Medical Center GABINET DERMATOLOGICZNY TOMASZ WASY YSZYN, 25/421 Gen. Anders St., Warszawa, Poland 2

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1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 6 pp. 1455ñ1460, 2016 ISSN Polish Pharmaceutical Society STUDIES ON STABILITY OF 2,3-DIPHENYLCYCLOPROPENONE IN CONTACT WITH WATER AND AQUEOUS NaCl SOLUTIONS. CONCLUSIONS FOR PURPOSE OF TOPICAL THERAPY OF PATIENTS WITH ALOPECIA AREATA TOMASZ WASY YSZYN 1 * and KATARZYNA BOROWSKA 2,3 1 Medical Center GABINET DERMATOLOGICZNY TOMASZ WASY YSZYN, 25/421 Gen. Anders St., Warszawa, Poland 2 Medical Center CADERM, 87/113 Marsza kowska St., Warszawa, Poland 3 Department of Histology and Embryology with Experimental Cytology Unit, Medical University of Lublin, 11 Radziwi owska St., Lublin, Poland Abstract: Diphenylcyclopropenone (DCP) is a topically administered agent used for more than three decades for treatment of alopecia areata (AA). Moreover, numerous recent studies show itís efficiency in treatment of cutaneous metastatic melanoma. Despite being a potentially useful drug still very little is known about the pharmacokinetics of DCP. The authors investigated the stability of DCP solutions in propylene glycol with the addition of 0.9% aquous solution of natrium chloride (0.9% NaCl) or with water. DCP was prepared in two concentrations: 0.1% and 3%. Itís stability was then measured with different proportions of 0.9% NaCl or water added and in different temperatures. Contrary to common opinion that DCP solutions are extremely unstable, authors have found them to be relatively stable. DCP solutions with the addition of equal quantity of 0.9% NaCl decomposed slowly at the temperature of 37 C but after 70 h all solutions still contained more than 80% of initial DCP. Solutions of DCP with the addition of 1% of water at the temperature of 4 C are in the present study stable, containing more than 98% of initial DCP after 20 days. Authors discuss the results in spite of possible metabolism of DCP on the surface of human skin during topical immunotherapy. Keywords: alopecia areata, contact sensitizers, diphenylcyclopropenone, stability, topical immunotherapy, solvents 2,3-Diphenylcyclopropenone (DCP) is a topically administered agent intended primarily for treating alopecia areata (AA). AA is a T cell-mediated autoimmune disease involving hair follicles characterized by hair loss. Frequency of AA ranges from 0.7% to 3.8% of patients attending dermatology clinics (1). Etiopathogenesis of AA is not known but evidence exists to support genetic, immune and environmental factors (2, 3). DCP plays the role of a sensitizing agent. It is also being used as an immune-modulating therapeutic factor. Although the mechanism of action of DCP has not been clearly defined, DCP induces changes in the perifollicular CD41/CD81 T-lymphocyte ratio (4), apoptosis of perifollicular lymphocytes (5) and modulate proinflammatory cytokines (6). Recently, there are numerous studies suggesting DCPís good efficiency in treatment of cutaneous metastatic melanoma (7, 8) suggesting the growing importance of this potential drug. Unfortunately, very little is known about the pharmacokinetics of DCP. The aim of this study was to investigate the stability of DCP in an environment similar to human body fluids or in the situation of contamination DCP samples with water. For this purpose the authors have chosen, respectively, 0.9% sodium chloride aqueous solution and water that was added to DCP solutions. EXPERIMENTAL Materials and Methods The authors chose solutions of DCP in propylene glycol because these were used successfully in their previous studies regarding treatment of AA (8). Usually, the concentration of 3% DCP is being used during the process of sensitization, at the beginning * Corresponding author: vasylek@poczta.fm 1455

2 1456 TOMASZ WASY YSZYN and KATARZYNA BOROWSKA of the treatment. Later on, smaller concentrations are administered to maintain moderate allergic reaction to DCP (8) and most often it is 0.1%. Hence, these two concentrations were chosen for this study. It was arranged to mimic three situations found during topical treatment with DCP: the treatment with 0.1% DCP (A), the sensitization with 3% DCP (B) and the potential contamination of medication with a small amount of water (C). The authors made three series of measurements, respectively: 0.1% DCP with 0.9% NaCl in proportions 1 : 1 (A), 3% DCP with 0.9% NaCl in proportions 1 : 1 (B) and 3% DCP with 1% addition of water which means proportions of 99 : 1 (C). The first two series of measurements have been performed at the temperature of 37 O C as it resembles the temperature of the human body. These were measured up to 70 h, as the therapeutic solutions of DCP (both 0.1% and 3%) are being washed from the scalp after h. The last one was made at the temperature of 4 O C like in the refrigerator where the DCP preparations are stored. This one was observed up to 20 days as authors usually prepare new DCP solutions once every 2-3 weeks. The study was performed for the standard of diphenylcyclopropenone (No , Sigma Aldrich) dissolved in propylene glycol (No. W294004, Sigma Aldrich) and in 0.9% NaCl aqueous solution. Samples were analyzed by high performance liquid chromatography with diode array detection (HPLC-UV DAD) using internal normalization method. Method description: HPLC-DAD conditions: column: Phenomenex C18, 250 mm 4.6 mm; oven temperature: 37 O C; mobile phase: acetonitrile + water ( ); mobile phase flow: 1 ml/min; λ = 296 nm. Reagents: diphenylocyclopropenone (DCP), 99%; Alfa Aesar, batch No ; diphenylacetylene (DPA); 98%, Aldrich, batch No. STBC7355V; propylene glycol, Sigma-Aldrich; deionized water, ultra pure, Millipore; acetonitrile, 99.9%, POCh; sodium chloride, POCh. Table 1. Decomposition of 3% DCP solution in propylene glycol (without additions). Numbers show proportions of DCP (left side) to it's product of decomposition, diphenylacetylene - DPA (right side). Time 0 After 7 days After 14 days Temperature / / / O C 99.87/ / / O C Table % DCP solution in propylene glycol with the addition of 0.9% NaCl in proprtions 1 : 1, v/v. Amount of DCP shown in percentage of the initial amount at time "0". Temperature + 37 O C. Time of Area of DCP percentage measurement DCP sample according to time C [mmol/l] Note: Area of DCP sample (Tables 2-5) is area under the curve, measured as a product of peak's height in milivolts and it's length in minutes.

3 Studies on stability of 2,3-diphenylcyclopropenone in contact with water and Table 3. The amount of diphenylacetylene (DPA) in 0.1% DCP solution in propylene glycol with the addition of 0.9% NaCl in proportions 1 : 1, v/v, using the same scale as in Table 2. Here 100% would mean that solution contains 0.1% DPA and all DCP has been decomposed. Temperature + 37 O C. Time of Area of DPA percentage measurement DPA sample according to time C [mmol/l] Table 4. 3% DCP solution in propylene glycol with the addition of 0.9% NaCl in proprtions 1 : 1 v/v. Amount of DCP shown in percentage of the initial amount at time "0". Temperature + 37 O C. Time of Area of DCP percentage measurement DCP sample according to time C [mmol/l] Analysis of DCP stability at a concentration level of 3% dissolved in propylene glycol (no additions) There were two 3% DCP mixtures in propylene glycol: stored at 37 O C and 4 O C. In determined time intervals (after 7 and 14 days) the solution was analyzed using HPLC technique. Peak area of DCP measured at time zero (at the beginning of the experiment) was compared with data obtained after above mentioned storage period intervals. Analysis of DCP at a concentration level of 0.1% dissolved in propylene glycol and in 0.9% NaCl aqueous solution DCP at a concentration level of 0.1% was prepared by dissolving the standard of DCP in a mixture of propylene glycol and 0.9% aqueous NaCl (1 : 1, v/v). The obtained solution was stored at 37 O C with light exposure. At determined time intervals (after 4, 5, 8, 11, 15, 18, 28, 35, 44, 51 and 70 h) the solution was analyzed using HPLC technique. Peak area of DCP

4 1458 TOMASZ WASY YSZYN and KATARZYNA BOROWSKA Table 5. 3% DCP solution in propylene glycol with the addition of water in proportions 99 : 1, v/v. Amount of DCP shown in percentage of the initial amount at time "0". Temperature + 4 O C. Time of Area of DCP percentage measurement DCP sample according to time C [mmol/l] 0 h h h h h days days days Table 6. Analysis of seven individual DCP solutions in propylene glycol, at the same concentration level, injected into chromatography column. Area of Solution DCP sample No. (mv min) Mean S.D RSD [%] 0.33 RSD = Relative standard deviation measured at time zero (at the beginning of the experiment) was compared with data obtained after the above mentioned storage period intervals. Furthermore, content (percentage) of diphenylacetylene, the main decomposition product of DCP, was assessed. Analysis of DCP at a concentration level of 3% dissolved in propylene glycol and in 0.9% NaCl aqueous solution DCP at a concentration level of 3% was prepared by dissolving the standard of DCP in mixture of propylene glycol and 0.9% aqueous NaCl (1 : 1, v/v). The obtained solution was stored at 37 O C with light exposure. At determined time intervals (after 4, 5, 8, 11, 15, 18, 28, 35, 44, 51 and 70 h) the solution was analyzed using HPLC technique. The sample was diluted before injection (0.1 ml of sample in 1 ml of acetonitrile) not to exceed the detector linearity range for the examined analyte. Peak area of DCP measured at time zero (at the beginning of the experiment) was compared with data obtained after above mentioned storage period intervals. Furthermore, content of diphenylacetylene, the main decomposition product of DCP, was assessed in time zero (0.58%) and after 70 h (4.80%). Analysis of DCP at a concentration level of 3% dissolved in propylene glycol with addition of 1 mass percent of water Water content in 3% DCP solution in propylene glycol was analyzed using the Karl Fischer technique. The result (0.063%, n = 4) was below the assumed value 0.1%. For the other part of the experiment one mass percent of water was added to the sample. DCP solution was stored at 4 O C with no light exposure. At determined time intervals (after 4, 24, 28 and 48 h, 6, 12 and 20 days) the solution was analyzed using HPLC technique. The sample was diluted before injection not to exceed the detector linearity range for the examined analyte. Peak area of DCP measured at time zero (at the beginning of the experiment) was compared with data obtained after the above mentioned storage period intervals. Furthermore, content of diphenylacetylene, the main decomposition product of DCP, was assessed in time zero (0.45%) and after 20 days (0.58%). Validation of the method The HPLC method was developed for this analysis mainly because gas chromatography (GC) has lim-

5 Studies on stability of 2,3-diphenylcyclopropenone in contact with water and Figure 1. Graphical presentation of data from Tables 2-4 ited usage in water solutions. The precision (repeatability) was determined by analyzing seven individual DCP solutions in propylene glycol, at the same concentration level, injected into chromatography column (Table 6). Relative standard deviation (RSD) is 0.33% which proves the methodís precision. This denotes about reliability of the analytical method, especially when the relative concentrations of analytes are presented as results (internal normalization method). The chromatographic system was stable during the experiment. For the long-term analyses, the quality control (QC) standards where used to check the system stability before injection of DCP solution after storage period. The study was conducted in accredited analytical laboratory - Institute of Industrial Organic Chemistry, Warszawa, Poland using apparatus under qualification procedure. Statistical analysis For statistical analysis Pearsonís correlation coefficient has been applied. RESULTS AND DISCUSSION Initially, the authors measured a decomposition range of 3% DCP solution in propylene glycol without any additions. Results are shown in Table 1. The solution that was kept in a temperature of 4 O C turned out to be stable, the degree of decomposition is negligible at least up to 14 days of observation (0.12%). This observation suggests that if ever DCP preparations in propylene glycol would be available commercially they should be kept in a refrigerator because sample stored at room temperature will decompose slowly but visibly (2.38% after 14 days). Further analysis of DCP samples with the addition of 0.9% NaCl or water is shown in Tables 2-5 and Figure 1. The study included a series of measurements which ended after 70 h (except 3% DCP with 1% of water). In both concentrations: 0.1% and 3% with 1 : 1 proportions of 0.9% NaCl has been decomposing during 70 h of observation. It remained 86.4% and 81.1%, respectively, of itís initial amount (Table 2 and 4). Correlation coefficient for these data is statistically important measuring This means that 3% DCP decomposes quicker than 0.1%. Meanwhile, the amount of diphenylacetylene (DPA), the product of DCPís decomposition (0.1% DCP 1 : 1 0.9% NaCl) increased proportionally to DCPís decomposition (Table 2). Correlation coefficient for the DCP versus DPA levels (in 0.1% DCP solution) measures 0.989; hence. the more DCP disappears, the more DPA appears in linear relationship, according to chemical equation, in which each molecule of DCP decomposes giving one molecule of DPA and one of carbon monoxide: DCP DPA + CO (9). This proved accuracy of previous measurements of DCP. Above mentioned findings suggest that large amounts of aqueous solution accelerate decomposition of DCP though they decomposes slower than the authors have expected. Ultimately, the authors wanted to assign the tempo of DCP decomposition in a situation when a small amount of water contaminated the sample, which may happen in any lab. The 1% (one mass percent) amount of water did not accelerate decomposition drastically; after 20 days there was still 98.19% of initial DCP, which is below the error of measurement (Table 5). CONCLUSION Very little is known about the way DCP reacts with human skin. It is hypothesized that at least

6 1460 TOMASZ WASY YSZYN and KATARZYNA BOROWSKA some molecules of DCP reach deep inside hair follicles since there was peribulbar and perivascular infiltrate of lymphocytes after itís application (4, 8). Authors concluded that since human fluids contain about 0.9% NaCl (sweat a bit less) the addition of 0.9% NaCl to DCP samples might be the first step to investigate itís stability in vivo. Of course human skin is much more complex and it is to be expected that the stability of DCP in vivo would be diminished by other factors: enzymes, keratin, immune system and others. In fact, from a clinical point of view, it would be best if the solutions of DCP, stable in a solvent and in the refrigerator, would be less stable in vivo. The process of topical immunotherapy is to obtain mild allergic reaction which stops after a day or two, to avoid serious side effects. Paradoxically, an ìunstableî allergen is advised here. From clinical observation (4), DCP might be exactly that kind of allergen since serious allergic reactions to it are described rarely. Moreover, most authors observed an allergic reaction which usually disappeared after a day or two, therefore it is possible that there are mechanisms which inhibit or decompose DCP in vivo. The results showed that after 70 h both samples: with 0.1% DCP and 3% DCP still contain more than 80% of primary DCP when mixed 1 : 1 with 0.9% NaCl. It does explain the phenomenon of the disappearing reaction to DCP only partially. There may be two scenarios to explain this phenomenon. The first one assumes that the product of DCP decomposition ñ diphenylacetylene (DPA) acts as an inhibitor of DCP in vivo. That would be possible since many products of chemical reactions inhibit the reaction itself. The second scenario suggests that there are other mechanisms in vivo, as mentioned above, that decompose DCP or inhibit itís activity. They include: the role of biological barriers, the role of the immune system, reactions with proteins such as keratin and others. These are numerous and require further investigation in the near future. Acknowledgments Authors would like to thank Dr. Tomasz ZieliÒski from National Defense University, Warsaw, for preparing statistical analysis and Edyta πcka, M.Sc., Analytical Department, Institute of Industrial Organic Chemistry, Warsaw for chemical analysis. REFERENCES 1. Alkhalifah A., Alsantali A., Wang E., McElwee K.J., Shapiro J.: J. Am. Acad. Dermatol. 62, 177 (2010). 2. Damian D.L., Shannon K.F., Saw R.P., Thompson J.F.: Australas. J. Dermatol. 50, 266 (2009). 3. Gilhar A., Paus R., Kalish R.S.: J. Clin. Invest. 117, 2019 (2007). 4. Herbst V., Zˆller M., Kissling S., Wenzel E., Stutz N. et al.: Eur. J. Dermatol. 16, 537 (2006). 5. Hoffmann R., Wenzel E., Huth A., van der Steen P., Sch ufele M. et al.: J. Investig. Dermatol. 103, 530 (1994). 6. Hordinsky M., Ericson M.: J. Investig. Dermatol. Symp. Proc. 9, 73 (2004). 7. Kim Y.J.: Ann. Dermatol. 24, 373 (2012). 8. Wasy yszyn T., Koz owski W., Zabielski S.L.: Arch. Dermatol. Res. 299, 231 (2007). 9. Wilkerson M.G., Henkin J., Wilkin J.K.: J. Am. Acad. Dermatol. 11, 802 (1984). Received: