Journal of Clinical Pharmacy and Therapeutics (1990) 15,291-300.
PHOTODEGRADATION OF DOXORUBICIN, DAUNORUBICIN AND EPIRUBICIN MEASURED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY M. J. Wood,* W. J. Irwin and D. K. Scott Drug Development Research Group, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B 4 7 E T and *Pharmacy Department, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 ZTH, U . K .
SUMMARY
The degradation kinetics of doxorubicin, daunorubicin and epirubicin in aqueous solution under fluorescent light and sunlight were studied using high-performance liquid chromatographic (HPLC) methods. The rates of photodegradation of all three drugs were similar, they were inversely proportional to the drug concentration and were accelerated by an increase in the pH of the vehicle. Photodegradation followed first-order kinetics. At concentrations greater than or equal to 500 pg/ml no special precautions appeared to be necessary to protect freshly prepared solutions of these agents from light. Photolysis was very rapid, however, at concentrations in the low microgram range therefore, when these solutions are used for in-vitro work or stability studies, they should be protected from light at all times. In addition, adsorptive losses, which may also be pronounced in low concentration solutions, should be prevented by storage in polypropylene containers. INTRODUCTION Doxorubicin, daunorubicin and epirubicin have been used successfully to treat a wide range of neoplastic diseases. Information on the stability of these analogues is available (1-6) but data are limited and contradictory. The stability of the anthracyclines is affected by many factors including the storage temperature, the pH of the vehicle and light. The large differences in stability, which have been reported by different groups for virtually identical experiments (2,7), may be partially explained by poor control, or a lack of control, of photodegradation. There are no published data concerning the photodegradation of daunorubicin and epirubicin. In addition, the photodegradation of doxorubicin has only been quantified fluorimetrically(7). Fluorescence does not differentiate between parent compounds and metabolites (8). Therefore, if, as some authors have postulated, aglycones are formed Correspondence: M. J. Wood, Pharmacy Department, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, U.K.
29 1
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during photolysis of the anthracyclines (9), the extent of photodegradation measured in these studies may have been underestimated by fluorescence techniques. The purpose of these investigations was to study and compare the degradation kinetics of doxorubicin, daunorubicin and epirubicin in natural and artificial light using HPLC methods. The conditions which facilitated or prevented photodegradation were identified by investigation of the effect of drug concentration, the solvent pH and the type of storage container on the rate of photodegradation. MATERIALS AND M E T H O D S
Chemicals and reagents Doxorubicin, daunorubicin and epirubicin were purchased as the commercially available pharmaceutical dosage forms Adriamycin, Pharmorubicin (both Farmitalia) and Cerubicin (Rhijne-Poulenc). All reagents were of analytical grade, or HPLC grade, as appropriate (BDH Ltd, Poole, U.K.). Equipment The HPLC system consisted of an Altex lOOA pump which was used to deliver eluent to a 10 cm x 4.6 mm, stainless steel, Shandon column which was packed with ODS Hypersil5 pm reversed-phase material. Injections were made with a Rheodyne model 7125 sample injector equipped with a lop1 injection loop. A Pye Unicam variable wavelength detector was operated at 290nm, (0-16 a.u.f.s.) and connected to a JJ instruments CR452 chart recorder. Assay conditions The HPLC methods were developed from a method reported by Beijnen et a l ( l 0 ) . The stability-indicating capacity of these methods was demonstrated by comparison of standard solutions, partially degraded and fully degraded solutions. Decomposition was accelerated by subjecting the solutions to extremes of temperature and pH. The mobile phases for doxorubicin, daunorubicin and epirubicin contained acetonitri1e:water40:60,55:45 and 5 0 5 0 (v/v), respectively. Ten drops of diethylamine were added to each litre of mobile phase which was then adjusted to pH 2.5 with 10% orthophosphoric acid (v/v). The flow rates were 1.4, 1.5 and 1.3 ml/min, respectively, and the chart speed was 5 mm/min. Preparation of solutions for photodegradation studies Vials of doxorubicin, epirubicin and daunorubicin were reconstituted with Waterfor-Injections and standard solutions, between 10 and 500 pg/ml, were prepared by simple dilution with Water-for-Injections using a Gilson fixed-volume pipette. Photodegradation was investigated directly after transfer of these solutions to either 20-ml clear glass, amber glass or opaque polyethylene bottles. Admixtures were prepared for the study at increased pH by combination of equal parts of single strength Tris buffer (pH 7.2 and 8.0) and standard solutions of doxorubicin (50 pg/ml). Duplicates of each container were sealed with plastic screw caps and stored at 25°C either covered with aluminium foil in a darkened room (doxorubicin only) or exposed to room light. In the room light studies, constant illumination was provided by four 65/80 W ceiling fluorescent tubes (Thorn EMI) mounted approximately 1 m above the
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samples, which were placed upright on the laboratory bench. The solutions were unprotected from sunlight. Two-millilitre samples were removed for chromatographic analysis at appropriate time intervals over a period of 168 h. An equal amount of 0.1 M HCl was added to all Tris buffer samples to quench the reaction. All samples were immediately frozen and stored at -20°C prior to assay using the described conditions. All samples were protected from light prior to initiation of the study, during thawing at ambient temperature and prior to assay. Experimental runs for each drug fluid admixture were duplicated. Duplicate samples (10 pl) were injected directly using the conditions described above. Treatment of data The remaining concentrations were determined by interpolation of the calculated peak areas on calibration curves which were constructed daily. All concentrations, which were expressed as a percentage of the original concentration at time zero, were the mean of duplicate values. Significant degradation was defined as a loss of 2 10% of the original concentration. Data were evaluated for a first-order kinetic model by construction of plots of the natural logarithm of the percentage parent drug remaining versus time. The rate constant (k)for drug loss was calculated by linear regression analysis of the first-order plot. Differences in the extent of degradation in the analysed samples were tested for significance by means of a two-way analysis of variance, with replicates. Where it was necessary only to test the difference between two means for significance, Student's t-test was employed. The pooled standard deviation and the standard error of the difference were calculated according to the usual equations (11). R E S U L T S AND DISCUSSION Chromatograms obtained for aqueous solutions of doxorubicin, (pH 6. l), stored in clear glass and polyethylene containers exposed to room light, showed a decrease in the concentration of the parent compound with time together with the emergence of three other peaks, which eluted close to the solvent front. Figure 1 shows a typical chromatogram obtained for a solution of doxorubicin (10 pg/ml) which was stored in clear glass. There was no evidence of these additional peaks on chromatograms obtained from solutions which were stored in amber glass and exposed to light, or in any of the control solutions which were stored in the dark. For this reason the three peaks, which eluted at 1.2, 1.4 and 1.6 min, were postulated to represent photodegradation products. Chromatograms obtained for solutions of 25 pg/ml doxorubicin in Tris buffer at pH 7.2 (exposed to light for 8 h) and pH 8-0(exposed to light for 2 h) both showed one broad peak (DII), which eluted after the parent compound and which was postulated to be an aglycone (Fig. 2). Chromatograms obtained for the same solutions after prolonged exposure to light (Fig. 3) showed almost total disappearance of both the parent compound and aglycone peaks and the presence of peaks which were similar to those observed after photodegradation of aqueous solutions of doxorubicin (Fig. 1). This suggests that hydrolysis in alkaline solution initiated aglycone formation while further exposure to light resulted in the breakdown of these aglycones to other products. The
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J 1
0
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J 0
Fig. 1. Chromatogramof doxorubicin (10 pg/ml in clear glass) showing 70% degradation after exposure to room light for 168 h, D =doxorubicin.
data also indicate that the final photodegradation products formed in alkaline solution were of a similar nature to those formed in aqueous solution. It has been suggested that the major photoadduct produced in weakly alkaline solution is the aglycone, 7,8-dehydro-9,10-desacetyldaunorubicinone (DII) (Fig. 4) and that the disappearance of this product after prolonged exposure to light involves an oxygen-dependent photobleaching process (9). This is in accordance with the data presented in Figs 2 and 3, respectively. It is also possible that a direct photobleaching process could occur without proceeding via an aglycone. This observation would account for the emergence of the peaks observed at 1.2, 1.4 and 1.6 min, shown in Fig. 1,which were not preceded by aglycone formation.
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1
0.002 A U
I NJ
6
4
2
Time (min)
0
6
4 2 Time (min)
0
Fig. 2. Chromatogram of doxorubicin (25 pg/ml in half strength Tris buffer pH 7.2 and 8.0)after exposure to room light for (a) 8 h and (b) 2 h, respectively, D =doxorubicin, DII = aglycone degradationproduct.
Aglycones were produced at pH 7.2 and 8.0 in the dark as well as in room light, but were not evident in any of the solutions stored at pH 6.1. This indicates that aglycones are not solely photodegradation products, but that their formation was dependent on the pH of the solution. Calculated rates of degradation and tgOs/, values for solutions of doxorubicin at pH 6.1, 7.2 and 8.0 are presented in Table 1.These results show that both photodegradation and degradation of solutions of doxorubicin, which were stored in the dark, were facilitated by an increase in the pH of the solvent. Data from previous studies, in which a pH profile was constructed for this drug, supported this statement as the rate of degradation of doxorubicin was shown to increase rapidly as the pH of the medium increased (pH 6.0-11.98) (12,13). Drug loss from aqueous solutions (pH 6.1), which were stored in the dark, increased as a proportion of the total amount as the concentration of the solution decreased. The amount of drug loss also appeared to be related to the container type (Tables 2 and 3). As chromatograms showed no evidence of degradation products it was concluded that adsorption onto the container had occurred. Adsorption of doxorubicin onto glass (14, 15) and polyethylene (14) has been observed. In addition, doxorubicin, daunorubicin and epirubicin have been reported to sorb to polyvinylchloride (PVC) in a process which involves a rapid adsorptive phase followed by a slower dissolution and migration of the drugs into the plastic matrix (13). Adsorptive and sorptive losses may be largely prevented by storage in polypropylene containers (13). The rates of photodegradation of doxorubicin, daunorubicin and epirubicin in aqueous solution were similar (Table 4) and were inversely proportional to the drug
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6
4
2
T i m e (rnin)
0
1
6
4
2
0
Time (min)
Fig.3. Chromatogram of doxorubicin (25 pg/ml in half strength Tris buffer pH 7.2 and 8.0)after exposure to room light for (a) 96 h and (b) 48 h, respectively.
concentration. Photodegradation was more rapid in solutions stored in clear glass than in solutions stored in polyethylene or amber glass. This observation was supported by data from ultraviolet (uv) transmission curves, which indicated that the opaque polyethylene afforded some protection from light as a gradual decrease in transmittance from 40% at 750 nm to 10% at 280 nmwasobserved. Theamberglass containers effectivelyprevented photodegradation as they conformed with the USP recommendations which state that transmittance should be less than 12% between 290 and 450 nm.
Doxorubicin, daunorubicin and epirubicin
CH,O
0
OH
0
OH
297
Fig. 4. Structure of 7,8-dehydro-9,1O-desacetyldaunorubicinone.
Table 1. Rate constants and rw,,ovalues for doxorubicin (25pg/ml in half strength Tris buffer at pH 7.2 and pH 8-0)in clear glass containers pH of admixture
Rate constant (h-')
6.1 (room light) 6.1(dark) 7.2(room light) 7.2(dark) 8.0(room light) 8.0(dark)
2.85x 10-3 1.40x 10-4 7.65x 1.41 x 1.23x lo-' 3.90x
two,
value (h)
32 > 168 1.3 6 0.8 1.8
Table 2. Percentage doxorubicin remaining after exposure to room light for 168h Percentage doxorubicin remaining j z i SD (n = 3)(pg/ml)
Clear glass Amber glass Polyethylene
500
250
100
50
25
10
98.6i0.5 96.1i0.7 98.8i0.8
94.8i0.6 97.3i0.5 101.8+0.8
93.3i 1.4 96.4f 1.1 96.8k0.7
84-9iO.6 94.6k1.6 96.9f 1.7
615f1.5 82.1f1.6 83.3f1.5
31.8f1.4 35.8f1.6 39.5f1.8
Table 3. Percentage doxorubicin remaining after storage in the dark for 168h Percentage doxorubicin remaining a+ SD (n = 3)(pglml)
500 Clear glass Amber glass Polyethylene
98.5i1.1 98.1k 1.3 98.9k1.0
250
100
97.8fl.2 97.0k1.4 98.3k1.3 98.7k1.4 101.5f1.8 102.4k1.1
50
25
10
98.3k1.1 98.4f1.1 98-4k1.4
965f1.9 96.6f2.0 98.1k1.0
90.9k1.3 90.5k1.5 96.9F1.5
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Table 4. Rate constants for photodegradationof doxorubicin, daunorubicinand epirubicin in clear glass Rate constant (k)(h-')(pgiml) Drug Epirubicin Daunorubicin Doxorubicin
100
50
25
10
3.75x 10-4 4.92x 3.93 x 10-4
1.41 x 10-3 1.23x 10-3 1.01 x 10-3
2.69 x 10-3 2.78x 10-3 2-85x 10-3
5.35x 10-3 5.13x 10-3 6.50 x 10-3
I
40
0
24
48
72
96
I20
144
I68
Time ( h )
Fig. 5. First-order plot for the photodegradationof epirubicin in clear glass containers. (A)100 pg/ml, (X) 50 pg/ml, (0) 25 pg/ml, (0) 10 pg/ml.
Plots of the natural logarithm of the percentage parent drug remaining versus time were linear ( r = >0.999), which indicated that photodegradation followed first-order kinetics. The first-order plots for epirubicin (Fig. 5) showed that rapid photolysis
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occurred over the concentration range 10-50 pg/ml. At concentrations 2 100 pg/ml, little or no photodegradation occurred in either glass or plastic containers (Table 2). These data indicate that in the clinical situation, where these drugs are used in concentrations 2 500 pg/ml, it is not necessary to protect solutions of these drugs from light during intravenous administration.
CONCLUSION At concentrations, such as those used for cancer chemotherapy ( 2500 pg/ml), no special precautions are necessary to protect freshly prepared solutions of these agents from light during intravenous administration. The experimental conditions of the present study are similar to those in which these agents are handled in low concentrations in the laboratory. The present findings are of practical importance and indicate that decomposition of these anthracyclines at concentrations in the low microgram and nanogram range may be very rapid if solutions are exposed to high intensity artificial light, or sunlight, for sufficient time. Photodegradation was also facilitated by an increase in the pH of the solvent. Concerning the possibility of preventing photolysis, keeping solutions in absolute darkness effectively protects the drugs from photodegradation. Drug loss due to adsorption onto the container, which may also be a problem in low concentration solutions, may be minimized by storage in polyethylene containers, or largely prevented by storage in polypropylene containers. REFERENCES 1. Benvenuto, J.A., Anderson, R., Kerkof, K., Smith, R.G. & Loo, T.L. (1981) Stability and compatibility of antitumour agents in glass and plastic containers. AmericanJournal of Hospital Pharmacy, 38, 1914-1918. 2. Poochikian, G.K., Cradock, J.C. & Flora, K.P. (1981) Stability of anthracycline antitumour agents in four infusion fluids. AmericanJournal of Hospital Pharmacy, 38,483-486. 3. Karlsen, J., Hjort Thonnesen, H., Resberg Olsen, I., Horne Sollien, A. & Skobba, T.J. (1983) Stability of cytotoxic intravenous infusions subjected to freezethaw treatment. Norwegica Pharmaceutica Acta, 45,61-67. 4. Keusters, L., Stolk, L.M.L., Umans, R. &Van Asten, P. (1986) Stability of solutions of doxorubicin and epirubicin in plastic minibags for intravesical use after storage at -20°C and thawing by microwave radiation. Pharmaceutisch Weekblad [Scientific Edition], 8, 194-197. 5. Beijen, J.H., Rosing, H., De Vries, P.A. & Underberg, J.M. (1985) Stability of anthracycline antitumour agents in infusion fluids. Journal of Parenteral Science and Technology, 39,220-222. 6. Hoffman, D.M., Grossano, D.D., Damin, L.-A. & Woodcock, T.M. (1979) Stability of refrigerated and frozen solutions of doxorubicin hydrochloride. American Journal of Hospital Pharmacy, 36, 1536-1538. 7. Tavoloni, N., Guarino, A.M. & Berk, P.D. (1980) Photolytic degradation of adriamycin. Communications. Journal of Pharmacy and Pharmacology, 32,860-862. 8. Rahman, A., Goodman, A., Foo, W., Harvey, J., Smith, F.P. & Schein, P.S. (1984) Clinical pharmacology of daunorubicin in phase I patients with solid turnours: development of an analytical methodology for daunorubicin and its metabolites. Seminars in Oncology, 11,36-44. 9. Gray, P.J.& Phillips, D.R. (1981) Ultraviolet photoirradiation of daunomycin and DNA-daunomycin complexes. Photochemistry and Photobiology, 33,297-303. 10. Beijnen, J.H., Wiese, G. & Underberg, W.J.M. (1985) Aspects of the chemical stability of doxorubicin and seven other anthracyclines in acidic solution. Pharmaceutisch Weekblad [Scientific Edition], 7, 109-1 16. 11. Neville, A.M. & Kennedy, J.B. (1 964) Basic Statistical Methods for Engineers and Scientists. Intertext Books, London.
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12. Wood, M.J. (1988)Stability of anthracycline cytotoxic agents in solution and infusionpuids. MPhil thesis, Aston University, Aston. 13. Wood, M.J., Irwin, W.J. & Scott, D.K. (1990)Stability of doxorubicin, daunorubicin and epirubicin in plastic syringes and minibags, Journal of Clinical Pharmacy and Therapeutics, IS, 279-289. 14. Bosanquet, A.G. (1986)Stability of antineoplastic agents during preparation and storage for in vitro assays. 11. Assay methods, adriamycin and the other antitumour antibiotics. Cancer Chemotherapy and Pharmacology, 17,l-10. 15. Tomlinson, E. & Malpeis, L. (1982)Concomitant adsorption and stability of some anthracycline antibiotics.Journal of Pharmaceutical Sciences, 71, 1121-1 125.
PHOTODEGRADATION OF DOXORUBICIN, DAUNORUBICIN AND EPIRUBICIN MEASURED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY M. J. Wood,* W. J. Irwin and D. K. Scott Drug Development Research Group, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B 4 7 E T and *Pharmacy Department, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 ZTH, U . K .
SUMMARY
The degradation kinetics of doxorubicin, daunorubicin and epirubicin in aqueous solution under fluorescent light and sunlight were studied using high-performance liquid chromatographic (HPLC) methods. The rates of photodegradation of all three drugs were similar, they were inversely proportional to the drug concentration and were accelerated by an increase in the pH of the vehicle. Photodegradation followed first-order kinetics. At concentrations greater than or equal to 500 pg/ml no special precautions appeared to be necessary to protect freshly prepared solutions of these agents from light. Photolysis was very rapid, however, at concentrations in the low microgram range therefore, when these solutions are used for in-vitro work or stability studies, they should be protected from light at all times. In addition, adsorptive losses, which may also be pronounced in low concentration solutions, should be prevented by storage in polypropylene containers. INTRODUCTION Doxorubicin, daunorubicin and epirubicin have been used successfully to treat a wide range of neoplastic diseases. Information on the stability of these analogues is available (1-6) but data are limited and contradictory. The stability of the anthracyclines is affected by many factors including the storage temperature, the pH of the vehicle and light. The large differences in stability, which have been reported by different groups for virtually identical experiments (2,7), may be partially explained by poor control, or a lack of control, of photodegradation. There are no published data concerning the photodegradation of daunorubicin and epirubicin. In addition, the photodegradation of doxorubicin has only been quantified fluorimetrically(7). Fluorescence does not differentiate between parent compounds and metabolites (8). Therefore, if, as some authors have postulated, aglycones are formed Correspondence: M. J. Wood, Pharmacy Department, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2TH, U.K.
29 1
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during photolysis of the anthracyclines (9), the extent of photodegradation measured in these studies may have been underestimated by fluorescence techniques. The purpose of these investigations was to study and compare the degradation kinetics of doxorubicin, daunorubicin and epirubicin in natural and artificial light using HPLC methods. The conditions which facilitated or prevented photodegradation were identified by investigation of the effect of drug concentration, the solvent pH and the type of storage container on the rate of photodegradation. MATERIALS AND M E T H O D S
Chemicals and reagents Doxorubicin, daunorubicin and epirubicin were purchased as the commercially available pharmaceutical dosage forms Adriamycin, Pharmorubicin (both Farmitalia) and Cerubicin (Rhijne-Poulenc). All reagents were of analytical grade, or HPLC grade, as appropriate (BDH Ltd, Poole, U.K.). Equipment The HPLC system consisted of an Altex lOOA pump which was used to deliver eluent to a 10 cm x 4.6 mm, stainless steel, Shandon column which was packed with ODS Hypersil5 pm reversed-phase material. Injections were made with a Rheodyne model 7125 sample injector equipped with a lop1 injection loop. A Pye Unicam variable wavelength detector was operated at 290nm, (0-16 a.u.f.s.) and connected to a JJ instruments CR452 chart recorder. Assay conditions The HPLC methods were developed from a method reported by Beijnen et a l ( l 0 ) . The stability-indicating capacity of these methods was demonstrated by comparison of standard solutions, partially degraded and fully degraded solutions. Decomposition was accelerated by subjecting the solutions to extremes of temperature and pH. The mobile phases for doxorubicin, daunorubicin and epirubicin contained acetonitri1e:water40:60,55:45 and 5 0 5 0 (v/v), respectively. Ten drops of diethylamine were added to each litre of mobile phase which was then adjusted to pH 2.5 with 10% orthophosphoric acid (v/v). The flow rates were 1.4, 1.5 and 1.3 ml/min, respectively, and the chart speed was 5 mm/min. Preparation of solutions for photodegradation studies Vials of doxorubicin, epirubicin and daunorubicin were reconstituted with Waterfor-Injections and standard solutions, between 10 and 500 pg/ml, were prepared by simple dilution with Water-for-Injections using a Gilson fixed-volume pipette. Photodegradation was investigated directly after transfer of these solutions to either 20-ml clear glass, amber glass or opaque polyethylene bottles. Admixtures were prepared for the study at increased pH by combination of equal parts of single strength Tris buffer (pH 7.2 and 8.0) and standard solutions of doxorubicin (50 pg/ml). Duplicates of each container were sealed with plastic screw caps and stored at 25°C either covered with aluminium foil in a darkened room (doxorubicin only) or exposed to room light. In the room light studies, constant illumination was provided by four 65/80 W ceiling fluorescent tubes (Thorn EMI) mounted approximately 1 m above the
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samples, which were placed upright on the laboratory bench. The solutions were unprotected from sunlight. Two-millilitre samples were removed for chromatographic analysis at appropriate time intervals over a period of 168 h. An equal amount of 0.1 M HCl was added to all Tris buffer samples to quench the reaction. All samples were immediately frozen and stored at -20°C prior to assay using the described conditions. All samples were protected from light prior to initiation of the study, during thawing at ambient temperature and prior to assay. Experimental runs for each drug fluid admixture were duplicated. Duplicate samples (10 pl) were injected directly using the conditions described above. Treatment of data The remaining concentrations were determined by interpolation of the calculated peak areas on calibration curves which were constructed daily. All concentrations, which were expressed as a percentage of the original concentration at time zero, were the mean of duplicate values. Significant degradation was defined as a loss of 2 10% of the original concentration. Data were evaluated for a first-order kinetic model by construction of plots of the natural logarithm of the percentage parent drug remaining versus time. The rate constant (k)for drug loss was calculated by linear regression analysis of the first-order plot. Differences in the extent of degradation in the analysed samples were tested for significance by means of a two-way analysis of variance, with replicates. Where it was necessary only to test the difference between two means for significance, Student's t-test was employed. The pooled standard deviation and the standard error of the difference were calculated according to the usual equations (11). R E S U L T S AND DISCUSSION Chromatograms obtained for aqueous solutions of doxorubicin, (pH 6. l), stored in clear glass and polyethylene containers exposed to room light, showed a decrease in the concentration of the parent compound with time together with the emergence of three other peaks, which eluted close to the solvent front. Figure 1 shows a typical chromatogram obtained for a solution of doxorubicin (10 pg/ml) which was stored in clear glass. There was no evidence of these additional peaks on chromatograms obtained from solutions which were stored in amber glass and exposed to light, or in any of the control solutions which were stored in the dark. For this reason the three peaks, which eluted at 1.2, 1.4 and 1.6 min, were postulated to represent photodegradation products. Chromatograms obtained for solutions of 25 pg/ml doxorubicin in Tris buffer at pH 7.2 (exposed to light for 8 h) and pH 8-0(exposed to light for 2 h) both showed one broad peak (DII), which eluted after the parent compound and which was postulated to be an aglycone (Fig. 2). Chromatograms obtained for the same solutions after prolonged exposure to light (Fig. 3) showed almost total disappearance of both the parent compound and aglycone peaks and the presence of peaks which were similar to those observed after photodegradation of aqueous solutions of doxorubicin (Fig. 1). This suggests that hydrolysis in alkaline solution initiated aglycone formation while further exposure to light resulted in the breakdown of these aglycones to other products. The
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J 1
0
I
L
I
6 4 2 T i m e (min)
J 0
Fig. 1. Chromatogramof doxorubicin (10 pg/ml in clear glass) showing 70% degradation after exposure to room light for 168 h, D =doxorubicin.
data also indicate that the final photodegradation products formed in alkaline solution were of a similar nature to those formed in aqueous solution. It has been suggested that the major photoadduct produced in weakly alkaline solution is the aglycone, 7,8-dehydro-9,10-desacetyldaunorubicinone (DII) (Fig. 4) and that the disappearance of this product after prolonged exposure to light involves an oxygen-dependent photobleaching process (9). This is in accordance with the data presented in Figs 2 and 3, respectively. It is also possible that a direct photobleaching process could occur without proceeding via an aglycone. This observation would account for the emergence of the peaks observed at 1.2, 1.4 and 1.6 min, shown in Fig. 1,which were not preceded by aglycone formation.
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1
0.002 A U
I NJ
6
4
2
Time (min)
0
6
4 2 Time (min)
0
Fig. 2. Chromatogram of doxorubicin (25 pg/ml in half strength Tris buffer pH 7.2 and 8.0)after exposure to room light for (a) 8 h and (b) 2 h, respectively, D =doxorubicin, DII = aglycone degradationproduct.
Aglycones were produced at pH 7.2 and 8.0 in the dark as well as in room light, but were not evident in any of the solutions stored at pH 6.1. This indicates that aglycones are not solely photodegradation products, but that their formation was dependent on the pH of the solution. Calculated rates of degradation and tgOs/, values for solutions of doxorubicin at pH 6.1, 7.2 and 8.0 are presented in Table 1.These results show that both photodegradation and degradation of solutions of doxorubicin, which were stored in the dark, were facilitated by an increase in the pH of the solvent. Data from previous studies, in which a pH profile was constructed for this drug, supported this statement as the rate of degradation of doxorubicin was shown to increase rapidly as the pH of the medium increased (pH 6.0-11.98) (12,13). Drug loss from aqueous solutions (pH 6.1), which were stored in the dark, increased as a proportion of the total amount as the concentration of the solution decreased. The amount of drug loss also appeared to be related to the container type (Tables 2 and 3). As chromatograms showed no evidence of degradation products it was concluded that adsorption onto the container had occurred. Adsorption of doxorubicin onto glass (14, 15) and polyethylene (14) has been observed. In addition, doxorubicin, daunorubicin and epirubicin have been reported to sorb to polyvinylchloride (PVC) in a process which involves a rapid adsorptive phase followed by a slower dissolution and migration of the drugs into the plastic matrix (13). Adsorptive and sorptive losses may be largely prevented by storage in polypropylene containers (13). The rates of photodegradation of doxorubicin, daunorubicin and epirubicin in aqueous solution were similar (Table 4) and were inversely proportional to the drug
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0*001 AU
I
0.0005 A U
J IN J
6
4
2
T i m e (rnin)
0
1
6
4
2
0
Time (min)
Fig.3. Chromatogram of doxorubicin (25 pg/ml in half strength Tris buffer pH 7.2 and 8.0)after exposure to room light for (a) 96 h and (b) 48 h, respectively.
concentration. Photodegradation was more rapid in solutions stored in clear glass than in solutions stored in polyethylene or amber glass. This observation was supported by data from ultraviolet (uv) transmission curves, which indicated that the opaque polyethylene afforded some protection from light as a gradual decrease in transmittance from 40% at 750 nm to 10% at 280 nmwasobserved. Theamberglass containers effectivelyprevented photodegradation as they conformed with the USP recommendations which state that transmittance should be less than 12% between 290 and 450 nm.
Doxorubicin, daunorubicin and epirubicin
CH,O
0
OH
0
OH
297
Fig. 4. Structure of 7,8-dehydro-9,1O-desacetyldaunorubicinone.
Table 1. Rate constants and rw,,ovalues for doxorubicin (25pg/ml in half strength Tris buffer at pH 7.2 and pH 8-0)in clear glass containers pH of admixture
Rate constant (h-')
6.1 (room light) 6.1(dark) 7.2(room light) 7.2(dark) 8.0(room light) 8.0(dark)
2.85x 10-3 1.40x 10-4 7.65x 1.41 x 1.23x lo-' 3.90x
two,
value (h)
32 > 168 1.3 6 0.8 1.8
Table 2. Percentage doxorubicin remaining after exposure to room light for 168h Percentage doxorubicin remaining j z i SD (n = 3)(pg/ml)
Clear glass Amber glass Polyethylene
500
250
100
50
25
10
98.6i0.5 96.1i0.7 98.8i0.8
94.8i0.6 97.3i0.5 101.8+0.8
93.3i 1.4 96.4f 1.1 96.8k0.7
84-9iO.6 94.6k1.6 96.9f 1.7
615f1.5 82.1f1.6 83.3f1.5
31.8f1.4 35.8f1.6 39.5f1.8
Table 3. Percentage doxorubicin remaining after storage in the dark for 168h Percentage doxorubicin remaining a+ SD (n = 3)(pglml)
500 Clear glass Amber glass Polyethylene
98.5i1.1 98.1k 1.3 98.9k1.0
250
100
97.8fl.2 97.0k1.4 98.3k1.3 98.7k1.4 101.5f1.8 102.4k1.1
50
25
10
98.3k1.1 98.4f1.1 98-4k1.4
965f1.9 96.6f2.0 98.1k1.0
90.9k1.3 90.5k1.5 96.9F1.5
M . J . Wood, W .J. Irwin and D . K . Scott
298
Table 4. Rate constants for photodegradationof doxorubicin, daunorubicinand epirubicin in clear glass Rate constant (k)(h-')(pgiml) Drug Epirubicin Daunorubicin Doxorubicin
100
50
25
10
3.75x 10-4 4.92x 3.93 x 10-4
1.41 x 10-3 1.23x 10-3 1.01 x 10-3
2.69 x 10-3 2.78x 10-3 2-85x 10-3
5.35x 10-3 5.13x 10-3 6.50 x 10-3
I
40
0
24
48
72
96
I20
144
I68
Time ( h )
Fig. 5. First-order plot for the photodegradationof epirubicin in clear glass containers. (A)100 pg/ml, (X) 50 pg/ml, (0) 25 pg/ml, (0) 10 pg/ml.
Plots of the natural logarithm of the percentage parent drug remaining versus time were linear ( r = >0.999), which indicated that photodegradation followed first-order kinetics. The first-order plots for epirubicin (Fig. 5) showed that rapid photolysis
Doxorubicin, daunorubicin and epirubicin
299
occurred over the concentration range 10-50 pg/ml. At concentrations 2 100 pg/ml, little or no photodegradation occurred in either glass or plastic containers (Table 2). These data indicate that in the clinical situation, where these drugs are used in concentrations 2 500 pg/ml, it is not necessary to protect solutions of these drugs from light during intravenous administration.
CONCLUSION At concentrations, such as those used for cancer chemotherapy ( 2500 pg/ml), no special precautions are necessary to protect freshly prepared solutions of these agents from light during intravenous administration. The experimental conditions of the present study are similar to those in which these agents are handled in low concentrations in the laboratory. The present findings are of practical importance and indicate that decomposition of these anthracyclines at concentrations in the low microgram and nanogram range may be very rapid if solutions are exposed to high intensity artificial light, or sunlight, for sufficient time. Photodegradation was also facilitated by an increase in the pH of the solvent. Concerning the possibility of preventing photolysis, keeping solutions in absolute darkness effectively protects the drugs from photodegradation. Drug loss due to adsorption onto the container, which may also be a problem in low concentration solutions, may be minimized by storage in polyethylene containers, or largely prevented by storage in polypropylene containers. REFERENCES 1. Benvenuto, J.A., Anderson, R., Kerkof, K., Smith, R.G. & Loo, T.L. (1981) Stability and compatibility of antitumour agents in glass and plastic containers. AmericanJournal of Hospital Pharmacy, 38, 1914-1918. 2. Poochikian, G.K., Cradock, J.C. & Flora, K.P. (1981) Stability of anthracycline antitumour agents in four infusion fluids. AmericanJournal of Hospital Pharmacy, 38,483-486. 3. Karlsen, J., Hjort Thonnesen, H., Resberg Olsen, I., Horne Sollien, A. & Skobba, T.J. (1983) Stability of cytotoxic intravenous infusions subjected to freezethaw treatment. Norwegica Pharmaceutica Acta, 45,61-67. 4. Keusters, L., Stolk, L.M.L., Umans, R. &Van Asten, P. (1986) Stability of solutions of doxorubicin and epirubicin in plastic minibags for intravesical use after storage at -20°C and thawing by microwave radiation. Pharmaceutisch Weekblad [Scientific Edition], 8, 194-197. 5. Beijen, J.H., Rosing, H., De Vries, P.A. & Underberg, J.M. (1985) Stability of anthracycline antitumour agents in infusion fluids. Journal of Parenteral Science and Technology, 39,220-222. 6. Hoffman, D.M., Grossano, D.D., Damin, L.-A. & Woodcock, T.M. (1979) Stability of refrigerated and frozen solutions of doxorubicin hydrochloride. American Journal of Hospital Pharmacy, 36, 1536-1538. 7. Tavoloni, N., Guarino, A.M. & Berk, P.D. (1980) Photolytic degradation of adriamycin. Communications. Journal of Pharmacy and Pharmacology, 32,860-862. 8. Rahman, A., Goodman, A., Foo, W., Harvey, J., Smith, F.P. & Schein, P.S. (1984) Clinical pharmacology of daunorubicin in phase I patients with solid turnours: development of an analytical methodology for daunorubicin and its metabolites. Seminars in Oncology, 11,36-44. 9. Gray, P.J.& Phillips, D.R. (1981) Ultraviolet photoirradiation of daunomycin and DNA-daunomycin complexes. Photochemistry and Photobiology, 33,297-303. 10. Beijnen, J.H., Wiese, G. & Underberg, W.J.M. (1985) Aspects of the chemical stability of doxorubicin and seven other anthracyclines in acidic solution. Pharmaceutisch Weekblad [Scientific Edition], 7, 109-1 16. 11. Neville, A.M. & Kennedy, J.B. (1 964) Basic Statistical Methods for Engineers and Scientists. Intertext Books, London.
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12. Wood, M.J. (1988)Stability of anthracycline cytotoxic agents in solution and infusionpuids. MPhil thesis, Aston University, Aston. 13. Wood, M.J., Irwin, W.J. & Scott, D.K. (1990)Stability of doxorubicin, daunorubicin and epirubicin in plastic syringes and minibags, Journal of Clinical Pharmacy and Therapeutics, IS, 279-289. 14. Bosanquet, A.G. (1986)Stability of antineoplastic agents during preparation and storage for in vitro assays. 11. Assay methods, adriamycin and the other antitumour antibiotics. Cancer Chemotherapy and Pharmacology, 17,l-10. 15. Tomlinson, E. & Malpeis, L. (1982)Concomitant adsorption and stability of some anthracycline antibiotics.Journal of Pharmaceutical Sciences, 71, 1121-1 125.
