Pharmaceuticals have been used by humans and livestock for the purpose of prevention or treatment of various diseases, all over the world. It is well-known that ultraviolet light (UV) irradiation, which is present in sunlight, provokes the loss of beneficial effects and the gain of adverse effects for photosensitive pharmaceuticals [1
]. Our previous reports also indicated that photodegradation of some pharmaceuticals, containing analgesic and antiepilepsy drugs, induced ecotoxicological effects on them [4
]. The energy of UV irradiation induces the excitation of chemical compounds, followed by elimination reaction, addition of the functional group, rearrangement and isomerization, and so on. Various photochemical reaction might change the physical or biological properties of photosensitive pharmaceuticals. Furthermore, UV irradiation makes a contribution to the generation of reactive oxygen species derived from the oxygen molecule, which might be a trigger of the oxidative reaction. The variety of photochemical reaction and their output as photoproducts is dependent on the variety of the chemical structure of UV-irradiated compounds and the wavelength of the UV. Additionally, UV irradiation has an effect on the content of active compounds in medicine. In the case of naproxen (NX), which is a non-steroidal anti-inflammatory drug (NSAIDs), the active compound in the tablet, its powder, and suspension was degraded by UV irradiation [5
]. This report suggests that photo-degradability of pharmaceuticals is a major determinant of their quality and quantity.
To overcome the photostability problem, many efforts were made to develop photostabilization strategies in recent years. A number of reports indicate that the encapsulation is the most used approach, followed by the addition of antioxidants and solar filters [6
]. In the encapsulation strategy, cyclodextrin and its modified form are major photoprotective carriers. However, in some cases, encapsulation is not able to stabilize photosensitive compounds, due to the difficulty of inclusion [7
]. The report indicates that various photoprotective methods are needed for the photostabilization of various pharmaceuticals. Our previous reports showed that selected antioxidants, such as ascorbic acid derivatives and some polyphenols (quercetin, catechin, and curcumin), are protective for the photodegradation of NX, and both the antioxidative potency and the photostability of antioxidants are needed for an efficient photostabilizer [8
]. Further study focused on researching the good photostabilizer is essential for the protection of photosensitive pharmaceuticals, especially those that are not protected by encapsulation.
In this study, the protective effects of selected amino acids on NX photodegradation were evaluated. NX is photosensitive for UV irradiation at longer wavelength and its degradation is suppressed by addition of some antioxidants, as shown in a previous report [8
], therefore, NX is used as a test compound. Properties of amino acids are dependent on their functional groups. Comparison of protective effects of amino acids on NX photodegradation is informative for clarifying the nature of a good photostabilizer. To the best of our knowledge, there are few reports focused on the protective effects of amino acids on the photodegradation of pharmaceuticals. First, photoprotective effects of 20 amino acids were investigated. NX was photo-exposed with each amino acid, and the residual amount of NX was determined by a high-performance liquid chromatography (HPLC) system, equipped with a reverse-phase column. Second, the photoprotective mechanisms of the tested amino acids were evaluated by means of a test kit for the potential antioxidant (PAO test) and UV absorption spectral analysis. The aim of this research was to determine the protective potencies of the tested amino acids for NX photodegradation, and to elucidate the importance of its nature for a good photostabilizer. The results of this experiment might make it possible to protect photosensitive pharmaceuticals from degradation, based on various mechanisms.
3. Materials and Methods
NX, Glycine, alanine, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, asparagine, phenylalanine, tyrosine, tryptophan, lysine, histidine, aspartic acid, glutamic acid, methanol, ethanol, hydrochloric acid (HCl), and acetic acid were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Proline, glutamine, and arginine were purchased from the Tokyo Chemical Industry Corporation (Tokyo, Japan). All amino acids used in this experiment were of DL-form. Milli-Q water (18.2 mΩ/cm) was prepared by using a Milli-Q water purification system (Merck, Darmstadt, Germany).
3.2. Preparation of the Test Solution
NX (10 mg) was initially dissolved in methanol (1 mL), and this solution was diluted with Milli-Q water to make a concentration of 10 mg/L (86.9 µmol/L). A volume of 9 mL of the diluted solution in a glass vial was used for the UV irradiation experiment. Additionally, selected amino acids were dissolved in 50% methanol to make a concentration of 50 mmol/L. When these did not dissolve easily, 10 µL of 1 mol/L HCl was added but the pH was not changed in comparison to pre-addition levels. In both conditions, the pH values of the test solutions were 6–7. In the photostabilization experiment, a test solution (9 mL) was prepared using a methanol solution of NX (43.4 mmol/L), a 50% methanol solution of amino acids (50 mmol/L) and Milli-Q water, to make a concentration of 86.9 µmol/L of NX and 100 µmol/L of each amino acid. Additionally, cysteine, tyrosine, and tryptophan solutions of 5 mol/L, 500 mmol/L, 500 µmol/L, and 50 µmol/L were prepared to evaluate the dose dependency of these amino acids. Each solution was added to the NX solution to achieve concentrations of cysteine, tyrosine, and tryptophan of 10 mmol/L, 1 mmol/L, 10 µmol/L, and 1 µmol/L.
3.3. UV Irradiation Experiment
UV irradiation was carried out using a light cabinet equipped with a 20 W FL20S BLB black light lamp (Toshiba, Tokyo, Japan). The most abundant wavelength of the emission light from this lamp was 365 nm, and its irradiation intensity value was 500 µW/cm2/s. The irradiation intensity was measured by a digital radiometer with a 365 nm sensor (UVX-36, UVP, Upland, CA, USA). The irradiation time was up to 3 h at 20 ℃. Water depth was 3.5 cm, and distance from the light source was about 15 cm. Control samples were also prepared for the same condition but covered with an aluminum foil to interrupt UV irradiation. All experiments were carried out in tetraplicates.
3.4. Evaluation of the Residual Amount of NX
The degradation of NX was monitored with a high-performance liquid chromatography (HPLC) system, which was composed of an LC-20 AD pump with an analytical column (Shim-pack VP-ODS, 5 µm, 4.6 × 150 mm), a SPD-20A UV detector, a CTO-20A column oven, and a C-R8A chromate-integrator (Shimadzu Corporation, Kyoto, Japan). Separation type of the HPLC system was reverse-phase chromatography. The column was kept at 40 ℃. A mixture of methanol and acetic acid (50% methanol containing 0.1% acetic acid, v/v) was used as an isocratic mobile phase at a flow rate of 1.0 mL/min. A mobile phase was prepared as follows; methanol (500 mL) and water (500 mL) were mixed, and acetic acid was added to make its concentration at 0.1% (v/v), followed by the shaking and the degas, using a sonicator and an aspirator. Detection wavelength was 254 nm. A volume of 20 µL of irradiated sample solutions were injected into an HPLC system. Retention times of NX and its main photoproduct were 22.2 min and 18.4 min. Amount of NX evaluated by HPLC are shown as % of initial compound before UV irradiation.
3.5. Evaluation of Antioxidative Activities
The antioxidative activities of selected amino acids were evaluated by means of the PAO test (Nikken SEIL Corporation, Shizuoka, Japan). This assay evaluated the Cu+ level derived from the reduction of Cu2+, induced by the antioxidative activities using the spectrophotometer. From these results, the antioxidative potencies of the tested samples were calculated as copper-reducing power (µmol/L). The tested amino acids were dissolved in 50% methanol to make a concentration at 100 µmol/L for the PAO test. All experiments were carried out in triplicates.
3.6. UV Spectral Analysis
Tested aromatic amino acids (phenylalanine, tyrosine, and tryptophan) were dissolved in ethanol at the final concentration of 100 µmol/L to 1 mmol/L. When there was difficulty in dissolving these acids, 10 µL of 0.1 mol/L HCl was added. UV absorption spectra were recorded with a V-670 UV/Vis spectrophotometer (JASCO, Tokyo, Japan), interfaced to a PC for data processing. The absorption-maximum wavelength (λmax, nm) of each amino acid was obtained from these results. The molar absorption coefficients (ε, L·mol−1·cm−1) were calculated from the absorption of λmax.
3.7. Statistical Analysis
Data are expressed as mean ± standard deviation (S.D.). The homogeneity of variance was established using a one-way ANOVA. Statistical significance was estimated by Tukey’s test. The threshold for assessing significance was p < 0.05 (vs. control).
From the results of the photostabilization experiments herein, cysteine, tyrosine, and tryptophan had protective effects on the photodegradation of the studied pharmaceutical. Cysteine suppressed NX photodegradation through antioxidative activity, and tyrosine and tryptophan suppressed it by UV filtering activity. Furthermore, dose dependencies of their protective effects were determined. However, in this experiment, the residual amount of the amino acids after UV irradiation, were not examined sufficiently. It is important for a good photostabilizer to have protective potencies and to be stable for photo-irradiation. Further research including investigation of other substances that have better protective potencies, and additional evaluation, are required to develop the stabilization strategy for photosensitive compounds.