SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—XL PHOTOLYSIS OF CHLORPROMAZINE METABOLITES: A SPIN-TRAPPING STUDY

Abstract. The photochemistry of chlorpromazine (CPZ) and its metabolites, 7-hydroxychlorpromazine (7OHCPZ), desmethylchlorpromazine (DCPZ), didesmethylchlorpromazine (DDCPZ) and chlorpromazine sulfoxide (CPZSO) was studied by the spin trapping technique with 2-methyl-2-nitrosopropane and 5,5-dimethyl-l-pyrroline- N -oxide. 7-Hydroxychlorpromazine generated hydroxyl radicals when excited at 330 nm under either anaerobic or aerobic conditions. 7-Hydroxychlorpromazine, DCPZ and DDCPZ all underwent dechlorination upon photoexcitation which was enhanced in the absence of air. Chlorpromazine sulfoxide did not undergo photodechlorination but instead generated a high yield of the hydroxyl radical. A comparison among CPZ and its derivatives shows that the yield of the photodechlorinated product is directly related to the degree of phototoxicity. This suggests photodechlorination is an important factor in the phototoxicity of CPZ and its metabolites.     

Phototoxicity testing by online irradiation and HPLC

A high-performance liquid chromatography (HPLC) system was developed for the determination of drug photostability and phototoxicity based on an automated column-switching system with aqueous online UV-A irradiation and hyphenated organic separation of the drug and its photoproducts. The photoreactor is built with an poly(ethylene-co-tetrafluoroethylene) (ETFE) reaction coil knitted around a UV-A light source. The chromatographic separation was performed with two special C₁₈ columns, which are also suitable for using with pure water as eluent. Degradation of chlorpromazine (CPZ) by ultraviolet light was investigated at pH 7 and pH 3. Furthermore chlorpromazine was irradiated in the presence of guanosine-5-monophosphate (GMP) in pH 7 buffered solution, leading to a new photoproduct. In the pH 3 irradiation studies of CPZ and GMP, no reaction was detected between the molecules.     

ENHANCEMENT OF UV-INDUCED SKIN CARCINOGENESIS BY AZATHIOPRINE: ROLE OF PHOTOCHEMICAL SENSITISATION

Abstract The phototoxicity of various drugs (chlorpromazine (CPZ), metronidazole (MET), 8-methoxypsoralen (8-MOP), azathioprine (AZA) and the azathioprine metabolites, 6-mercaptopurine (6-MP), methylnitrothio-imidazole (MNTI), methylnitroimidazole (MNI)) was determined in hairless (Skh-hrl) mice exposed to a light source with broad emission (290–700 nm). Chlorpromazine, MET, 8-MOP, AZA, MNI and 6-MP were phototoxic, as determined by the oedematous response of skin, at doses comparable to those used clinically.
The effects of long-term drug therapy and UV radiation on skin carcinogenesis were then determined. Chlorpromazine and MET, and to a lesser extent AZA, MNTI and 6-MP, enhanced photocarcinogen-esis. In each case, both the tumour yield and the proportion of malignant:benign skin tumours were increased. The results of this study imply that prolonged treatment of mice with low-level phototoxins predisposes to photocarcinogenesis.     

毛细管电泳电致化学发光法同时测定氯丙嗪、异丙嗪及其主要代谢物

建立了同时检测氯丙嗪、异丙嗪及其主要代谢物的毛细管电泳电致化学发光新方法。最佳实验条件为: 检测电位1.20 V,钌联吡啶浓度5 mmol/L,检测池磷酸缓冲溶液40 mmol/L (pH 6.5),分离磷酸缓冲溶液18 mmol/L (pH 4.8),进样电压11 kV,进样时间8 s,分离电压13.5 kV。方法的检出限(3σ)分别为氯丙嗪8.3×10~7 g/L、异丙嗪7.2×10~6 g/L、氯丙嗪亚砜1.9×10~5g/L和异丙嗪亚砜3.7×10~6g/L,各组分的电化学发光强度和迁移时间的相对标准偏差(RSD)分别不超过3%和1%。本方法具有简便、快速、灵敏、进样量少和不受共存物干扰等特点,可在不必预分离的情况下直接同时连续测定家犬尿样中的氯丙嗪、异丙嗪、氯丙嗪亚砜和异丙嗪亚砜。     

Photochemical Reactions Involved in the Phototoxicity of the Anticonvulsant and Antidepressant Drug Lamotrigine (Lamictal®)

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal^®, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (¹O₂) with a quantum yield of 0.22 in CH₂Cl₂, 0.11 in MeCN and 0.01 in D₂O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak ¹O₂ quencher ( k _q = 3.2 × 10⁵  m ⁻¹ s⁻¹ in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched ¹O₂ very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated ¹O₂ twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of ¹O₂ prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.     

NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN PRODUCED BY PHOTOSENSITIZATION WITH PROMAZINE and CHLORPROMAZINE

A sensitive near-infrared detection system has been used to study the steady-state emission of ¹O₂ at 1268 nra produced by promazine (PZ) and chlorpromazine (CPZ) during photo-illumination. Singlet molecular oxygen could be detected in a variety of ordinary and perdeuterated organic solvents, but was not detectable in water or deuterium oxide. The emission was enhanced in the perdeuterated organic solvents and could be eliminated by rigorous degassing or by addition of the singlet oxygen scavenger 2,3-dimethylfuran. Singlet oxygen could not be detected in any of the solvents during irradiation of the sulfoxides of PZ and CPZ. We conclude that in biological systems ¹O₂ production is not a major pathway to phototoxicity for the sulfoxides, while for the parent phenothiazines the formation of ¹O₂ is much more likely to be important in nonpolar environments such as cell membranes than in the aqueous parts of the cell.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

Electrochemical and Photoelectrochemical Sensing of NADH and Ethanol Based on Immobilization of Electrogenerated Chlorpromazine Sulfoxide onto Graphene‐CdS Quantum Dot/Ionic Liquid Nanocomposite

Here, a simple one‐step solvothermal procedure was employed to synthesize a nanocomposite containing graphene‐nanosheets and CdS quantum dots (GNs‐CdS QDs). The electrochemical oxidation of chlorpromazine (CPZ) to chlorpromazine‐sulfoxide (CPZ‐SO) onto a GNs‐CdS QDs/ionic liquid (IL) nanocomposite modified glassy carbon (GC) electrode give rise to redox‐active products which showed excellent electrocatalytic and photoelectrocatalytic activity toward NADH oxidation at reduced overpotential. A linear response up to 200 µM was obtained for photoamperometric determination of NADH with detection limit 1 µM. Immobilizing alcohol dehydrogenase(ADH) onto the modified electrode via a simple cross linking procedure, the photoelectrochemical capability of the proposed system toward ethanol biosensing was clearly shown.     

STRUCTURE AND PHOTOBIOLOGICAL ACTIVITY OF 7-CHLORO-1,4-BENZODIAZEPINES. STUDIES ON THE PHOTOTOXIC EFFECTS OF CHLORDIAZEPOXIDE, DESMETHYLCHLORDIAZEPOXIDE AND DEMOXEPAM USING A BACTERIAL INDICATOR SYSTEM

The photobiological activity of chlordiazepoxide, an active ingredient of the drug Librium, which is known to induce phototoxic effects, and two of its metabolites, desmethylchlordiazepoxide and demoxepam, was investigated. Upon irradiation of these biologically active compounds with longwave UV light, the main decomposition product formed is an oxaziridine. Using a strain of Salmonella typhimurium as a test organism for cytotoxicity, it could be demonstrated that not only the drug itself, but also the major mammalian metabolites are phototoxic and, furthermore, that the respective oxaziridines are responsible for the toxic effects found upon irradiation. A close relationship appears to exist between the phototoxicity of the nitrones and the toxicity in the dark of their respective oxaziridines. Investigations of the photobiological activity of a few closely structurally related benzodiazepines could establish that a 4-oxide moiety in the benzodiazepine nucleus is the structural characteristic responsible for the appearance of phototoxicity; in those compounds which contain a 4-oxide in the benzodiazepine nucleus, photo-decomposition to a toxic oxaziridine is observed, while the analogues lacking the 4-oxide moiety do not show this characteristic and, therefore, no phototoxic effects can be observed. Finally, mutagenicity tests performed with the same bacterial indicator as used for phototoxic studies, and including chlorpromazine as a positive reference compound, indicate that under the present experimental conditions photoproducts formed upon irradiation of chlordiazepoxide and its metabolites with longwave UV light do not exert a mutagenic effect.     

In vitro PHOTODEGRADATION OF CHLORPROMAZINE

The in vitro photodecomposition of chlorpromazine (CPZ) was investigated with the aim to evaluate possible reactive determinants that could play a role in the occurrence of the in vivo -observed photosensitivity. In view of the in vivo situation, CPZ was dissolved in low concentration in buffered aqueous solution (pH 7.4) or in dilute human serum and irradiated with low intensity (5–7 W m^-2) UV-A and UV-B. No distinct difference was found between UV-A or UV-B irradiation as far as photoproduct formation is concerned. This suggests the same degradation mechanism at both wavelength ranges. In buffered aqueous solution, irradiation of CPZ resulted in 65 and 90% 2-hydroxypromazine (PZOH), 5 and 7% promazine (PZH) and 2 and 0% chlorpromazinesulfoxide (CPZSO) under aerobic and anaerobic conditions, respectively. In dilute human serum, there was only a shift in the PZH/PZOH ratio, probably as a result of H-atom or electron donation by sulfur containing groups present in proteins. The results demonstrate that photodegradation of CPZ in vitro , under conditions relevant to the in vivo situation, proceeds almost entirely by dechlorination rather than by radical cation formation (the essential pathway of CPZSO production). Thus we conclude that the thiyl radical cation probably does not play a major role in the in vivo -observed phototoxic reactions.     

PHOTOPRODUCTS OF CHLORPROMAZINE WHICH CAUSE RED BLOOD CELL LYSIS

A mechanism for chlorpromazine (CPZ) phototoxicity has been proposed that attributes the response to formation of stable, toxic photoproducts which cause cell membrane disruption. We have characterized these toxic photoproducts as dimers and higher multimers of CPZ. Chlorpromazine solutions (3 or 10 mA/) were irradiated with a medium pressure Hg lamp filtered to exclude λ < 280 nm. Five low mol wt photoproducts were separated by high performance liquid chromatography. Two were identified as CPZ-sulfoxide and promazine. Higher mol wt photoproducts were separated by Sephadex G-50 chromatography into 3 broad bands which were characterized by their absorption and fluorescence spectra. Band A (mol wt > 800) had λ_max^abs= 263 nm, λ_max^fl= 490 nm and Band B (mol wt = 350-800) had λ_max^abs= 255 nm, λ_max^fl= 450 nm. Based on the mol wt of CPZ, Band A contained trimers and higher mol wt compounds and Band B was composed of dimeric structures. BandC(λ_max^abs= 255,310 nm; λ_max^fl= 445 nm) was composed of CPZ (mol wt = 315) and the low mol wt photoproducts. Red blood cell lysis was used as an assay for the ability of photoproducts to cause membrane disruption. Bands A and B, but not Band C, caused cell lysis. These data indicate that the CPZ photoproducts which cause cell membrane disruption are dimers (Band B) and higher multimers (Band A).     

Phototoxicity, distribution and kinetics of association of UVA-activated chlorpromazine, 8-methoxypsoralen, and 4,6,4'-trimethylangelicin in Jurkat cells

Extracorporeal phototherapy (ECP) is a therapeutic approach based on photobiological effects of 8-methoxypsoralen (8-MOP) on white blood cells isolated from the blood, exposed to UVA and then reinfused into the patient. 8-MOP is presently the only drug approved for clinical application of ECP; therefore, identification of other photosensitizers with better photochemical and pharmacokinetic properties might enhance the efficacy of this treatment modality. Among such alternative drugs are 4,6,4'-trimethylangelicin (TMA) and chlorpromazine (CPZ), which have previously been studied in an animal model for ECP. In this current study, cellular bioavailability of 8-MOP, TMA and CPZ was investigated in vitro, using low doses of UVA relevant for the clinical setting of ECP. Our fluorescence microscopy study revealed that 8-MOP and CPZ penetrated readily into the cells, where they accumulated with similar kinetics. No distinct fluorescence was observed in cells incubated with TMA. We found that the phototoxic efficiency of 8-MOP was an order of magnitude greater than that of CPZ, i.e., to obtain a similar reduction in survival of cells subjected to photosensitization by the drugs, the concentration of CPZ needed to be 10 times higher than that of 8-MOP. The photoactivated TMA exhibited the highest pro-apoptotic efficiency. A clear indication of photoinduced formation of reactive oxygen species and peroxidation of lipids was observed only in CPZ-sensitized cells, suggesting different mechanisms for phototoxicity mediated by CPZ and by the two furocoumarins.     

INVESTIGATIONS ON THE MECHANISM OF CHLORPROMAZINE PHOTOTOXICITY: EFFECTS ON LYSOSOMES OF CULTURED HUMAN FIBROBLASTS

Abstract The effect of chlorpromazine (CPZ) and UVA on lysosomes of cultured normal human fibroblasts has been investigated. Acid phosphatase (ACPase) activity in 12 000 g pellet of cells treated with CPZ (10 μg/m l ) and UVA (6 × 10⁴ J/m²) was found to be decreased as compared with non-treated, CPZ or UVA treated control cells. This decrease, however, was not accompanied by a concomitant increase in ACPase activity in the 12 000 g supernatant. The addition of Triton X-100 to cells pretreated with CPZ + UVA resulted in only a moderate increase in ACPase activity of the 12 000 g supernatant. ACPase activity of the cells incubated in media containing preirradiated CPZ was also found to he decreased. These results indicate that CPZ + UVA directly inactivate lysosomal enzymes, possibly without affecting the membrane.     

FREE RADICAL PRODUCTION BY CHLORPROMAZINE SULFOXIDE, AN ESR SPIN-TRAPPING AND FLASH PHOTOLYSIS STUDY

Abstract— Using the spin-trapping technique we have investigated the photolysis of chlorpromazine sulfoxide and promazine sulfoxide. Photolysis of these sulfoxides in aqueous solution resulted in a species which is capable of oxidizing ascorbate, cysteine, glutathione, NADH, and azide by one electron, in addition to extracting hydrogen atoms from ethyl alcohol and dimethyl sulfoxide. These oxidations were not dependent on the presence of dissolved oxygen. The oxidizing species is proposed to be the hydroxyl free radical arising from the homolytic cleavage of the S-O bond of the sulfoxide. Flash photolysis of the chlorpromazine and promazine sulfoxides demonstrated the formation of cation radicals consistent with the loss of the hydroxyl radical from the sulfoxides. In addition we present a simple direct method for the quantitative synthesis of promazine and chlorpromazine sulfoxides from the parent promazine derivatives.     

INVOLVEMENT OF SINGLET OXYGEN IN THE PHOTOTOXICITY MECHANISM FOR A METABOLITE OF PIROXICAM

It has been previously shown that a metabolite of piroxicam but not piroxicam itself causes phototoxicity to cells in vitro after exposure to UVA (320–400 nm) radiation. The phototoxicity mechanism for this metabolite, 2-methyl-4-oxo-2H-l,2-benzothiazine-l,l-dioxide (Compound I), was investigated. In vitro phototoxicity to human mononuclear cells was assayed using 0.5 m M Compound I and UVA radiation. The UVA fluence required for phototoxicity of Compound I was lower by a factor of 2-3 in D₂O buffer compared to H₂O buffer. Superoxide dismutase and mannitol, which remove O₂^- and OH", respectively, do not decrease the phototoxicity. The photodecomposition of Compound I was inhibited by sodium azide, enhanced by human serum albumin and unaffected by mannitol. Stable photoproducts of Compound I were not toxic to the cells. The quantum yield of singlet oxygen based on its emission at 1270 nm was 0.19 and 0.35 for Compound I and s2 ± 10^-3 and 10^-2 for piroxicam in D₂O and C₆H₆, respectively. While the extremely low quantum yield for singlet oxygen from piroxicam appears to account for its lack of phototoxicity, the phototoxicity mechanism for its metabolite, Compound I, most likely does involve singlet oxygen.     

Structural changes by sulfoxidation of phenothiazine drugs

Summary The side-chain conformations of psychoactive phenothiazine drugs in crystals are different from those of biologically inactive ring sulfoxide metabolites. This study examines the potential energies, molecular conformations and electrostatic potentials in chlorpromazine, levomepromazine (methotrimeprazine), their sulfoxide metabolites and methoxypromazine. The purpose of the study was to examine the significance of the different crystal conformations of active and inactive phenothiazine derivatives, and to determine why phenothiazine drugs lose most of their biological activity by sulfoxidation. Quantum mechanics and molecular mechanics calculations demonstrated that conformations with the side chain folded over the ring structure had lowest potential energy in vacuo, both in the drugs and in the sulfoxide metabolites. In the sulfoxides, side chain conformations corresponding to the crystal structure of chlorpromazine sulfoxide were characterized by stronger negative electrostatic potentials around the ring system than in the parent drugs. This may weaken the electrostatic interaction of sulfoxide metabolites with negatively charged domains in dopamine receptors, and cause the sulfoxides to be virtually inactive in dopamine receptor binding and related pharmacological tests.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–XII. SPIN TRAPPING STUDY OF THE FREE RADICALS GENERATED DURING THE PHOTOLYSIS OF PHOTO ALLERGENS BITHIONOL and FENTICHLOR

Free radicals were trapped and observed by ESR when photoallergens bithionol and fentichlor were irradiated in the presence of spin traps N- t -butyl-α-phenylnitrone (PBN) and 5,5-dimethyl-pyrroline-N-oxide (DMPO). In the absence of air, both PBN and DMPO trapped a carbon-centered radical. The carbon-centered radical, which was capable of abstracting a hydrogen atom from cysteine, glutathione, ethanol and formate, was identified as an aryl radical derived from the homolytic cleavage of the carbon-chlorine bond. In the presence of air, both carbon-centered radicals and hydroxyl radicals were trapped by DMPO. Under similar conditions, the yield of the hydroxyl radicals was greater from bithionol than from fentichlor. The presence of the hydroxyl radical was confirmed by kinetic experiments employing hydroxyl radical scavengers (ethanol, formate). Superoxide and H₂O₂ were not involved. Experiments with oxygen-¹⁷O indicated that the hydroxyl radicals came exclusively from dissolved oxygen. The precursor of the hydroxyl radical is postulated to be a peroxy intermediate (ArOO*) derived from the reaction of an aryl radical (Ar*) with molecular oxygen. Both bithionol and fentichlor photoionized only when excited in the UVC (<270 nm) region. Free radicals have long been postulated in the photodechlorination of bithionol and fentichlor and the present study provides supporting evidence for such a mechanism. Aryl and hydroxyl radicals are reactive chemical species which may trigger a series of events that culminate in photoallergy.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

In vivo PHOTODEGRADATION OF CHLORPROMAZINE

The in vivo photodegradation of chlorpromazine (CPZ) in the skin was investigated after systemic administration of 3H-CPZ to shaven Wistar rats and exposure to UV-A. Promazine (PZ) and 2-hydroxy-promazine (2-OH-PZ) appeared to be formed in irradiated rats, but not in the skin of rats kept in the dark. This indicates that upon irradiation with UV-A the PZ-radical is formed which can be held responsible for the photobinding to eye and skin constituents as observed earlier [Schoonderwoerd and Beijersbergen von Henegouwen (1987) Photochem. Photobiol. 46, 501-505]. Chlorpromazine-sulfoxide (CPZSO) is a major metabolite of CPZ. Less CPZSO was found in the skin of irradiated rats compared to those kept in the dark. As this appeared not to be caused by photobinding or photodegradation of CPZSO it can be concluded that CPZSO is not a photoproduct of CPZ under these experimental conditions. This study shows that the in vivo photodegradation of CPZ proceeds via the promazinyl radical rather than via the radical cation.     

Determination Of Chlorpromazine And Its Sulfoxide And 7-Hydroxy Metabolites By Ion-Pair High Pressure Liquid Chromatography

Abstract

An ion-pair HPLC approach with ordinary silica has been applied, with detection by ultraviolet absorption, to the assay of plasma for chlorpromazine and its sulfoxide on the one hand, and for 7-hydroxychlorpromazine (an active metabolite) on the other hand. The respective sample-preparation procedures entail extraction of the plasma with heptane at strongly alkaline pH, or else with diethyl ether at a less alkaline pH and with ensuing back-extraction and re-extraction. For each of the compounds, levels as low as 10 ng. ml^?1 are measurable. The conditions adopted are such that specificity and reproducibility are satisfactory although chlorpromazine and its various metabolites, especially 7-hydroxychlorpromazine, are chemically unstable and, moreover, are readily lost onto glass. With the unorthodox separation system adopted, adsorption rather than partition appears to be the dominant mechanism.