Anti-staphylococcal activity and mode of action of thioridazine photoproducts.

Antibiotic resistance became an increasing risk for population health threatening our ability to fight infectious diseases. The objective of this study was to evaluate the activity of laser irradiated thioridazine (TZ) against clinically-relevant bacteria in view to fight antibiotic resistance. TZ in ultrapure water solutions was irradiated (1-240 min) with 266 nm pulsed laser radiation. Irradiated solutions were characterized by UV-Vis and FTIR absorption spectroscopy, thin layer chromatography, laser-induced fluorescence, and dynamic surface tension measurements. Molecular docking studies were made to evaluate the molecular mechanisms of photoproducts action against Staphylococcus aureus and MRSA. More general, solutions were evaluated for their antimicrobial and efflux inhibitory activity against a panel of bacteria of clinical relevance. We observed an enhanced antimicrobial activity of TZ photoproducts against Gram-positive bacteria. This was higher than ciprofloxacin effects for methicillin- and ciprofloxacin-resistant Staphylococcus aureus. Molecular docking showed the Penicillin-binding proteins PBP3 and PBP2a inhibition by sulforidazine as a possible mechanism of action against Staphylococcus aureus and MRSA strains, respectively. Irradiated TZ reveals possible advantages in the treatment of infectious diseases produced by antibiotic-resistant Gram-positive bacteria. TZ repurposing and its photoproducts, obtained by laser irradiation, show accelerated and low-costs of development if compared to chemical synthesis.

Experimental and in silico assessment of fate and effects of the antipsychotic drug quetiapine and its bio- and phototransformation products in aquatic environments.

The antipsychotic drug quetiapine (QUT) has been frequently detected in sewage treatment plants. However, information on the fate of QUT in aquatic environments and its behavior during UV treatment is limited. In this study, QUT is shown not to be readily biodegradable in the Closed Bottle Test and the Manometric Respirometry Test according to OECD guidelines. The main biotransformation product (BTP) formed in the tests, a carboxylic acid derivative, was identified by means of high-resolution mass spectrometry. This BTP is presumably a human metabolite and showed higher detection rates than QUT in a river sampling campaign conducted in northern Germany. UV elimination kinetics of QUT at different initial concentrations (226.5, 45.3, 11.3, and 2.3 µmol L-1) were faster at lower initial concentrations. All seven phototransformation products (PTPs) could be still identified at initial concentration of 11.3 µmol L-1. The photolytic mixture generated after 128 min of photolysis of QUT was not better biodegradable than QUT. Initial UV treatment of QUT led to the formation of several additional BTPs. Four of them were identified. The bacterial cytotoxicity and genotoxicity before and after phototransformation of QUT in a modified luminescent bacteria test (LBT) and the umu-test (ISO/FDIS 13829) showed cytotoxic effects in the LBT for QUT. Furthermore, PTPs had similar cytotoxic effects on luminescent bacteria. The umu-test did not reveal any genotoxic activity for QUT or PTPs. In conclusion, the release of QUT into sewage treatment plants and aquatic environments could result in the formation of a main BTP. Additional UV treatment of QUT would lead to the formation of additional BTPs. Moreover, treatment did not result in lower toxicity to tested organisms. In conclusion, UV treatment of QUT should be considered critically as a potential treatment for QUT in aquatic systems.

Environmental fate and effect assessment of thioridazine and its transformation products formed by photodegradation.

An experimental and in silico quantitative structure-activity relationship (QSAR) approach was applied to assess the environmental fate and effects of the antipsychotic drug Thioridazine (THI). The sunlight-driven attenuation of THI was simulated using a Xenon arc lamp. The photodegradation reached the complete primary elimination, whereas 97% of primary elimination and 11% of mineralization was achieved after 256 min of irradiation for the initial concentrations of 500 µg L(-1) and 50 mg L(-1), respectively. A non-target approach for the identification and monitoring of transformation products (TPs) was adopted. The structure of the TPs was further elucidated using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proposed photodegradation pathway included sulfoxidation, hydroxylation, dehydroxylation, and S- and N-dealkylation, taking into account direct and indirect photolysis through a self-sensitizing process in the higher concentration studied. The biodegradability of THI and photolytic samples of THI was tested according to OECD 301D and 301F, showing that THI and the mixture of TPs were not readily biodegradable. Furthermore, THI was shown to be highly toxic to environmental bacteria using a modified luminescent bacteria test with Vibrio fischeri. This bacteriotoxic activity of THI was significantly reduced by phototransformation and individual concentration-response analysis confirmed a lowered bacterial toxicity for the sulfoxidation products Thioridazine-2-sulfoxide and Thioridazine-5-sulfoxide. Additionally, the applied QSAR models predicted statistical and rule-based positive alerts of mutagenic activities for carbazole derivative TPs (TP 355 and TP 339) formed through sulfoxide elimination, which would require further confirmatory in vitro validation tests.

Chlorpromazine transformation by exposure to ultraviolet laser beams in droplet and bulk.

Multiple drug resistance requires a flexible approach to find medicines able to overcome it. One method could be the exposure of existing medicines to ultraviolet laser beams to generate photoproducts that are efficient against bacteria and/or malignant tumors. This can be done in droplets or bulk volumes. In the present work are reported results about the interaction of 266nm and 355nm pulsed laser radiation with microdroplets and bulk containing solutions of 10mg/ml Chlorpromazine Hydrochloride (CPZ) in ultrapure water. The irradiation effects on CPZ solution at larger time intervals (more than 30min) are similar in terms of generated photoproducts if the two ultraviolet wavelengths are utilized. The understanding of the CPZ parent compound transformation may be better evidenced, as shown in this paper, if studies at shorter than 30minute exposure times are made coupled with properly chosen volumes to irradiate. We show that at exposure to a 355nm laser beam faster molecular modifications of CPZ in ultrapure water solution are produced than at irradiation with 266nm, for both microdroplet and bulk volume samples. These effects are evidenced by thin layer chromatography technique and laser induced fluorescence measurements.

Photodegradation of psychiatric pharmaceuticals in aquatic environments--kinetics and photodegradation products.

Benzodiazepines are widely consumed psychiatric pharmaceuticals which are frequently detected in the environment. The environmental persistence and fate of these pharmaceuticals as well as their degradation products is of high relevance and it is, yet, scarcely elucidated. In this study, the relevance of photodegradation processes on the environmental persistence of four benzodiazepines (oxazepam, diazepam, lorazepam and alprazolam) was investigated. Benzodiazepines were irradiated under simulated solar irradiation and direct and indirect (together with three different fractions of humic substances) photodegradation kinetics were determined. Lorazepam was shown to be quickly photodegradated by direct solar radiation, with a half-life time lower than one summer sunny day. On the contrary, oxazepam, diazepam and alprazolam showed to be highly resistant to photodegradation with half-life times of 4, 7 and 228 summer sunny days, respectively. Apparent indirect and direct photodegradation rates are of the same order of magnitude. However, humic acids were consistently responsible for a decrease in the photodegradation rates while fulvic acids and XAD4 fraction caused an enhancement of the photodegradation. Overall, the results highlight that photodegradation might not be an efficient pathway to prevent the aquatic environmental accumulation of oxazepam, diazepam and alprazolam. Also, nineteen direct photodegradation products were identified by electrospray mass spectrometry, the majority of which are newly identified photoproducts. This identification is crucial to a more complete understanding of the environmental impact of benzodiazepines in aquatic systems.

Effect of cyclodextrin derivation and amorphous state of complex on accelerated degradation of ziprasidone.

Inclusion complexes of ziprasidone with several ß-cyclodextrins [ß-CDs; sulfobutylether-ß-cyclodextrins (SBEßCD), hydroxypropyl-ß-cyclodextrins (HPßCD), methyl-ß-cyclodextrins (MßCD), and carboxyethyl-ß-cyclodextrins (CEßCD)] were prepared and solution stability was evaluated at elevated temperature. Solid-state stability was assessed by subjecting various CD complexes of ziprasidone, spray-dried dispersion (SDD), partially crystalline ziprasidone-SBEßCD salts, and the physical mixture of ziprasidone-SBEßCD to γ-irradiation. Degradant I was formed by oxidation of ziprasidone, which upon aldol condensation with ziprasidone formed degradant II in both solution and solid states. In the solution state, CD complexes with electron-donating side chains, such as SBEßCD and CEßCD, produced the highest oxidative degradation followed by HPßCD with 6, 3, and 4 degrees of substitution. In the solid state, crystalline drug substance and physical mixture of crystalline drug-SBEßCD showed very little to no degradation. In contrast, amorphous ßCD, MßCD, CEßCD, and SBEßCD complexes as well as the amorphous SDD exhibited greatest extent of oxidative degradation. Results suggest that electron-donating side chains of the derivatized CD interact with transition state of the oxidation reaction and catalyze drug degradation in solution, However, higher mobility in the amorphous state of CD-drug complexes promoted chemical instability of ziprasidone under accelerated conditions irrespective of the chemical nature of the side chain on CD.

Development of a stability-indicating UPLC method for determining olanzapine and its associated degradation products present in active pharmaceutical ingredients and pharmaceutical dosage forms.

A simple, sensitive and reproducible ultra performance liquid chromatography (UPLC) coupled with a photodiode array detector method was developed for the quantitative determination of olanzapine (OLN) in API and pharmaceutical dosage forms. The method is applicable to the quantification of related substances and assays of drug substances. Chromatographic separation was achieved on Acquity UPLC BEH 100-mm, 2.1-mm, and 1.7-µm C-18 columns, and the gradient eluted within a short runtime, i.e., within 10.0 min. The eluted compounds were monitored at 250 nm, the flow rate was 0.3 mL/min, and the column oven temperature was maintained at 27°C. The resolution of OLN and eight (potential, bi-products and degradation) impurities was greater than 2.0 for all pairs of components. The high correlation coefficient (r(2)>0.9991) values indicated clear correlations between the investigated compound concentrations and their peak areas within the test ranges. The repeatability and intermediate precision, expressed by the RSD, were less than 2.4%. The accuracy and validity of the method were further ascertained by performing recovery studies via a spike method. The accuracy of the method expressed as relative error was satisfactory. No interference was observed from concomitant substances normally added to the tablets. The drug was subjected to the International Conference on Harmonization (ICH)-prescribed hydrolytic, oxidative, photolytic and thermal stress conditions. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.

Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects.

By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

Primary steps of the photochemical reactions of 2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine, the photoproduct of cyamemazine, a phototoxic neuroleptic: comparison with the sulfoxide.

The UVA-absorbing photoproduct resulting from the oxidation of the sulfur atom and of the side chain nitrogen of the phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-phenothiazine) is a potent photodynamic photosensitizer. The photophysical and photochemical properties of this photoproduct (P) (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine)) have been investigated in neutral buffered aqueous solutions and in ethanol and compared to those of the sulfoxide (S) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-5-oxide-phenothiazine), a CMZ oxidation product of cells. The fluorescence quantum yield (PhiF) of P is 0.25 and 0.21 in pH 7 phosphate buffer and ethanol, respectively. By contrast, S (PhiF = 0.14 in buffer) is practically unfluorescent in alcohol. In buffer, the fluorescence lifetimes of P and S are 10.5 and 11.8 ns, respectively. The transient absorbance of the first excited triplet state (3P1) with a characteristic absorption band peaking at 660 nm (epsilon = 5,300 M(-1) cm(-1)) has been observed by 355 nm laser flash spectroscopy of deaerated phosphate buffer or ethanol solutions. In buffer, the 3P1 lifetime is 0.5 micros. The energy transfer which occurs from the 3P1 to naproxen suggests that the 3P1 energy is greater than 62 kcal mol(-1). Triplet quenching by dioxygen occurs at rate 2.3 x 10(9) M(-1) s(-1). With the triplet benzophenone as actinometer, the 3P1 formation quantum yield is found to be 0. 40 in buffer. The 3P1 state is quenched by ethanol and 2-propanol with bimolecular reaction rate constants of 1.6 and 2.4 x 10(6) M(-1) s(-1), respectively. In buffer, P and S triplet states react with tryptophan, indole and cysteine at rate constants of the order of 10(9) M(-1) s(-1) for Trp and indole and 10(8) M(-1) s(-1) for Cys.

Excited-state properties and in vitro phototoxicity studies of three phenothiazine derivatives.

This work concerns a combined photophysical, photochemical and photobiological study of three drugs (psychotherapeutic agents) of the phenothiazine series: perphenazine, fluphenazine hydrochloride and thioridazine hydrochloride. The excited-state properties were first investigated by stationary and time-resolved fluorimetry and by laser flash photolysis. The spectral description was assisted by quantum-mechanical calculations with the INDO/1-CI method. In organic media the lowest excited singlet state was found to decay by fluorescence (small quantum yield) and mainly by intersystem crossing to the lowest triplet state, which is responsible for oxygen photosensitization (high yields of singlet oxygen production) and photodegradation. A further decay pathway in aqueous solutions was the photoionization process, which led to the formation of the phenothiazine radical cations and the solvated electron. After the preliminary study of the photobehavior in organic solvents and in water, the phototoxicity of the three drugs was investigated on various biological substrates through a series of in vitro assays under UVA irradiation. Photohemolysis of mouse erythrocytes and phototoxicity on cultured murine fibroblasts were observed for all three compounds. Lipid photoperoxidation was then investigated using linoleic acid as the unsaturated lipid model and isolated red blood cell membranes. The drug-induced photodamage was also evaluated on proteins by measuring the photosensitizing cross-linking in erythrocyte ghosts. The combined approach proved to be useful in understanding the mechanism by which these phenothiazine derivatives induce skin photosensitization. In particular, the photophysical properties of the compounds under investigation and the results of the study on their phototoxicity are in agreement with a mechanism that involves the radical cation of the drugs as a main intermediate.

The photochemical stability of cis- and trans-isomers of tricyclic neuroleptic drugs.

The irradiation of the tranquillizers flupenthixol, clopenthixol and chlorprothixene has been found to induce rapid cis-trans isomerization. The composition of the photosatitionary mixture is not that of the batch drug and hence this process may affect the activity. Further decomposition to a thioxanthone derivative occurs rapidly in the presence of air. Exclusion of oxygen, however, does not prevent further degradation and a slower secondary isomerization is observed on prolonged irradiation. Doxepin and dothiepin also undergo analogous reactions but the isomerizations are much slower and the oxidative degradation yields many products.

Kinetics of drug decomposition. Part 46. Photooxidation and photolysis of some perazine derivatives.

The rate and type of the photochemical degradation of perazine derivatives in acidic aqueous solutions depends upon the nature of the substituent at C2 atom. Free perazine degrades by two parallel reactions, namely a fast reversible first-order photooxidation and a slow zero-order photolysis. An introduction of the substituent in the C2 position results frequently in the elimination of one of these reactions.

Kinetics of drug decomposition. Part 47. Effect of substituents on photochemical stability of perazine derivatives.

A quantum yield phi of photooxidation of perazine derivatives estimated actinometrically and the pKa, values were used for the correlation with the substituent volumes. The Hammett type plots of phi= f(sigma) and phi= f(pKa10--pKa1) are discussed.