Role of type-II pathway in apoptotic cell death induction by photosensitized CDRI-97/78 under ambient exposure of UV-B.

Novel trioxane 97/78, developed by Central Drug Research Institute (CDRI), Lucknow has shown promising antimalarial activity. Clinical experience of anti-malarial drugs registered the occurrence of phototoxicity in patients exposed with sunlight subsequent to medication. Photodegradation study has identified one photo-product up to 4h under UV-B/Sunlight by LC-MS/MS. UV-B irradiated 97/78 compound produced ¹O2 via type-II dependent reaction mechanism, corroborated by its specific quencher. 2'-dGuO degradation and % tail development in photochemical as well as comet test, advocated the genotoxic potential of 97/78. The photocytotoxicity assays (MTT and NRU) on HaCaT cell line revealed the considerable decline in cell viability by 97/78. Cell cycle and Annexin V/PI double stain along with AO/EB demonstrated the G2/M phase arrest and apoptosis. Significant caspase-3 activity was measured in photoexcited 97/78 by colorimetric assay. Fluorescence stain with AO/JC-1 confirmed the lysosomal disruption and mitochondrial membrane destabilization by UV-B irradiated 97/78. Gene expression by RT-PCR showed significant upregulation of p21 and pro-apoptotic Bax, but no change observed in Bcl-2. In conclusion, the study highlights ROS mediated DNA damage, lysosomal and mitochondrial destabilization via upregulation of Bax and activation of caspase-3 which further leads to apoptosis.

Considerations on the quinine actinometry calibration method used in photostability testing of pharmaceuticals.

This paper addresses two critical issues concerning the guidelines adopted by the ICH on the photostability testing: the quinine actinometry method and the light/radiation exposure map distribution of the photostability chamber. Using a qualified non-commercial photostability chamber tests were performed using quinine and physical actinometry and compared the results to those which are used as the basis of the ICH guidelines. The statistical analysis on the results showed that: (i) the calibration curve of the quinine solution depends on its concentration and on its location in the chamber; (ii) the quinine actinometry method currently recommended by the ICH guidelines should not be generalized to any photostability chamber.

Photoreactivity of biologically active compounds. XIX: excited states and free radicals from the antimalarial drug primaquine.

The formation and reactivity of excited states and free radicals from primaquine, a drug used in the treatment of malaria, was studied in order to evaluate the primary photochemical reaction mechanisms. The excited primaquine triplet was not detected, but is likely to be formed with a short lifetime (<50 ns) and with a triplet energy <250 kJ/mol as the drug is an efficient quencher of the fenbufen triplet and the biphenyl triplet, and forms (1)O(2) by laser flash photolysis ((PQ)Phi(Delta)=0.025). Primaquine (PQ) exists as the monocation (PQH(+)) in aqueous solution at physiological pH. PQH(+) photoionises by a biphotonic process and also forms the monoprotonated cation radical (PQH(2+)*) by one electron oxidation by HO* (k(q)=6.6 x 10(9) M(-1) s(-1)) and Br*(2)(-) (k(q)=4.7 x 10(9) M(-1) s(-1)) at physiological pH, detected as a long-lived transient decaying essentially by a second order process (k(2)=7.4 x 10(8) M(-1) s(-1)). PQH(2+)* is scavenged by O(2), although at a limited rate (k(q)=1.0 x 10(6) M(-1) s(-1)). The reduction potential (E degrees) of PQH(2+)*/PQH(+) is < +1015 mV, as measured versus tryptophan (TRP*/TRPH). Primaquine also forms PQH(2+)* at pH 2.4, by one electron oxidation by Br*(2)(-) and proton loss (k(q)=2.7 x 10(9) M(-1) s(-1)). The non-protonated cation radical (PQ(+)*) is formed during one electron oxidation with Br*(2)(-) at alkaline conditions (k(q)=4.2 x 10(9) M(-1) s(-1) at pH 10.8). The estimated pK(a)-value of PQH(2+)*/PQ(+)* is pK(a) approximately 7-8. Primaquine is not a scavenger of O*(2)(-) at physiological pH. Thus self-sensitization by O*(2)(-) is eliminated as a degradation pathway in the photochemical reactions. Impurities in the raw material and photochemical degradation products initiate photosensitized degradation of primaquine in deuterium oxide, prevented by addition of the (1)O(2) quencher sodium azide. Photosensitized degradation by formation of (1)O(2) is thus important for the initial photochemical decomposition of primaquine, which also proceeds by free radical reactions. Formation of PQH(2+)* is expected to play an essential part in the photochemical degradation process in a neutral, aqueous medium.

Photoreactivity of biologically active compounds. XVII. Influence of solvent interactions on spectroscopic properties and photostability of primaquine.

The influence of solvent interactions on absorption properties, fluorescence properties (emission spectra and quantum yields) and relative photochemical degradation rates of primaquine has been investigated, in order to evaluate photochemical reaction mechanisms and chemical properties of the compound. The first absorption band (n - pi*) of primaquine is only slightly dependent on properties of the solvent, which can be ascribed to a strong, intramolecular hydrogen bond between the quinoline N and amine group in the ground state (S0). Amphiprotic solvents with predominant acidic properties (water and methanol) will to some extent stabilize the molecule and initiate hypsochromic shifts of the absorption band by protic interactions, while the other solvents (amphiprotic, basic and neutral) influence the absorption spectrum by general solvent effects only. The excited singlet (S1*) state of primaquine interacts more efficiently with the surrounding solvents than the S0 state, as evaluated by the Stokes shifts. The pKa value of the quinoline N is likely to increase in the S1* state, which is important for the observed protic interactions with amphiprotic solvents of predominant acidity. Specific solvent effects are highly important for the efficiency of the fluorescence (fluorescence quantum yields; phi f). The fluorescence is quenched by amphiprotic solvents, likely due to a rupture of the intramolecular bond and protonation of the quinolone N, and enhanced by polar, non-protic (basic) solvents, probably by stabilization of the delta intramolecular hydrogen bond. The observed photochemical degradation rates of primaquine in amphiprotic media are positively correlated with phi f, indicating that the photochemical degradation of primaquine is dependent on intramolecular hydrogen bonding and non protonated lone-pair electrons at the quinoline N. The intramolecular ring-formation with a subsequent increased lipophilic character and (lack of) interactions with the surroundings, are important factors for biological behavior as well as pharmaceutical properties of primaquine. Knowledge about solvent interactions with primaquine in the S0 and S1* states is essential for the proceeding evaluation of photostability and phototoxicity of the drug.

Photophysical and photobiological behavior of antimalarial drugs in aqueous solutions.

This article describes the results of a combined photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs quinine (Q), quinacrine (QC) and mefloquine (MQ). Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed on irradiation. The results obtained showed that fluorescence and intersystem crossing account for all the adsorbed quanta for Q and MQ (quantum yield of about 0.1 and 0.9, respectively) and only for 24% in the case of QC, which has a negligible fluorescence quantum yield (0.001). Laser flash photolysis experiments evidenced, for QC and MQ, the occurrence of photoionization processes leading to the formation of the radical cations of the drugs. The effects of tryptophan and histidine on the excited states and transient species of the three drugs were also investigated. In parallel, the photoactivity of the antimalarial drugs was investigated under UV irradiation on various biological targets through a series of in vitro assays in the presence and in the absence of oxygen. Phototoxicity on 3T3 cultured fibroblasts and lipid photoperoxidation were observed for all the drugs. The photodamage produced by the drugs was also evaluated on proteins by measuring the photosensitized cross-linking of spectrin. The combined approaches were proven to be useful for understanding the mechanism of phototoxicity induced by the antimalarial drugs.

Photophysical studies on antimalarial drugs.

Most drugs used in the treatment of malaria produce phototoxic side effects in both the skin and the eye. Cutaneous and ocular effects that may be caused by light include changes in skin pigmentation, corneal opacity, cataract formation and other visual disturbances including irreversible retinal damage (retinopathy) leading to blindness. The mechanism for these reactions in humans is unknown. We irradiated a number of antimalarial drugs (amodiaquine, chloroquine, hydroxychloroquine, mefloquine, primaquine and quinacrine) with light (lambda > 300 nm) and conducted electron paramagnetic resonance (EPR) and laser flash photolysis studies to determine the possible active intermediates produced. Each antimalarial drug produced at least one EPR adduct with the spin-trap 5,5-dimethyl-1-pyrroline N-oxide in benzene: superoxide/hydroperoxyl adducts (chloroquine, mefloquine, quinacrine, amodiaquine and quinine), carbon-centered radical adducts (all but primaquine), or a nitrogen-centered radical adduct only (primaquine). In ethanol all drugs except primaquine produced some superoxide/hydroperoxyl adduct, with quinine, quinacrine, and hydroxychloroquine also producing the ethoxyl adduct. As detected with flash photolysis and steady-state techniques, mefloquine, quinine, amodiquine and a photoproduct of quinacrine produced singlet oxygen ([symbol: see text]delta = 0.38; [symbol: see text]delta = 0.36; [symbol: see text]delta = 0.011; [symbol: see text]delta = 0.013 in D2O, pD7), but only primaquine quenched singlet oxygen efficiently (2.6 x 10(8) M-1 s-1 in D2O, pD7). Because malaria is a disease most prevalent in regions of high light intensity, protective measures (clothing, sunblock, sunglasses or eye wraps) should be recommended when administering antimalarial drugs.

Photoreactivity of biologically active compounds, XIV: influence of oxygen on light induced reactions of primaquine.

The influence of molecular oxygen and oxygen radicals on the photoreactivity of the antimalarial drug primaquine (PQ) has been investigated. Oxygen is directly involved in photodecomposition of the drug. Flushing with helium gas prior to and during irradiation to suppress the oxygen level of the medium, retards the degradation rate of PQ (followed by HPLC) and leads to the formation of only two degradation products (identified by MS) compared to eight main- and several minor products under normal atmospheric conditions. Flushing with oxygen gas prior to and during irradiation to increase the oxygen content of the medium accelerates the degradation rate of PQ. PQ produces oxygen radicals (hydroxyl and superoxide) during photolysis, while the photoproducts of PQ seem likely to induce singlet oxygen formation (detected by addition of radical scavengers). Sensitization reactions involving singlet oxygen lead to decomposition of PQ (followed by HPLC). On the basis of our results, photochemical reaction mechanisms of PQ are postulated and discussed. At physiological conditions (aqueous, neutral pH, oxygen rich) PQ has a large potential to decompose after light absorption. The photoreaction seems to be initiated at the quinoline nitrogen. The ability to form an intramolecular hydrogen bond seems to be essential for the luminescence properties of the drug. Phosphorescence lifetime of PQ is about 5 microseconds. Fast chemical reactions may occur from the short-lived triplet state of the drug, but the excited compound can diffuse only a limited distance prior to deexcitation. This can be important concerning light-induced adverse effects which may appear after medication with PQ.

Photodegradation studies on chloroquine phosphate by high-performance liquid chromatography.

A method based on high-performance liquid chromatography (HPLC) was developed to study degraded chloroquine samples produced after exposure to sunlight in the Sudan. The method was also used to investigate chloroquine photodegradation after irradiation by UV and sunlight at ambient temperature. The study showed that the photodecomposition of chloroquine was pH and solvent dependent. Moreover, the extent of reaction was found to increase in the absence of oxygen. At pH 8, where the reaction rate was high, the photodecomposition was found to follow pseudo-first-order reaction kinetics. The HPLC method developed was also employed to analyse chloroquine and its degradation products in two commercially available brands of chloroquine injections which had been stored under local conditions in the Sudan. A number of degradation products were separated and examined by photodiode array spectroscopy and preparative TLC.

Photolytic degradation of alpha-[(dibutylamino)methyl]-6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline methanol: an experimental antimalarial.

A study of the effects of various storage conditions on the rate and products of degradation of the quinoline methanol antimalarial agent, alpha-[(dibutylamino)methyl]-6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline methanol, was undertaken. The degradation was followed by high-pressure liquid chromatography and TLC in oxygenated and deoxygenated methanol, ethanol, chloroform, and chloroform-heptane mixtures under UV and laboratory fluorescent lighting irradiation, as well as in the absence of light. The kinetics of degradation confirmed the major catalyzing factor to be UV irradiation. The compound was stable in the absence of light and reasonably stable under fluorescent lighting both in the presence and absence of oxygen. The degradation resulted in a major product, 6,8-dichloro-2-(3',4'-dichlorophenyl)-4-quinoline-carboxaldehyde, whose structure was confirmed by elemental analysis and IR, NMR, and mass spectral data.

An in vitro and in vivo investigation of the phototoxic effect and its amelioration with radioprotective compounds.

Electron spin resonance spectroscopy has been used to demonstrate that the phototoxic antimalarial drug, 6,8-dichloro-2-phenyl-a-2-piperidnylquinolinemethanol (WR 7930), when irradiated with long-wave ultraviolet (UV) light (lambda greater than 320 nm) while held in a glassy matrix at 73 degrees K, enters a triplet state and releases hydrogen atoms in its environment. The steady-state concentration of triplet WR 7930 molecules and of hydrogen atoms is reduced 2 to 3 times when mercaptoethylamine (MEA) is also present in the UV-irradiated glass. Organosulfur radicals form on MEA while hydrogen atoms and triplet-state molecules are reduced in number. Hydrogen atoms and triplet WR 7930 molecules are considered as mediators of the phototoxicity of the antimalarial drug. Thus, hydrogen atom scavanging and chemical quenching of the triplet state are possible mechanisms by which protection against phototoxic effects could be gained. Protection is demonstrated in mice receiving 20 mg per kg WR 7930 intraperitoneally and exposed to long-wave UV for 20 hr when the radioprotective aminothiol-forming compound, 2-(3-aminopropylamino) ethyl dihydrogen phosphorothioate (WR 2721), is administered at 400 mg per kg immediately before irradiation. When no protective drug is administered concurrently, WR 7930 administration results in intense erythema, edema, and eventual necrosis of ear tissues.