Effect of superoxide derived from lucifer yellow CH on voltage-gated currents of mouse taste bud cells.

Lucifer yellow CH (LY), a fluorescent membrane-impermeable cell marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation, without considering its pharmacological effect. Recently, we showed that the exposure of cells loaded with LY to light for microscopy produced unidentified radical species that retarded the inactivation of voltage-gated Na+ currents irreversibly (Higure Y et al. 2003). Here, we show that superoxide dismutase, an enzyme that decomposes superoxide, reverses the retardation effect, which assures that superoxide is the unidentified radical species. The estimated mean lifetime of superoxide in recording electrodes (in the absence of cytoplasm) is approximately 6 min, and hence, the Na+ currents are retarded even in the dark, when LY is exposed to light before being loaded into the cell. Superoxide has no effect on voltage-gated Cl- currents. These results show that superoxide action on ion channels is rather selective. The breakdown of superoxide inside cells and the effect of endogenous superoxide on the superoxide-susceptible channels are discussed.

Superoxide modifies AMPA receptors and voltage-gated K+ channels of mouse hippocampal neurons.

Lucifer Yellow CH (LY), a fluorescent membrane-impermeable cell-marker dye, has been routinely loaded into cells through recording electrodes to visualize these cells after electrophysiological investigation. Recently we showed that LY produced superoxide when LY was exposed to light at ordinary intensities for microscopy, and that the resultant superoxide retarded the inactivation of voltage-gated Na+ channels even in the dark. Here, we show that superoxide produced by exposure to light prolongs the duration of action potentials, and increases the magnitude of outward rectifier K+ currents and inward rectifier K+ currents in cultured mouse hippocampal neurons. Superoxide also increases the current response of AMPA receptors, but has no effect on that of kainate and NMDA receptors, GABA(A) receptors, high-voltage activated Ca2+ channels of the hippocampal neurons, nor on 5HT3 receptors of N1E-115 cells. These superoxide effects are irreversible. The addition of superoxide dismutase, an enzyme that selectively decomposes superoxide, to LY-loaded recording electrodes reverses the superoxide effects, but addition of heat-inactivated superoxide dismutase fails to reverse the effects. The application of dithiothreitol, a free radical scavenger, to a bathing solution also reverses the superoxide effects. This shows that superoxide rather selectively modifies ion channels. The effects of exogenous and endogenous superoxide on the superoxide-susceptible channels are discussed.

Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840.

In order to understand its mechanism of action we have begun an effort to better define the cellular target of action of the experimental antitumor agent DMP 840 (NSC D640430; (R,R)-2,2'-(1,2-ethanediylbis(imino-(1-methyl-2,1-ethanediyl)))-bi s(5- nitro-1H-benz(de)isoquinoline-1,3-(2H)-dione) dimethanesulfonate). Using a combination of gentle cell fractionation procedures and a previously unidentified photochemical crosslinking reaction, we have shown that after the drug is added to cultured Clone A cells, more than 80% of the drug that is found associated with cells partitions to the chromatin-containing structural framework of the cell and that the primary target after crosslinking with 360 nm light is DNA. While DMP 840 photoreacts quite efficiently with purified RNA in vitro, no photoattachment of the drug to RNA was observed in cells. In vitro photochemical studies also reveal that while GC-rich DNA is a preferred target for drug interaction, AT-rich DNA is more active in the photochemical crosslinking reaction. These results suggest that DMP 840 probably kills cells by interfering with DNA-metabolic processes, and that the drug and its derivatives are likely to be useful photoactive molecular probes for investigating higher order chromatin structures in cells.

Two-photon ionisation of the antitumor drug pazelliptine (BD40) by 355 nm laser photolysis.

The effect of 2 ns pulses of 355 nm laser light on aqueous solutions of pazelliptine (PZE) was investigated and biphotonic ionization was observed. The absorption spectrum corresponding to the pazelliptine radical cation (PZE+) and the hydrated electron simultaneously formed in this process was determined. In the absence of oxygen, eaq- reacted with unexcited PZE (k = 1.6 x 10(10) M-1 s-1) to give the pazelliptine radical anion (PZE-). This latter species was identified by separate pulse radiolysis experiments. The radicals cation and anion disappeared by recombination on the millisecond time range. In presence of oxygen, eaq- was scavenged by O2 leading to the formation of the superoxide radical (O2.-) in competition to the formation of the radical PZE.-.PZE+ reacted with O2.- to produce H2O2 (k = 9 x 10(9) M-1 s-1). The spectral analysis revealed that PZE triplet was also formed during the laser pulse. In the absence of oxygen, the triplet-triplet absorption decreased on the microsecond time scale (2k = 1.5 x 10(10) M-1 s-1). In oxygenated solutions, eaq- and the pazelliptine triplet decayed exponentially in the same time range.

Development and validation of a stability-indicating LC method for determining palonosetron hydrochloride, its related compounds and degradation products using naphthalethyl stationary phase.

A selective and simple reversed phase HPLC method using naphthalethyl stationary phase was developed and validated for the quantitative determination of palonosetron hydrochloride (PALO), its related compounds and degradation products. Chromatographic separation (R(s)>2) was achieved with linear gradient mode of elution at a flow rate of 1 mL/min and with UV detection at 210 nm. The intra and inter-day coefficients of variation were less than 1.0% (RSD). Consistent recoveries were obtained for PALO (99.2-100.5%) and its impurities (90.0-104.8%). All the analytes exhibited excellent linearity with R² value greater than 0.998. Limit of detection (LOD) and limit of quantification (LOQ) were determined to be in the range 0.011-0.013 µg/mL and 0.035-0.046 µg/mL respectively. The test solution was found to be stable up to 5 days. Induced degradation methods were applied to study the degradation behavior of the drug. LC-MS was used to analyze the degraded samples and possible structural identifications were assigned based upon known reactivity of the drug and molecular weights. The m/z values matched with the hydroxylated, keto and N-oxide metabolites of PALO. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.9%.

PATIC: a conformationally constrained photoisomerizable amino acid.

The synthesis of a conformationally constrained photoisomerizable amino acid, phenylazo-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (PATIC), is described. This amino acid can be incorporated into peptides using standard Fmoc procedures and can be accommodated within alpha-helical structures albeit with some loss of stability of the structure. PATIC can serve as a useful building block for the synthesis of photoregulated peptides and proteins.

Photolability evaluation of the new cytostatic drug mitonafide.

A qualitative and quantitative study on the stability of the new cytostatic drug mitonafide (N-[2-(dimethyl-amino) ethyl]-3-nitronaphthalimide, CAS 54824-17-8) against UVA, UVC and visible radiations was carried out. Initially a test with controlled lighting on samples from mitonafide solution is carried out. This test include the determination of the protector effect of different kinds of glasses (clear and amber glass). The results achieved are verified by means of a test in normal lighting conditions (direct sun light, normal laboratory lighting and darkness). High mitonafide photodecomposition, deeper against UVA radiation, requires conservation of raw material in darkness. Similarly UV sterilizing radiations must be avoided in sterile rooms during manufacture. The use of amber glass ampoules is not enough to protect parenteral solutions from radiations. Direct sunlight must be avoided in the manufacture, control tests and administration in perfusion of pharmaceutical dosage forms, although artificial light can be used during short periods of time.

Laser chemotherapy of human carcinoma cells with three new anticancer drugs.

A new experimental therapy for squamous carcinoma was tested by sensitizing human tumor cells with light-sensitive anticancer drugs followed by laser illumination at visible or infrared wavelengths. The anthrapyrazole DUP-941 and the isoquinoline derivative DUP-840 were compared with the dianthraquinone hypericin. P3 human squamous carcinoma cells were incubated for 2 h with the drugs at escalating doses ranging from 5 to 100 micrograms/ml, then exposed to visible green 532-nm or infrared 1064-nm light at 300 J output from a KTP/Nd:YAG laser. Tumor cell toxicity measured by in vitro MTT viability assays was minimal after DUP-840 uptake but was slightly enhanced by infrared laser emissions. By contrast, the strong tumoricidal effects seen after DUP-941 uptake were amplified over 10-fold by 532-nm light and up to 2-fold by 1064-nm light. Hypericin-sensitized tumor cells were killed after 532 nm irradiation even at the lowest drug dose but were not affected by 1064-nm illumination. The results suggest that laser chemotherapy with drugs sensitive to photothermal energy could become a useful new treatment modality for cancer.