Interaction of procarbazine with calf thymus DNA-a biophysical and molecular docking study.

Interaction of procarbazine (PCZ) with calf thymus DNA was studied using biophysical and molecular docking studies. Procarbazine was to interact with DNA with a binding constant of 6.52 × 103  M-1 as calculated using ultraviolet-visible spectroscopy. To find out the binding mode, molecular docking was performed that predicted PCZ to interact with DNA through groove binding mode with binding affinity of -6.7 kcal/mole. To confirm the groove binding nature, different experiments were performed. Dye displacement assays confirmed the non-intercalative binding mode. Procarbazine displaced Hoechst dye from the minor groove of DNA while it was unable to displace intercalating dyes. There was no increase in the viscosity of DNA solution in presence of PCZ. Also, negligible change in the secondary structure of DNA was observed in presence of PCZ as evident by circular dichroism spectra. Procarbazine caused decrease in the melting temperature of DNA possibly because of decrease in the stability of DNA caused by groove binding interaction of PCZ with DNA.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC. Also it is known as an in vitro and in vivo mutagen and genotoxicant. However, the molecular mechanism by which procarbazine induces mutations is not thoroughly understood and the spectrum of procarbazine-induced in vivo mutations is described insufficiently. We employed flow cytometry-based erythrocyte and T lymphocyte assays in order to quantify the frequencies of cells deficient in glycosylphosphatidyl inositol-anchored surface markers CD59 and CD48 (presumed mutants in the endogenous X-linked Pig-a gene) in rats. The rats were treated once daily with 100 mg/kg procarbazine HCl for 3 days. In addition, we sorted mutant-phenotype spleen T cells and immediately analysed their Pig-a gene using next generation sequencing of dual-indexed multiplex libraries and error-correcting data filtering. More than 100-fold increase in the frequencies of CD59-deficient RBCs was observed at Day 29 after the last administration, and a 10-fold increase in the frequency of CD48-deficient T cells was observed at Days 45 to 50. Sequencing revealed that, in T cells from procarbazine-treated rats, mutations in the Pig-a gene occurred predominantly at A:T basepairs when A was located on the non-transcribed DNA strand. A→T transversion was the most common mutation. Our results suggest that, at least for the transcribed X-linked Pig-a gene, in vivo methyl guanine adducts are not the major contributors to mutations induced by procarbazine.

Ultra-trace speciation analysis of Cr(III) and Cr(VI) in rice using species-specific isotope dilution and HPLC-ICP-MS.

The study aims to clarify the current controversy related to conflicting reports on whether presence of Cr(VI) in rice is possible or not. For this purpose, a method was employed for the single run speciation analysis of Cr(III) and Cr(VI) in rice samples using species-specific isotope dilution (SS-ID) and high performance liquid chromatography coupled to inductively coupled mass-spectrometry (HPLC-ICP-MS) and selective single run species complexation/derivatisation. The quantification limits (LOQs) were 0.014 µg kg-1 for Cr(III) and 0.047 µg kg-1 for Cr(VI), while the detection limits (LODs) were 0.004 and 0.014 µg kg-1 for Cr(III) and Cr(VI), respectively. A total of 10 rice samples of different origin and colour (depending on the type of industrial processing) were analysed in this study. The content of Cr(VI) was below the limit of quantification in all of the rice samples analysed, while the Cr(III) levels ranged between 0.59 (whole grain rice) up to 104 µg kg-1 (brown rice). All samples were also analysed for their total Cr (Crtotal) content by ICP-MS solely and the results were in all cases comparable with the Cr(III) levels determined in the same samples. To assess the stability of Cr(III) and Cr(VI) in rice, one sample was spiked with Cr(III) and Cr(VI) (individually) at different levels (5.0, 10, 15 and 20 µg kg-1), held for 2 h, and then analysed by SS-ID HPLC-ICP-MS. The results showed a complete reduction of Cr(VI) to Cr(III), while Cr(III) remained stable at all spiking levels. These findings support the general statement from the European Food Safety Authority related to the complete absence of Cr(VI) in foods and confirms that Cr in rice is found solely as Cr(III) species.

New Acylhydrazone Photoswitches with Quantitative Conversion and High Quantum Yield but without Hydrogen Bond Stabilizing ( Z)-Isomer.

Hydrazones are recently attracting increasing interest because of their facile synthesis and high addressability, fatigue resistance, and modifiability as molecular switches. However, this new class of switches generally suffers from low conversion from E- to Z-configuration. Here, novel benzoylhydrazones were synthesized by condensation of 2-methoxynaphthaldhyde and benzoylhydrazine. In this hydrazone system, both sides of the imine double bond had large steric hindrance, so that the ( E)-isomer of the benzoylhydrazones was less stable and easily converted into the ( Z)-isomer even without an intramolecular hydrogen bond. Up to 99% conversion efficiency and 89% quantum yield were obtained, in addition to excellent addressability and high fatigue resistance. Outstandingly, the crystal structure of one ( Z)-isomer disclosed no intermolecular hydrogen bonds between the molecules of the ( Z)-isomer but strong and sequential hydrogen bonds between those of the ( E)-isomer. Therefore, the ( E)-isomer was less soluble in solvents than the ( Z)-isomer. This molecular switch system could be easily modified by both hydrophilic pentaethylene glycol chains and hydrophobic octyl chains. Under light irradiation, the resultant amphiphilic acylhydrazone could be transferred from ( E)-isomer to ( Z)-isomer in more than 90% yield even in water after light irradiation. Meanwhile, the self-assembled big nanospheres could rearrange into much smaller vesicles because of the solubility difference of ( Z)- and ( E)-isomers. After the anticancer drug procarbazine was loaded by this kind of acylhydrazone in water, it could be released by light irradiation, showing potential application in photocontrollable drug release.

Chemical reactivity and adsorption properties of pro-carbazine anti-cancer drug on gallium-doped nanotubes: a quantum chemical study.

In this study, we propose new armchair single-walled nanotubes (SWNTs) for stable adsorption, increasing drug delivery performance and decreasing side effects of pro-carbazine (Pro-CB) anti-cancer in the framework of B3LYP/6-31 g*/Lanl2DZ level of theory. Indeed, doping gallium (Ga) metal in SWNTs is naturally followed by changing of geometry, increasing dipole moment, and creating one site with high reactivity in order to better adsorption of the drug molecule. Chemical reactivity descriptors show that SWNTs and Pro-CB have electrophile and nucleophile roles in interaction, respectively. More importantly, high local and dual softness in Ga-doped SWNTs indicate improvement of drug adsorption. Parallel and perpendicular complexes result from their interaction in the N and the O sites. Negative values of binding energy (Ebind) show that composed complexes are energetically stable especially in the O site in comparison with the N site. On the other hand, more negative value of the Ebind in SWCNTs shows that these nanotubes are more effective for drug adsorption than their boron nitride counterparts. Graphical abstract The Ga dopping results in reducing of HOMO-LUMO gap and increasing charge transfer between SWNTs and Pro-CB, and formation better complex, especially SWCNT.

Procarbazine--a traditional drug in the treatment of malignant gliomas.

The methylhydrazine derivative Procarbazine (PCZ) as monotherapy or in combination with CCNU and vincristine (PCV) was evaluated in a vast number of clinical trials and is still used in patients with high-grade and low-grade gliomas. The compound is an antineoplastic agent with multiple sites of action. It inhibits incorporation of small DNA precursors, as well as RNA and protein synthesis. PCZ can also directly damage DNA through an alkylation reaction. The drug is not cross-resistant with other mustard-type alkylating agents. As PCZ was in almost all trials used in a combination with CCNU and Vincristin, the efficacy can only be evaluated in the view of the PCV regimen. The published data suggest a role of PCV as a salvage regimen, especially in oligodendroglial tumors; however, well designed studies with high evidence are rare in all entities. This article summarizes the existing data with the goal to define the role of PCZ/PCV in modern neurooncology.

Indirect oxidation of the antitumor agent procarbazine by tyrosinase--possible application in designing anti-melanoma prodrugs.

The interaction of tyrosinase with the anticancer drug procarbazine has been investigated. In the presence of the enzyme alone no oxidation of this dialkylhydrazine above the background level was observed. However, when phenolic substrates (4-tert-butylcatechol or N-acetyl-l-tyrosine) were included in the reaction mixture, procarbazine was rapidly degraded. Oxygen consumption measurements showed that in a mixture both the phenolic substrate and the drug were oxidized. The major product of procarbazine degradation was isolated and identified as azoprocarbazine, the first active metabolite of this drug detected in previous in vivo and in vitro studies. This indirect oxidation of the hydrazine group in this anticancer agent indicates possible application of a hydrazine linker in construction of tyrosinase-activated anti-melanoma prodrugs.

Reactions of OH-radicals with procarbazine. A pulse radiolysis and computer simulation study.

Pulse radiolysis was applied to study the reactivity of *OH radicals with procarbazine (PC), a cytostatic agent widely used in radiation- and chemotherapy. An overall rate constant of k(*OH+PC) = 3.7 x 10(9) l.mol(-1).s(-1) was determined. The thereby formed transients had a strong absorption at 350 nm, epsilon350 = 4.46 x 10(3) l.mol(-1).cm(-1), and a weak absorption band around 530 nm. Computer simulation studies to elucidate the most probable sites of *OH attack on the PC molecule showed that *OH radical addition to the aromatic ring had the highest probability. These transients decayed by a first order reaction, k = 1.75 x 10(3) s(-1), whereby species having a maximum absorption at 300 nm and broad shoulder at 340-380 nm were formed. Similar absorptions were observed after gamma radiolysis of PC. A reaction mechanism is suggested. For the reaction of H-atoms with PC, a rate constant k(*H+PC) = 6.4 x 10(8) l.mol(-1).s(-1) was determined.

Procarbacine radiolysis and experiments in vitro.

Steady-state radiolysis of aqueous procarbazine (PC) was studied in air-free, aerated and solutions saturated with N2O. The corresponding Gi(-PC)-values obtained at pH=7.4 were: 2.85, 5.60 and 3.45, respectively. The investigations in vitro, using E. coli (AB 1157) as a model for living systems, demonstrated that PC acts as a cytostatic in air-free as well as in aerated media. However, it shows radiation protecting ability in the presence of N2O, where OH-radicals are the predominant reactants. Similar results were observed at pH=6.2. The experimental data contribute to a better understanding of the many-sided and frequently contradictory behavior of PC.

Methoxyphenylcipro induces antitumor activity in human cancer cells.

To examine the antitumor activity of a new derivative of ciprofloxacin called methoxyphenylcipro (CMPP). Cell viability was assessed using the MTT assay and apoptotic cells and reactive oxygen species were evaluated using flow cytometry. Results revealed that CMPP induces antiproliferative activity against breast cancer cells and melanoma and to a lesser extent against colorectal cancer cells. Interestingly, compared to ciprofloxacin, CMPP-induced a selective cytotoxicity against human cancer cells but not human normal fibroblasts. The potential of CMPP to inhibit cellular growth in MD-MB-486 breast cancer cells and MV3 melanoma cells was largely due to induction of caspase-dependent apoptosis, as confirmed by caspase-3 activation and cleavage of its substrate PARP. In addition, results indicated that CMPP-induced apoptosis is mediated by generation of reactive oxygen species. These findings revealed that CMPP has a selective antitumor activity against cancer cells and warrants further clinical evaluation.

Functionalization of PEGylated Fe3O4 magnetic nanoparticles with tetraphosphonate cavitand for biomedical application.

In this contribution, Fe3O4 magnetic nanoparticles (MNPs) have been functionalized with a tetraphosphonate cavitand receptor (Tiiii), capable of complexing N-monomethylated species with high selectivity, and polyethylene glycol (PEG) via click-chemistry. The grafting process is based on MNP pre-functionalization with a bifunctional phosphonic linker, 10-undecynylphosphonic acid, anchored on an iron surface through the phosphonic group. The Tiiii cavitand and the PEG modified with azide moieties have then been bonded to the resulting alkyne-functionalized MNPs through a "click" reaction. Each reaction step has been monitored by using X-ray photoelectron and FTIR spectroscopies. PEG and Tiiii functionalized MNPs have been able to load N-methyl ammonium salts such as the antitumor drug procarbazine hydrochloride and the neurotransmitter epinephrine hydrochloride and release them as free bases. In addition, the introduction of PEG moieties promoted biocompatibility of functionalized MNPs, thus allowing their use in biological environments.

Determination of terephthalic acid isopropylamide in urine with a liquid chromatography/mass spectrometry (LC/MS) method.

A sensitive and simple liquid chromatography/mass spectrometry (LC/MS) method was developed for the determination of terephthalic acid isopropylamide, the final metabolite of procarbazine in human urine. A solid-phase extraction with C(18) cartridges was used followed by LC/MS with a single mass spectrometer (SSQ 7000 from Finnigan). Terephthalic acid isobutylamide was the internal standard. The quantification limit was 30 ng/mL in urine (6 x noise). This assay was applied for drug monitoring of terephthalic acid isopropylamide in urine after oral administration of procarbazine in children and adolescents with Hodgkin lymphomas.

Isolation, purification, and characterization of two new chemical decomposition products of methylazoxyprocarbazine.

We have previously reported that the antineoplastic agent, procarbazine, in aqueous solutions was chemically oxidized to its azoxy metabolites (methylazoxy and benzylazoxy). To determine if there was additional metabolism of the most active metabolite, methylazoxyprocarbazine, it was incubated in the presence and absence of CCRF-CEM human leukemia cells. Incubations were extracted, and potential metabolites were detected by HPLC with UV detection and by combined HPLC and thermospray mass spectrometric analysis. The major metabolite identified by HPLC with UV detection of the extracts was N-isopropyl-p-formylbenzamide; this was identified by comparison of its retention time with that of a synthesized standard. This identification was further corroborated by HPLC/thermospray mass spectrometry (LC/MS). Analysis of the extracts by LC/MS also showed the presence of a closely eluting peak that had a protonated molecular ion at m/z 207. This new metabolite was identified as N-isopropyl-(benzene-1,4-bis-carboxamide) by 1H NMR and gas chromatography/ion trap mass spectrometry. This metabolite is postulated to arise from breakage of the N-N bond in the hydrazine portion of the molecule. Reconstructed ion (m/z 236) current profiles from the analysis of the cell extracts indicated that there was only a trace amount of methylazoxyprocarbazine left after a 72-hr incubation. Interestingly, a peak with the same molecular weight as the parent compound (methylazoxyprocarbazine) was observed in the cellular incubations and also in extracts of control incubations in which methylazoxyprocarbazine was incubated in medium without cells. This unknown was silylated and identified as a hydroxyazo compound by an ion trap mass spectrometer operated under both single and multiple-stage mass analysis. Formation of this decomposition product appears to involve a novel intramolecular rearrangement of methylazoxyprocarbazine in solution. This pathway may be responsible for the formation of the ultimate cytotoxic species by chemical decomposition of procarbazine.