The Chemistry and Pharmacology of Taxol and its Derivatives
`V Farina editor
`© 1995 Elsevier Science BV All rights reserved
`
`103
`
`3 P
`
`ACLITAXEL TAXOL®
`FORMULATION AND PRODRUGS
`
`Dolatrai M Vyas
`
`BristolMyers Squibb Company Pharmaceutical Research Institute
`5 Research Parkway Wallingford CT 064927660 USA
`
`31
`
`INTRODUCTION
`
`Interest
`
`Paclitaxel 111 Figure 11 a natural diterpene isolated from the bark of
`Taxus brevifolia Pacific yew 1 has been heralded as the antitumor agent of
`the 1990s because of its promising clinical activity against a variety of human
`solid tumors such as ovary breast lung head and neck and melanoma 24
`in various aspects of this drug has mushroomed due to the clinical
`significance of its antitumor profile in treating common human cancers 5 6
`is an FDA approved cancer agent
`in the United States for
`Currently paclitaxel
`second line treatment against cisplatinum refractory ovarian cancer and
`It has
`metastatic breast cancer which is refractory to anthracycline treatment
`for similar indications On the basis of
`
`also been approved in other countries
`
`first line chemotherapeutic
`
`broader ongoing clinical trials there is a good likelihood of paclitaxel becoming a
`in the very near future 7
`Within the current arsenal of cancer chemotherapeutics 8 paclitaxel is a
`
`agent
`
`unique tubulininteracting agent Unlike other clinical antimitotic agents such
`as the vinca alkaloids 9 101 which inhibit the microtubule assembly process
`
`Taxol® is a registered trademark of the BristolMyers Squibb corporation The generic name paclitaxel
`throughout
`this chapter
`
`is used
`
`Abraxis EX2003
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`104
`
`paclitaxel
`
`promotes tubulin polymerization and stabilizes the resulting
`microtubules toward depolymerization 11 This shift in the normal dynamics in
`the cellular tubulinmicrotubule system by paclitaxel
`is currently widely
`recognized as its mode of action for cell cytotoxicity
`Owing in part to the
`in 1979 11 of paclitaxels
`discovery by Susan Horwitz
`and coworkers
`mechanism of action the National Cancer Institute NCI decided to accelerate
`the human clinical
`trials for this drug Eventual marketing of paclitaxel
`for
`oncology clinical use was facilitated by a Cooperative Research and Development
`Agreement CRADA between the NCI and the Bristol Myers Squibb BMS
`Company in 1989 and the subsequent massive efforts by BMS to secure ample
`clinical supplies of paclitaxel 12
`
`BzNH 0
`
`Ph
`
`5H
`
`Ac0
`
`OH
`
`fee
`
`HO
`
`Bz0
`
`Aco
`
`Boc
`
`NH
`
`0
`
`Ph
`
`b
`
`OH
`
`111 Paclitaxel
`Taxol
`
`Figure 1 Clinically relevant
`
`taxanes
`
`HO
`
`OH
`
`HO
`
`= Ac0
`Bz0
`
`112 Docetaxel
`Taxotere
`
`In spite of paclitaxels promising clinical antitumor profile and the ongoing
`
`efforts to optimize its utility
`
`for curative cancer
`in multimodality treatment
`problems during its intravenous iv
`therapy the drug has presented
`administration to patients 13 These are formulation related problems
`stemming from the use of the excipient Cremophor EL® 14 as a solubilizing
`detergent for iv administration Interestingly a close analog of paclitaxel
`namely docetaxel 112Taxotere® 15 Figure 1 which is currently awaiting
`FDA approval
`for clinical use is devoid of such problems Docetaxel
`possesses a
`slightly better aqueous solubility than paclitaxel and is administered as a
`Tween80 polyoxyethylene sorbitan monooleateethanol
`formulation Several of
`formulation related patient care issues such
`as
`have been successfully addressed and managed in
`the clinic 16 However the pharmaceutical
`issues 17 related to Cremophor
`ELs use still persist during intravenous administration and demand precautions
`
`paclitaxels
`
`alleged
`
`hypersensitivity reactions
`
`

`

`current clinical
`
`alleviate or circumvent
`
`105
`
`to
`
`and adherence to strict pharmacy protocols The main intent of this chapter is to
`document and highlight all of the formulation related issues stemming from the
`formulation and briefly discuss possible solutions sought
`them The quest
`toward the development
`of a widely
`formulation devoid of Cremophor EL is
`and safe intravenous
`continuing in several research laboratories and is discussed herein
`
`acceptable
`
`32
`
`PHARMACEUTICAL DEVELOPMENT SUMMARY
`
`collection of
`
`Paclitaxels early pharmaceutical development history 12 18 19 is
`mainly confined to the NCI laboratories since it was through a joint NCI USDA
`United States Department of Agriculture initiative in the early 1960s to screen
`plant material for novel cytotoxic agents that paclitaxel was first discovered
`The isolation and identification of this agent were accomplished in 1966 at the
`NCI contract
`laboratories of the Research Triangle Institute North Carolina by
`M Wani and M Wall approximately four years after the initial
`Taxus Brevifolia plant material 18 The isolation of pure paclitaxel was
`facilitated through a bioassay guided fractionation protocol
`following in vitro
`cytotoxicity in KB cells and in vivo antitumor activity against murine tumor
`models such as leukemia P1534 and carcinosarcoma Walker 256 During the
`in several of the
`next few years the predinical antitumor profile of paclitaxel
`NCIs murine hematological
`tumor models namely leukemias L1210 P388 and
`P1354 was established Also activity against the Walker 256 sarcoma model
`was demonstrated
`These efficacy evaluations were carried out
`in mice by
`administering paclitaxel as a suspension in the peritoneal ip cavity against
`ipimplanted tumors Due to the extremely hydrophobic character of paclitaxel
`20 it was difficult
`to obtain a purely water based formulation for iv or ip
`administration Consequently the vehicles employed for ip administration
`included steroidal suspensions vegetable oils peanut sesame olive normal
`cellulose CMC It was not until 1974 that the first
`saline and carbomethoxy
`evidence of paditaxels efficacy in an ipip murine solid tumor B16 melanoma
`was obtained Until this time paclitaxel was considered an unexciting cytotoxic
`agent with solubility problems and in vivo efficacy confined mainly to ipip
`localized tumor models Consequently there was little enthusiasm in pushing
`this drug further toward human clinical trials However by 1980
`several new
`findings had made paclitaxel a prime candidate for human clinical
`
`trials
`
`In
`
`

`

`106
`
`the introduction of human tumor xenograft models to the NCI
`1978 after
`screening program paclitaxel was shown for the first
`time to possess distal
`the LX 1 lung the MX1 breast and the CX1 colon
`tumor efficacy against
`xenografts 18 In all
`these tumor models the drug was administered
`to the human tumor implanted in the sub renal capsule kidney
`subcutaneously
`of mice More importantly the excitement and interest about
`this drug was
`in 1979 when a report by SHorwitz and coworkers 11 disclosed
`heightened
`that paclitaxel acted via a novel tubulininteracting mechanism Unfortunately
`
`at this juncture a suitable formulation for intravenous administration of the
`the outset 21 it was
`drug was lacking and remained to be developed
`determined at the NCI
`that paclitaxel was totally devoid of any activity when
`administered orally to mice up to a dosage of 160 mgKg
`
`At
`
`321 Early Formulation Studies
`the NCI was directed at developing
`an
`Since 1978 a major effort at
`intravenous formulation for paclitaxel in order to initiate human clinical studies
`Paclitaxels extremely low water solubility < 001mgm1 coupled with the
`functionality amine or carboxylic acid for salt
`absence of a suitable chemical
`formation led to the evaluation of cosolvents and excipients as the first strategy
`for developing an iv formulation Much of this effort is described in detail in a
`recent report 21 The approximate solubility of paclitaxel
`and certain organic solvents are reported in Table 1
`
`in aqueous vehicles
`
`Table 1 Solubility of Paclitaxel
`
`in Various Solventsa
`
`Solvent
`
`Solubility mgml
`
`Methylene Chloride
`
`Ethanol
`75 Propylene glycol
`75 Polyethylene glycol 400 PEG 400
`35 PEG 400
`Soybean oil
`
`a adapted from ref 21
`
`Triacetin
`
`> 19
`
`ca 39
`<14
`
`31
`
`003
`03
`
`75
`
`

`

`107
`
`Paclitaxel has substantial solubility in organic solvents such as ethanol
`and methylene chloride Highly concentrated millimolar paclitaxel solutions
`in some of these solvents were attainable However diluting paclitaxel solutions
`of water miscible solvents such as ethanol with water presented problems of
`precipitation The solubility of paclitaxel
`
`in lipids such as soybean oil
`
`intralipid
`
`was also not quite adequate for formulation considerations
`The two intravenous formulations which received indepth evaluation at
`the NCI were those involving cosolvent PEGpolyethylene glycol 400 75 and
`surfactant Cremophor EL® 19 21 The 75 PEG 400 solution in water
`containing 16 mgml paclitaxel was found to be chemically stable by HPLC
`analysis and free of particulate material for up to 14 days at 25°C However
`infusion precipitation cloudiness was discernible
`upon dilution for
`
`Formulation with solubilizing surfactant Cremophor EL® 5 in ethanol 5
`and 09 saline under equilibrium conditions
`gave a solution with paclitaxel
`concentration of about 01 mgml With this formulation concentrations greater
`than 06 mgml were achievable by dilution of a 6 mgml solution of paclitaxel in
`11 Cremophor ELEt0H Fortunately this solution had adequate physical and
`chemical stability over periods as long as 24 h >96 of drug was found in
`solution over this time period Thus the Cremophor formulation provided some
`
`assurance against
`
`the possibility of drug precipitating during the infusion
`
`period Also this formulation was the best on an efficacy basis since paclitaxel
`administered ip as an aqueous suspension containing Cremophor EL® was
`found to be more efficacious against an ipimplanted B16 melanoma tumor than
`paclitaxel administered ip in PEG 400 Due to all these considerations in 1980
`the Cremophor formulation was selected for clinical trials The current clinical
`dosage form of paclitaxel consists of a 5m1 size vial containing 30 mg of
`paclitaxel 264 g of Cremophor EL® and 497 Et0H 11 vv This
`concentrated solution required further dilution with injectable fluids such as 5
`dextrose 09 sodium chloride and 5 dextrose in Ringers solution The intact
`vial shelf life is estimated to be 5 years under refrigeration 21
`
`322 Formulation Issues
`With the launching
`into clinical
`trials
`of paclitaxel
`pharmaceutical and patient care issues surfaced at the very outset
`These
`apparently stemmed from the use of Cremophor EL® as an excipient in the
`intravenous formulation Cremophor EL® is a chemically and physiologically
`
`several
`
`

`

`108
`
`active surfactant 14 Thus the formulation is associated with potential medical
`depending upon the amount of Cremophor EL® present and the route
`by which this formulation is administered to patients As it currently stands
`
`liabilities
`
`formulation has the highest amount of this surfactant
`the paclitaxel clinical
`among all currently marketed drugs 14 Other drugs with Cremophor in their
`formulations are teniposide 22 and cyclosporine 23 The potential for harmful
`that
`effects of Cremophor to patients is further augmented by the fact
`quantities of this solubilizing surfactant are required for intravenous delivery of
`doses of paclitaxel ranging from 150 to 300 mgm2
`issues 17 stemming from the
`Pharmaceutical
`Issues Pharmaceutical
`intravenous administration of the Cremophor ELObased formulation have
`mainly affected hospital health care professionals such as pharmacists and
`nurses who are required to exercise caution and care during iv infusion
`Chemical stability studies have shown that the paclitaxel
`
`therapeutic
`
`large
`
`formulation diluted
`
`with standard infusion fluids is stable for up to 27 hours at room temperature
`This has facilitated the preparation and storage of iv solutions in hospital
`pharmacies
`However due to the leaching properties of Cremophor toward
`phthalate plasticizers from PVC infusion bags and intravenous administration
`set tubings use of polyolefin plastic containers glass bottles or vinyl acetate
`type bags has been mandated
`
`Also to guard against
`precipitation during infusion paclitaxel is administered with an in line filtration
`device containing a 022 µm membrane filter eg IVEX2 and IVEXHP
`The issue of paclitaxel compatibility with other medications has also been
`studied in some detail 24 25 An examination of visual and turbidimetric
`compatibility of paclitaxel with 59 other drugs was carried out The list of drugs
`included antimicrobials anticancer agents analgesics antiemetics and
`involved the mixing of 12 mgml
`The experiment
`antiallergy medications
`solution 4 ml of paclitaxel
`in D5W with a 4 ml sample of the test drug at
`clinically relevant concentrations
`All of the drugs but four were compatible with
`
`the perils of drug
`
`paclitaxel based on the turbidity measurement criteria These included two
`cancer drugs mitoxantrone hydrochloride and doxorubicin
`In the case of
`doxorubicin a loss of 12 paclitaxel
`in 96 hours was determined by HPLC
`measurements It was stressed in this study that the absence of turbidity effects
`does not automatically imply chemical compatibility with paclitaxel and caution
`
`should be exercised particularly when need arises for combination therapy with
`
`other drugs
`
`

`

`109
`
`Patient Care Issues In clinical
`
`trials paclitaxel has manifested some of the
`classical drug related toxicities associated with an anticancer agent 16 These
`The
`include neutropenia neurotoxicity mucositis GI toxicities and alopecia
`
`cumulative dose paclitaxel
`
`major dose limiting toxicity was demonstrated to be neutropenia which was
`usually severe at doses >200 mgm2 The incidence of severe neutropenia was
`similar at all dose levels and recovery was complete after 510 days Since
`hematopoietic growth factors eg GCSF are now commonly used to ameliorate
`remains a major concern in high single and
`neutropenia neurotoxicity
`therapy The neurotoxicity manifested is sensory
`neuropathy which is common when paclitaxel is administered by a 24 h infusion
`and exceeds a dose of 200 mgm2
`signs and
`Hypersensitivity reactions particularly
`anaphylactoid
`liability and patient care issue in the clinical
`symptoms remain a major medical
`use of paclitaxel 26 The hypersensitivity
`reaction is alleged to be due to
`Cremophor EL® contained
`in the formulation The symptoms include rapid
`onset of hypotension respiratory distress eg bronchospasms urticaria and
`rash These appear to be classical histamine release mediated reactions which
`have also been observed in dogs treated with Cremophor EL® alone 27 These
`allergic reactions due to the Cremophor threatened the continuation of earlier
`paclitaxel clinical trials due to the death of a patient 7 However
`they were
`infusion and emergency treatment of
`controlled by discontinuation of paclitaxel
`patients with antihistamines H2blockers and oral steroids 13
`and the severity of
`Currently in order to minimize the occurrence
`hypersensitivity reactions in patients two routine measures have been
`instituted These include the extension of the infusion duration time to 6 or 24 h
`and the prophylactic use of antihistamines and corticosteroids prior to paclitaxel
`administration 7 These measures do not completely eliminate the histamine
`
`they certainly reduce the
`release effects of paclitaxel administration but
`episodes and severity of hypersensitivity reactions In spite of the introduction
`
`of these safeguards working with paclitaxel
`in the hospital setting remains a
`challenge A nonCremophor based intravenous formulation should alleviate the
`above pharmaceutical and patient care concerns However a suitable clinical
`substitute has not yet been identified Several NCI funded labs and independent
`
`research groups are currently actively engaged in research
`
`to either develop a
`
`safer parenteral administration of paclitaxel through watersoluble prodrugs or
`to identify a suitable carrier system for iv administration
`
`

`

`110
`
`33 PRODRUGS OF PACLITAXEL
`
`A thoroughly explored preclinical strategy for iv administration of
`paclitaxel has involved the use of prodrugs in a nonCremophorcontaining
`preferably 100 aqueous formulation 28 Initially this effort has involved the
`synthesis and evaluation of prodrugs with moieties carrying a solubilizing group
`In this context an ideal handle for prodrug synthesis has been the C2 hydroxyl
`functionality since derivatives masked at this position are devoid of activity
`until unmasked in vivo through hydrolysis by enzymatic or chemical means A
`free hydroxyl group at C2 seems to be a prerequisite for tubulin polymerization
`and consequently for cytotoxicity 29 In contrast
`the C7 hydroxyl
`is not such an ideal site for prodrug design since masked derivatives eg esters
`at this position are usually much more stable than their C2 counterparts to in
`vivo cleavage 30 Consequently only special C7 derivatives with a rapid in
`vivo unmasking pathway have the potential
`to serve adequately
`as a prodrug
`Pro moieties which are slow to unmask in vivo may simply act as paclitaxel
`analogs 31
`
`functionality
`
`as Prodrugs
`
`331 Acvl Derivatives
`In 1984 Mellado et al 32 during their investigation on the biological
`activity of paclitaxel acetates established that the C2 hydroxyl can be readily
`the C7 hydroxyl
`and selectively acetylated over
`functionality Furthermore
`they found the C2 acetate to be more labile toward hydrolysis than its C7
`counterpart These earlier observations formed the basis for considering the C2
`for future research efforts on
`hydroxyl group as the preferred functionality
`watersoluble prodrugs 28
`Paclitaxel C2 Esters The basic strategy in designing C2 esters as water
`soluble prodrugs has been the incorporation of ionizable groups such as amines
`amino acids and sulfonic acid groups In this vein a plethora of C2 derivatives
`have been synthesized and evaluated
`for their suitability as watersoluble
`prodrugs of paclitaxel These include Figure 2 succinate eg 311 and
`glutarate derivatives eg 312 33 34 sulfonic acid derivatives eg 313 and
`314 35 and amino acid derivatives eg 315 and 316 30 34 Although
`these derivatives eg 311 and 312
`possess adequate water
`solubility up to 1 for an iv formulation and good biological activity in vivo
`
`several of
`
`

`

`111
`
`their chemical
`
`This was attributed to
`they were found unsuitable as prodrugs of paclitaxel
`instability in aqueous solution a property deemed unacceptable
`for intravenous administration of a highly insoluble agent such as paclitaxel To
`date among this class of prodrugs 243NNdiethylaminopropionylpaclitaxel
`316 prepared by Stella and coworkers 30 appears to be best suited for
`It has acceptable solution stability at pH 35
`half life >400 h However the half life at physiological
`pH 74 was <30 mm
`life was even shorter < 5 min The aqueous
`In human plasma the half
`In vivo 316 showed
`solubility of this prodrug was determined to be >10 mgml
`complete tumor remission in the MX1 mammary tumor model
`
`prodrug delivery of paclitaxel
`
`Ac0
`
`OH
`
`BzNH 0
`Ph
`
`OR
`
`HO
`
`E Ac0
`OBz
`
`311 R= COCH22COOH HOCH2CH23N
`312 R= COCH23COONa
`313 R= COCH22SO3Na
`314 R= COCH22CONHCH2SO3Na
`315 R= COCH22NH2 HCO2H
`316 R= COCH22NEt2 CH3S03H
`317 R= COCH2OCH2CO2H
`318 R= COCH2SCH2CO2H
`319 R= COCH2S02CH2CO2H
`3110 R= COCH22CONHPEG
`
`Figure 2 Watersoluble C2 esters as paclitaxel prodrugs
`
`More recently a Scripps group led by Nicolaou has reported another series
`of C2 esters named protaxols 36 These are monoesters 317 318 and
`319 of dicarboxylic acids incorporating a heteroatom functionality oxygen or
`sulfur for the purpose of imparting further water solubility These compounds
`have been reported to have acceptable water solubility ca 1 mgml and stability
`at neutral pH and room temperature Under basic conditions 319 rapidly
`
`

`

`112
`
`The authors envisage that such a mechanism may be
`generated paclitaxel
`relevant in vivo in the basic microenvironment of certain tumor cells However
`in vivo antitumor activity of these protaxols in murine tumor models have not
`
`yet been reported by the Scripps group
`Thus far all of the above approaches have only yielded prodrugs with
`moderate up to 10 mgml water solubility However
`conjugates of hydrophobic
`glycol PEG of molecular weight of 2kD or greater
`molecules with polyethylene
`are known to impart even greater water solubility
`In this context Greenwald and coworkers 37 have recently reported the
`synthesis and limited biological evaluation of a variety of C2 polyethyleneglycol
`esters as highly watersoluble prodrugs of paclitaxel 3110 The PEG esters
`reported are claimed to have water solubility of >666 mgml at ambient
`temperature The half life of 3310 was determined to be approximately 40 h
`and 11 h in pH 74 buffer and rat plasma respectively The authors have not
`reported any in vivo antitumor activity but the approach seems very promising
`Paclitaxel C7 Esters In general C7 esters have found little utility as water
`To date several C7 derivatives have been
`soluble prodrugs of paclitaxel
`reported 30 and in most cases
`they have been the counterparts of their
`corresponding C2 derivatives
`Their poor prodrug properties have
`attributed to enhanced in vivo stability
`toward esterases and hydrolytic
`
`been
`
`cleavage
`For example the cationic watersoluble derivative 3111Figure 3 had a
`life of 378 h at pH 38 compared with a half
`life of of 96 h for its
`half
`corresponding C2 ester derivative A similar trend was observed
`physiological pH 74 where 3111 had a half life of 34 h compared to 6 h for its
`C2 counterpart Half lives in human plasma for 3111 and its C2 counterpart
`were reported to be 3 h and <30 mm respectively
`The enhanced hydrolytic chemical and enzymatic stability of C7 esters
`over their C2 counterparts
`can be ascribed to the steric congestion surrounding
`this position Biologically unlike the C2 esters cationic watersoluble C7
`esters such as 3111 were shown to promote microtubule assembly as effectively
`as paclitaxel but have been found to be poorly bioactive in whole cell assays
`This was attributed to poor cell membrane penetration
`
`at
`
`

`

`113
`
`Bz NHO
`
`Ph
`
`OH
`
`Figure 3 Typical C7 ester prodrug of paclitaxel
`
`Paclitaxel C2 Carbonates Recently the BMS group and the Scripps group
`have demonstrated that C2 carbonates like their ester counterparts behave as
`paclitaxel prodrugs in vivo Carbonates and carbamates have so far found little
`
`utility as prodrugs because they are more stable than the corresponding esters
`toward hydrolytic and enzymatic cleavage Notable exceptions in this area are
`38 and
`carbamate derivatives of drugs such as 10hydroxycamptothecin
`CC1065 39 which have been shown to be effective prodrugs in vivo
`The BMS group has recently disclosed the synthesis and in vivo biological
`profile of C2 carbonates derivatives 311217 Figure 4401
`
`Ac0
`
`OH
`
`H 0
`
`Bz
`
`Ph
`
`0
`
`HO
`
`CO
`
`E
`
`OBz
`
`RO
`
`3112 R= CH3
`3113 R= CH2CH3
`3114 R= CHCH32
`3115 R= CH2CI
`3116 R= CH2Ph
`3117 R=CH=CH2
`
`Figure 4 C2 carbonate derivatives synthesized by the BMS group
`
`When evaluated in the cytotoxicity assay paclitaxel2carbonates were 2
`line HCT
`10 times less cytotoxic than paclitaxel against human colon cancer cell
`116 Also they were found to be inactive in the tubulin polymerization assay
`However after incubation in rat plasma at 37 °C for 18 hrs some of these
`carbonate derivatives particularly 3112 and 3113 were found to promote
`microtubule assembly indicating the generation of
`the parent compound
`
`paclitaxel
`
`in rat plasma All of the above observations were fully substantiated
`
`

`

`114
`
`by their in vivo antitumor activity in the Madison 109 murine lung carcinoma
`M109 tumor model 41 as summarized in Table 2 In this tumor model both
`the prodrug and the tumor were localized intraperitoneally ip and as such
`represented an in vivo testtube model All carbonates were effective TC >
`125 is considered to be active in increasing life span The methyl carbonate
`3112 was less active than paclitaxel but all
`the other carbonates exhibited
`comparable in vivo antitumor activity with respect
`results indicated that paclitaxel2carbonates
`are converted to the parent drug
`
`These in vivo
`
`to paclitaxel
`
`under the in vivo conditions and act as true prodrugs of paclitaxel
`
`Table 2 In vivo Antitumor Activity of Paclitaxel2Carbonates 311217
`
`Compound
`
`3112
`3113
`3114
`3115
`3116
`3117
`
`In vivo Antitumor Activitya
`TC mgKginjection
`Carbonate Deny
`
`Paclitaxel
`
`162 90 mgKgb
`>475 60c
`247 100b
`275 60c
`310 50c
`>475 60c
`
`276 75 mgKgb
`275 30c
`197 50b
`275 30c
`270 50c
`275 30c
`
`a Madison 109 murine lung carcinoma M109 ipimplant model Drugs administered ip in 10
`in 10 DMSOsaline 311317 or in 10 DMSOH20
`Tween 80 in saline paclitaxel
`80 3112 TC refers to the percentage
`few drops of Tween
`of the median survival
`drug treated mice six per dose vs saline treated control The TC values are determined at
`the maximum tolerated dose shown in brackets TC >125 is defined as active in this tumor
`model b Dose administered ip on days 5 and 8c Dose administered ip on days 1 5 and 9
`
`plus a
`
`time of
`
`One obvious drawback of the above carbonate derivatives is their extreme
`
`release in vivo
`
`insolubility in water As will be discussed later
`a watersoluble prodrug
`strategy for one of these carbonates was successfully developed by attaching
`the C7 hydroxyl functionality
`In a similar vein
`soluble pro moieties at
`have recently reported novel watersoluble C2
`Nicolaou and coworkers
`arylsulfonyl ethylcarbonates 311820 Figure 5 36 These compounds were
`prepared in order to evaluate a novel mechanism of paclitaxel
`in vivo through a base
`
`Accordingly they were conceived
`
`to generate paclitaxel
`
`

`

`induced 13 elimination reaction However these authors have not yet reported
`
`whether these putative prodrugs are actually effective in vivo
`
`115
`
`NH
`
`0
`
`Ph
`
`0
`
`RO
`
`3118 R
`
`3119 R =
`
`3120 R =
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`NO2
`
`NH2
`
`Figure 5 Paclitaxel 2 carbonates prepared by the Scripps group
`
`332 Phosphate Esters Derivatives
`The Bristol Myers Squibb BMS group elected for reasons of novelty to
`focus on the synthesis and evaluation of watersoluble phosphatasecleavable
`prodrugs of paclitaxel 28 This strategy mandated the synthesis and
`evaluation of hitherto unknown phosphate esters of paclitaxel Use of phosphate
`
`as Prodrugs
`
`derivatives at
`
`derivatives as water solubleprodrugs of clinically useful drugs such as etoposide
`42 clindamycin 43 and mustards 44 has been well documented
`The
`rationale behind their use as prodrugs stems from the ubiquitous nature of
`phosphatase enzymes in mammalian systems 45 Interestingly certain tumors
`are shown to express high levels of membrane bound alkaline phosphatases and
`and accumulation of these
`thus provide an opportunity for selective cleavage
`the tumor site 46 In the BMS prodrug program the initial
`targets synthesized were the obvious prototypic C2 and C7 phosphate
`derivatives 321 and 322 Figure 6 47
`the sodium salts of 321 and 322 were endowed with
`Although
`impressive water solubility 10 mgml their in vitro and in vivo evaluation
`indicated that they were poor prodrugs of paclitaxel The first
`indication of this
`was evident when both derivatives failed to generate paclitaxel
`in vitro upon
`treatment with purified preparations of bovine intestinal alkaline phosphatase
`
`

`

`116
`
`BzNH 0
`
`Ph
`
`OR1
`
`Ac0
`
`9 R2
`
`321 R1=P0OH2 R2=H
`322 Ri=H R2=P0OH2
`
`=
`
`HO
`
`Ac0
`0 Bz
`Figure 6 Prototypical paclitaxel phosphates
`
`This was further corroborated by their extreme stability and failure to generate
`in rat plasma Also both were inactive in promoting microtubule
`paclitaxel
`assembly in vitro In vivo evaluation of sodium salts of 321 and 322 against
`the intraperitoneal ip Madison 109 murine M109 lung tumor model
`in
`comparison with paclitaxel demonstrated see Table 3 that
`these analogs
`possessed marginal antitumor activity at best in this model TC 125 is
`
`considered active
`
`Table 3 In vivo Activity of Sodium Salts of 321 and 322 in the M109 Tumor
`Modela
`
`Maximum TCb mgkginj
`
`Experiment
`
`Compound
`
`Phosphate
`
`Paclitaxelc
`
`id
`
`2d
`
`321 Na salt
`322 Na salt
`
`14025
`123 30
`
`27050e
`19010
`
`aMurine lung carcinoma ip implant model bTC refers to the percent of the median survival
`time of drug treated mice 6 per dose us saline treated controls at the maximum tolerated dose
`in brackets cAdministered in 10 Tween 80 in saline dDose administered ip on days 1 5 and
`9 eAlso a dose of 25 mgkginj achieved a TC of 240 with 16 mice cured
`
`The lack of in vitro enzymatic cleavage and inferior in vivo activity of 321
`and 322 indicated that
`these phosphates
`are poor substrates for alkaline
`phosphatase Sterically both the C2 phosphate and the C7 phosphate moieties
`in 321 and 322 are quite close to the congested
`taxane core Consequently it
`enzymes are not able to easily bind these
`is likely that
`the phosphatase
`functionalities and process them effectively
`In order to
`
`to generate paclitaxel
`
`

`

`in prototypes 321 and 322 toward
`address the steric barrier encountered
`phosphatase cleavage the BMS chemists pursued a more complex strategy ie
`the synthesis of proprodrugs of paclitaxel which can be activated by
`phosphatase Figure 7
`
`117
`
`Bz
`
`NH
`
`0cLPh
`
`OH
`
`Bz
`
`Ph
`
`NH 0
`
`oH
`
`spont
`
`00P0OH
`
`phosphatase
`
`0
`
`Ac0
`
`HO
`
`= Ac0
`OBz
`
`323
`
`Ac0
`
`OH
`
`= Ac0
`OBz
`
`HO
`
`111
`
`OH
`
`324
`
`4= self immolating
`
`linker
`
`Figure 7 Proprodrug strategy based on phosphatase
`
`enzymes
`
`Proprodrug strategies have been sucessfully applied in medicinal chemistry
`48 The two most widely employed concepts in the design of proprodrugs have
`been the exploitation of the fragmentation cascade 49 and of the lactonization
`reaction 50 Adaptation of these strategies to paclitaxel entails the design and
`use of appropriate selfimmolating linkers at either C2 or C7 as depicted in
`Figure 7 The concept
`involves dephosphorylation of the proprodrug 323 by
`phosphatases in vivo followed by the rapid collapse through a fragmentation
`cascade reaction
`step of
`transient dephosphorylated
`lactonization
`intermediate 324 to yield paclitaxel 111 The lactone approach
`previously exploited by medicinal chemists in prodrug design since the factors
`
`or
`
`has been
`
`

`

`118
`
`that influence the rates of lactonization reactions are generally well understood
`51
`
`NH2
`
`OMe
`
`327
`
`325
`
`326
`
`Figure 8 A selfimmolative linker approach to drug delivery
`
`A prototypic proprodrug design to demonstrate the proof of principle was
`lock linker recently reported by Borchardt and co
`based on the ltrimethyl
`workers 52 in the design of an esterase cleavable proprodrug of an amine
`Figure 8 It was elegantly demonstrated by this group through in vitro
`experiments with isolated enzyme preparations and plasma studies that 325
`was an effective proprodrug of pmethoxy aniline 327 The success
`of this
`lactonization rate enhancement of the
`approach was ascribed to the phenomenal
`order of >105 achieved by the presence of methyl substituents on the alkyl chain
`and the aromatic ring The estimated half life of lactonization for the desacetyl
`analog of 325 is of the order of 1 min
`Extension of this strategy to phosphate derivatives
`required the synthesis
`of acid linker 328 Figure 9 This was then utilized in the synthesis of the
`desired targets 329 and 3210 53 The sodium salts of 329 and 3210 were
`found to possess acceptable water solubility ca 10 mgml for iv evaluation of
`these prodrugs in murine tumor models Solution stability determinations in pH
`74 Tris buffer 50mM at 37 °C were substantially different Tgo values defined
`as the time required for the compound to undergo 10 degradation were
`determined to be ca 20 h for 329 and >100 h for 3210 Thus 329 may lack
`the stability necessary for intravenous administration without encountering
`precipitation problems during iv infusion An indepth in vitro evaluation of
`these two proprodrugs in biological
`fluids and with pure enzyme was
`undertaken
`the necessary parameters Also kinetic
`the cleavage process using bovine intestinal AP and p
`
`investigations
`
`of
`
`to establish
`
`all
`
`

`

`119
`
`nitrophenol phosphate PNP as a reference standard were performed
`The
`VmaxKm ratio for the prodrugs was determined and compared with that of PNP
`It was found that
`as a measure of enzyme efficiency with these substrates
`3210 had VmaxKm = 06 vs PNP=1 whereas VmaxKm for 329 was only
`013 This may indicate that the enzyme is confronted with lesser steric
`congestion around C7 than C2 Interestingly the selfimmolation process for
`by the AP in both cases
`the linker to generate paclitaxel after dephosphorylation
`was rapid t12<5 min in agreement with literature precedents
`
`Ph26
`
`NH
`
`0
`
`Bz
`
`OH20P9
`
`9
`
`OH
`
`328
`
`Bz
`
`NH
`
`0
`
`1111
`
`6H
`
`329
`
`Figure 9 Phosphate proprodrugs of paclitaxel
`
`The above in vitro observations for the two phosphate derivatives were
`further substantiated by their in vivo antitumor performance When evaluated
`the C2 esterphosphate 329 was found
`against the ipip M109 tumor model
`to be marginally active whereas the C7 esterphosphate 3210 was as active as
`paclitaxel Table 4
`The above results led to the exploration of other