Paweł Grobelny

Photostability of pitavastatin—A novel HMG-CoA reductase inhibitor

The photostability of pitavastatin, an HMG-CoA reductase inhibitor used in the treatment of hypercholesterolemia, was investigated. The sample solution was exposed to UV-A radiation and the photodegradation process was monitored by means of spectrophotometric method and HPLC-DAD. Pitavastatin was shown to be photolabile and its photodegradation reaction followed the first-order kinetics with the rate constant k=3.54 x 10(-4)+/-9.43 x 10(-6)s(-1). The chromatograms revealed the presence of four major photoproducts (PP-1-PP-4). The separated and isolated photolytic products were identified using a mass spectrometer coupled with a time of flight (TOF) analyzer. The main reaction observed during exposure to radiation of pitavastatin was photocyclisation leading to formation of four-ring photoproducts.

DFT study on hydroxy acid–lactone interconversion of statins: the case of fluvastatin

Fluvastatin is a member of the HMG-CoA reductase inhibitor family of drugs, commonly referred to as statins. It is generally known that, under physiological conditions, statins are susceptible to pH-dependent interconversion between their active (hydroxy acid) and inactive (lactone) forms. The mechanism of this interconversion, under both acidic and basic conditions, was investigated theoretically using the density functional theory (DFT) method. Regardless of the conditions, the lactone form was always higher in energy by 6-19 kcal mol(-1). However, under basic conditions, the activation barrier for the hydrolysis was significantly lower (9 kcal mol(-1)) than for the reverse reaction (28 kcal mol(-1)), making the lactone form unstable. The activation barriers under acidic conditions were of comparable height in both directions (22 and 28 kcal mol(-1)), making the occurrence of both forms equally probable. Due to the high activation barrier (>40 kcal mol(-1)), a one-step, direct interconversion between the two forms turned out to be unfavourable. Moreover, the potential energy surface of fluvastatin was briefly inspected, revealing relatively small energetic differences (<5 kcal mol(-1)) between the key conformers.

The Phototoxicity of Fluvastatin, an HMG-CoA Reductase Inhibitor, Is Mediated by the formation of a Benzocarbazole-Like Photoproduct

In this paper, we have investigated the mechanism of phototoxicity of fluvastatin, an 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in human keratinocytes cell line NCTC-2544. Fluvastatin underwent rapid photodegradation upon Ultraviolet-A (UVA) irradiation in buffered aqueous solution as shown by the changes in absorption spectra. Interestingly, no isosbestic points were observed but only a fast appearance of a spectral change, indicative of the formation of a new chromophore. The isolation and characterization of the main photoproduct revealed the formation of a polycyclic compound with a benzocarbazole-like structure. This product was also evaluated for its phototoxic potential. Cell phototoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide test after 72 h from the irradiation in the presence of fluvastatin. The results showed a reduction of the cell viability in a concentration and UVA dose-dependent manner. Surprisingly, the photoproduct showed a dramatic decrease of the cell viability that occurred at concentrations of an order of magnitude lower than the parent compound. Flow cytometric analysis indicated that fluvastatin and its main photoproduct induced principally necrosis as revealed by the large appearance of propidium iodide-positive cells and confirmed also by the rapid drop in cellular adenosine triphosphate levels. Interestingly, a rapid increase of intracellular calcium followed by an extensive cell lipid membrane peroxidation and a significant oxidation of model proteins were induced by fluvastatin and its photoproduct, suggesting that these compounds exerted their toxic effect mainly in the cellular membranes. On the basis of our results, the phototoxicity of fluvastatin may be mediated by the formation of benzocarbazole-like photoproduct that acts as strong photosensitizer.

Pitavastatin, a new HMG-CoA reductase inhibitor, induces phototoxicity in human keratinocytes NCTC-2544 through the formation of benzophenanthridine-like photoproducts

This study reports the results of an investigation of the phototoxicity mechanism induced by pitavastatin and its photoproducts, namely 6-cyclopropyl-10-fluoro-7,8-dihydrobenzo[k]phenanthridine (PP3) and 6-cyclopropyl-10-fluorobenzo[k]phenanthridine (PP4). The phototoxicity was tested in human keratinocytes cell lines NCTC-2544, and the results proved that under the same conditions, all three compounds exhibited phototoxic effects in the model tested. The reduction in cell viability was found to be both concentration- and UVA dose-dependent. A point of note is that both the photoproducts produced a dramatic decrease in cell viability with GI50 values one order of magnitude lower compared to the parent compound. In particular, the fully aromatic derivative (PP4) showed the highest antiproliferative activity. Flow cytometric analysis indicated that pitavastatin and the photoproduct PP4 principally induced necrosis, as revealed by the large appearance of propidium iodide-positive cells and also confirmed by the rapid drop in cellular ATP levels. Further studies committed to better understanding of photoinduced cell death mechanism(s) revealed that neither pitavastatin nor PP4 induced mitochondrial depolarization or lysosomal damage, but, interestingly, extensive cell lipid membrane peroxidation along with a significant oxidation of model proteins occurred, suggesting that pitavastatin and PP4 exert their phototoxic effect mainly in the cellular membranes. The present results suggest that the phototoxicity of pitavastatin may be mediated by the formation of benzophenanthridine-like photoproducts that appear to have high potential as photosensitizers.

Identification of photoproducts of fluvastatin in solutions

Photochemical decomposition of fluvastatin, a medical therapy drug from the group of HMG-CoA reductase inhibitors, has been analyzed. Photodegradation of fluvastatin has been studied in methanol or water solution, in accordance with to the recommendations of the first version of the ICH document. The process has been examined by HPTLC and spectrophotometry. The products of photochemical decomposition of fluvastatin have been separated by HPTLC on a nonpolar octadecyl stationary phase with a mixture of phosphate buffer and methanol as mobile phase. The retention factors of the compounds were described with the aid of the modified Soczewiński-Snyder equation. The UV spectra of unseparated photoproducts were acquired. The photodegradation of fluvastatin, either in water or methanol solutions, has been found to lead to the formation of three photoproducts.

Identification of Photoproducts of Hexahydroquinoline Derivatives by GC-EI-MS and HPLC-ESI-MS

Photoproducts of hexahydroquinoline derivatives have been analyzed with gas chromatography electro ionization-mass spectrometry (GC-EI-MS) and high performance chromatography electrospray ionization-mass spectrometry (HPLC-ESI-MS). The study was performed on four HHQ derivatives: 2,6,6-trimethyl-3-carbomethoxy-5-oxo-4-(R-phenyl)-1,4,5,6,7,8-hexahydroquinoline; R=2′-Me, 3′-Me, 2′-MeO, and 3′-MeO. The photochemical degradation of each of the HHQ derivatives led to the appearance of one product. The photoproducts were identified as the corresponding tetrahydroquinoline analogues, which were formed by dehydrogenation of dihydropyridine moiety. In GC-mass spectra, the most frequent way of fragmentation was elimination of CH3* or CH3O* radical of the ester group. In the photoproducts substituted at 2′-position of the phenyl ring, elimination of isobutene (C4H8) was observed. In the photoproducts with 3′-position substituents, elimination of COOCH3* radical was noted.

Indigotine, azorubine, and cochineal red as photoprotectors of manidipine.

BACKGROUND The calcium antagonists from the group of 1,4-dihydropyridine (DHP) derivatives are photolabile, and the products of their photochemical decomposition have no pharmacological activity. Like the other DHP derivatives, after exposure to light, manidipine undergoes decomposition leading to a weakening of the pharmacological activity. METHOD The protective influence of selected dyes on manidipine has been tested. The photodegradation of manidipine was performed according to the first version of the ICH Guideline for photostability testing. The solutions were illuminated by a high-pressure lamp HBO-200, and the methods used for evaluation of manidipine photodegradation were HPLC, HPLC-MS, and UV-vis spectrophotometry. Quantitative evaluation of photodegradation was made on the basis of the quantum yields of the processes with the use of Reinecke salt as a chemical actinometer. RESULTS Results have shown that all the dyes studied, indigotine, azorubine, and cochineal red, slow down the process of manidipine photodegradation, and the most effective is azorubine. The role of the dyes has been determined on the basis of the Stern-Volmer dependence. CONCLUSION The dyes studied, indigotine, azorubine, and cochineal red, can be potential photoprotectors of manidipine as they quench the excited states of the compound.