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Radiation sterilization of anthracycline antibiotics in solid state.

1. Introduction

Anthracycline antibiotics belong to the group of anticancer drugs. They were originally isolated from cultures of Streptomyces. Anthracyclines are widely used in the treatment of neoplastic diseases such as leukemia, breast cancer, and AIDS-related Kaposi's sarcoma. They also demonstrate activity against tumors of the ovaries, lung, testes, prostate, cervix, bladder, and Ewing's sarcoma [1-3]. The most widely used anthracyclines are doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) [4-6] (Figure 1). Their low bioavailability necessitates parenteral administration [7, 8], which requires sterility [9] obtained mainly by filtration. Given the considerable exposure of medical personnel to anthracycline antibiotics and their adsorption to most surfaces, especially those of sterilization filters, there is a need to find new sterilization methods that do not rely on filtration. Although radiation sterilization is an effective alternative, the structure of drugs may alter as a consequence of exposure to irradiation. The aglycone attached to the aminosugar with a glycoside bond may be prone to cleavage resulting from electron transfer within the molecule. Stability studies demonstrated degradation of anthracycline antibiotics in solutions, under the influence of increased temperature and when exposed to light in the solid state as well as a consequence of combining chemotherapy with radiotherapy [10-15]. Regarding the effects of radiation sterilization on anthracycline antibiotics, only DOX was studied in that respect with a focus on the impact of a standard dose of 25 kGy on its stability [16]. As DAU and EPI are widely used in anticancer pharmacotherapy, those antibiotics also require analysis of their vulnerability to ionizing radiation.

The aim of this work was to assess the possibility of applying radiation sterilization to DOX, DAU, and EPI as active substances or conversion-induced impurities.

2. Materials and Methods

2.1. Samples. DOX, DAU, and EPI were synthesized at the Institute of Biotechnology and Antibiotics, Department of Modified Antibiotics, Warsaw, Poland. They were reddish powders, freely soluble in water and methanol. Sodium lauryl sulfate, phosphoric acid, and all other chemicals were obtained from Merck KGaA (Germany) and were of analytical or high-performance liquid chromatographic grade.

2.2. Irradiation. 0.025 g samples of each substance were placed in 3mL colorless glass vials that were closed with plastic stoppers. The samples in the vials were exposed to beta irradiation in a linear electron accelerator LAE 13/9 (9.96 MeV electron beam and 6.2 [micro]A current intensity) until they absorbed doses of 25, 50,100, 200, and 400 kGy.

2.3. Electron Paramagnetic Resonance (EPR) Spectroscopy. Detection of free radicals and determination of their concentration were carried out using a Bruker ELEXSYS 500 spectrometer (X-band) at 297 K. EPR spectra were recorded as a first derivative of the absorption signal. The number of free radicals was calculated using the integration procedure described elsewhere [17].

2.4. UV-Vis Spectroscopy. Chemical changes in nonirradiated and irradiated samples were analyzed by using a UV-Vis Varian Carry 100 spectrophotometer. 2 mg of each sample was dissolved in 20.0 mL of methanol. 1.0 mL of the so-obtained solution was diluted to 10.0 mL with methanol. The final concentration of the solutions was 0.01mg/mL. Absorption spectra of the so-prepared solutions were recorded in the wavelength range 190-900 nm.

2.5. IR Spectroscopy. IR spectra of nonirradiated and irradiated anthracyclines were taken with the use of a Thermo Scientific Nicolet iS10 spectrophotometer with the Omnic software. The infrared transmittance spectra of the crystalline samples were recorded after a time necessary to achieve plateaus in EPR study, in the frequency range from 400 to 7500 cm-1, at room temperature.

2.6. HPLC Analysis. An HPLC Waters Alliance e2695 system was used for chromatographic separation of the degradation products of nonirradiated and irradiated DOX, DAU and EPI samples. All the samples (1mg/mL) were dissolved in the mobile phase. A Symmetry C18 (250 x 4.6 mm, 5 [micro]m) analytical column was employed as a stationary phase. The mobile phase consisted of solution A (acetonitrile) and solution B (2.88 g of sodium lauryl sulfate and 2.25 g of phosphoric acid(V) 85% in 1000 mL) (50:50, v:v). UV detection was performed at 254 nm. The flow rate was 1.0mL/min. The injected volume was 5 [micro]L.

2.7. Theoretical Analysis. All the calculations were made by using the Gaussian 03 package [18]. In order to interpret the experimental results of IR absorption scattering, quantum chemical calculations were performed based on a density functional theory (DFT) method with the B3LYP hybrid functional and 6-31G(d,p) basis set.

3. Results and Discussions

The first EPR analysis was performed 1 day after irradiation for samples exposed to a dose of 25 kGy. DOX, DAU, and EPI irradiated at 25 kGy contained about 3.94 x [10.sup.15] spins/g, 1.37 x [10.sup.15] spins/g, and 2.44 x [10.sup.15] spins/g, respectively. Exponential decay of unstable free radical was observed with the half-time of 4 days in DAU and DOX and 7 days in EPI after irradiation. The EPR signals of the sterilized anthracycline antibiotics were very weak. The plateaus of free radical concentrations versus time for DOX and DAU appeared after about 10 days, whereas for EPI after 20 days. EPR spectra after these periods consisted approximately of only stable free radicals.

The analytical study was conducted during a period when only stable free radicals were detected by EPR. The impact of an irradiation dose size on the structure of DAU, dOx, and EPI was studied at 0, 25, 50, 100, 200, and 400 kGy without the presence of unstable free radical in EPR spectra. By using UV-Vis spectroscopy the location and the intensity of the absorption maximum were determined, whereas IR spectroscopy was employed to establish the intensity, location, and type of characteristic vibrations. For the DAU, DOX, and EPI samples no significant changes in the location (~289 nm, ~233 nm, and 221 nm) or intensity of the absorption maximum were recorded (Figures 2,3, and 4). The spectra of their nonirradiated and irradiated samples did not show any essential differences in the value of absorbance. All samples exhibited two absorption maxima, at 233 nm and 251 nm. The IR spectra of DOX, DAU, and EPI were compared with the theoretical spectra based on the density functional theory. The main characteristic vibrations obtained from the IR spectra are collected in Table 1. The conformation between the calculated and experimental spectra is quite good. The most significant is the region between 700 and 1800 cm-1, where intense and characteristic bands related to intramolecular vibrations of the molecules are observed, including the deformation of rings as well as stretching of various C-C bonds (Figures 5, 6, and 7). Vibrational spectra of the nonirradiated and irradiated samples of the three samples are very similar. We did not observe any change in the position and shape of the bands. This suggests that the radiation sterilization does not influence the stability of the DOX, DAU, and EPI. Similar results were received by comparing SEM images of the nonirradiated and irradiated samples. Taking into account the biological activity of the most important impurities specified by Ph. Eur. [9], changes in the concentration of the main substances in the presence of those impurities were analyzed (Figures 8, 9, 10, 11, 12, and 13). By separating the compounds to be examined from the impurities, it was possible to assess changes in their content before and after irradiation at 25 kGy. It was found that exposure to such a dose of radiation did not produce any changes in the concentrations of the main substances or the impurities. Slight alterations were registered when the samples of DAU, DOX, and EPI were exposed to greater doses of radiation. Under such conditions, EPI demonstrated the greatest content change, and the presence of unstable free radicals was noted for the longest period of time. It was also proved, by observing the mass balance, that the main substances did not convert into unknown impurities. Similar studies of some tetracycline analogs showed that the aglycon was stable when irradiated at 25 kGy and that changes occurred when greater radiation doses were applied [19]. It may therefore be proposed that not only a modification of the aglycon structure but also its ability to bind with a sugar moiety of specific stereoisomerism are the factors that stabilize the structures of analogs of anthracycline antibiotics.

4. Conclusions

The current study of the impact of radiation sterilization on the stability of DAU, DOX, and EPI demonstrates that this kind of sterilization may be an alternative to filtration recommended for sterilizing analogs of anthracycline antibiotics. The effect of radiation sterilization on the stability of DAU, DOX, and EPI depends on the structure of a particular compound. With regard to those analogs, it is important to assay the postirradiation content of the main substance in the presence of all possible related substances in order to determine whether other degradation products or postirradiation conversion occur. 10.1155/2013/258758


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A. Kaczmarek, (1) J. Cielecka-Piontek, (2) P. Garbacki, (2) K. Lewandowska, (3) W. Bednarski, (3) B. Barszcz, (3) P. Zalewski, (2) W. Kycler, (4) I. Oszczapowicz, (5) and A. Jelinska (2)

(1) Biofarm Sp. z o.o., Walbrzyska 13, 60-198 Poznan, Poland

(2) Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland

(3) Institute of Molecular Physics, Polish Academy of Sciences, Smoluchowskiego 17, 60-179 Poznan, Poland

(4) Department of Oncological Surgery II, Great Poland Cancer Centre, Garbary 15, 61-866 Poznan, Poland

(5) Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, Staroscimka 5, 02-516 Warsaw, Poland

Correspondence should be addressed to P Garbacki;

Received 23 August 2013; Accepted 19 September 2013

Academic Editors: J. McHowat and S. J. Rajput

Table 1: Main characteristic vibrational modes of daunorubicin
(DAU), doxorubicin (DOX), and epidoxorubicin (EPI) observed in
experimental and calculated spectra.


DAU         DOX     EPI

723         736     736

752         754     749

776         773     774

931         930     935

959         957     951
984          --      --

1010        1008    1006

1021        1029    1032

1079        1078    1065

1105        1104    1104

1123        1123    1122

1137        1139    1136

1157        1157    1156

1206        1205    1200

1226        1225    1221

--          1239    1239

1278        1275    1275

1291        1290    1293

1317        1317    1318

1329        1329    1330

1367        1367    1366
1404         --      --

1425        1427    1423

1476        1476    1478

1502        1502    1505

1524        1524    1527

1614        1615    1615

1640        1640    1642

1661        1663    1663

1688        1688    1688

1744        1744    1745

1791        1808    1807


3009        3010    2960

3488        3488    3485

3566        3566    3571

3630        3634    3639

3791        3788    3785

3809        3801     --

--          3831    3831


DAU         DOX     EPI

764         761     761

795         795     795

816         804     805

940         939     939

955         949     949

986         990     989

1008        1005    1005

1070        1072    1072

1085        1089    1089

1109        1114    1114

1109        1114    1114

1153        1143    1143

1194        1201    1201

1205        1211    1211

            1235    1235

1262        1263    1263

1289        1284    1285

1289        1284    1285

1317        1318    1318

1374        1374    1374

1404        1413    1413

1474        1471    1472

1506        1507    1507

            1525    1524

1576        1582    1581

1576        1582    1581

1617        1616    1616

1617        1616    1616

1707        1717    1717

1716        1730    1730


2878        2896    2896

3161        3326    3329

            3527    3527

            3545    3545

Band assignment

C-C-C b in a ring + def. aglycone group + breathing
tetrahydropyran ring in aminosugar group + N[H.sub.2] w

N[H.sub.2] w + C-C-C b in aminosugar group + C-H w at d ring and
in aminosugar group

C-C-C b in a ring + def. aglycone group + breathing
tetrahydropyran ring in aminosugar group + C[H.sub.2] r at d ring

C-O-H b at c ring + C-H w in aminosugar group

C-C-C b in d ring + N[H.sub.2] w + CH w at d ring and aminosugar
group C[H.sub.3] w in COC[H.sub.3] group

Breathing aglycone group + C[H.sub.2] r + N[H.sub.2] r +
C[H.sub.3] w in COC[H.sub.3] + C[H.sub.2] w in COC[H.sub.2]OH

Breathing a ring + def. aglycone group + C-O s at a ring + C-O-H
b at c ring + N[H.sub.2] w + C[H.sub.3] w in COC[H.sub.3] group

C-O s between tetrahydropyran ring and O-H group in aminosugar

C-O s in metoxy group at a ring + C-C-C b in a ring + C[H.sub.2] r

C-O s in tetrahydropyran ring + C-O s in glycosidic bond + C-C s
in d ring + C-H w in C[H.sub.3] in aminosugar group

C-C s in d ring and in tetrahydropyran ring in aminosugar group +
C-H b at d ring and in tetrahydropyran ring in aminosugar group +
C[H.sub.3] w in COC[H.sub.3] group + C-O s in COC[H.sub.2]OH

C-O s in glycosidic bond + C-O s between tetrahydropyran ring and
O-H group in aminosugar group + C-H r in C[H.sub.3] in aminosugar
group + def. d ring

C-O s in glycosidic bond + C-C s Id ring + C-H b at d ring and
aminosugar group

C[H.sub.2] t at d ring + breathing a ring + C-O-H b at c ring

C-O-H b in COC[H.sub.2]OH group

C-O-H b at c ring + C-C s in aglycone group + C[H.sub.2] t in
COC[H.sub.2]OH group

C-O-H b at c ring + breathing a and c ring

Breathing c ring + C-O s at c ring + C-O s at a ring + C[H.sub.2]
w at a ring +C-H w at d ring

C-O s at a ring + breathing a and c ring

C-C-C b in d ring + C-C s in a and b ring + C-O s at c ring +
C-O-b at c ring C[H.sub.3] sc in COC[H.sub.3] group

O-C-H b + N-C-H b

C-O s at c ring + def. c ring + C[H.sub.3] umbrella mode at a
ring + C-O-H b at c ring

C-O-H b at c ring + C[H.sub.3] sc at a ring + C-C s in aglycone

C-O-H b at c ring + C[H.sub.3] sc at a ring + C-C s in aglycone

C-C s in c ring + C-O-H b at c ring + C=O s at b ring + C-C s in
a ring

C-C s in a ring + C-O-H b at c ring + C[H.sub.3] w at a ring

N[H.sub.2] sc

C=O s at b ring + C-O-H b at c ring

C=O s at b ring

C=O s at d ring

C-H s

O-H s at c ring

N-H s symmetric

N-H s antisymmetric

O-H s

O-H s

O-H s

O-H s in COC[H.sub.2]OH group

Vibrational modes-s: stretching, b: bending, w:
wagging, sc: scissoring, r: rocking, and t: twisting.
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Title Annotation:Research Article
Author:Kaczmarek, A.; Cielecka-Piontek, J.; Garbacki, P.; Lewandowska, K.; Bednarski, W.; Barszcz, B.; Zale
Publication:The Scientific World Journal
Article Type:Report
Date:Jan 1, 2013
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