WO2006090259A2

WO2006090259A2 - A method for screening photosensitive compound

Info

Publication number: WO2006090259A2
Application number: PCT/IB2006/000402
Authority: WO
Inventors: Satomi Onoue; Yoshiko Tsuda
Original assignee: Pfizer Japan Inc; Pfizer Inc
Current assignee: Pfizer Japan Inc; Pfizer Inc
Priority date: 2005-02-24
Filing date: 2006-02-15
Publication date: 2006-08-31
Anticipated expiration: 2007-08-24
Also published as: WO2006090259A3

Abstract

The present invention provides a method for screening photosensitive compounds including active pharmaceutical compounds, cosmetic compounds, food additives, excipients and the like, wherein the compound is capable of generating photo excitation radical species. The method of the present invention comprises the steps; adding solutions of a test compound and a reacting agent for reactive oxygen species (singlet oxygen and/or superoxide) to a reaction container; photo irradiating the solution mixture of the above step by a light source; monitoring an reactive oxygen species generated by irradiation in each reaction container; and evaluating the possibility of photosensitivity of the test compound by comparison of the data generated with data for a known phototoxic compound. Additionally, the method of present invention is applicable to the simulation of the physiological environment and for use in high-through-put system.

Description

A METHOD FOR SCREENING PHOTOSENSITIVE COMPOUND

FIELD OF THE INVENTION

The present invention relates to a method for screening photosensitive compounds, wherein the compound is capable of generating photo excitation radical species. More particularly, the invention relates to a method for screening compounds such as active pharmaceutical compounds, cosmetic compounds, food additives, excipients, and the like.

BACKGROUND OF THE INVENTION

Photo hypersensitivity is a skin disease wherein the skin's sensitive reaction to light is induced by pharmaceutical compounds, cosmetic compounds, food additives, excipients and the like. Photo hypersensitivity leads to dermatitis, maculopapule, erythema, vesicular eruption, pigmentation and the like. Since photo hypersensitivity is undesirable for a pharmaceutical compound, photo hypersensitivity should be carefully investigated during pharmaceutical development. The screening of new chemical entities for photo hypersensitivity is required by Food and Frug Administration, FDA during pharmaceutical development.

Photo biological reactions normally occur when a chemical compound is able to absorb UV or visible light. A chemical compound that absorbs UV or visible light with a wavelength of from 290 nm to 700 nm may cause phototoxicity and/or photosensitivity. For such compounds, further investigations for phototoxicity, photoallergy and photogenotoxicity are required during pharmaceutical development. FDA. Known methods of photochemical assessment suffer form the problem of false-positive results.

Furthermore, it is known that some compounds which absorb UV light of low wavelength less than 290 nm bind to drug-binding proteins such as lipoprotein or albumin, and the resulting bound molecule may cause photosensitivity. In such cases, the known methods of photochemical assessment cannot predict photosensitivity.

Thus, accurate, simple, rapid, and low cost screening methods of photosensitive compounds in the early stages of pharmaceutical development are required.

Published Japanese Unexamined Patent publication No. 2001-91510 discusses an evaluation method for phototoxic compounds using an optical stability examination of polysaccharides. However, it has been reported that often no relationship between photostability and phototoxicity is observed. Thus, this method not sufficiently reliable for predicting of phototoxicity.

Published Japanese Unexamined Patent publication No. 07-190936 discusses a detection method using leukocyte activation prevention.

US Patent No. 6,171 ,858 discusses processes for determining the phototoxicity and/or photosensitivity of substances or mixtures thereof using non-human vertebrate embryo or tissues. US Patent No. 5,5053,340 discusses in vitro tests for derma! toxic properties using artificial membrane support and regents.

However, the above-mentioned methods are not suitable for high throughput screening because of the peculiarity of the methods. There is a need for screening methods for photosensitive compound, which are predictive of the effects in humans, and which are applicable to high-through-put screening systems. Furthermore, there is a need for screening methods for photo toxicity in the physiological environment, since, as discussed hereinabove, some compounds show phototoxicity only when bound with drug-binding proteins in the physiological environment. There is also a need for methods for screening compounds for photo stability since stability under photo irradiation is desirable for pharmaceutical compounds.

SUMMARY OF THE INVENTION

The present invention provides a method for screening photosensitive compounds, including active pharmaceutical compounds, cosmetic compounds, food additives, excipients and the like, wherein the compound is capable of generating photo excitation radical species.

The method of the present invention comprises the steps;

(1) adding solutions of a test compound and reacting agent for a reactive oxygen species (singlet oxygen and/or superoxide) to a reaction container;

(2) photo irradiating the mixture solution by light source;

(3) monitoring the generation of reactive oxygen species by irradiation in each reaction container; and

(4) photosensitivity of the test compound is evaluated compared with a data of known photosensitive drug.

Generation of a reactive oxygen species by photosensitive compounds is considered to play an important role in the phototoxic effect. It has been found by the inventors that known photosensitive compounds tend to generate reactive oxygen species such as singlet oxygen and super oxygen and the like when exposed to UVA radiation.

Furthermore, the present invention provides a method for screening photosensitive compounds comprising detecting both singlet oxygen and superoxide simultaneously. Singlet oxygen and superoxide are independently related with phototoxicity. This method is predictive of phototoxicity induced by test compound.

Yet, furthermore, the present invention provides a method for screening photogenotoxic compounds comprising the use of plasmid DNA.

The present invention provides a method to predict photosensitivity and photosafety easily and rapidly by monitoring reactive oxygen induced by exposing the test compound to UV light. A further merit of this invention is the easy and efficient evaluation of a number of samples simultaneously. Thus, the method of the present invention is applicable to high-through-put screeningsystem.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows the protective effect of radical scavengers, on the photo-induced degradation of quinine. Quinine was dissolved in 20 mM sodium phosphate buffer (final concentration, 0.25 mg/mL) with or without radical scavengers, including butylated hydroxyanisole (BHA, 1 mM), reduced gluthatione (GSH, 1 mM), 1 ,4-diazobicyclo[2,2,2]octane (DABCO, 1 mM), sodium azide (NaN₃, 1 mM), and superoxidedismutase (SOD, 200 U). After exposure to Xe lamp (30,000 lux) for the indicated periods, intact quinine was determined by HPLC analysis. Data indicates the mean±SD for four determinations.

Figure 2 shows the reaction container for reactive oxygen scavengers (ROS) assay, consisting of quartz plate, multiwell plate, and the retainer.

DETAILED DESCRIPTION OF THE INVENTION

The goal of the screening method of the present invention is to detect the phototoxicity, photosensitivity, and/or photo instability of compounds in the presence of light and to be applicable to high-through-put screening systems. This type of testing is particularly relevant for pharmaceutical compounds that enter the skin via dermal penetration or systemic circulation. Thus, it is also a requirement of the present invention functions in simulated physiological environments. The present invention provides a method for screening photosensitive compounds including active pharmaceutical compounds, cosmetic compounds, food additives, excipients and the like, wherein the compound is capable of generating photoexcitation radical species.

It is well-established that photo-irradiation of photosensitive compounds results in significant photochemical and photobiochemical reactions, which are tightly associated with the photoinstability and phototoxicity of the compounds.

Phototoxic skin response occurs via cascade reactions mediated by energy transfer. The photosensitive compound is excited by light irradiation, and excited singlet oxygen and superoxide species are generated. These singlet oxygen and superoxide species cause toxic reactions with the skin cell composition, oxidation or peroxidation of bimolecular (lipid, protein) to damage cell components. Furthermore, resulting singlet oxygen and superoxide may change the characterisitics of the compound; this change is considered to be related to stability during light irradiation.

An embodiment of the present invention is provided a method comprising the steps;

(1 ) adding the solution of a test compound and reacting agents for colorimetric assay of reactive oxygen species (singlet oxygen and/or superoxide) to a reaction container;

(2) irradiating the mixture solution by light source for designated time;

(3) monitoring the reactive oxygen species generated by photo irradiation in each reaction container;

The term "photosensitive compound" as used herein means a compound which is capable of generating photo excitation radical species, and which may show phototoxicity and/or photoinstability.

The term "known photosensitive drug" as used herein means an active pharmaceutical compound which is known to show photosensitivity, phototoxicity and/or photoinstability. Examples of phototoxic drugs include, but not limited to, Chlorpromazine, Naproxen, Ketoprofen, Norfloxacin, Nalidixic acid, Indomethacin, Ibuprofen, Furosemide, Benzoyl peroxide, Amiodarone, Oxytetracycline, Diclofenac sodium, Sulfamethoxazole, Retinol, 8-Methoxy psoralen, Omeprazole, Nitrendipine, Quinine.

Suitable compounds for use as test materials in the present invention include, without limitation, active pharmaceutical compounds from the following classes: abortifacients, angiotensin-converting enzyme inhibitors, adrenergic agonists, adrenergic blockers, adrenocortical suppressants, adrenocorticotropic hormones, alcohol deterrents, aldose reductase inhibitors, aldosterone antagonists, anabolics, analgesics (including narcotic and non-narcotic analgesics), androgens, angiotensin Il receptor antagonists, anorexics, antacids, anthelminthics, antiacne agents, antiallergics, antialopecia agents, antiamebics, antiandrogens, antianginal agents, antiarrhythmics, antiarteriosclerotics, antiarthritic/antirheumatic agents (including selective cyclooxygenase-2 inhibitors), antiasthmatics, antibacterials, antibacterial adjuncts, anticholinergics, anticoagulants, anticonvulsants, antidepressants, antidiabetics, antidiarrheal agents, antidiuretics, antidotes to poison, antidyskinetics, antieczematics, antiemetics, antiestrogens, antifibrotics, antifiatulents, antifungals, antiglaucoma agents, antigonadotropins, antigout agents, antihistaminics, antihyperactives, antihyperlipoproteinemics, antihyperphosphatemics, antihypertensives, antihyperthyroid agents, antihypotensives, antihypothyroid agents, anti-infiammatories, antimalarials, antimanics, antimethemoglobinemics, , antimigraine agents, antimuscarinics, antimycobacterials, antineoplastic agents and adjuncts, antineutropenics, antiosteoporotics, antipagetics, antiparkinsonian agents, antipheochromocytoma agents, antipneumocystis agents, antiprostatic hypertrophy agents, antiprotozoals, antipruritics, antipsoriatics, antip sychotics, antipyretics, antirickettsials, antispasmodics, antisyphylitics, antithrombocythemics, antithrombotics, antitussives, antinlceratives, antiurolithics, antivenins, antiviral agents, anxiolytics, aromatase inhibitors, astringents, benzodiazepine antagonists, bone resorption inhibitors, bradycardic agents, bradykinin antagonists, bronchodilators, calcium channel blockers, calcium regulators, carbonic anhydrase inhibitors, cardiotonics, chelating agents, cholelitholytic agents, choleretics, cholinergics, cholinesterase inhibitors, cholinesterase reactivators, CNS stimulants, contraceptives, debriding agents, decongestants, depigmentors, dermatitis herpetiformis suppressants, digestive aids, diuretics, dopamine receptor agonists, dopamine receptor antagonists, ectoparasiticides, emetics, enkephalinase inhibitors, enzymes, enzyme cofactors, estrogens, expectorants, fibrinogen receptor antagonists, fluoride supplements, gastric and pancreatic secretion stimulants, gastric cytoprotectants, gastric proton pump inhibitors, gastric secretion inhibitors, gastroprokinetics, glucocorticoids, glucosidase inhibitors, growth hormone inhibitors, growth hormone releasing factors, growth stimulants, hematinics, hematopoietics, hemolytics, hemostatics, heparin antagonists, hepatic enzyme inducers, hepatoprotectants, histamine H₂ receptor antagonists, human immunodeficiency virus (HIV) protease inhibitors, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors, immunomodulators, immunosuppressants, insulin sensitizers, ion exchange resins, keratolyses, lactation stimulating hormones, laxatives/cathartics, leukotriene antagonists, LH-RH agonists, lipotropics, lipoxygenase inhibitors, lupus erythematosus suppressants, matrix metalloproteinase inhibitors, mineral corticoids, miotics, monoamine oxidase inhibitors, mucolytics, muscle relaxants, mydriatics, narcotic antagonists, neuroprotectives, nootropics, ovarian hormones, oxytocics, pepsin inhibitors, pigmentation agents, plasma volume expanders, potassium channel activators, progestogens, prolactin inhibitors, prostaglandins, protease inhibitors, reductase inhibitors, respiratory stimulants, reverse transcriptase inhibitors, sedatives/hypnotics, serenics, serotonin noradrenaline reuptake inhibitors, serotonin receptor agonists, serotonin receptor antagonists, serotonin uptake inhibitors, somatostatin analogs, thrombolytics, thromboxane A₂ receptor antagonists, thyroid hormones, thyrotropic hormones, tocolytics, topoisomerase I and Il inhibitors, uricosurics, vasomodulators including vasodilators and vasoconstrictors, vasoprotectants, xanthine oxidase inhibitors, and combinations thereof.

The term "reactive oxygen species" means a oxygen which causes a radical reaction easily. Examples of reactive oxygen species include, but are not limited to, singlet oxygen, superoxide, hydrogen peroxide and hydroxy radical.

The term "reacting agent for singlet oxygen" means a reagent which reacts with singlet oxygen as reactive oxygen species to form the absorbable compound. Examples of reacting agents for singlet oxygen include, but are not limited to, para-nitroso dimethylaniline with imidazole, para-nitroso dimethylaniline with histidine, cholesterol, dimethylfuran, and diphenylfuran. Preferable reacting agent for singlet oxygen is para-nitroso dimethylaniline with imidazole.

The term "reacting agent for superoxide" means a reagent which reacts with superoxide as reactive oxygen species to form the absorbable compound. Examples of reacting agents for superoxide include, but are not limited to, nitro blue tetrazolium, cytochrome c, epinephrine, lactose dehydrogenase, 6- hydroxy dopamine, ascorbic acid, tetranitromethane, lactoperoxidase, horseradish peroxidase, 2-methyl- 6-[p-methoxyphenol]-3,7-dihydroimidazo[1 ,2-a]pyrazine-3-one, and 2-methyl-6-phenyl-3,7- dihydroimidazo[1 ,2-a]pyrazine-3-one.

Photo irradiation is conducted by light source. The light sources for irradiation include, but are not limited to, sunlight, Xe lamp, low-pressure mercury lamp, high-pressure mercury lamp, super high- pressure mercury lamp, fluorescent mercury lamp, Na lamp, and laser with or without optical filter. The preferred light source is Xe lamp. The exposure time of photo irradiation is variable depending on the condition of the test materials. An example of the exposure time is from 2 minutes to 3 days, preferably from 2 hours to 24 hours.

The term "monitoring the reactive oxygen species" means measurement of (1) UV transition with the use of spectrophotometer and/or UV plate reader, (2) fluorescence using spectrofluorometer, (3) chemiluminescence using chemiluminescence detector, and (4) structural changes in reacting reagent(s) with the use of HPLC or other chromatographic tchnologies.

The method of the present invention is conducted in a reaction container. The transparent reaction container is preferable for colorimetric determination. Examples of reaction containers include glass tube, glass vial, crystal tube, crystal vial, multiple well plates made of crystal. A container preferred for use in the present invention is a glass vial or multiple well plate such as 96 well plates. It should be noted that size, shape, and material of the vial sometimes affect the photoreactivity of irradiated compounds, and the sample setting, e.g. standing, laying, and skewing, may be important for the reproducibility of the assay data, possibly due to the changes of light penetration. In this context, a multiwell plate may be suitable for this assay, since all the tested samples could be exposed to light in the same manner. It has been found that the use of multiwell plates resulted in the significant improvement of precision and productivity of assay. The cover of multiwell plate may be tightly associated with the light penetration, therefore the use of a quartz cover plate is suitable. Thus, the preferred container for use in the present invention is a multiple well plate such as 96 well plates with quartz cover plate.

The concentration of test material is variable depend on the material. An example of the concentration of test material is from 1 nM to 100 mM. Preferably the concentration is from 100 nM to 1 mM. Most preferably the concentration is 200 nM.

The concentration of the reacting agent is varible depend on the reacting agent used. An example of the concentration of reacting agent is from 0.1 nM to 100 mM. Preferably the concentration is from 0.1 nM to 100 mM. Most preferably the concentration is from 50 μM to 0.2 mM.

The PH of the testing solution is variable for a purpose of a testing. PH 7.4 is generally used for the purpose of simulation of the physiological environment. PH is controlled by the use of buffer solutions as known by those who are skilled in the art. Preferably the buffer solution used for the purpose of simulation of thephysiological environment is sodium phosphate buffer (pH 7.4).

Another embodiment of the present invention is the simulated test condition of the physiological environment. The addition of micelle, including sodium dodecyl sulfate (SDS), sodium laurate, Tween, sodium cholate, triton, 3- [ ( 3-Cholamidopropyl ) dimethylammonio ] -1 -propanesulfonate (CHAPS), cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), Zwittergent 3-14, and digitonin, organic solvents including methanol, ethanol, and trifluoroethanol to the assay system is effective to mimic the environment on the surface of cell membrane. Some photosensitive compounds are known to show photosensitivity when bound with drug-binding protein. This invention is applicable to simulating the physiological emviroments using drug-binding protein.

Another embodiment of the present invention is provided a method comprising the steps;

(1) adding the solution of a test compound, drug-binding protein and reacting agents for colorimetric assay of reactive oxygen species (singlet oxygen and/or superoxide) to a reaction container;

(2) irradiating the mixture solution by light source fqr designated time;

The term "drug-binding protein " means a protein which is capable of binding with an active pharmacetucial compound, include physiological molecules originated in animals including humans. Examples of drug-binding proteins include, but are not limited to, serum, plasma, blood cell, lipid, and albumin. Preferred drug-binding proteins are bovine serum albumin or newborn calf serum.

The concentration of drug-binding protein is variable depending on the drug-binding protein used. An example of the concentration of drug-binding protein is from 0.0001 % to 10%. Preferably the concentration is from 0.001% to 1%. Most preferably the concentration is 0.5%.

Another embodiment of the present invention provides a method for screening photogenotoxic compounds comprising the use of plasmid DNA. In general, plasmid DNA tends to form a supercoiled structure, however significant damage, including enzymatic digestion, oxidative stress, heat, and chemical reactions, results in the transformation into the open circular form. Further damage will lead to the cleavage and fragmentation of DNA. Thus, DNA damage may be easily assessed by the determination of plasmid structural changes. In this context, plasmid DNA may be generally used for the assessment of photogenotoxicity [Photochemistry and Photobiology, 2005, 81 :89-95].

Another embodiment of the present invention is provided a method for screening of photogenotoxic compounds comprising the steps;

(1 ) adding a solution of a test compound and plasmid DNA to a reaction container;

(2) photo irradiating the mixture solution by light source;

(3) monitoring the photocleavage of DNA or generation of photoadducts by electrophoretic analysis;

(4) photogenotoxicity of the test compound is evaluated in comparison with a data of known photogenotoxic drug.

The productivity of photogenotoxicity assay was improved as follows. Plasmid DNA and photosensitizers are dissolved in buffer solutions, and transferred into multiwell plate on the quartz reaction container. Mixtures are exposed to light from solar simulator, and then subjected to electrophoretic analyses, including gel electrophoresis and capillary gel electrophoresis. ./

> The term "Electrophoretic analysis" means a analysis for the transport process of charged analytes under the influence of an electric field. Nucleic acids are generally electrophoresed in neutral or basic buffers as anions with their negatively charged phosphate groups. Preferably determination is carried out using capillary electrophoresis because of its high productivity and precision. This assay system is applicable to high-throughput screening systems.

The term "Plasmid DNA" means a circular piece of DNA that exists apart from the chromosome and replicates independently. Plasmid DNA is often used in generic engineering to carry desired genes into organisms. Examples of plasmid DNA include, but not limited to, CoI El, pBR322, pBR325, pMW119, pMW218, pMW219, pkc7, and pEI14. Preferably the reacting agent for the photogenotoxicity assay of the present invention is pBR322.

When using the multiple well plates, the processes of filling the test materials and reacting agent, irradiating, and monitoring the reactive oxygen species may be automated, thus being applicable to high- throughout screening systems..

EXAMPLES

The following examples are given by way of illustration of the present invention. These examples are not to be construed as a limitation in any way of the present invention. Many variations of these examples are possible within the scope of the present invention. Example 1 Generation of sinαlet oxygen from photo-irradiated quinine

Quinine and sodium dodecyl sulfate (SDS) were used as a photosentisizer and a well-characterized photostable and non-phototoxic compound, respectively.

Quinine was dissolved in 20 mM sodium phosphate buffer (pH 7.4), containing 50 μM imidazole and 50 μM para-nitroso dimethylaniline, giving a final concentration of 0.2 mM. 1 ml_ of the solution in clear glass vial (Waters, Milford, MA, USA) was irradiated by Xe lamp (30,000 lux) for 2, 4, 8, 12, 18, and 24 hours. Then, UV absorbance of the tested solution mixture at 440 nm (A440) was measured, and the decrease of A440 indicated the existence of singlet oxygen in the solution. The same experiment using sodium dodecyl sulfate (SDS) instead of quinine was also conducted.

Photo-irradiation of the quinine solution (0.2 mM) resulted in a significant generation of singlet oxygen in a time-dependent manner (Table 1), whereas photo-irradiated SDS (0.2 mM) did not show any generation of singlet oxygen.

Table 1 Photo-induced eneration of sin let ox en

Data represents the mean for 4 independent experiments. N.D. means "not detected".

Example 2 Generation of superoxide from photo-irradiated quinine

Quinine was dissolved in 20 mM sodium phosphate buffer (pH 7.4), containing 50 μM imidazole and 0.2 mM nitro blue tetrazolium, giving a final concentration of 0.2 mM. 1 mL of the solution in clear glass vial (Waters, Milford, MA, USA) was irradiated by Xe lamp (30,000 lux) for 2, 4, 8, 12, 18, and 24 hours. Then, UV absorbance of the tested solution mixture at 560 nm (A560) was measured. The increase of A560 indicated the existence of superoxide in the solution.

Photo-irradiation of the quinine solution (0.2 mM) resulted in a significant generation of superoxide in a time-dependent manner (Table 2), whereas photo-irradiated SDS (0.2 mM) did not show any generation of superoxide.

Table 2 Photo-induced eneration of su eroxide

Example 3 Generation of reactive oxygen species from photo-sensitizers and photostable compounds

Generation of reactive oxygen species from photo-irradiated compounds (0.2 mM), including 17 photo-sensitizers and 9 non-photoreactive compounds, was examined using the above-referenced colorimetric assay.

After 18 hours exposure to Xe lamp (30,000 lux), all tested photosensitizers showed the significant induction of singlet oxygen, superoxide, or both as evidenced by the bleaching of para-nitrosoaniline and reduction of nitro blue tetrazolium. The yields and type of reactive oxygen species differed, and may depend on the structures and physicochemical properties of the test compounds. On the contrary, the generation of reactive oxygen species from photo-irradiated non-phototoxic compounds was negligible. These results provided further insight into the mechanism of phototoxicity, suggesting that measurements of reactive oxygen species after photo-irradiation may be effective for the prediction of phototoxic potential of organic and/or inorganic compounds.

Table 3 Photo-induced eneration of reactive ox en s ecies

Example 4 Inhibitory effect of ROS scavengers on the photo-induced degradation of furosemide and ketoprofen

An Inhibition study using reactive oxygen scavengers (ROS) was conducted to investigate the effect of ROS degradation on the test compound. Furosemide and ketoprofen, typical photosensitizers, were dissolved in 20 mM sodium phosphate buffer (pH 7.4), containing (1 ) superoxide scavenger; superoxide disumtase (SOD, 200 U), (2) radical scavenger; butylated hydroxyanisole (BHA, 1 mM) or gluthatione (GSH, 1 mM), (3) singlet oxygen scavenger; 1 ,4-Diazobicyclo[2,2,2]octane (DABCO, 1 mM). The solution of photosensitizer was photo-irradiated with Xe lamp (30,000 lux) for 12 hours. After irradiation, the purity of tested compound was evaluated by reversed phase HPLC (Waters Alliance system, Waters, Milford, MA, USA). Both furosemide and ketoprofen showed significant photo-degradation, whereas addition of some scavenger resulted in the dramatical attenuation of photo-degradation (Table 4). These results suggested the involvement of radical generation in the photo-induced degradation.

Table 4 Inhibitory effect of ROS scavengers on the photo-induced degradation of furosemide and ketoprofen

Scavengers Inhibition (%)

Data represents the mean for 3 independent experiments. N. D. means "not detected".

Example 5 Inhibitory effect of ROS scavengers on the photo-induced degradation of Quinine

Quinine, an anti-malarial drug, was dissolved in 20 mM sodium phosphate buffer giving a final concentration of 0.25 mg/mL, and then exposed to Xe lamp (30,000 lux). After exposure to light, HPLC analysis was conducted for the determination of intact quinine. The results showed the gradual photo- degradation of quinine in a time-dependent manner, and quinine was undetectable after 18 hr-exposure to Xe lamp. Interestingly, addition of radical scavengers to quinine solution resulted in the significant attenuation of photo-degradation of quinine (Figure 1 ). Among tested scavengers, sodium azide, a typical singlet oxygen scavenger, showed the most potent protective effect, whereas SOD, a specific scavenger for superoxide, failed to inhibit the photo-degradation. These results suggested the possible, involvement of singlet oxygen-generation in the photoinstable property of quinine.

Example 6 Photo-induced peroxidation of linoleic acid in the presence of photo-sensitizers and non-phototoxic compounds

Peroxidation of lipid was considered as a causative agent for photo-induced dermatitis, and it is well- established that reactive oxygen species also induce and enhance the lipid peroxidation. To clarify the involvement of reactive oxygen generation in the dermatitis induced by the photosensitizers, occurrence of lipid peroxidation was evaluated during photo-exposure of photosensitizers. Photo-sensitizers and non-phototoxic compounds were dissolved in 20 mM sodium phosphate buffer containing 0.05%(v/v) Tween 20 and linoleic acid (1 mM), and stored in the clear glass vial (Waters). After exposure to Xe lamp (30,000 lux) for 18 hours, oxidized fatty acid was quantitatively determined using 2-thiobarbituric acid reactive substrates (TBARS).

As shown in Table 5, all tested photosensitizers, especially benzoyl peroxide, showed the significant peroxidation of linoleic acid (P < 0.05), whereas non-phototoxic compounds showed no or weak effect.

Interestingly, SOD (200 U) inhibited the peroxidation of linoleic acid induced by furosemide (P < 0.01 ).

These results, taken together with a previous report indicating the relationship between lipid peroxidation and photo-dermatitis (H. Bagheri, et al. Drug Saf, 2000; 22(5):339-49), suggest that reactive oxygen plays the important role in the photosensitizers-induced lipid peroxidation and dermatitis.

Table 5 The effect of photosensitizers and non-phototoxic compounds on peroxidation of linoleic acid fter photo-irradiation

Data represents the mean+SD for 4 independent experiments. **, P < 0.01 ; *, P < 0.05 with respect to irradiated linoleic acid. ^#, P < 0.05 between indicated groups.

Example 7 Simulation for physiological environment using Bovine serum albumin

Sulfamethoxazole and benzocaine were used as test compounds. Test compounds (at a final concentration of 0.02 mM) and albumin (at a final concentration of 0.5%) were dissolved in 20 mM sodium phosphate buffer, containing 50 μM imidazole, 0.2 mM nitro blue tetrazolium and 50 μM para-nitroso dimethylaniline. The solution mixture, 1 mL, in clear glass vial (Waters, Milford, MA, USA) was irradiated by Xe lamp (30,000 lux) for 24 hours. Then, UV absorbances of the tested solution mixture at 440 nm and at 560nm were measured for singlet oxygen and superoxide, respectively.

The effect of drug-binding protein on the photo-induced generation of reactive oxygen species was investigated. As shown in Table 6, albumin (0.5%) enhanced the generation of singlet oxygen and superoxide in the presence of sulfamethoxazole and benzocaine. These results, taken together with UV spectral analyses, suggested that drug-binding protein in the reaction mixture is effective to simulate physiological environment.

Table 6 The effect of albumin on Photo-induced generation of reactive oxygen species

Example 8 Simulation of the physiological environment using micellar solutions

Anthracene, Chlorothiazide and Tamoxifen were used as test compounds. Test compounds (at a final concentration of 0.02 mM) and micelle (at a final concentration of 1.0% for Tween 20, and 100 mM for sodium laurate or sodium dodecyl sulfate) were dissolved in 20 mM sodium phosphate buffer, containing 50 μM imidazole and 50 μM para-nitroso dimethylaniline. The solution mixture, 1mL, in clear glass vial (Waters, Milford, MA, USA) was irradiated by Xe lamp (250 W/m²) for 1 hours. Then, UV absorbances of the tested solution mixture at 440 nm were measured for singlet oxygen.

The effect of micellar solutions on the photo-induced generation of singlet oxygen was investigated. As shown in Table 7, the generation of singlet oxygen from photo-irradiated compounds was variable depending on the type of compound and micelle. Anthracene, a neutral compound, showed a similar photoreactivity in all micellar solutions, however there are significant changes in the photoreactivity of chlorothiazide (pKa: 6.8, 9.4) and tamoxifen (pKa: 8.9). The membrane of epidermal cells contains a lot of acidic phospholipids, so the assay with the use of sodium laurate or sodium dodecyl sulfate reflects the physiological environment.

Table 7 The effect of albumin on Photo-induced generation of reactive oxygen species

Data represents the mean for 4 independent experiments.

Example 9 Evaluation of phototoxic and photosensitive potential of nifedipine and amlodipine

Generation of reactive oxygen species from photo-irradiated compounds (0.2 mM), including nifedipine and amlodipine, was examined by above-referenced colorimetric assay. After 18 hours exposure to Xe lamp (30,000 lux), mainly superoxide was detected in nifedipine-treated group (Increase of A560; 0.064), whereas amlodipine showed relatively low generation of superoxide (Increase of A560; 0.009).

Additionally, the photostability of the test compounds was investigated in solid and solution state. Exposure of nifedipine to Xe lamp (5,000 lux, solid state: 4 days; solution state: 6 hours) resulted in significant photo-degradation (Remaining nifedipine, not detected in solid state; not detected in solution state). On the contrary, amlodipine was not sensitive to the light exposure (Remaining oamlodipine, 93% in solid state; 98% in solution state). These findings indicate the probability that levels of reactive oxygen species from photo-activated compounds reflect their photosensitivity/photoinstability, as well as their phototoxic potential.

Example 10. Design of reaction container for high throughput screening

To improve the productivity of the assay, assay methodology was optimized with the use of a multiwell plate. Although the multiwell plate requires the use of a tight-fitting cover in order to inhibit the vaporization of solution and the increase of mixture concentration, glass and polymer covers tend to cut off the light depending on the thickness. This may have some effect on the reproducibility and precise of assay. Hence, for the multiwell plate cover, a quartz plate was chosen, because of its high light transparency. Thus, an effective reaction container was designed as shown in Figure 2.

Example 11 Generation of reactive oxygen species from photo-sensitizers and photoinactive compounds using quartz reaction container

Generation of reactive oxygen species from photo-irradiated compounds (0.2 mM), including 30 photo-sensitizers and 6 non-photoreactive compounds, was examined using the above-referenced colorimetric assay using a quartz reaction container.

After 18 hours exposure to Xe lamp (30,000 lux), all tested photosensitizers showed the significant induction of singlet oxygen, superoxide, or both as evidenced by the bleaching of para-nitrosoaniline and reduction of nitro blue tetrazolium. The yields and type of reactive oxygen species differed and may depend on the structures and physicochemical properties of the test compounds(Table 8).

Table 8: Generation of reactive oxygen species from photoirradiated compounds

On the contrary, the generation of reactive oxygen species from photo-irradiated non-phototoxic compounds was weak or negligible. These results clearly indicate that use of the quartz reaction container with multiwell plate is effective for the rapid and reproducible screening of photosensitizers. Example 12 Screening method for photogenotoxic compound

For the screening of photogenotoxic potential of the test compounds, pBR322 DNA and quinine were dissolved in 20 mM sodium phosphate buffer. The final concentration of pBR322 DNA and quinine were 0.2 μg/ml and 200 μM, respectively. The solution was exposed to light at the illuminance of 250 W/m² for 5, 10, 20 and 30 min. After light exposure, the reaction mixture was subjected to the agarose gel (1%) electrophoresis, and then gel was stained with ethidium bromide (Table 9).

Table 9: Structure of pBR322 DNA after light exposure with or without quinine (200 μM)

*Band detection; +++, Very strong; ++, Strong; +, weak; - , negligible

Without quinine, light exposure of pBR322 for at least 30 min did not show any changes. On the contrary, pBR322 in the presence of quinine displayed the photocleavage and conversion into open circular form in a time-dependent manner, and complete cleavage of pBR322 was confirmed at 30 min- exposure. These results suggested that quinine has photogenotoxic potentials.

Claims

WHAT IS CLAIMED IS:

1. A method for screening photosensitive compound comprising the steps;

(1) adding a solution of a test compound and reacting agents for singlet oxygen and/or superoxide to a reaction container;

(2) photo irradiating the mixture solution by light source;

(3) monitoring the reactive oxygen species generated by photo irradiation in each reaction container; and

2. A method of Claim 1 , wherein a drug-binding protein is also added in step (1 ).

3. A method for screening of photogenotoxic compounds comprising the steps;

(1) adding a solution of a test compound and plasmid DNA to a reaction container;

(2) photo irradiating the mixture solution by light source;

(3) monitoring the photocleavage of DNA or generation of photoadducts by electrophoretic analysis; and

(4) photogenotoxicity of the test compound is evaluated in comparison with a data of known photogenotoxic drug

4. A method of any one of Claims 1 to 2, wherein reacting agent for singlet oxygen is para-nitroso dimethylaniline with imidazole, para-nitroso dimethylaniline with histidine, cholesterol, dimethylfuran, or diphenylfuran.

5. A method of any one of Claims 1 to 2, wherein reacting agents for singlet oxygen are para-nitroso dimethylaniline with imidazole.

6. A method of any one of Claims 1 to 2, wherein reacting agent for superoxide is nitro blue tetrazolium, cytochrome c, epinephrine, lactose dehydrogenase, 6-hydroxy dopamine, ascorbic acid, tetranitromethane, lactoperoxidase, horseradish peroxidase, 2-methyl-6-[para-methoxyphenol]-3,7- dihydroimidazo[1 ,2-a]pyrazine-3-one, or 2-methyl-6-phenyl-3,7-dihydroimidazo[1 ,2-a]pyrazine-3-one.

7. A method of any one of Claims 1 to 2, wherein reactive agent for superoxide is nitro blue tetrazorium.

8. A method of Claim 2, wherein drug-binding protein is lipoprotein or albumin.

9. A method of any one of Claims 1 to 8, wherein the concentration of the test compound is from 1 nM to 10O mM.

10. A method of any one of Claims 1 to 8, wherein the concentration of the test compound is from 100 nM to 1 mM.

11. A method of any one of Claims 1 , 2, 4, 5, 6, 7, 8, 9 and 10, wherein the concentration of the reacting agent for reactive oxygen species is from 0.1 nM to 100 mM.

12. A method of any one of Claims 2 and 8, wherein the concentration of the drug-binding protein is from 0.001% to 1 %.

13. A method of Claim 3, wherein plasmid DNA is CoI El, pBR322, pBR325, pMW119, pMW218, pMW219, pkc7, or pEI14.

14. A method of Claim 3, wherein plasmid DNA is pBR322.

15. A method of any one of Claims 1 to 14, wherein reaction mixture is exposed to light in the quartz reaction container for high throughput screening, consisting of multiwell plate, quartz cover, and holding fixture.