Research ArticleStability of Epidoxorubicin Hydrochloride in Aqueous SolutionsExperimental and Theoretical Studies
Agnieszka Sobczak1 Monika A Lesniewska-Kowiel1 Izabela Muszalska1
Artur Firlej1 Judyta Cielecka-Piontek1 Szymon Tomczak1 BolesBaw Barszcz2
Irena Oszczapowicz3 and Anna JeliNska1
1Department of Pharmaceutical Chemistry Faculty of Pharmacy Poznan University of Medical SciencesGrunwaldzka 6 60-780 Poznan Poland2Institute of Molecular Physics Polish Academy of Sciences Smoluchowskiego 17 60-179 Poznan Poland3Department of Modified Antibiotics Institute of Biotechnology and Antibiotics Staroscinska 5 02-515 Warszawa Poland
Correspondence should be addressed to Agnieszka Sobczak asobczakumpedupl
Received 4 January 2017 Accepted 14 June 2017 Published 30 July 2017
Academic Editor Paula G De Pinho
Copyright copy 2017 Agnieszka Sobczak et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The first-order degradation kinetics of epidoxorubicin were investigated as a function of pH temperature and buffers concentra-tions The degradation was followed by HPLC Buffer catalysis was observed in acetate and phosphate buffers The pH-rate profileswere obtained at 333 343 353 and 363KThe pH-rate expression was 119896pH = 1198961times119886H+ times1198911+1198962times1198911+1198963times1198912+(1198964times1198912+1198965times1198913)times119886OHminus where 1198961 1198964 and 1198965 are the second-order rate constants (molminus1 L sminus1) for hydrogen ion activity and for hydroxyl ion activity respec-tively and 1198962 and 1198963 are the first-order constants (s
minus1) for spontaneous reaction under the influence ofwater Epidoxorubicin demon-strates the greatest stability in the pH range 3ndash5The electrostatic molecular potential orbitals HOMO-LUMOwere also defined inorder to determine the cause of the reactivity of particular epidoxorubicin molecule domains in solutions with various pH values
1 Introduction
Epidoxorubicin hydrochloride (EP) is the most commonlyused anthracycline antitumor antibiotic consisting of a tetra-cyclic quinolid aglycone connected by a glycoside bondwith an amino sugarmdashL-acosamine The drug is structurallyrelated to doxorubicin and the difference between them isin the stereochemistry of the C-41015840 hydroxyl group of thesugar moiety Due to its structure EP demonstrates lowercardiotoxicity and so it can be used in higher doses [1ndash3]The lower efficiency and higher toxicity of drugs can be alsoassociated with degradation during their storage or clinicaluse (eg long-term infusions) EP is produced as powderor solutions for injections and administered as long-terminfusions Therefore it is important to investigate its stabilityboth in the solid phase and in solutions
The influence of temperature and relative air humid-ity on the stability of EP as well as of doxorubicin and
daunorubicin in the solid state was previously reported [45] The degradation of EP in the atmosphere of increasedrelative air humidity was a first-order reaction dependingon the substrate concentration and in dry hot air (RH 0393K) a reversible first-order reaction relative to the substrateconcentration The dependences ln 119896 = 119891(1119879) and ln 119896 =119891(RH)were described by the equations ln 119896 = (351plusmn109)minus(16250 plusmn 3823)(1119879) and ln 119896 = (379 plusmn 334) times 10minus2(RH) minus(129plusmn24) respectively and the kinetic and thermodynamicparameters of the above processes were calculated [4]
The photodegradation [6] and hydrolysis of EP underacidic (005M 363K) and basic (005M 303K) conditions[7] were first-order reactionsThe photodegradation rate wasinversely proportional to the concentration of the drug androse as the solvent pH increased In the case of concentrationswhich are used in chemotherapy (gt500mcgmL) protectionfrom light was not necessary [6]
HindawiJournal of ChemistryVolume 2017 Article ID 8107140 6 pageshttpsdoiorg10115520178107140
2 Journal of Chemistry
The stability of EP was also assessed in sodium chloridesolutions (09NaCl pH 520 and 647) and in dextrose (5pH436) at 25∘C 4∘C andminus20∘C stored in polyvinyl chloride(PCV) minibags which are no longer in clinical use and inwater for injections stored at 4∘C in polypropylene syringes[8] The drug was stable (loss below 10) under the aboveconditions with the exception of storage in 09 NaCl (pH647) at 25∘C when degradation exceeding 10was observedafter 43 days It was also found that the repeated freezing anddefrosting of these solutions stored in minibags did not causesignificant degradation and that polypropylene appeared tobe the best material for EP storing [8]
During the storage of EP injections at pH 3 and a temper-ature of 2ndash8∘C for 12 months or at 25∘C for 6 months somedegradation products were observed [9]Three products wereisolated and characterized byNMRandLC-MSMSmethodsProduct I (dimer of EP) was formed due to the condensationof two EP molecules Product III (4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2512-trihydroxy-7-me-thoxy-611-dioxo-1234611-hexahydro-naphthacene-2-car-boxylic acid hydroxymethyl ester) was produced by oxida-tive rearrangement of EP in the side chain under acidicconditions After hydrolysis of the ester group of degradationproduct III product IV (4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2512-trihydroxy-7-methoxy-611-dioxo-1234611-hexahydro-naphthacene-2-carboxylicacid) was formed which can also be directly produced dueto the oxidation of the side chain of EP and the eliminationof the ester moiety [9]
The literature does not refer to comprehensive kineticstudies of EP in aqueous solutions in a wide range of pHvalues and temperatures Thus the aim of this study was toevaluate the degradation of EP under the influence of pH andtemperature and to develop equations permitting the estima-tion of kinetic and thermodynamic parameters To separateEP and its degradation products our previously designedHPLC method was used [4] The geometric structure of EPmolecule FMOs orbitals (HOMO-LUMOHighest OccupiedMolecular Orbital Lowest Unoccupied Molecular Orbital)and molecular electrostatic potential were also determined
2 Experimental
21 Materials and Reagents Epidoxorubicin hydrochloridewas synthesized at the Department of Modified AntibioticsInstitute of Biotechnology and Antibiotics Warsaw PolandSodium laurilsulfate (AC reagent Sigma-Aldrich LogistikGmbH Germany) and all other chemicals and solvents wereobtained fromMerck KGaA Germany andwere of analyticalor high-performance liquid chromatographic grade
22 Chromatographic Conditions Chromatographic separa-tion and quantitative analysis were performed by using theHPLC method [4] The analytical system consisted of a Shi-madzu SPD-20A Prominence UVVIS detector a Rheodynewith a 50 120583L loop An LiChrospher RP-18 column (250mmtimes 4mm 5 120583m particle size Merck Germany) was used asthe stationary phase The mobile phase consisted of equalvolumes of acetonitrile and a solution containing 288 gL of
sodium laurilsulfate and 225mLL of phosphoric acid Theflow rate was 10mLmin andUV detection was performed at254 nm As the HPLC method was previously evaluated andvalidated for a stability study of epidoxorubicin in the solidstate [4] selectivity was examined during stability studiesin aqueous solutions in this work Papaverine hydrochloridewas used as an internal standard at a concentration of005mgmL
23 Kinetic Procedures The degradation of EP in aqueoussolutions was studied at 333 K 343K 353K and 363K in thepH range 042ndash995 The pH values of the reaction solutionsand those of the buffer standards were measured at reactiontemperatures The pH values of the reaction solutions in HClwere calculated from the equation pH = minus log119891HCl [HCl]The activity coefficient 119891HCl was obtained or calculated fromthe literature data [10] The ionic strength of all the solutionswas adjusted to 050M with a solution of sodium chloride(4M) Solutions of the desired pH and ionic strength of050M were heated to the required temperatures and then asample of EP was added to obtain the initial concentrationof 02mgmL At selected times determined by the rate ofdegradation samples of the solutions (10mL) were collectedand instantly cooled with a mixture of ice and water Sampleswith pH above 75 were neutralized by using HCl solutions atconcentrations ensuring that their pH was approximately 2To each such sample 10mL of the internal standard solution(papaverine hydrochloride solution) was added 50 120583L of thesolution sampleswas injected into the columnDue to the factthat anthracyclines are photolabile all samples to be studiedwere protected from light
Microsoft Excel 2000 was used for the calculation ofregression parameters
24 Theoretical Studies In order to interpret the FMOs(frontier molecular orbitals) and EP quantum-chemical cal-culations were performed by using the Gaussian 03 package[11] The GaussView software was utilized to propose theinitial geometry of the investigated moleculesThemoleculargeometries were optimized by means of a density functionaltheory (DFT) method with the B3LYP hybrid functional anda 6-31G(dp) basis set
3 Results and Discussion
31 Observed Rate Constants The observed rate constants ofEP degradation in aqueous solutions were determined in apH range of 042ndash995 and described by the equation of apseudo-first-order reaction
ln 119888119905 = ln 1198880 minus 119896obs times 119905 (1)
where 119888119905 and 1198880 are the time-dependent concentration andthe initial concentration of EP at times 119905 gt 0 and 119905 = 0respectively 119896obs is the observed rate constant of the pseudo-first-order reaction of EP degradation
The number of measurements of 119888119905 for each series rangedfrom 8 to 12
Journal of Chemistry 3
242
283
337
00E + 00
20E minus 05
40E minus 05
60E minus 05
80E minus 05
10E minus 04
12E minus 04
01 02 03 04 050
kＩ＜Ｍ(Ｍ
minus1)
[B]T
Figure 1 The plots of 119896obs = 119891([119861]119879) for the degradation of EP inphosphate buffer at 353 K
32 Buffer Catalysis Under the conditions of this study therate constants (119896obs) depended on the total concentrationsof the phosphate and acetate buffers which indicated thatthe components of the buffers catalyzed the degradationof EP (general acid-base catalysis) In order to verify thatthe differences between 119896obs determined at different bufferconcentrations were statistically significant the parallelismtest was used Under the conditions of general acid-basecatalysis 119896obs were calculated from the following equation
where [119861]119879 is the total buffer concentration 119896pH is the rateconstant at zero buffer concentration and 119896119861 describes thecatalytic effect of the buffer components The catalytic effectof the acetate buffer was investigated at pH 402ndash589 whilethose of phosphate buffers were investigated at pH 215ndash340(Figure 1) and pH 591ndash745 Since in the reaction solutions ofEP in HCl and borate buffer general acid-base catalysis wasnot observed in that pH range the values of 119896obs = 119896pH
33 pH-Rate Profiles The values 119896pH calculated from (2)under the conditions of general acid-base catalysis (acetateand phosphate buffers) and those obtained for hydrochloricacid as well as borate buffer (119896obs = 119896pH) were used to calculatethe relationship log 119896pH = 119891(pH) (Figure 2)
The semilogarithmic relationship 119896pHndashpH (Figure 2)indicated that in water solutions at pH 042ndash995 the follow-ing reactions occurred
(i) Degradation of the protonated molecules of EP cat-alyzed by hydrogen ions (1198961)
(ii) Spontaneous hydrolysis of protonated (1198962) andunprotonated (1198963) molecules of EP under theinfluence of water
(iii) Degradation of unprotonated molecules andmonoanions of EP catalyzed by hydroxide ions (1198964and 1198965)
20 40 60 80 10000
pH
10E minus 07
10E minus 06
10E minus 05
10E minus 04
10E minus 03
10E minus 02
10E minus 01
343K353K 333K
363KkＪ（(Ｍ
minus1)
Figure 2The pH-rate profiles for the degradation of EP at 333 343353 and 363KThe symbols represent experimental points the lineswere calculated from (3)
The total reaction rate was equal to the sum of partialreaction rates
119896pH = 1198961 times 119886H+ times 1198911 + 1198962 times 1198911 + 1198963 times 1198912
+ (1198964 times 1198912 + 1198965 times 1198913) times 119886OHminus (3)
where 119886H+ is the hydrogen ion activity and 1198911ndash1198913 are thefractions of protonated and unprotonated molecules andmonoanions of EPThe 1198911ndash1198913 values were calculated by usingthe values of pK119886 of EP that were about 74 and 95
The catalytic rate constants (1198961) were calculated from theplots 119896pH = 119891(119886H+) by using the values of 119896pH in the pH range042minus340 The plots were linear with the positive slope thatequaled 1198961 (Figure 3)
The catalytic rate constants 1198964 and 1198965 were calculatedfrom the equation 1198961015840pH = 119896pH minus (1198961 times 119886H+ times 1198911) = (1198964 times 1198912 +1198965 times 1198913) times 119886OHminus using 119896pH values above pH 85 where theconcentrations of 1198912 + 1198913 rarr 1 The plots 1198961015840pH(119886OHminus times 1198913) =119891(11989121198913)were linearwith positive slopes equal to the catalyticrate constant 1198964 and the value 119887 corresponded to the catalyticrate constant 1198965 The catalytic rate constants 1198962 and 1198963 werecalculated from the following equation 1198961015840pH = 119896pHminus(1198961times119886H+times1198911+(1198964times1198912+1198965times1198913)times119886OHminus) = 1198962times1198911+1198963times1198912 using the 119896pHvalues at pH 28ndash7 where the concentrations of 1198911 + 1198912 rarr 1
The plots 1198961015840pH1198911 = 119891(11989121198911) were linear with positiveslopes equal to the catalytic rate constant 1198963 and the value 119887corresponded to the catalytic rate constant 1198962
The calculated theoretical profile of log 119896 = 119891(pH)and that obtained from the experimental results were nearlyidentical indicating that the choice of the equation describingthe total rate of EP degradation was correct (Figure 2)
4 Journal of Chemistry
Table 1 Catalytic rate constants and thermodynamic parameters for the degradation of EP in aqueous solutions
Catalytic rate constant Temperature (K) (119896 plusmn Δ119896) Parameters of regressionln 119896119894 = 119891(1119879)
Thermodynamic parameters
1198961(molminus1 L sminus1) 333 (102 plusmn 007) times 10minus3 119903 = minus09997 119864119886 = 1188 plusmn 94 (kJmolminus1)
Δ119867= and Δ119878 = were calculated for 293K 119864119886 = minus119886119877 (Jmolminus1) Δ119867 = = 119864119886 minus 119877119879 (Jmolminus1) Δ119878 = = 119877[ln119860 minus ln(119896119879ℎ)] (J Kminus1molminus1) where 119896 is Boltzmannrsquos
constant (13807 times 10minus23 J Kminus1) ℎ is Planckrsquos constant (6626 times 10minus34 J s) 119877 is the universal gas constant (8314 J Kminus1molminus1) 119879 is temperature in K 119886 is thevectorial coefficient of the Arrhenius relationship and 119860 is the frequency coefficient
01 02 03 04000E + 00
20E minus 03
40E minus 03
60E minus 03
80E minus 03
10E minus 02
333K343K
353K363K
kＪ（(Ｍ
minus1)
a（+
Figure 3 The plots of 119896pH = 119891(119886H+ ) for the degradation of EP inaqueous solutions at the indicated temperatures
Based on the Arrhenius relationship ln 119896 = ln119860minus119864119886119877119879linear plots of ln 119896 = 119891(1119879)were used to calculate the energyof activation (119864119886) the entropy (Δ119878
=) the enthalpy (Δ119867=) and
the preexponential coefficient (119860) for the five partial reactions(Table 1)
The lowest energy of activation was observed in thereactions of unprotonated molecules and monoanions ofEP catalyzed by hydroxide ions whereas the highest energyof activation was observed in the reaction of protonatedmolecules of EP catalyzed by hydrogen ions
34 Theoretical Studies Based on theoretical calculationsthe places in EP molecules that might be connected withthe differences in EP reactivity in solutions with various pHvalues were proposedThe electrons in EP were concentratedin the tetracyclic quinolid aglycone especially in the C ringThat domain was responsible for the reactivity of EP duringbase hydrolysis (Figure 4)
The calculations also indicated that the A ring of thetetracyclic quinolid aglycone played a part in EP susceptibilityto degradation during acidic hydrolysis A comparison ofelectron distribution in the HOMO and LUMO of EPdemonstrated that it was similar to electron distributionin daunorubicin and doxorubicin (Figure 5) Previousstability studies of those three anthracycline antibiotics didnot show any statistically significant differences between
Journal of Chemistry 5
Figure 4 ESP of epidoxorubicin
OrbitalsHOMO LUMO
Epidoxorubicin
E = minus577 eV E = minus267 eV
Daunorubicin
E = minus261 eV
Doxorubicin
E = minus574 eV E = minus264 eV
E = minus571 eV
Figure 5 FMOs of EP DOX and DAU
their degradation rate constants in the solid state [4 5]and revealed a similarity between the relationships log 119896pH= 119891(pH) which correlates with theoretical study results[12 13] The values of energy gap for the three derivativeswere 31 eV proving their similar chemical stability
Although the stereoisomerismof theC-41015840 hydroxyl groupof L-acosamine in EP differs from that observed for DOXthe difference does not significantly influence EP reactivityand consequently the susceptibility of EP to degradationduring acid-base hydrolysis is not affected That difference
6 Journal of Chemistry
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
The stability of EP was also assessed in sodium chloridesolutions (09NaCl pH 520 and 647) and in dextrose (5pH436) at 25∘C 4∘C andminus20∘C stored in polyvinyl chloride(PCV) minibags which are no longer in clinical use and inwater for injections stored at 4∘C in polypropylene syringes[8] The drug was stable (loss below 10) under the aboveconditions with the exception of storage in 09 NaCl (pH647) at 25∘C when degradation exceeding 10was observedafter 43 days It was also found that the repeated freezing anddefrosting of these solutions stored in minibags did not causesignificant degradation and that polypropylene appeared tobe the best material for EP storing [8]
During the storage of EP injections at pH 3 and a temper-ature of 2ndash8∘C for 12 months or at 25∘C for 6 months somedegradation products were observed [9]Three products wereisolated and characterized byNMRandLC-MSMSmethodsProduct I (dimer of EP) was formed due to the condensationof two EP molecules Product III (4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2512-trihydroxy-7-me-thoxy-611-dioxo-1234611-hexahydro-naphthacene-2-car-boxylic acid hydroxymethyl ester) was produced by oxida-tive rearrangement of EP in the side chain under acidicconditions After hydrolysis of the ester group of degradationproduct III product IV (4-(4-amino-5-hydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-2512-trihydroxy-7-methoxy-611-dioxo-1234611-hexahydro-naphthacene-2-carboxylicacid) was formed which can also be directly produced dueto the oxidation of the side chain of EP and the eliminationof the ester moiety [9]
The literature does not refer to comprehensive kineticstudies of EP in aqueous solutions in a wide range of pHvalues and temperatures Thus the aim of this study was toevaluate the degradation of EP under the influence of pH andtemperature and to develop equations permitting the estima-tion of kinetic and thermodynamic parameters To separateEP and its degradation products our previously designedHPLC method was used [4] The geometric structure of EPmolecule FMOs orbitals (HOMO-LUMOHighest OccupiedMolecular Orbital Lowest Unoccupied Molecular Orbital)and molecular electrostatic potential were also determined
2 Experimental
21 Materials and Reagents Epidoxorubicin hydrochloridewas synthesized at the Department of Modified AntibioticsInstitute of Biotechnology and Antibiotics Warsaw PolandSodium laurilsulfate (AC reagent Sigma-Aldrich LogistikGmbH Germany) and all other chemicals and solvents wereobtained fromMerck KGaA Germany andwere of analyticalor high-performance liquid chromatographic grade
22 Chromatographic Conditions Chromatographic separa-tion and quantitative analysis were performed by using theHPLC method [4] The analytical system consisted of a Shi-madzu SPD-20A Prominence UVVIS detector a Rheodynewith a 50 120583L loop An LiChrospher RP-18 column (250mmtimes 4mm 5 120583m particle size Merck Germany) was used asthe stationary phase The mobile phase consisted of equalvolumes of acetonitrile and a solution containing 288 gL of
sodium laurilsulfate and 225mLL of phosphoric acid Theflow rate was 10mLmin andUV detection was performed at254 nm As the HPLC method was previously evaluated andvalidated for a stability study of epidoxorubicin in the solidstate [4] selectivity was examined during stability studiesin aqueous solutions in this work Papaverine hydrochloridewas used as an internal standard at a concentration of005mgmL
23 Kinetic Procedures The degradation of EP in aqueoussolutions was studied at 333 K 343K 353K and 363K in thepH range 042ndash995 The pH values of the reaction solutionsand those of the buffer standards were measured at reactiontemperatures The pH values of the reaction solutions in HClwere calculated from the equation pH = minus log119891HCl [HCl]The activity coefficient 119891HCl was obtained or calculated fromthe literature data [10] The ionic strength of all the solutionswas adjusted to 050M with a solution of sodium chloride(4M) Solutions of the desired pH and ionic strength of050M were heated to the required temperatures and then asample of EP was added to obtain the initial concentrationof 02mgmL At selected times determined by the rate ofdegradation samples of the solutions (10mL) were collectedand instantly cooled with a mixture of ice and water Sampleswith pH above 75 were neutralized by using HCl solutions atconcentrations ensuring that their pH was approximately 2To each such sample 10mL of the internal standard solution(papaverine hydrochloride solution) was added 50 120583L of thesolution sampleswas injected into the columnDue to the factthat anthracyclines are photolabile all samples to be studiedwere protected from light
Microsoft Excel 2000 was used for the calculation ofregression parameters
24 Theoretical Studies In order to interpret the FMOs(frontier molecular orbitals) and EP quantum-chemical cal-culations were performed by using the Gaussian 03 package[11] The GaussView software was utilized to propose theinitial geometry of the investigated moleculesThemoleculargeometries were optimized by means of a density functionaltheory (DFT) method with the B3LYP hybrid functional anda 6-31G(dp) basis set
3 Results and Discussion
31 Observed Rate Constants The observed rate constants ofEP degradation in aqueous solutions were determined in apH range of 042ndash995 and described by the equation of apseudo-first-order reaction
ln 119888119905 = ln 1198880 minus 119896obs times 119905 (1)
where 119888119905 and 1198880 are the time-dependent concentration andthe initial concentration of EP at times 119905 gt 0 and 119905 = 0respectively 119896obs is the observed rate constant of the pseudo-first-order reaction of EP degradation
The number of measurements of 119888119905 for each series rangedfrom 8 to 12
Journal of Chemistry 3
242
283
337
00E + 00
20E minus 05
40E minus 05
60E minus 05
80E minus 05
10E minus 04
12E minus 04
01 02 03 04 050
kＩ＜Ｍ(Ｍ
minus1)
[B]T
Figure 1 The plots of 119896obs = 119891([119861]119879) for the degradation of EP inphosphate buffer at 353 K
32 Buffer Catalysis Under the conditions of this study therate constants (119896obs) depended on the total concentrationsof the phosphate and acetate buffers which indicated thatthe components of the buffers catalyzed the degradationof EP (general acid-base catalysis) In order to verify thatthe differences between 119896obs determined at different bufferconcentrations were statistically significant the parallelismtest was used Under the conditions of general acid-basecatalysis 119896obs were calculated from the following equation
where [119861]119879 is the total buffer concentration 119896pH is the rateconstant at zero buffer concentration and 119896119861 describes thecatalytic effect of the buffer components The catalytic effectof the acetate buffer was investigated at pH 402ndash589 whilethose of phosphate buffers were investigated at pH 215ndash340(Figure 1) and pH 591ndash745 Since in the reaction solutions ofEP in HCl and borate buffer general acid-base catalysis wasnot observed in that pH range the values of 119896obs = 119896pH
33 pH-Rate Profiles The values 119896pH calculated from (2)under the conditions of general acid-base catalysis (acetateand phosphate buffers) and those obtained for hydrochloricacid as well as borate buffer (119896obs = 119896pH) were used to calculatethe relationship log 119896pH = 119891(pH) (Figure 2)
The semilogarithmic relationship 119896pHndashpH (Figure 2)indicated that in water solutions at pH 042ndash995 the follow-ing reactions occurred
(i) Degradation of the protonated molecules of EP cat-alyzed by hydrogen ions (1198961)
(ii) Spontaneous hydrolysis of protonated (1198962) andunprotonated (1198963) molecules of EP under theinfluence of water
(iii) Degradation of unprotonated molecules andmonoanions of EP catalyzed by hydroxide ions (1198964and 1198965)
20 40 60 80 10000
pH
10E minus 07
10E minus 06
10E minus 05
10E minus 04
10E minus 03
10E minus 02
10E minus 01
343K353K 333K
363KkＪ（(Ｍ
minus1)
Figure 2The pH-rate profiles for the degradation of EP at 333 343353 and 363KThe symbols represent experimental points the lineswere calculated from (3)
The total reaction rate was equal to the sum of partialreaction rates
119896pH = 1198961 times 119886H+ times 1198911 + 1198962 times 1198911 + 1198963 times 1198912
+ (1198964 times 1198912 + 1198965 times 1198913) times 119886OHminus (3)
where 119886H+ is the hydrogen ion activity and 1198911ndash1198913 are thefractions of protonated and unprotonated molecules andmonoanions of EPThe 1198911ndash1198913 values were calculated by usingthe values of pK119886 of EP that were about 74 and 95
The catalytic rate constants (1198961) were calculated from theplots 119896pH = 119891(119886H+) by using the values of 119896pH in the pH range042minus340 The plots were linear with the positive slope thatequaled 1198961 (Figure 3)
The catalytic rate constants 1198964 and 1198965 were calculatedfrom the equation 1198961015840pH = 119896pH minus (1198961 times 119886H+ times 1198911) = (1198964 times 1198912 +1198965 times 1198913) times 119886OHminus using 119896pH values above pH 85 where theconcentrations of 1198912 + 1198913 rarr 1 The plots 1198961015840pH(119886OHminus times 1198913) =119891(11989121198913)were linearwith positive slopes equal to the catalyticrate constant 1198964 and the value 119887 corresponded to the catalyticrate constant 1198965 The catalytic rate constants 1198962 and 1198963 werecalculated from the following equation 1198961015840pH = 119896pHminus(1198961times119886H+times1198911+(1198964times1198912+1198965times1198913)times119886OHminus) = 1198962times1198911+1198963times1198912 using the 119896pHvalues at pH 28ndash7 where the concentrations of 1198911 + 1198912 rarr 1
The plots 1198961015840pH1198911 = 119891(11989121198911) were linear with positiveslopes equal to the catalytic rate constant 1198963 and the value 119887corresponded to the catalytic rate constant 1198962
The calculated theoretical profile of log 119896 = 119891(pH)and that obtained from the experimental results were nearlyidentical indicating that the choice of the equation describingthe total rate of EP degradation was correct (Figure 2)
4 Journal of Chemistry
Table 1 Catalytic rate constants and thermodynamic parameters for the degradation of EP in aqueous solutions
Catalytic rate constant Temperature (K) (119896 plusmn Δ119896) Parameters of regressionln 119896119894 = 119891(1119879)
Thermodynamic parameters
1198961(molminus1 L sminus1) 333 (102 plusmn 007) times 10minus3 119903 = minus09997 119864119886 = 1188 plusmn 94 (kJmolminus1)
Δ119867= and Δ119878 = were calculated for 293K 119864119886 = minus119886119877 (Jmolminus1) Δ119867 = = 119864119886 minus 119877119879 (Jmolminus1) Δ119878 = = 119877[ln119860 minus ln(119896119879ℎ)] (J Kminus1molminus1) where 119896 is Boltzmannrsquos
constant (13807 times 10minus23 J Kminus1) ℎ is Planckrsquos constant (6626 times 10minus34 J s) 119877 is the universal gas constant (8314 J Kminus1molminus1) 119879 is temperature in K 119886 is thevectorial coefficient of the Arrhenius relationship and 119860 is the frequency coefficient
01 02 03 04000E + 00
20E minus 03
40E minus 03
60E minus 03
80E minus 03
10E minus 02
333K343K
353K363K
kＪ（(Ｍ
minus1)
a（+
Figure 3 The plots of 119896pH = 119891(119886H+ ) for the degradation of EP inaqueous solutions at the indicated temperatures
Based on the Arrhenius relationship ln 119896 = ln119860minus119864119886119877119879linear plots of ln 119896 = 119891(1119879)were used to calculate the energyof activation (119864119886) the entropy (Δ119878
=) the enthalpy (Δ119867=) and
the preexponential coefficient (119860) for the five partial reactions(Table 1)
The lowest energy of activation was observed in thereactions of unprotonated molecules and monoanions ofEP catalyzed by hydroxide ions whereas the highest energyof activation was observed in the reaction of protonatedmolecules of EP catalyzed by hydrogen ions
34 Theoretical Studies Based on theoretical calculationsthe places in EP molecules that might be connected withthe differences in EP reactivity in solutions with various pHvalues were proposedThe electrons in EP were concentratedin the tetracyclic quinolid aglycone especially in the C ringThat domain was responsible for the reactivity of EP duringbase hydrolysis (Figure 4)
The calculations also indicated that the A ring of thetetracyclic quinolid aglycone played a part in EP susceptibilityto degradation during acidic hydrolysis A comparison ofelectron distribution in the HOMO and LUMO of EPdemonstrated that it was similar to electron distributionin daunorubicin and doxorubicin (Figure 5) Previousstability studies of those three anthracycline antibiotics didnot show any statistically significant differences between
Journal of Chemistry 5
Figure 4 ESP of epidoxorubicin
OrbitalsHOMO LUMO
Epidoxorubicin
E = minus577 eV E = minus267 eV
Daunorubicin
E = minus261 eV
Doxorubicin
E = minus574 eV E = minus264 eV
E = minus571 eV
Figure 5 FMOs of EP DOX and DAU
their degradation rate constants in the solid state [4 5]and revealed a similarity between the relationships log 119896pH= 119891(pH) which correlates with theoretical study results[12 13] The values of energy gap for the three derivativeswere 31 eV proving their similar chemical stability
Although the stereoisomerismof theC-41015840 hydroxyl groupof L-acosamine in EP differs from that observed for DOXthe difference does not significantly influence EP reactivityand consequently the susceptibility of EP to degradationduring acid-base hydrolysis is not affected That difference
6 Journal of Chemistry
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
Figure 1 The plots of 119896obs = 119891([119861]119879) for the degradation of EP inphosphate buffer at 353 K
32 Buffer Catalysis Under the conditions of this study therate constants (119896obs) depended on the total concentrationsof the phosphate and acetate buffers which indicated thatthe components of the buffers catalyzed the degradationof EP (general acid-base catalysis) In order to verify thatthe differences between 119896obs determined at different bufferconcentrations were statistically significant the parallelismtest was used Under the conditions of general acid-basecatalysis 119896obs were calculated from the following equation
where [119861]119879 is the total buffer concentration 119896pH is the rateconstant at zero buffer concentration and 119896119861 describes thecatalytic effect of the buffer components The catalytic effectof the acetate buffer was investigated at pH 402ndash589 whilethose of phosphate buffers were investigated at pH 215ndash340(Figure 1) and pH 591ndash745 Since in the reaction solutions ofEP in HCl and borate buffer general acid-base catalysis wasnot observed in that pH range the values of 119896obs = 119896pH
33 pH-Rate Profiles The values 119896pH calculated from (2)under the conditions of general acid-base catalysis (acetateand phosphate buffers) and those obtained for hydrochloricacid as well as borate buffer (119896obs = 119896pH) were used to calculatethe relationship log 119896pH = 119891(pH) (Figure 2)
The semilogarithmic relationship 119896pHndashpH (Figure 2)indicated that in water solutions at pH 042ndash995 the follow-ing reactions occurred
(i) Degradation of the protonated molecules of EP cat-alyzed by hydrogen ions (1198961)
(ii) Spontaneous hydrolysis of protonated (1198962) andunprotonated (1198963) molecules of EP under theinfluence of water
(iii) Degradation of unprotonated molecules andmonoanions of EP catalyzed by hydroxide ions (1198964and 1198965)
20 40 60 80 10000
pH
10E minus 07
10E minus 06
10E minus 05
10E minus 04
10E minus 03
10E minus 02
10E minus 01
343K353K 333K
363KkＪ（(Ｍ
minus1)
Figure 2The pH-rate profiles for the degradation of EP at 333 343353 and 363KThe symbols represent experimental points the lineswere calculated from (3)
The total reaction rate was equal to the sum of partialreaction rates
119896pH = 1198961 times 119886H+ times 1198911 + 1198962 times 1198911 + 1198963 times 1198912
+ (1198964 times 1198912 + 1198965 times 1198913) times 119886OHminus (3)
where 119886H+ is the hydrogen ion activity and 1198911ndash1198913 are thefractions of protonated and unprotonated molecules andmonoanions of EPThe 1198911ndash1198913 values were calculated by usingthe values of pK119886 of EP that were about 74 and 95
The catalytic rate constants (1198961) were calculated from theplots 119896pH = 119891(119886H+) by using the values of 119896pH in the pH range042minus340 The plots were linear with the positive slope thatequaled 1198961 (Figure 3)
The catalytic rate constants 1198964 and 1198965 were calculatedfrom the equation 1198961015840pH = 119896pH minus (1198961 times 119886H+ times 1198911) = (1198964 times 1198912 +1198965 times 1198913) times 119886OHminus using 119896pH values above pH 85 where theconcentrations of 1198912 + 1198913 rarr 1 The plots 1198961015840pH(119886OHminus times 1198913) =119891(11989121198913)were linearwith positive slopes equal to the catalyticrate constant 1198964 and the value 119887 corresponded to the catalyticrate constant 1198965 The catalytic rate constants 1198962 and 1198963 werecalculated from the following equation 1198961015840pH = 119896pHminus(1198961times119886H+times1198911+(1198964times1198912+1198965times1198913)times119886OHminus) = 1198962times1198911+1198963times1198912 using the 119896pHvalues at pH 28ndash7 where the concentrations of 1198911 + 1198912 rarr 1
The plots 1198961015840pH1198911 = 119891(11989121198911) were linear with positiveslopes equal to the catalytic rate constant 1198963 and the value 119887corresponded to the catalytic rate constant 1198962
The calculated theoretical profile of log 119896 = 119891(pH)and that obtained from the experimental results were nearlyidentical indicating that the choice of the equation describingthe total rate of EP degradation was correct (Figure 2)
4 Journal of Chemistry
Table 1 Catalytic rate constants and thermodynamic parameters for the degradation of EP in aqueous solutions
Catalytic rate constant Temperature (K) (119896 plusmn Δ119896) Parameters of regressionln 119896119894 = 119891(1119879)
Thermodynamic parameters
1198961(molminus1 L sminus1) 333 (102 plusmn 007) times 10minus3 119903 = minus09997 119864119886 = 1188 plusmn 94 (kJmolminus1)
Δ119867= and Δ119878 = were calculated for 293K 119864119886 = minus119886119877 (Jmolminus1) Δ119867 = = 119864119886 minus 119877119879 (Jmolminus1) Δ119878 = = 119877[ln119860 minus ln(119896119879ℎ)] (J Kminus1molminus1) where 119896 is Boltzmannrsquos
constant (13807 times 10minus23 J Kminus1) ℎ is Planckrsquos constant (6626 times 10minus34 J s) 119877 is the universal gas constant (8314 J Kminus1molminus1) 119879 is temperature in K 119886 is thevectorial coefficient of the Arrhenius relationship and 119860 is the frequency coefficient
01 02 03 04000E + 00
20E minus 03
40E minus 03
60E minus 03
80E minus 03
10E minus 02
333K343K
353K363K
kＪ（(Ｍ
minus1)
a（+
Figure 3 The plots of 119896pH = 119891(119886H+ ) for the degradation of EP inaqueous solutions at the indicated temperatures
Based on the Arrhenius relationship ln 119896 = ln119860minus119864119886119877119879linear plots of ln 119896 = 119891(1119879)were used to calculate the energyof activation (119864119886) the entropy (Δ119878
=) the enthalpy (Δ119867=) and
the preexponential coefficient (119860) for the five partial reactions(Table 1)
The lowest energy of activation was observed in thereactions of unprotonated molecules and monoanions ofEP catalyzed by hydroxide ions whereas the highest energyof activation was observed in the reaction of protonatedmolecules of EP catalyzed by hydrogen ions
34 Theoretical Studies Based on theoretical calculationsthe places in EP molecules that might be connected withthe differences in EP reactivity in solutions with various pHvalues were proposedThe electrons in EP were concentratedin the tetracyclic quinolid aglycone especially in the C ringThat domain was responsible for the reactivity of EP duringbase hydrolysis (Figure 4)
The calculations also indicated that the A ring of thetetracyclic quinolid aglycone played a part in EP susceptibilityto degradation during acidic hydrolysis A comparison ofelectron distribution in the HOMO and LUMO of EPdemonstrated that it was similar to electron distributionin daunorubicin and doxorubicin (Figure 5) Previousstability studies of those three anthracycline antibiotics didnot show any statistically significant differences between
Journal of Chemistry 5
Figure 4 ESP of epidoxorubicin
OrbitalsHOMO LUMO
Epidoxorubicin
E = minus577 eV E = minus267 eV
Daunorubicin
E = minus261 eV
Doxorubicin
E = minus574 eV E = minus264 eV
E = minus571 eV
Figure 5 FMOs of EP DOX and DAU
their degradation rate constants in the solid state [4 5]and revealed a similarity between the relationships log 119896pH= 119891(pH) which correlates with theoretical study results[12 13] The values of energy gap for the three derivativeswere 31 eV proving their similar chemical stability
Although the stereoisomerismof theC-41015840 hydroxyl groupof L-acosamine in EP differs from that observed for DOXthe difference does not significantly influence EP reactivityand consequently the susceptibility of EP to degradationduring acid-base hydrolysis is not affected That difference
6 Journal of Chemistry
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
Δ119867= and Δ119878 = were calculated for 293K 119864119886 = minus119886119877 (Jmolminus1) Δ119867 = = 119864119886 minus 119877119879 (Jmolminus1) Δ119878 = = 119877[ln119860 minus ln(119896119879ℎ)] (J Kminus1molminus1) where 119896 is Boltzmannrsquos
constant (13807 times 10minus23 J Kminus1) ℎ is Planckrsquos constant (6626 times 10minus34 J s) 119877 is the universal gas constant (8314 J Kminus1molminus1) 119879 is temperature in K 119886 is thevectorial coefficient of the Arrhenius relationship and 119860 is the frequency coefficient
01 02 03 04000E + 00
20E minus 03
40E minus 03
60E minus 03
80E minus 03
10E minus 02
333K343K
353K363K
kＪ（(Ｍ
minus1)
a（+
Figure 3 The plots of 119896pH = 119891(119886H+ ) for the degradation of EP inaqueous solutions at the indicated temperatures
Based on the Arrhenius relationship ln 119896 = ln119860minus119864119886119877119879linear plots of ln 119896 = 119891(1119879)were used to calculate the energyof activation (119864119886) the entropy (Δ119878
=) the enthalpy (Δ119867=) and
the preexponential coefficient (119860) for the five partial reactions(Table 1)
The lowest energy of activation was observed in thereactions of unprotonated molecules and monoanions ofEP catalyzed by hydroxide ions whereas the highest energyof activation was observed in the reaction of protonatedmolecules of EP catalyzed by hydrogen ions
34 Theoretical Studies Based on theoretical calculationsthe places in EP molecules that might be connected withthe differences in EP reactivity in solutions with various pHvalues were proposedThe electrons in EP were concentratedin the tetracyclic quinolid aglycone especially in the C ringThat domain was responsible for the reactivity of EP duringbase hydrolysis (Figure 4)
The calculations also indicated that the A ring of thetetracyclic quinolid aglycone played a part in EP susceptibilityto degradation during acidic hydrolysis A comparison ofelectron distribution in the HOMO and LUMO of EPdemonstrated that it was similar to electron distributionin daunorubicin and doxorubicin (Figure 5) Previousstability studies of those three anthracycline antibiotics didnot show any statistically significant differences between
Journal of Chemistry 5
Figure 4 ESP of epidoxorubicin
OrbitalsHOMO LUMO
Epidoxorubicin
E = minus577 eV E = minus267 eV
Daunorubicin
E = minus261 eV
Doxorubicin
E = minus574 eV E = minus264 eV
E = minus571 eV
Figure 5 FMOs of EP DOX and DAU
their degradation rate constants in the solid state [4 5]and revealed a similarity between the relationships log 119896pH= 119891(pH) which correlates with theoretical study results[12 13] The values of energy gap for the three derivativeswere 31 eV proving their similar chemical stability
Although the stereoisomerismof theC-41015840 hydroxyl groupof L-acosamine in EP differs from that observed for DOXthe difference does not significantly influence EP reactivityand consequently the susceptibility of EP to degradationduring acid-base hydrolysis is not affected That difference
6 Journal of Chemistry
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
their degradation rate constants in the solid state [4 5]and revealed a similarity between the relationships log 119896pH= 119891(pH) which correlates with theoretical study results[12 13] The values of energy gap for the three derivativeswere 31 eV proving their similar chemical stability
Although the stereoisomerismof theC-41015840 hydroxyl groupof L-acosamine in EP differs from that observed for DOXthe difference does not significantly influence EP reactivityand consequently the susceptibility of EP to degradationduring acid-base hydrolysis is not affected That difference
6 Journal of Chemistry
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
in stereoisomerism was crucial for ensuring the greatertherapeutic safety of EP
Conflicts of Interest
The authors declare that there are no conflicts of interestregarding the publication of this paper
References
[1] G Minotti S Licata A Saponiero et al ldquoAnthracyclinemetabolism and toxicity in human myocardium Comparisonsbetween doxorubicin epirubicin and a novel disaccharideanalogue with a reduced level of formation and [4Fe-4S] reac-tivity of its secondary alcohol metaboliterdquo Chemical Research inToxicology vol 13 no 12 pp 1336ndash1341 2000
[2] A Mordente E Meucci G E Martorana B Giardina and GMinotti ldquoHuman heart cytosolic reductases and anthracyclinecardiotoxicityrdquo IUBMB Life vol 52 no 1-2 pp 83ndash88 2001
[3] M Piekarski and A Jelinska ldquoAnthracyclines still prove effec-tive in Anticancer Therapyrdquo Mini-Reviews in Medicinal Chem-istry vol 13 no 5 pp 627ndash634 2013
[4] A Sobczak A Jelinska M Lesniewska A Firlej and I Oszcza-powicz ldquoStability of epidoxorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 54 no 4 pp 869ndash872 2011
[5] J Cielecka-Piontek A Jelinska M Zajac M Sobczak ABartold and I Oszczapowicz ldquoA comparison of the stabilityof doxorubicin and daunorubicin in solid staterdquo Journal ofPharmaceutical and Biomedical Analysis vol 50 no 4 pp 576ndash579 2009
[6] M J Wood W J Irwin and D K Scott ldquoPhotodegradationof doxorubicin daunorubicin and epirubicin measured byhigh-performance liquid chromatographyrdquo Journal of ClinicalPharmacy andTherapeutics vol 15 no 4 pp 291ndash300 1990
[7] A Jelinska M Zając K Krzyston A Firlej and I Oszczapow-icz ldquoThe use ofHPLC for determining the stability of epirubicinand the influence of ionic strength on the stability of epirubicinrdquoAnnalesUniversitatisMariae Curie-Skłodowska Lublin - Poloniavol 19 no 2 pp 45ndash50 2006
[8] M J Wood W J Irwin and D K Scott ldquoStability of dox-orubicin daunorubicin and epirubicin in plastic syringes andminibagsrdquo Journal of Clinical Pharmacy and Therapeutics vol15 no 4 pp 279ndash289 1990
[9] D Kumar R S Tomar S K Deolia R Srivastava M Mitraand S Tyagi ldquoIsolation and characterization of degradationimpurities in epirubicin hydrochloride injectionrdquo Journal ofChromatography B vol 869 no 1-2 pp 45ndash53 2008
[10] E Pawełczyk and T HermannThe Fundamentals of Stability ofDrugs Warsaw Poland 1982
[11] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Revision D01 Gaussian Inc Wallingford Conn USA 2004
[12] J H Beijnen O A G J van der Houwen and W J MUnderberg ldquoAspects of the degradation kinetics of doxorubicinin aqueous solutionrdquo International Journal of Pharmaceuticsvol 32 no 2-3 pp 123ndash131 1986
[13] J H Beijnen O A G J van der Houwen M C H Voskuilenand W J M Underberg ldquoAspects of the degradation kineticsof daunorubicin in aqueous solutionrdquo International Journal ofPharmaceutics vol 31 no 1-2 pp 75ndash82 1986
