V. Nastasa

Exposure of Chlorpromazine to 266 nm Laser Beam Generates New Species with Antibacterial Properties: Contributions to Development of a New Process for Drug Discovery

Introduction Phenothiazines when exposed to white light or to UV radiation undergo a variety of reactions that result in degradation of parental compound and formation of new species. This process is slow and may be sped up with exposure to high energy light such as that produced by a laser. Methods Varying concentrations of Chlorpromazine Hydrochloride (CPZ) (2–20 mg/mL in distilled water) were exposed to 266 nm laser beam (time intervals: 1–24 hrs). At distinct intervals the irradiation products were evaluated by spectrophotometry between 200–1500 nm, Thin Layer Chromatography, High Pressure Liquid Chromatography (HPLC) - Diode Array Detection, HPLC tandem mass spectrometry, and for activity against the CPZ sensitive test organism Staphylococcus aureus ATCC 25923. Results CPZ exposure to 266 nm laser beam of given energy levels yielded species, whose number increased with duration of exposure. Although the major species produced were Promazine (PZ), hydroxypromazine or PZ sulfoxide, and CPZ sulfoxide, over 200 compounds were generated with exposure of 20 mg/mL of CPZ for 24 hrs. Evaluation of the irradiation products indicated that the bioactivity against the test organism increased despite the total disappearance of CPZ, that is due, most probably, to one or more new species that remain yet unidentified. Conclusions Exposure of CPZ to a high energy (6.5 mJ) 266 nm laser beam yields rapidly a large number of new and stable species. For biological grade phenothiazines (in other words knowing the impurities in the samples: solvent and solute) this process may be reproducible because one can control within reasonably low experimental errors: the concentration of the parent compound, the laser beam wavelength and average energy, as well as the duration of the exposure time. Because the process is “clean” and rapid, it may offer advantages over the pyrogenically based methods for the production of derivatives.

Direct modification of bioactive phenothiazines by exposure to laser radiation

Whereas exposure of combinations of a phenothiazine and bacterium to incoherent UV increases the activity of the phenothiazine, exposure of the phenothiazine alone does not yield an increase of its activity. Because the laser beam energy is greater than that produced by the incoherent UV sources, exposure of phenothiazines to specific lasers may yield molecules with altered activities over that of the unexposed parent. Chlorpromazine, thioridazine and promethazine active against bacteria were exposed to two distinct lasers for varying periods of time. Absorption and fluorescence spectra were conducted prior to and post-exposure and the products of laser exposure evaluated for activity against a Staphylococcus aureus ATCC strain via a disk susceptibility assay. Exposure to lasers alters the absorption/fluorescence spectra of the phenothiazines; reduces the activity of thioridazine against the test bacterium; produces a highly active chlorpromazine compound against the test organism. Exposure of phenothiazines to lasers alters their structure that results in altered activity against a bacterium. This is the first report that lasers can alter the physico-chemico characteristics to the extent that altered bioactivity results. Exposure to lasers is expected to yield compounds that are difficult to make via chemical manipulation methods. A survey of selected patents of interest, even co-lateral for the subject of this article is shortly made.

Characterization of mixtures of compounds produced in chlorpromazine aqueous solutions by ultraviolet laser irradiation: their applications in antimicrobial assays

Abstract. The study reports an investigation of the photoproducts obtained by exposure of chlorpromazine hydrochloride in ultrapure water (concentration 2  mg/mL) to a 266-nm laser beam obtained by fourth harmonic generation from a Nd:YAG laser (6-ns full time width at half maximum, 10-Hz pulse repetition rate). The photoproducts were analyzed by steady-state UV-Vis absorption, laser-induced fluorescence, Fourier transform infrared spectroscopy, and liquid chromatography–tandem time-of-flight mass spectroscopy. Two figures showing pathways that take place during irradiation for obtaining the final products are shown. The quantum yield of singlet oxygen generation by chlorpromazine (CPZ) was determined relative to standard Zn-phthalocyanine in dimethyl sulfoxide. To outline the role of fluorescence in photoproducts formation rates, fluorescence quantum yield of CPZ during exposure to 355-nm radiation (third harmonic of the fundamental beam of Nd:YAG laser) was investigated relative to standard Coumarin 1 in ethanol. The CPZ solutions exposed 60 and 240 min to 266-nm laser beam, respectively, were tested against Staphylococcus aureus ATCC 25923 strain. For 25  μL of CPZ samples irradiated 240 min, a higher diameter of inhibition has obtained against the tested strain than for the 60-min exposed ones.

Enhanced fluorescence emitted by microdroplets containing organic dye emulsions

In this paper, laser beam resonant interaction with pendant microdroplets that are seeded with a laser dye (Rhodamine 6G (Rh6G)) water solution or oily Vitamin A emulsion with Rhodamine 6G solution in water is investigated through fluorescence spectra analysis. The excitation is made with the second harmonic generated beam emitted by a pulsed Nd:YAG laser system at 532 nm. The pendant microdroplets containing emulsion exhibit an enhanced fluorescence signal. This effect can be explained as being due to the scattering of light by the sub-micrometric drops of oily Vitamin A in emulsion and by the spherical geometry of the pendant droplet. The droplet acts as an optical resonator amplifying the fluorescence signal with the possibility of producing lasing effect. Here, we also investigate how Rhodamine 6G concentration, pumping laser beam energies and number of pumping laser pulses influence the fluorescence behavior. The results can be useful in optical imaging, since they can lead to the use of smaller quantities of fluorescent dyes to obtain results with the same quality.

Optical investigation of medicine solutions in micro-droplets form at interaction with laser radiation

One of the alternatives to the existing medicines and treatment procedures in fighting multi drug resistance (MDR) is strengthening the effects of medicines by modifying their molecular structures through exposure to laser radiation. A method associated with this, is the generation of micro-droplets which contain medicines solutions; the droplets are utilized/produced as vectors to transport the medicines to targets. In our studies we try to combine these two methods in order to obtain a new technique to deliver the efficient medicines to targets that can be applied for a relative large number of chemicals. For this purpose we have developed an experimental set-up containing a liquid droplets generator, a tunable laser source used to irradiate droplets, a subunit to measure the laser induced fluorescence (LIF) signals and a real time recording system for droplet image analysis. Measurements on different probes, like ultrapure water, commercial grade medicines, newly developed medicines and laser dyes were performed.. All these measurements were performed on waterbased solutions. We present in this paper the laser induced fluorescence measurements results on medicine solutions (in bulk or in a micro-droplet form) that exhibit important modifications after the exposure at laser radiation. It was evidenced that the exposures to laser beams/coherent optical radiation of some medicines solutions in ultrapure water may produce molecular modifications in solutions. These slight modifications of the molecules made them more efficient against bacteria strains.

Laser Optofluidics in Fighting Multiple Drug Resistance

The interaction of laser modified medicine solutions with hydrophilic and hydrophobic target surfaces has been investigated under the effect of simulated hypergravity conditions, employing the Large Diameter Centrifuge (LDC) facility, developed by the European Space Agency (ESA). Experiments have been performed within the HyperMed project under the aegis of the ESA “Spin Your Thesis!” 2015 programme. During centrifugation, real-time video files have been recorded regarding generation of ultrapure water, unexposed and laser exposed chlorpromazine aqueous pendant droplets, followed by their detachment due to the exerted high gravitational accelerations and finally by the formation of sessile droplets on target surfaces. In this way, information about the volume of the generated droplet, the degree of wetting and its time evolution at different hypergravity levels has been obtained. Phenothiazine solutions irradiated with UV laser radiation indicate reduced surface tension, thus presenting better wetting properties. Target surfaces impregnated with medicine solutions may constitute an unconventional tool and even vector in developing new drug delivery systems. Such a wetting process under high g-level conditions may be useful in space medicine applications. Microorganisms can survive, grow and even proliferate under the effect of increased gravity. Therefore, upon launching of a spacecraft, during a long-term mission in microgravity conditions, astronauts and spacecraft surfaces may require treatment and decontamination, respectively, against onboard infectious microbes. Since non-terrestrial gravity may alter drug properties, medicine droplets behaviour in interaction with target surfaces under hypergravity conditions is the aim of the present study.

Stability studies on Promethazine unexposed and exposed to UV laser radiation

As a phenothiazine derivative, Promethazine may undergo structural modifications when it is exposed to light. This process consists in the degradation of the initial compound and in the generation of new photoproducts with possible anti-infectious qualities. Stability studies are necessary in order to establish the proper use of drug solutions in different applications. At the same time, these investigations are important in the context of the generation of side-products induced by environmental conditions that bring new benefits to the compound. This study reports the stability of Promethazine aqueous solutions, based on their absorption spectra acquired before and after Nd:YAG laser irradiation sessions or under different temperature and illuminating storage conditions. Samples of Promethazine solutions in ultrapure water, at a concentration range between 10-6 M – 10-2 M, were kept in dark at 22°C, and 4°C as well as at 22°C in ambient light up to a time interval of three months. Absorption spectra were recorded periodically in order to determine any changes of the optical properties. Also, solutions of 20 mg/mL were exposed for different time intervals to laser radiation emitted at 266 nm by the Nd:YAG laser. The stability of the optical properties of irradiated Promethazine solutions for 4 h was investigated up to two months. The laser irradiated samples show similar but more rapid and intense changes compared to solutions exposed to ambient light, suggesting molecular modifications that could be due to the production of more polar phenothiazine derivatives.

Generation and biological evaluation of the products formed from the exposure of Phenothiazine to a 266nm laser beam

Phenothiazine exposed to white light or UV radiation undergoes a variety of reactions that result in the degradation of the parental compound and the formation of new species. Chlorpromazine exposed to the 266 nm laser beam of given energy levels yielded species derived from it, whose number increased with the exposure duration. At distinct time intervals the irradiation products were evaluated by spectrophotometry between 200-1500 nm, Thin Layer Chromatography, and for antimicrobial activity of Chlorpromazine against different test organisms such as Staphylococcus aureus.

Study of Commercial Grade Aetoxisclerol by Optical Means, in View of Its Use in Varicose Vein Treatment

Aetoxisclerol (or Polidocanol in usual medical terms) is an intermediate sclerosing agent that contains a hydrophilic and a hydrophobic pole and acts by altering the surface tension at the interface between the endothelial cells and their environment. The understanding of the interaction between the Polidocanol and the target veins (tissues) becomes an important factor in utilizing it in varicose veins disease. More, it seems that the exposure of the tissues impregnated with this medicine to laser radiation emitted at 1.06 μm improves the efficiency of the treatment. Because the commercially available Aetoxisclerol is a mixture of substances, when it is exposed to laser radiation, one should consider, actually the interaction with all the compounds. The absorption spectra in UV‐VIS‐NIR spectral ranges are measured for Aetoxisclerol. We exposed it at pulsed Nd:YAG laser beam between 2–30 min at known irradiation doses. The possible mechanisms implied in improvement of the efficiency of the irradiated Polid...

Generation of micro- and nano-droplets containing immiscible solutions in view of optical studies

The multiple resistances to treatment, developed by bacteria and malignant tumors require finding alternatives to the existing medicines and treatment procedures. One of them is strengthening the effects of cytostatics by improving the delivery method. Such a method is represented by the use of medicines as micro/nano-droplets. This method can reduce the substance consumption by generating drug micro-droplets incorporated in substances that can favour a faster localization, than the classical mode of medicine administration, to the tumor tissues. This paper contains the results concerning the generation and study of micro/nano-droplets and the generation of micro-droplets with an inner core (medicine) and a thin layer covering it. We have measured the surface tension at water/air interface and water/oil interface for a medicine (Vancomycin) and we have generated and measured droplets of medicine containing a layer of Vitamin A by using a double capillary system. The micro/nano-droplets may be produced by mixing of two immiscible solutions in particular conditions (high rotating speed and/or high pressure difference). For this we have studied the generation of emulsions of vitamin A diluted in sunflower oil and a solution of a surfactant Tween 80 in distilled water. The concentration of surfactant in water was typically 4*10-5M. We have studied in a batch stirred tank system the dependence of the droplet dimensions in emulsion, function of the mixing rotation speed, agitation time and components ratio. The droplet diameters were measured using a Malvern light scattering instrument type Mastersizer Hydro 2000M. We have obtained droplets with diameters smaller than 100 nm; the diameters distribution exhibited a peak at 65 nm.