BLM. BLM might induce delayed chromosomal instability, but no effect of ELF-EMF was observed on the BLM-induced delayed chromosomal instability in fibroblast cells.

Near infrared light mediated release of doxorubicin using upconversion nanoparticles.

Lanthanide doped upconversion nanoparticles grafted with a photocaged analog of doxorubicin allow near IR-release of doxorubicin.

Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical.

The activation of the antitumor protein antibiotic neocarzinostatin (NCS) by the carboxyl radical CO-2, a one-electron donor obtained selectively from gamma-ray irradiation of nitrous oxide-saturated formate buffer, has been investigated in the presence and in the absence of DNA at pH 4.7 and pH 7.0. The reaction of NCS with CO-2 in the absence of DNA is followed by a marked red shift (420----441 nm) and a pronounced increase (X 8.8) of the fluorescence emission corresponding to the naphthalene moiety of the NCS chromophore. The light absorption spectrum shows in parallel a hypochromic change with considerable fine structure throughout the 250-400 nm wavelength range. When DNA is present, the fluorescence intensity at completion of the reaction is slightly reduced (by 5 to 15 per cent) and the maximum emission wavelength shifted to 436-438 nm. However, the bulk rate of reaction is not altered by DNA and is independent of the pH, of the temperature and of the concentration of NCS. The NCS concentration-independence of the reaction rate is consistent with a high intrinsic rate (k greater than 10(8)M-1 . s-1) for the reaction of CO-2 with the NCS chromophore. Complete reduction of the NCS chromophore involves a total of three electron-equivalents. The final product does not react with oxygen, shows no odd electron spin, and is unable to induce DNA strand scission. Its molecular state, however, is fundamentally different when gamma-ray irradiations are performed with DNA. This bears evidence of short-lived one electron or two-electrons reduced intermediates decaying via non-identical routes depending on the presence of the acceptor DNA. Actually, dose-related strand breaks appear in DNA exposed to the action of NCS and CO-2. Some NCS chromophore-DNA covalent adducts are also found. DNA strand breakage by CO-2-activated NCS is correlated with thymine release and is inhibited by a redox-stable intercalating agent. The DNA-nicking process thus bears resemblance to that reported by other authors using mercaptans to initiate reductive activation of the NCS chromophore. However, some spectral differences are observed between the CO-2-reacted and the thiol-treated chromophores. Moreover, thymine release and strand scission in DNA incubated with CO-2 and NCS proceed under anaerobic conditions. It is proposed that the strict oxygen requirement for DNA damage by NCS in the presence of mercaptans is due, at least partly, to competition between oxygen and thiols for reaction with the same primary deoxyribose radical resulting from DNA attack by the reductively activated NCS chromophore.

The riboflavin-mediated photooxidation of doxorubicin.

PURPOSE: Previously, it was shown that exposing doxorubicin (ADR) to 365 nm light resulted in the loss of its cytotoxic activity as well as its absorbance at 480 nm. These processes were much enhanced when mediated by riboflavin. In the present study we investigated the quantitative and qualitative aspects of riboflavin-mediated photodegradation of ADR. METHODS: ADR solutions containing variable concentrations of riboflavin and other agents were exposed to 365 nm light for variable time periods and then the absorbance spectrum of ADR was measured by a double beam spectrophotometer. These measurements were used to calculate the half-time of the ADR degradation process. The degraded ADR solutions were analyzed by chromatography and mass spectrometry. RESULTS: Analysis of the riboflavin effect indicated that a maximal rate of photolytic degradation of ADR was obtained only after most of the ADR molecules had formed bimolecular complexes with riboflavin. The retardation of lumichrome formation by ADR and the inhibition of ADR bleaching by excess of ascorbic acid suggested that ADR was degraded by a photooxidation process. Similar spectral changes occurred when ADR was exposed to strong oxidizers such as sodium hypochlorite and dipotassium hexachloroiridate. Cyclic voltammetry revealed that the oxidation-reduction process of ADR was not electrochemically reversible and therefore the oxidation potential could not be determined accurately; however its value should be between 0.23 and 0.78 V. Analysis of the photooxidative process revealed that it was not mediated by the formation of singlet oxygen, superoxide anion radicals, hydrogen peroxide or hydroxyl radicals, and it is suggested that ADR was oxidized directly by the excited triplet riboflavin. The mass spectrograms and the HPLC chromatograms of photooxidized ADR indicate that the central ring of ADR was opened and that 3-methoxysalicylic acid was produced by this cleavage. CONCLUSIONS: The riboflavin-mediated photodegradation of ADR is an oxidative process resulting in the cleavage of the anthraquinone moiety. 3-Methoxysalicylic acid was identified as one of the resulting fragments. It is possible that some of the large fractions of the ADR metabolites that are non-fluorescent are the result of an in vivo oxidation of ADR and that 3-methoxysalicylic acid may play a role in the different biological activities of ADR.

The inactivation of doxorubicin by long ultraviolet light.

PURPOSE: Irradiation of doxorubicin (DOX) dissolved in RPMI medium 1640 by long ultraviolet (UVA) light results in a rapid decline in the cytotoxic activity of the drug. The present study was designed to sort out which component(s) of this medium are associated with the UVA inactivation of DOX. METHODS: The effects of UVA irradiation of DOX in solutions of various compositions were evaluated by measuring the changes in the drug growth inhibitory activity in P388 cells and in the DOX absorbance spectrum. RESULTS: Riboflavin seemed to be the major photosensitizing component in the medium and the effect was enhanced by the presence of histidine, methionine, tryptophan and tyrosine but not by other amino acids. The changes in DOX resulting from UVA irradiation in the presence of riboflavin, were not blocked by 1,4-diazabicyclo [2.2.2]octane (5 mM), superoxide dismutase (300 units/ml), catalase (150 units/ml) or sodium benzoate (50 mM). The effects of UVA light on doxorubicin could be prevented by excess ascorbic acid. CONCLUSIONS: The effects of UVA on DOX are mediated by riboflavin. The photoexcited riboflavin apparently interacts directly with DOX rather than by first forming reactive oxygen species. The results suggest that the photoinactivation of DOX may involve an oxidation step. The mechanism by which certain amino acids facilitate the photoinactivation of DOX is not known. It is suggested that patient intake of riboflavin and exposure to the sun and fluorescent light could affect the outcome of anthracycline treatment.

A novel concept to activate enediynes for DNA cleavage.

A novel concept is presented to activate enediynes via a biscumulenic intermediate using photoinduced electron transfer (PET).

An ESR study of the visible light photochemistry of gilvocarcin V.

Photolysis of gilvocarcin (GV) at 405 nm in argon saturated dimethylsulfoxide (DMSO) or 50% DMSO-water solutions in the presence of the sodium salt of 3,5-dibromo-2,6-dideutero-4-nitrosobenzene sulfonic acid (DBNBS-d2) generates the CH3-DBNBS-d2.spin adduct. It is postulated that this spin adduct is produced by photoreduction of DMSO by GV and the consequent formation and trapping of the generated methyl radicals. Gilvocarcin V also photoreduces oxygen and methyl viologen with quantum yields of 0.019 and 0.0012 respectively. The quantum yield for singlet oxygen formation by GV in DMSO, determined by measuring the rate of production of the nitroxyl radical produced by the reaction of 2,2,6,6-tetramethylpiperidinol with singlet oxygen, was found to be 0.15. Thus, GV photochemistry proceeds by both Type I and Type II pathways which could contribute to the reported GV phototoxicity in biological systems.

A comparative study of the visible light photochemistry of gilvocarcins V and M.

Quantum yields for the formation of superoxide ions, O2-., and singlet oxygen, 1O2, were determined during the photolyses of gilvocarcin M (GM) in air-saturated dry dimethylsulfoxide (DMSO) and in 45:55 (vol/vol) DMSO-water mixtures. The quantum yield for the photoreduction of methyl viologen by GM in nitrogen-saturated dry DMSO was also determined. These values are not different, within experimental error, from those corresponding to gilvocarcin V (GV). Because GV is a strong photocytotoxic agent and GM is not, these results imply that Type I and Type II mechanisms are not important pathways in the cytotoxicity of GV.

The photodeactivation of hedamycin, an antitumor antibiotic of the pluramycin type.

The cytotoxicities of hedamycin and photohedamycin A as well as of kidamycin and isokidamycin were determined using HeLa cell cultures. Photohedamycin A proved to be 15 times less cytotoxic than hedamycin thus explaining the loss of biological activity observed for solutions of hedamycin left in daylight. The fact that photohedamycin A is less active than hedamycin, and isokidamycin less than kidamycin points to the important role the rings E and F play in the biological activity of hedamycin and kidamycin.

Aminopeptidase activity of an antitumor antibiotic, C-1027.

An antitumor antibiotic C-1027, a complex protein consisting of an apoprotein and a non-covalently bound chromophore, showed some aminopeptidase activity, 1/15 (on the basis of activity per mg protein) that of porcine kidney enzyme [E.C. 3.4.11.2] by use of L-phenylalanyl 4-methyl-coumaryl-7-amide as the substrate. Neither the apoprotein alone nor the chromophore alone were active. Amastatin and bestatin but not leupeptin inhibited the activity. The enzyme activity of the holo-antibiotic, as opposed to that of the porcine kidney enzyme, was readily lost by UV irradiation, indicating that the intact structure of the chromophore was needed to maintain the native conformation of the holo-antibiotic. The cytotoxicity of the holo-antibiotic, but not that of the chromophore, to Ehrlich carcinoma cells in vitro was reduced to 1/5 by 1 microgram/ml of amastatin which alone had no effect on cell growth. The porcine aminopeptidase was not cytotoxic at all even at higher concentrations (higher enzyme activities/ml). Amastatin possibly occupied the catalytic domain of the holo-antibiotic, interfering with the binding of the holo-antibiotic with some cell-surface protein(s). Amastatin did not inhibit the holo-antibiotic to cleave isolated DNA.

[Stability of high molecular weight anticancer agent SMANCS and its transfer from oil-phase to water-phase. Comparative study with neocarzinostatin].

SMANCS is a conjugate protein of copolymer of styrene-maleic acid [SMA] (molecular weight: 1,500) and an antitumor protein neocarzinostatin [NCS] (molecular weight: 11,700). It has an approximate molecular weight of 15,000. We report here stability of SMANCS in oil and in water, and NCS in water, under various physical conditions such as exposure to heat, UV, pH, and ultrasonic treatment. Then, we carried out an experiment of transfer of SMANCS in lipid contrast medium [lipiodol] (oil phase) to water phase (blood and physiological saline) in vitro. Results are summarized as follows: In aqueous condition, SMANCS is far more stable than NCS against the exposure to heat and UV, though it is inactivated by excessive exposures. SMANCS in an oily medium was found much more stable even at higher temperatures than in the aqueous phase. Both SMANCS and NCS are the most stable at pH 4.9-6.0. SMANCS dissolved in oil transferred to water phase slowly, having T1/10 of 24 hours (in case of lipiodol). This helps maintaining the anticancer effect of the drug in vivo for a long period of time. SMANCS in lipiodol was found to exert its action against cultured tumor cells as in an aqueous solution.