Degradation of Paclitaxel and Related Compounds in
`Aqueous Solutions Ill Degradation Under Acidic pH
`Conditions and Overall Kinetics
`
`JIAHER TIAN12 VALENTINO J STE11A1
`
`1Department of Pharmaceutical Chemistry The University of Kansas Lawrence Kansas 66047
`
`2Forest Laboratories
`
`Inc 220 Sea Lane Farmingdale NewYork 11 735
`
`Received 11 July 2008 revised 22 July 2009 accepted 24 July 2009
`
`Published online 9 September 2009 in Wiley InterScience wwwintersciencewileycom DOI 101002 Ijps21910
`
`ABSTRACT
`are complex molecules with numerous
`Paclitaxel and related taxanes
`hydrolysable ester groups possible epimerization at the 7position and possessing a
`strained oxetane ring a possible site for acid catalyzed cleavage Presented here is the
`III baccatin III and Nbenzoy13phenyliso
`stability of paclitaxel 10deacetylbaccatin
`serine ethyl ester in aqueous solution over a pH range of 15 at various temperatures
`Analysis of various samples was by HPLCUV and LCMS Baccatin III 10deacetyl
`ethyl ester were found to undergo acid
`baccatin III and Nbenzoy13phenylisoserine
`catalysis since pH rate profiles all
`followed a first order dependency
`in hydrogen ion
`concentration No evidence of any epimerization was noted at acidic pH values Baccatin
`III and 10deacetylbaccatin
`III showed similar degradation rates with possible products
`being possible dehydration around the 13hydroxy group and cleavage of the oxetane
`III was a minor initial pathway
`ring Cleavage of the 10 acetyl group of baccatin
`ethyl ester degraded significantly slower than both 10
`NBenzoy13phenylisoserine
`III and baccatin III At pH 2 paclitaxel degraded at a rate between that
`deacetylbaccatin
`III The pH of
`ethyl ester and 10deacetylbaccatin
`of Nbenzoy13phenylisoserine
`maximum stability for all compounds appeared to be around pH 4 © 2009 Wiley Liss
`Inc and the American Pharmacists Association J Pharm Sci 9912881298
`2010
`Keywords
`III oxetane ring cleavage
`III baccatin
`paclitaxel
`10deacetylbaccatin
`hydrolysis degradation pH stability acid catalysis
`
`INTRODUCTION
`
`Earlier articles in this series examined the
`and mechanisms of
`pathways
`the
`kinetics
`epimerization and base catalyzed degradation of
`paclitaxel and related taxanes in aqueous solution
`at near neutral and basic pH values The purpose
`of the present study was to explore the degrada
`tion of these compounds under acidic pH condi
`tions To our knowledge the kinetics and major
`
`to Valentino J Stella Telephone 785864
`Correspondence
`3755 Fax 7858645736 Email stellakuedu
`Journal of Pharmaceutical Sciences Vol 99 12881298 2010
`0 2009 Wiley Liss Inc and the American Pharmacists Association
`
`routes of aqueous degradation of paclitaxel and
`pH conditions
`under
`related taxanes
`acidic
`have not been published A number of articles
`did report the effect of acids and electrophiles on
`the stability of paclitaxel and related materials
`in non aqueous systems These earlier studies
`explored the biological activity of various pacli
`fragments formed under acidic reaction
`taxel
`conditions35
`The structures of paclitaxel 1 and its related
`compounds 27 used in this study are shown in
`Figure 1 These were the same as those reported
`in our earlier studies By studying the kinetics
`and pathways to degradation of
`fragments of
`paclitaxel and taxotere it was hoped that
`the
`
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`DEGRADATION OF PACLITAXEL AND RELATED COMPOUNDS
`
`1289
`
`could undergo ringopening reaction under acidic
`condition as was seen with some electrophilic
`reagents35
`
`EXPERIMENTAL
`
`Materials
`
`All of
`
`as
`
`the
`
`solvents were
`and
`the chemicals
`in detail earlier The pH of
`described
`solution was controlled throughout
`the reaction
`by using dilute hydrochloric acid and appropriate
`at pH 1 2 and 3 were
`buffers Reactions
`performed in dilute solutions of hydrochloric acid
`Acetic acidsodium acetate buffer was used for pH
`4 and 5 and the buffer concentration was 10 mM
`No
`of pH was
`observed
`change
`significant
`the reaction The ionic strength was
`throughout
`maintained at 015 with sodium chloride
`
`HC
`
`0
`
`OH
`
`CH
`
`= CH3C0 R2 = OH
`= CH3C0 R2 = H
`R2 = OH
`= H
`= H
`R2 = H
`
`R3 = H
`R3 = OH
`R3 = H
`R3 = OH
`
`1234
`
`5 R1 = CH3C0 R2 = OH
`6 R1 = H
`R2 = OH
`
`R3 = H
`R3 = H
`
`H2
`
`Figure 1 The structures of paclitaxel and related
`paclitaxel 1 7epitaxol 2 10deacetyl
`compounds
`taxol 3 7epi10deacetyltaxol 4 baccatin III 5 10
`III 6 and Nbenzoy13phenyliso
`serine ethyl ester 7
`
`deacetylbaccatin
`
`degradation complexity seen with larger mole
`cules like paclitaxel would be better understood
`Paclitaxel has four hydrolysable ester bonds
`which might be expected to undergo hydrolysis
`promoted by acid In addition to possible ester
`hydrolysis the oxetane ring D ring of paclitaxel
`
`HPLC and Mass Spectrometry Assays
`HPLCUV was
`employed
`simultaneously
`to
`detect and quantify the presence of the starting
`and its degradation products The
`compound
`isocratic HPLCUV assay
`indicating
`stability
`and the HPLC system operating
`conditions
`were
`described previously as were mass
`spectrometer conditions and are not
`here
`
`repeated
`
`Kinetics
`
`The kinetics of
`the degradation reaction was
`investigated in aqueous solutions at pH 15 For
`the kinetic experiments at 25°C 25 mL of
`the
`appropriate buffer solutions were equilibrated in
`a water bath at 250 ± 01°C and the hydrolysis
`study initiated by adding 08 mL stock solution
`125 µgmL of the appropriate substrate in acet
`onitrile into the reaction buffer This resulted in
`of 20 ptgmL
`an initial
`reaction concentration
`64 x 106M for Nbenzoy13phenylisoserine
`ethyl ester 37 x 106M for 10deacetylbaccatin
`III 34 x 106M for baccatin III and 23 x 106M
`for paclitaxel At various time intervals aliquots
`08 mL of the reaction solutions were withdrawn
`and assayed immediately by HPLC
`For the stability studies at elevated tempera
`tures at 435 500 and 700°C respectively
`vials containing reaction solutions were placed
`controlled ovens Solutions
`in thermostatically
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`100
`
`zoo
`
`300
`
`400
`
`Time hr
`
`100
`
`10
`
`ResidualPercent
`
`1290
`
`TIAN AND STELLA
`
`were prepared at 25°C and the reported pH values
`were those determined at 25°C No corrections for
`temperature effects on pH were applied The
`reaction solutions were maintained at the desired
`the kinetic study The
`temperature throughout
`vials were removed from the ovens at appropriate
`time intervals and quickly cooled in ice water to
`quench the reaction and followed by immediate
`HPLC analysis
`values were
`The
`individual
`rate constant
`obtained from the best multivariance regression
`of experimental data to various equations using
`the SigmaPlot program v 7101 SPSS Inc
`Chicago IL Numbers are reported ± standard
`deviation ±SD where statistics could be deter
`mined
`
`RESULTS AND DISCUSSION
`Paclitaxel 1 is a complex molecule capable of
`undergoing both epimerization and hydrolysis of
`to basic pH
`various ester bonds under neutral
`conditions in aqueous buffers Tian and Stella
`were better able to understand the degradation of
`paclitaxel and taxotere under these conditions by
`also studying the hydrolysis of 10deacetylpacli
`III
`
`taxel 3 baccatin III 5 10deacetylbaccatin
`6 and Nbenzoy13phenylisoserine
`7 and some of their related epimers 2 and 4 see
`Fig 1
`
`ethyl ester
`
`Degradation of 10Deacetylbaccatin
`
`III 6
`The degradation of the 10deacetylbaccatin
`the taxane fragment of taxotere and 10deacetyl
`
`III 6
`paclitaxel 3 was monitored in aqueous solutions
`of pH 109 199 305 453 and 518 at 25°C and
`higher temperatures No epimerization was noted
`in any studies under acidic pH conditions This
`observation
`held for all
`the substrates studied
`under these conditions In the pH range13 the
`loss of the compound demonstrated good
`overall
`linearity on the semilogarithmic plots as shown
`in Figure 2 For those reactions where only limited
`degradation occurred first order kinetics was
`assumed based on the observation
`over many
`the more completely
`halflives for
`degraded
`samples The reactions at pH 45 were too slow
`to follow at 25°C so no reliable results were
`achieved over
`the time period of study at
`this
`temperature and are therefore not reported here
`
`Figure 2 Semilog plots for the degradation of 10
`in aqueous solution at pH 109
`deacetylbaccatin
`
`III
`
`0 pH 199 V and pH 305 a T = 25°C showing
`
`the loss of 10deacetylbaccatin III
`order kinetics
`
`follows pseudo first
`
`Although ester hydrolysis might be expected to
`route of degradation HPLCUV
`be the major
`analysis showed the appearance of six quantifi
`able peaks with time The peak area for loss of
`III and formation of the six
`10deacetylbaccatin
`degradation peaks is shown in Figure 3 For
`example at pH 2 and 70°C two initial products
`were observed one eluting before the parent peak
`P2 and one eluting later than the parent peak
`P1 A sample HPLCUVMS chromatogram is
`shown in Figure 4 These products P1 and P2
`were formed by parallel competing reactions from
`as shown by the open
`10deacetylbaccatin
`III
`square and circle symbols These products were
`followed by other products with time open and
`filled upside down triangles and the filled circle
`symbols that are clearly secondary or tertiary
`the upside down triangle products
`as indicated
`by the delay in their appearance
`that
`is they
`were formed from one or both of
`the initial
`products An effort was only made to identify P1
`and P2 from HPLCUVMS data Unfortunately
`used and the
`because of the low concentrations
`small quantity of 10deacetylbaccatin III available
`to work with it was not possible to isolate enough
`of these degradation products to run NMR studies
`to confirm the proposed structures
`The late eluting peak P1 open square symbol
`in Fig 3 was not the epimer when compared to a
`
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`DEGRADATION OF PACLITAXEL AND RELATED COMPOUNDS
`
`1291
`
`60000
`
`50000
`
`40000
`
`30000
`
`20000
`
`10000
`
`03
`
`a
`
`0 y
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`Time hr
`Figure 3 Plot of the peak area versus time course for
`dation of 10deacetylbaccatin III M and the appear
`various peaks seen in the chromatogram for the degra
`ance of its initial degradation products P1 0 and P2
`0 in aqueous solution at pH 2 T= 70°C The HPLC
`UV chromatogram shows more decomposition
`products
`with time That is two primary products P1 El mass
`number = 527 and P2 0 mass number = 563 see
`for proposed structures were followed by
`indicated
`further degradation to secondary
`products
`by the symbols V and v
`
`Scheme 1
`
`the other
`
`standard earlier shown to also elute later than
`the parent peak of 10deacetylbaccatin III 6 but
`showed a loss of mass number 18 consistent with
`the loss of water supporting the conclusion that
`loss of acetate or benzoate
`ester hydrolysis
`was not responsible for this product The longer
`retention time indicated this intermediate having
`it was
`thus
`considered
`a
`reduced polarity
`possible dehydrated product
`By contrast
`the mass number of
`initial product P2 open circle symbol in Fig 3
`which eluted faster than 10deacetylbaccatin
`III
`gave a mass number for the addition of 18 mass
`units again not consistent with ester hydrolysis
`This structure was considered to be the product
`from the oxetane ring opening consistent with
`findings by others under acidic reaction condi
`tions35 With longer reaction time both initial
`degraded to further products Among
`products
`the later products analyzed no ester hydrolytic
`including the presence of benzoic acid
`products
`were observed From Figure 3 there did appear to
`be one major final product but this was an early
`front and may have
`eluting peak near the solvent
`
`hydrolytic
`
`opening of
`
`been made up of multiple products The contents
`of this peak were not characterized
`Based on the information gathered the pro
`posed initial degradation pathways of 10deace
`tylbaccatin III 6 in the acidic pH range 13 is
`shown in Scheme 1 The primary reactions were
`probable dehydration of the C130H P1 and
`the oxetane D ring P2
`Once the D ring is opened two additional OH
`groups are formed Due to the close proximity it
`likely the nearby C4 acetyl group is transferred to
`either of the two OH positions Consistent with
`this speculation was the observation
`two of
`that
`the additional but secondary products showed
`the same mass number but slightly different
`HPLC retention times as P2 Overall a simplified
`treatment of the kinetic data can be made for the
`III 6 in
`degradation of 10deacetylbaccatin
`low pH can be defined Scheme 1 where Eq 1
`can define the overall
`loss of 10deacetylbaccatin
`III 6
`
`is
`
`d Ddt = ki D + k2 D
`
`1
`and D ring
`where
`the proposed
`dehydration
`opening are parallel
`reactions with constants k1
`and k2 respectively
`D = Do exP ki + k2t = Do exp kobst 2
`The overall rate constant kohs is the sum of ki and
`k2 and can be obtained by slope of linear fitting of
`the semilog plot for the loss of starting material
`reaction products P1
`against time These initial
`and P2 degrade further to subsequent products
`k3 and k4 respectively
`with apparent constants
`As such all k values could be estimated from the
`peak area versus time profiles of products Pi and
`P2 see Fig 5 if
`the assumption were made that
`the response factors for Pi and P2 were identical
`If one makes the
`to that of 10deacetylbaccatin III
`assumption that the response factor for Pi and P2
`are similar it appears from peak area versus time
`the dehydration and
`plot seen in Figure 5 that
`opening of D ring have comparable
`hydrolytic
`rates at pH 2
`the oxetane ring structure of
`Theoretically
`paclitaxel should be much less reactive than an
`epoxide its three membered analogue However
`step to form a Dsecotaxol
`this ring opening
`derivatives appears to occur quite readily The
`is 10
`estimated acid catalyzed rate constant
`times faster
`than the estimated value for the
`opening of a similar epoxide structure based on
`reported data6 The
`ringopening
`previously
`reaction to form Dsecotaxols results in a tricyclic
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`Dabac 26pgimi 79C ilhr pH2 10VACNI
`
`106
`
`1 Sla
`
`Channul
`
`1 UV L 230flin
`Ant
`
`148135
`
`P2
`
`5 5S
`
`IS 1
`
`0$0
`
`10deacetylbaccatin
`
`Ill
`
`417
`
`888
`
`1002
`
`124
`
`1660
`
`P1
`
`IF 2z
`
`1827
`
`i
`
`j
`
`8can ES+
`TIC
`6 na
`
`Time
`
`Figure 4 HPLC chromatogram and MS data for the degradation of 10deacetyl
`baccatin III at 91mM and formation of the primary products Pi and P2 in aqueous
`solution at pH 2 T = 70°C t =11 h Two primary products early and late eluting 57 P2
`and 162 Pi mm respectively followed by further degradation to secondary products
`the peak at 64 mm was only seen after the appearance of Pi and P2
`
`k3
`
`P3 oxetane nrig cleavage
`
`P2
`
`III 6 at
`Scheme 1 Proposed
`degradation pathway
`for 10deacetylbaccatin
`low pH values in which the primary reaction pathways resulting in Pi and P2 are
`dehydration of C13 0H and hydrolytic opening of oxetane
`D ring respectively Once the
`D ring is opened transesterification is proposed with the acetyl group transferred to either of
`the two newly formed OH positions followed by other complex reactions
`
`P4 dehydration product
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`DEGRADATION OF PACLITAXEL AND RELATED COMPOUNDS
`
`1293
`
`the pH dependency for 10deacetyl
`Moreover
`baccatin III 6 and baccatin III 5 degradation
`Figure 6 shows that both compounds undergo
`apparent acid catalysis since the slopes of the pH
`to minus unity The
`rate profiles are close
`the two compounds
`are
`degradation rates for
`almost identical
`indicating the hydrolysis of the
`additional acetyl ester group on 10 position seen
`is minimal compared to the two
`with baccatin III
`other reactions seen with 10deacetylbaccatin III
`although a small peak for 10deacetylbaccatin III
`was observed This indicates that ester hydrolysis
`to the overall
`does not contribute significantly
`initial degradation in the acidic pH range That is
`the 10 acetate group was faster
`if hydrolysis of
`than the dehydration of the C130H group or the
`D ring opening seen with 10deacetylbaccatin
`III
`baccatin
`should exhibit
`faster degradation
`III
`III at the same acidic pH
`than 10deacetylbaccatin
`value
`From these results the concentration time
`profiles at acidic pH values are thought
`to be a
`consequence of reactions pathways illustrated in
`Scheme 2 where three reactions occur in parallel
`the C130H opening of oxetane
`dehydration at
`ring D ring and minor hydrolysis of the 10 acetyl
`group With longer reaction time these initial
`products P5 P6 and P7 6 degrade further to
`
`secondary products profiles not characterized
`
`0
`
`0
`
`pH
`
`20000
`
`1 5000
`
`10000
`
`5000
`
`P2
`
`PeakAreao
`
`f
`
`10
`
`20
`
`30
`
`40
`
`20
`
`40
`
`60
`
`80
`
`Time hr
`
`Time hr
`
`Figure 5 Experimental data and fitted lines describ
`131 0
`and P2 0 for the degradation of 10deacetylbaccatin
`ing the appearance of the two initial products
`III at pH 2 T=70°C
`
`flexibility
`
`ring system which is considerably more flexible
`than the rigid inverted cup shaped tetracyclic
`the 1HNMR
`ring system It was reported that
`signals of the protons of the A ring underwent a
`noticeable shift on opening of the D ring indicat
`and
`ing this increased
`structural
`conformational change5
`The pseudo first order degradation rate con
`stants for 10deacetylbaccatin III under acidic pH
`increased rapidly with increasing temperature
`An Eyring plot not shown of 10deacetylbaccatin
`III degradation at pH 196 was performed The
`enthalpy of degradation Alk was determined to
`be 28 ± 2 kcal mol 1 from the slope of the straight
`line and the entropy of activation
`was found
`to be 42 ± 06 eu
`
`10Deacetylbaccatin
`
`Degradation of Baccatin III 5 and Comparison to
`III 6
`The time courses of degradation of baccatin III 5
`were measured in aqueous solutions of pH 112
`and 198 at 25°C Linear semi logarithmic plots
`the overall degradation followed
`indicated that
`pseudo first order kinetics while again no epi
`merization was observed Upon analysis of the
`final products hydrolysis of the ester bonds was
`considered insignificant under
`these acidic pH
`conditions compared to other reactions
`as seen
`with 10deacetylbaccatin III with the products
`showing similar relative retention times to those
`seen with 10deacetylbaccatin III
`
`Figure 6 Partial pH rate profiles for the degradation
`III 0 and baccatin III 0 in
`of 10deacetylbaccatin
`the acidic pH range 13 T=25°C The solid line for
`to Eq 4 while
`is the best
`line joining the
`is the straight
`
`10deacetylbaccatin
`the line for baccatin III
`two points
`
`III
`
`fit
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`1294
`
`TIAN AND STELLA
`
`0
`
`k5
`
`CH3
`
`5
`
`minor pathway
`
`0
`
`OH
`
`06
`P7 6
`
`Scheme 2 Proposed degradation profile for baccatin III
`in the acidic pH range The
`degradation of baccatin III at low pH undergoes three pathways in parallel apparent
`dehydration D ring opening and hydrolysis of acetyl groups followed by a series of
`III constituted a minor initial
`reactions Formation of 10deacetylbaccatin
`secondary
`III was detected in the HPLC assay
`pathway as little 10deacetylbaccatin
`
`The overall
`loss of the starting compound follows
`pseudo first order kinetics
`D = Do exPk5 + k6 + k7t
`= Do expkobst
`
`3
`
`where the overall rate constant kohs the sum of k5
`k6 and k7 can be readily obtained from the slope
`
`JOURNAL OF PHARMACEUTICAL SCIENCES VOL 99 NO 3 MARCH 2010
`
`of the linear semi logarithmic plot of degradation
`of the starting material
`In the acidic pH range 13 acid catalyzed
`is dominant
`to water
`compared
`degradation
`and
`the
`total
`of
`shape
`catalysis
`pH profiles can
`versus
`following equation
`
`be
`
`expressed by
`
`log
`
`kob
`the
`
`kobskHaH
`
`4
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`DEGRADATION OF PACLITAXEL AND RELATED COMPOUNDS
`
`1295
`
`where kH is an acid catalysis second order rate
`constant and aH is the activity of hydrogen ion
`that was approximated
`to concentration
`In
`Figure 6 the solid line for 10deacetylbaccatin
`line calculated from a
`III represent the theoretical
`to Eq 4 while the data points are the
`linear fit
`experimental results The slope of this line is close
`the solid line is just the
`to unity For baccatin III
`line joining the two experimental values
`straight
`with again the slope being very close
`to the
`expected unity value The apparent second order
`rate constants for the acid catalyzed degradation
`kH are 77 x 102 and 68 x 102m1 h1 at 25°C
`III 6 and baccatin III 5
`for 10deacetylbaccatin
`respectively No statistical
`analysis was per
`formed due to the limited quantity of data
`Obviously the apparent dehydration reaction
`seen with baccatin III and 10deacetylbaccatin III
`cannot be a primary pathway for paclitaxel and
`taxotere degradation under acidic pH conditions
`without
`the side chain ester
`first hydrolysis of
`group Once the side chain is cleaved however
`the apparent dehydration reaction is possible and
`such a reaction pathway would be
`seen
`in
`secondary degradation products This points out
`in using model
`one of
`the occasional
`dangers
`compounds such as baccatin III and 10deacetyl
`and predicting the
`baccatin
`in studying
`III
`degradation rate of more complex molecules such
`as paclitaxel and taxotere that is a pathway seen
`in the model compounds that are not possible in
`the more complex molecule
`
`Degradation of NBenzoy13Phenylisoserine
`Ethyl Ester
`
`ethyl ester 7 is
`Nbenzoy13phenylisoserine
`selected as a mimic of the side chain of paclitaxel
`The loss of the compound was followed kinetically
`at pH 096 194 and 268 at 70°C as well as pH
`096 at 50°C The semi logarithmic plots of
`the
`the starting compound versus
`concentration
`of
`time were linear and indicated that
`the overall
`degradation followed pseudo first order kinetics
`Figure 7 is the pH rate profile for the degrada
`by plotting the rate constants
`tion generated
`determined at 70°C and the data point generated
`at 50°C In the pH range 13 acid catalyzed
`degradation seems the dominant pathway at 70°C
`and is expected to be similar at 25°C and is
`adequately defined by Eq4 The second order rate
`for the acid catalyzed degradation kH at
`constant
`70°C determined from the experimental data
`
`00
`
`05 I
`
`= 10
`
`2
`
`20 I
`
`25
`
`05
`
`10
`
`15
`
`20
`
`25
`
`30
`
`pH
`
`Figure 7 Partial pH rate profile for the degradation
`ethyl ester at acidic pH
`of Nbenzoy13phenylisoserine
`
`experimental data determined at 70°C while the open
`
`values at 70°C The tilled symbol 0 represents the
`symbol 0 represents the experimental point deter
`
`mined at 50°C
`
`was 0958 ± 0138 M1 h1 If one were to use the
`70 and 50°C data points generated at pH 096 a
`value 17 x 10 2M 1h1 for kH at 25°C can be
`estimated This value is lower compared to a value
`of 68 x 102M1 h1 for baccatin III degradation
`under similar acidic pH conditions
`
`Degradation of Paclitaxel Under
`Acidic pH Conditions
`Because of its low solubility <1 µgmL in water
`at 25°C it
`to follow the stability of
`is difficult
`this temperature When the time
`paclitaxel at
`course of paclitaxel at pH 203 was followed at 50
`and 70°C the
`increased
`these
`at
`solubility
`elevated temperatures was sufficient
`for quanti
`tative analysis Semilogarithmic plots of the total
`loss of the starting compound versus time were
`linear and indicated that the overall degradation
`followed pseudo first order kinetics unlike those
`pH conditions where
`seen under
`the
`basic
`reversible epimerization reaction leads to complex
`The
`kinetics12
`data
`experimental
`yielded
`values of 362 ± 019 x 103 and
`263 ± 013 x 102h at 50 and 70°C respec
`at pH 203 Figure 8
`illustrates the
`tively
`comparison of the degradation of 10deacetylbac
`catin III Nbenzoy13phenylisoserine
`ethyl ester
`
`rate constant
`
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`oxetane ring cleavage is probably the predomi
`nant reaction under this condition
`The acid catalyzed degradation of 10deacetyl
`and baccatin
`III were discussed
`baccatin
`III
`earlier Both compounds
`can undergo apparent
`and oxetane
`and
`ring opening
`dehydration
`reaction is fast By contrast
`the overall
`the
`Nbenzoy13phenylisoserine
`ester
`goes
`ethyl
`through a slower acid catalyzed ester hydrolysis
`shows
`a reaction rate in between
`Paclitaxel
`indicating multiple pathways that are faster than
`these
`side chain ester hydrolysis alone under
`fol
`conditions These results suggest paclitaxel
`lows the reaction pathways described in Scheme 3
`Degradation of paclitaxel under acidic pH under
`and D ring
`the side chain
`goes hydrolysis of
`opening simultaneously Once the side chain is
`cleaved the baccatin III being produced degrades
`through dehydration and hydrolytic Dring open
`ing and a small contribution from 10deacetyla
`tion
`Dehydration of C130H and the hydrolysis
`opening of oxetane ring D ring are the primary
`steps for the degradation of 10deacetylbaccatin
`III at pH values <3 while no
`III and baccatin
`epimerization is observed under these conditions
`These two reactions are acid catalyzed with
`comparable rate constants at the same pH value
`
`1296
`
`TIAN AND STELLA
`
`100
`
`10
`
`ResidualPercent
`
`20
`
`40
`
`60
`
`80
`
`Time hr
`
`Figure 8 Semilog plots showing the degradation of
`Nbenzoy13phenylisoserine
`
`ethyl ester V paclitaxel
`M and 10deacetylbaccatin III 0 in pH 2 aqueous
`
`solution maintained at 70°C
`
`and paclitaxel at pH 203 and 70°C If one were to
`correct for the fact that paclitaxel cannot undergo
`the dehydration reaction seen with 10deacetyl
`baccatin and baccatin III one would conclude that
`
`+ NbenzoyI3phenylisoserine
`
`OH
`
`hydrolysis
`
`1
`
`paclitaxel
`
`oxetane ring
`cleavage
`
`0
`
`NH
`
`OH
`
`Scheme 3 Proposed degradation scheme for paclitaxel
`in the acidic pH range
`Paclitaxel undergoes hydrolysis of the side chain and D ring opening simultaneously
`Once the side chain is cleaved the baccatin III being produced degrades further through
`apparent dehydration and hydrolytic D ring opening as indicated in Scheme 2
`
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`

`DEGRADATION OF PACLITAXEL AND RELATED COMPOUNDS
`
`1297
`
`Acid catalyzed hydrolyses of ester bonds on C2
`C4 and C10 positions are relatively slow under
`acidic pH conditions and make little to no major
`contribution to the overall degradation kinetics
`some hydrolysis of its side
`Paclitaxel undergoes
`chain and oxetane ring opening simultaneously
`without epimerization After the side chain
`cleaved the initial product baccatin III degrades
`further through the pathways described above
`
`is
`
`Overall pH Dependency of the Degradation of
`PaclitaxelRated Compounds
`
`and several
`The epimerization
`of paclitaxel
`related taxanes have been examined in aqueous
`solution and the rate constants of the intercon
`version of the R and Sepimers determined in
`near neutral and basic pH range
`Figure 9 shows the overall pH rate profile for
`the epimerization and degradation of baccatin III
`from pH 112 at 25°C The data for the neutral
`to
`
`6
`
`4
`
`2
`
`2
`
`4
`
`6
`
`8
`
`sE
`
`0
`
`g
`
`o
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`14
`
`pH
`
`first order rate constants forward and reverse respec
`tively for the base catalyzed reversible epimerization
`
`Figure 9 pH rate profile for baccatin III degradation
`in aqueous solution pH 112 at 25°C where k is the
`rate constant used to describe each of the pathways
`circle 0 and the filled circle 0 represent the pseudo
`defined by each of the appropriate symbols The open
`while the filled square M represents the total hydro
`lytic degradation of the ester groups from Refs 12
`The open square El represents the total degradation
`apparent dehydration oxetane ring opening and ester
`hydrolysis under acidic condition generated
`in the
`present study
`
`basic pH range was those determined experimen
`tally from earlier work A very similar plot
`to basic pH range for 10
`is seen in the neutral
`III not shown and from more
`deacetylbaccatin
`limited data for paclitaxel The plots show a
`Vshape profile with base catalyzed reversible
`epimerization to C7OH and hydrolytic degrada
`tion of ester groups at high pH Compared to basic
`condition the compounds are much more stable
`under acidic pH conditions in which the primary
`degradation appears to be through dehydration at
`the C130H and hydrolytic
`the
`opening
`of
`oxetane ring The maximal stability is obtained
`near pH 45
`Based on the similarity of the chemical struc
`ture the measured kinetic data of smaller partial
`and
`structures such as 10deacetylbaccatin III
`baccatin III allows one to make reasonable and
`reliable assumptions about the chemical stability
`tax
`of the more complex molecules Paclitaxel
`otere and their other derivatives likely have
`similar Vshape profiles The rates for epimeriza
`tion of paclitaxel and taxotere are comparable to
`baccatin III and 10deacetylbaccatin III
`respec
`tively while paclitaxel showed faster hydrolysis
`under basic pH due to the more labile side chain
`ester bond Lacking the dehydration reaction seen
`III paclitaxel showed
`with 10deacetylbaccatin
`better chemical stability at pH values <3
`
`ACKNOWLEDGMENTS
`
`The authors greatly appreciate the contribution
`and support by Dr Richard Schowen The authors
`the support of
`also gratefully acknowledge
`Dr Gunda Georg and Tapestry Pharmaceuticals
`Inc as suppliers of paclitaxel and other related
`compounds
`
`REFERENCES
`
`1 Tian J Stella VJ 2008 Degradation of paclitaxel and
`related compounds in aqueous solutions I Epimer
`ization J Pharm Sci 9712241235
`2 Tian J Stella VJ 2008 Degradation of paclitaxel and
`in aqueous solutions II None
`related compounds
`to basic pH
`pimerization degradation under neutral
`conditions J Pharm Sci 9731003108
`3 Kingston DGI Molinero AA Rimoldi JM 1993 The
`taxane diterpenoids Frog Chem Org Nat Prod 611
`206
`
`DOI 101002jps
`
`JOURNAL OF PHARMACEUTICAL SCIENCES VOL 99 NO 3 MARCH 2010
`
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`
`

`

`1298
`
`TIAN AND STELLA
`
`4 Samaranayake G Magri NF Jitrangsri C Kingston
`DGI 1991 Modified taxols 5 Reaction of taxol with
`and preparation of a rear
`electrophilic reagents
`ranged taxol derivative with tubulin assembly activ
`ity J Org Chem 5651145119
`
`5 Magri NF Kingston DGI 1986 Modified taxols 2
`Oxidation products of taxol J Org Chem 51797802
`6 Mabey W Mill T 1978 Critical review of hydrolysis
`of organic compounds in water under environmental
`conditions J Phys Chem Ref Data 7383415
`
`JOURNAL OF PHARMACEUTICAL SCIENCES VOL 99 NO 3 MARCH 2010
`
`DOI 101002jps
`
`Abraxis EX2030
`Apotex Inc. and Apotex Corp. v. Abraxis Bioscience, LLC
`IPR2018-00151; IPR2018-00152; IPR2018-00153
`
`