Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`2
`
`ISRN Pharmacology
`
`OH
`
`FIGURE 1 Structure of Paclitaxel 2a4a5p7p10p13 a 410bisacetyloxy13
`oyl oxy 17dihydroxy9oxo520epoxytax11en2y1
`benzoate
`
`2R3S3 benzoylamino2hydroxy3phenylpropan
`
`10
`
`6
`
`5
`
`8
`
`3
`
`1 21
`
`FIGURE 2 Taxane ring
`
`clinical problems related to paclitaxels solubility
`toxicity
`and development of drug resistance have led to innovative
`of alternative dosage
`research leading to the development
`forms that can overcome the above mentioned problems
`to discuss the challenges and various
`intends
`This paper
`related aspects of the formulation of paclitaxel
`
`2 The Paclitaxel Molecule and Its
`Delivery Challenges
`
`from its natural source Taxus brevi
`Extraction of paclitaxel
`folia causes the death of several plants to yield a few grams
`of the drug which is not a practical option A completely
`synthetic method of production of paclitaxel has not yet been
`developed due to the challenges presented by its structural
`complexity 1012 Current industrial production of pacli
`is done by plant cell fermentation technology whereby
`line and
`is extracted from a cultured
`Taxus cell
`
`taxel
`
`paclitaxel
`purified by chromatography
`is a diterpenoid centered around a bulky com
`and fused taxane ring Figure 2 composed
`of a
`plex
`number of hydrophobic substituents which makes it a highly
`lipophilic compound with a log P value of approximately 4
`and aqueous solubility of less than 001 mgmL 1315
`The compound lacks potentially ionizable functional groups
`which could lead to an increase in its solubility with alter
`ation in pH Among nonaqueous solvents the solubility is
`found to be 46 mM in ethanol 20 mM in methylene
`chloride or acetonitrile and 14 mM in isopropanol
`tertiarybutanol and dimethyl sul
`also soluble in methanol
`foxide 4 16
`The common approaches to increase aqueous solubility
`such as formation of salts and addition of charged com
`feasible options for paclitaxel 17
`pledng agents are not
`Synthesis of prodrugs to improve the aqueous solubility
`
`Paclitaxel
`
`It
`
`is
`
`has been attempted The 2 position on the
`of paclitaxel
`structure of paclitaxel may be the ideal position for the inser
`tion of functional groups to create prodrugs of paclitaxel
`because several derivatives of 2 acylpaclitaxel
`can quickly
`in the blood 18 A C7
`hydrolyze back into paclitaxel
`prodrug ester of paclitaxel has also been prepared because the
`arrangement of the C7 hydroxyl group does not appear to
`in influencing cytotoxicity 19 A paclitaxel ester
`be a factor
`in the presence of a strong electron withdrawing substituent
`the ester was
`like an alkoxy group in the a position of
`synthesized by Nicolaou et al to facilitate the quickening
`of the hydrolytic cleavage 20 These prodrugs have shown
`cytotoxic activity comparable to paclitaxel against cancer cell
`formulated using PEG
`lines in vitro Prodrugs of paclitaxel
`has been a promising approach because PEG imparts supe
`rior aqueous solubility 21 22 Nevertheless
`the pursuit of
`designing prodrugs of paclitaxel remains primarily academic
`focused on the design of water
`as industrial efforts have
`soluble derivatives or structural analogues of paclitaxel
`
`3 Commercially Available Dosage
`Forms of Paclitaxel
`
`The formulation of paclitaxel most widely used in the clinical
`setting is the solubilized form of
`the drug brand and
`generic which is diluted before intravenous administration
`castor oil Cre
`The formulation contains polyoxyethylated
`mophor EL in a 1 1 vv mixture with dehydrated ethanol a
`powerful solubilizer combo It
`remains stable in unopened
`vials for 5 years at 4°C 4 Polyoxyethylated
`castor oil
`
`is
`
`also used in the formulation of various other hydrophobic
`agents such as teniposide echinomycin and di
`anticancer
`demnin B 23 However
`is associated with side
`this excipient
`effects such as bronchospasms hypotension and symptoms
`of hypersensitivity 24 25 The constituents of polyoxyethy
`and cause
`induce histamine release
`lated castor oil can
`and hypersensitivity reactions following rapid
`hypotension
`administration of the infusion or as a result of an IV bolus
`administration 26 27 Therefore paclitaxel
`formulations
`must be slowly infused over a period of several hours to
`minimize the intensity and frequency of side effects The
`hypersensitivity reactions were evident at almost every phase
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`3
`
`testing there
`in development in both preclinical and clinical
`is recommended
`that patients be premedicated with
`fore it
`and antihistamines prior to drug infusion to
`corticosteroids
`prevent or minimize the anticipated reactions 26 28 29
`The commercially available paclitaxel solution formulation
`is diluted 520 fold in normal saline or 5 dextrose solution
`
`for administration as an intravenous
`
`infusion therefore
`the formulation after dilution is a concern
`stability of
`of 03
`The diluted formulation results in a concentration
`12 mgmL of drug which is considerably higher than the
`001 mgmL aqueous solubility of paclitaxel posing the risk
`of drug precipitation upon dilution 4 25
`
`4 Nanopartides and Nanosuspensions
`
`tional
`
`To decrease
`associated with the conven
`the toxic effects
`formulation described above and minimize the risk
`of precipitation of paclitaxel upon dilution a nanoparticle
`formulation for paclitaxel was introduced in 2005 under
`the
`for particle forma
`trade name Abraxane The technology
`tion involves a proprietary process that binds unmodified
`albumin to the paclitaxel molecule yielding conjugate masses
`of 130 nm size Following infusion these nanoparticles
`rapidly dissociate to yield an albumin bound drug complex
`Albumin paclitaxel molecules bind to an albumin receptor
`gp60 on endothelial cells which transports paclitaxel
`into
`via the formation caveolae 30
`the extravascular
`space
`An alternate transport pathway
`is considered to be via
`binding of
`the nanoparticles with secreted protein acidic
`rich in cysteine SPARC Since SPARC is overexpressed in
`many solid tumors including bladder and prostate cancers
`leads to a 33 increase in intratumoral
`the nabpaclitaxel
`and a 50 higher dose of paclitaxel delivered
`concentrations
`infusion Moreover
`compared with a conventional
`paclitaxel
`the nab paclitaxel being solvent
`free the infusion time is
`much shorter compared to paclitaxel with Cremophor EL
`31 Ibrahim et al reported on the pharmacokinetics
`of
`Abraxane known as ABI007 during development 32
`Among alternative
`experimental delivery systems of
`paclitaxel nanoparticles of various biodegradable polymers
`and bioadhesive materials have been considered as promis
`can help to improve the bioavailability
`ing Nanotechnology
`of poorly soluble drugs such as paclitaxel enhancing drug
`delivery 33 Nanoparticles
`also have the ability to permeate
`through certain tissues adding to their drug targeting poten
`the drug to the targeted tissue efficiently
`tial They deliver
`without clogging the capillaries while protecting the drugs
`bioactivity and stability 14 3436 Upon incorporation
`of paclitaxel
`into nanoparticles there is a demonstrable
`enhancement
`due to changes
`in drug action
`in tissue
`15 37 Additionally it
`distribution and pharmacokinetics
`has been shown that nanoparticles
`can avoid rapid clearance
`system and can preferentially
`by the reticuloendothelial
`accumulate in solid tumors by escaping the angiogenic vas
`culature permeating the neoplasm 3840 Studies indicate
`carriers result in an
`that drugs delivered in nanoparticle
`extended retention of the drug in the tumors diminution
`in tumor growth and prolonged survival of the test subject
`34 41 42 The multidrug resistance phenotype mediated
`
`100
`
`v
`
`75
`
`T
`
`Al
`
`5
`
`25
`
`0
`
`24
`
`72
`
`120
`
`168
`
`216
`
`Time hours
`
`from PLGA
`FIGURE 3 Cumulative in vitro release of paclitaxel
`Nps Resomer RG 502 PLGANps containing paclitaxel 1 ww
`
`at 37°C and shaken horizontally
`were diluted in PBS incubated
`At preselected time intervals the released drug was separated by
`ultracentrifugation and the residual amount of paclitaxel
`in the nanospheres was determined by HPLC reproduced with
`permission from Fonseca et al 33
`
`present
`
`at
`
`by pglycoprotein in the tumor cells can also be overcome
`drug delivery This is important because
`by nanoparticle
`acquired resistance to paclitaxel has been reported 4345
`An additional advantage
`is their enhanced
`of nanoparticles
`stability both during storage and in biological fluids 46
`Fonseca et al have prepared nanoparticles of paclitaxel by
`using the interfacial deposition method in which they added
`an organic solution of PLGA and paclitaxel
`in acetone
`to
`an aqueous poloxamer 188 solution under magnetic stirring
`room temperature followed by washing
`and harvest
`The in vitro
`ing of nanoparticles
`by ultracentrifugation
`studies were conducted by measuring the residual
`release
`amount of paclitaxel at specific time points after the PLGA
`containing paclitaxel were diluted in PBS and
`nanoparticles
`at 37°C in a horizontal shaker The particles
`incubated
`exhibited a biphasic pattern for paclitaxel
`release during the first
`by an early fast
`release Figure 3 Two different
`by a slower and constant
`copolymers were studied and the in vitro antitumoral
`activity of the drug loaded nanoparticles
`was determined
`by the colorimetric MTT assay after
`incubating
`with paclitaxelloaded nanoparticles
`for various time periods
`Figure 4 33
`Mu and Feng investigated
`the use of doctocopheryl
`succinate vitamin E TPGS or
`1000
`polyethylene
`glycol
`TPGS as a novel emulsifier and matrix material for the
`preparation of nanoparticles 34 The nanoparticles
`loaded
`with paclitaxel were prepared by a modified oil in water
`single emulsion solvent evaporationextraction technique
`The size of the particles ranged from 3001000 nm In vitro
`release from the nanoparticles was studied by suspending the
`in PBS and shaking the tubes hor
`drug loaded nanoparticles
`izontally At specific time points the tubes were centrifuged
`and the supernatant was collected for analysis using HPLC
`nanoparticles were resuspended in the buffer
`Precipitated
`and shaken When different
`types of PLGA were used as
`polymers with 003 wt TPGS as emulsifier an initial burst
`
`release illustrated
`24 hrs followed
`
`the cells
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`being suitable for parenteral administration The suspension
`form permits targeting of paclitaxel
`to organs like
`dosage
`liver spleen lungs and lymphatic circulation to a certain
`extent by the use of particles of varying size and through
`administration through various routes 4 49 MeriskoLiv
`ersidge et al evaluated the process of wet milling in the for
`mulation of a nanocrystalline
`suspension of poorly soluble
`anticancer drugs including paclitaxel 24 The nanoparticle
`formulation containing 2 wv drug suspension and 1
`wv surfactant stabilizer Pluronic F127 for paclitaxel was
`wet milled on a low energy mill until
`size of
`a final
`less than 400 nm was achieved
`as evaluated by photon
`correlation spectroscopy These suspensions were suitable for
`intravenous bolus injection without any incidence of acute
`toxicity in vivo and the tumor
`regression expressed as the
`percentage of tumor weight was significant 24
`
`5 Liposome Formulations of Paditaxel
`
`from
`
`Liposomes
`lipoid vesicles
`are microscopic
`ranging
`250 A to >20 ium in diameter and offer
`a flexible platform
`to encapsulate both lipophilic and hydrophilic drugs The
`lipophilic drugs incorporate in the lipid bilayer while the
`hydrophilic drugs locate in the vesicle cavity Encapsulation
`in their pharma
`of drugs in liposomes causes a change
`cokinetic and pharmacodynamic
`properties resulting in a
`in toxicity and increase in the potency 4 50
`decrease
`Liposomes
`can be used for specific targeting of cytotoxic
`tissues but they can be rapidly cleared
`compounds
`to select
`by the mononuclear phagocytic system in the liver and spleen
`unless special modifications are made to the phospholipid
`surface 51 The major problems associated with liposomes
`are their limited longterm stability and difficulty in large
`scale manufacturing of sterile liposomes
`Crosasso et al have prepared and characterized sterically
`stabilized paclitaxelcontaining liposomes They compared
`conventional
`paclitaxelloaded liposomes with stealth or
`sterically stabilized
`long circulating paclitaxelcontaining
`these
`in the
`liposomes because
`can
`circulate
`liposomes
`periods The PEGylated liposomes
`blood for prolonged
`using paclitaxel with phospholipids and
`were prepared
`in the molar ratio of 1 30 mol drug mol lipid
`cholesterol
`The formulation containing
`egg yolk phosphatidylcholine
`PC and phosphatidylglycerol PG transesterified from egg
`PC in the ratio 9 1 and paclitaxel
`and lipids in the
`molar ratio of 1 30 was
`found to have
`an incorporation
`efficiency of 95 This formulation retained 90 of the drug
`content over a study period of 2 months in the hydrated
`at 4°C during which the liposomes were physically
`state
`stable Figure 6 51 Incorporation of more than 20
`the incorporation efficiency and
`of cholesterol
`decreased
`physical stability of the formulations The liver and spleen
`and PEGylated liposomes
`distributions of the conventional
`after extraction
`of
`paclitaxel were evaluated
`from the tissue using t butyl methyl ether Figures
`7a and 7b 51 Drug uptake
`and spleen
`in liver
`after 05 and 3 hrs
`was
`lower with PEGylated liposomes
`51
`
`containing
`
`paclitaxel
`
`24
`
`72
`
`120
`
`168
`
`0 25 µgmL
`0 25 tigmL
`025 tigmL
`
`Incubation time h
`x 0025 tigmL
`0 Drug free Nps
`
`a RG 502
`
`120
`
`80
`
`40
`
`0 A
`
`72
`
`4
`
`120
`
`80
`
`40
`
`77
`
`24
`
`72
`
`120
`
`168
`
`0 25 µgmL
`0 25 tigmL
`A6 025 ligmL
`
`Incubation time h
`x 0025 tigmL
`0 Drug free Nps
`
`b RG 755
`
`FIGURE 4 Viability of NCI H69 cells incubated with drug free Nps
`or PtxPLGANps prepared with the RG 502 copolymer a or with
`RG 755 b Cells were seeded into 96 well plates and incubated
`
`of both formulations for 24 72 120
`with different concentrations
`or 168 has described reproduced with permission from Fonseca et
`al 33
`
`release pattern was common to all
`formulations following
`which the release rate dropped to generate nearly constant
`release of drug up to one month Figure 5a 34 When
`TPGS was blended with PLGA in the nanoparticle matrix it
`became more hydrophilic and resulted in an increase in drug
`release rate with an increasing ratio of TPGS Figure 5b
`34
`Sharma et al reported the preparation of paclitaxel con
`crosslinked with
`
`taining polyvinylpyrrolidone nanoparticles
`
`effect
`
`N N methylene bisacrylamide MBA in inverse micro emul
`sion and determined their antitumor
`in murine
`melanoma The in vivo efficacy of these nanoparticles when
`measured using reduction in tumor volume and increased
`time as parameters was significantly greater com
`survival
`of free taxol 38 Si
`concentration
`pared to an equivalent
`Shen and Guofeng also prepared nanoparticles of paclitaxel
`using freeze drying and solvent extractionevaporation tech
`niques Their data indicated that short and saturated chains
`of phospholipids such as dipalmitoylphosphatidylcholine
`DPPC have good emulsifying effect 47
`Muller and Petes evaluated the process of highpressure
`homogenization for the formulation of nanosuspensions of
`poorly soluble drugs 48 Nanosuspension
`formulations of
`insoluble drugs such as paclitaxel offer
`
`the advantage
`
`of
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`5
`
`50
`
`40
`
`30
`
`20
`
`10
`
`A E5X E7
`
`ID Es
`
`10
`
`20
`
`30
`
`40
`
`0
`
`10
`
`20
`
`30
`
`40
`
`Released time days
`
`Released time days
`
`a
`
`M1
`
`M2A M3
`
`b
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`Qc`
`
`V
`
`r24
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Releasedpaclitaxel
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Releasedpaclitaxel
`
`10
`
`20
`
`30
`
`40
`
`10
`
`20
`
`30
`
`40
`
`Released time days
`
`E7
`
`E13
`
`e E14
`
`c
`
`9 E8
`
`EIS
`
`E16
`
`Released time days
`
`d
`
`prepared under various experiment parameters a E5 PLA E7 PLGA
`FIGURE 5 In vitro release curves of paclitaxel
`loaded nanoparticles
`75 25 E8 PLGA 50 50 b ratio for PLGATPGS M1 2 1 M2 1 1 M3 1 2 c PLGA 75 25 concentrationE70125
`E13 0188 E14 025 d PLGA 50 50 concentrationE8 0125 E15 0188 E16 025 reproduced with permission from Mu and Feng
`34
`
`liposome for
`Ceruti et al also compared conventional
`mulations of paclitaxel with stealth long circulating lipo
`somes The PEGylated liposomes were long circulating with
`an elimination halflife of nearly 50 hrs compared to less
`liposomes 52 The group
`than 10 hrs with conventional
`attempted to improve the solubilization and targeting of
`paclitaxel using watersoluble prodrugs and incorporated
`2 succinylpaclitaxel
`2 methylpyridinium acetate pacli
`taxel and 2mPEGpaclitaxel in liposomes The 2 succinyl
`liposomes had superior stability at 4°C at both pH
`58 or 74 for up to 6 days but at 37°C the percentage of
`released increased from 4 to 12 Figures 8a
`and 8b 52 The halflife of this liposomal prodrug was
`than the free drug liposome formulations and the
`cytotoxic activity was similar to that of the free drug 52
`Sharma and Straubinger prepared liposome formulations of
`
`longer
`
`paclitaxel
`
`paclitaxel
`
`paclitaxel using phospholipids in the molar ratio of 1 33
`with phosphatidylcholine PC and phosphatidylglycerol
`PG transestrified from egg PC in the ratio 9 1 The
`liposomes were stable for more than 2 months at 4°C
`and 1 month at 20°C The stability of these liposomes as
`a function of PG and storage temperature
`is shown
`Figure 9 53 The results are expressed as percentage of
`taxol concentration
`remaining in the liposomes at
`initial
`different time points The formulations were well tolerated
`in mice when given via intravenous IV and intraperitoneal
`IP bolus doses The maximum tolerated dose was found
`to be >200 mgkg for liposomal paclitaxel whereas it
`is only
`30 mgkg by IV and 50 mgkg by IP route for free paclitaxel
`53
`Sampedro et al prepared liposomes using mixtures of
`phospholipids such as Ldimyristoyl phosphatidylcholine
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`05
`
`3
`
`24
`
`Time after injection hours
`a
`
`25
`
`20
`
`0
`
`t4 15
`
`ac
`
`10
`
`40
`
`35
`
`30
`
`o 25
`00
`
`w
`
`20
`
`15
`
`10
`
`5
`
`05
`
`3
`
`24
`
`Time after injection hours
`b
`FIGURE 7 a Biodistribution in Balbc mice in plasma white
`liver black and spleen grey 05 3 and 24 hrs after
`injection of
`liposomes Standard deviations were below
`conventional
`5 of the mean values reproduced with permission from Crosasso
`et al 51 b Biodistribution in Balbc mice in plasma white
`liver black and spleen grey 05 3 and 24 hrs after injection of
`liposomes Standard deviations were below 5
`PEGylated paclitaxel
`of the mean values reproduced with permission from Crosasso et
`al 51
`
`paclitaxel
`
`paclitaxel
`
`phase 58 Paclitaxel
`has
`limited
`solubility in the oil
`solubility in soybean and similar vegetable oils therefore it
`prevents the use of simple 0W emulsion for formulation
`57
`Tarr and Yalkowsky prepared an 0W emulsion of pacli
`taxel using triacetin as the internal phase It was found that
`is highly soluble in triacetin 75 mgmL Regard
`less the drug precipitates when the emulsion is diluted
`approximately nine times with 5 dextrose 59 Tarr et al
`prepared the first parenteral emulsion of paclitaxel contain
`in a vehicle composed of 50
`ing 1015 mgmL paclitaxel
`triacetin 15 soylecithin 15 pluronic F68 and 20
`ethyl oleate About 10 of glycerol was added to prevent
`creaming 60 Although this system was stable for a period
`of 6 months when stored at 4°C the amount of vehicle
`triacetin needed for delivering the therapeutic
`containing
`dose of paclitaxel was found to be toxic when administered
`activity was
`intravenously to mice and no antitumor
`reported 60
`
`120
`
`100
`
`80
`
`60
`
`40
`
`initial
`
`of
`
`Encapsulateddrug
`
`20 H
`
`6
`
`10
`
`15
`
`
`
`2020
`
`25
`
`Time days
`
`FIGURE 6 Release of paclitaxel
`from conventional
`and
`squares
`triangles liposomes in storage conditions at 4°C
`PEGylated
`solid symbols or in human plasma at 37°C outline symbols
`reproduced with permission from Crosasso et al 51
`
`glycerol DMPG
`DMPC and Ldimyristoylphosphatidyl
`with or without
`cholesterol by the standard evapora
`tionhydration method Combination of the two phospho
`lipids in the ratio of 73 and 9 1 with the addition of 5
`cholesterol ww conferred optimal results The cytotoxicity
`of these liposome formulations in L1210 cells was found
`than that of free paclitaxel 54
`to be significantly greater
`in both
`Bartoli et al compared
`the efficacy of paclitaxel
`and liposome formulations They tested solu
`nanoparticle
`in DMSO and Cremophor EL against
`tions of paclitaxel
`liposomal formulations both in vitro and in vivo in P388 and
`L1210 leukemia models and concluded that
`the liposome
`formulation of paclitaxel was superior both in vitro and in
`formulation was found to be
`vivo whereas the nanoparticle
`toxic mainly because of its composition 55 Straubinger
`and
`a series of
`et al developed
`liposome formulations
`the in vitro tests revealed that
`retained
`the liposomes
`growth inhibitory activity while showing a delay of tumor
`progression against colon 26 a taxolresistant murine tumor
`56
`
`6 Emulsion Systems
`
`aqueous phase
`
`An emulsion is a heterogenous
`system comprising of an
`dispersed in an oil phase or vice
`versa
`stabilized by an emulsifier 0W emulsion formulations for
`the delivery of anticancer
`drugs have generated interest
`that many approved emulsifiers
`but are limited by the fact
`hemolytic reactions 57 The drug should
`be
`produce
`incorporated completely into the dispersed phase to form
`a stable emulsion therefore the formulation of a drug as
`0W emulsion is possible only when the drug has adequate
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`7
`
`100
`
`80 1
`
`60
`
`40
`
`20
`
`0
`
`14
`
`12
`
`Parlitaxelreleased
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`160
`
`0
`
`5
`
`I
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Time hours
`a
`
`Time hours
`b
`
`FIGURE 8 a Paclitaxel
`triangles pointing up and 2MPApaclitaxel
`release at 4°C from 2PEGpaclitaxel circles 2succinylpaclitaxel
`triangles pointing up and 2MPA
`triangles pointing down in PBS buffer pH 58 2PEGpaclitaxel squares 2succinylpaclitaxel
`triangles pointing down in PBS buffer pH 74 reproduced with permission from Ceruti et al 52 b Paclitaxel
`paclitaxel
`triangles pointing down and 2MPApaclitaxel hexagons
`in PBS buffer
`37°C from 2PEGpaclitaxel circles 2succinylpaclitaxel
`pH 74 2PEGpaclitaxel squares 2succinylpaclitaxel
`triangles pointing up and 2MPApaclitaxel diamonds in PBS buffer pH 58
`reproduced with permission from Ceruti et al 52
`
`release at
`
`t
`
`110
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`a n
`
`30
`
`20
`
`10
`
`110
`
`100
`
`90
`
`80
`
`70
`
`initial
`
`of
`
`60
`
`°o
`
`50
`
`Taw
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Time days
`Time days
`b
`a
`FIGURE 9 In these formulations the PG PC ratio was varied open squares PC only filled squares PG PC 19 open circles PG PC
`3 7 filled circles PG PC 5 5 open triangles PG PC 73 filled triangles PG onlyreproduced with permission from Sharma and
`Straubinger 53
`
`Kan et al prepared 0W emulsions in which triacylglyc
`erols were used to solubilize paclitaxel
`incorporated with
`various emulsifiers 61 The antitumor activity was tested
`both in vitro and in vivo The solubility of paclitaxel
`oils such as tributylin tricaproin tricaprylin soybean oil
`corn oil cottonseed oil and mineral oil was determined
`
`in
`
`Table 1 61 Paclitaxel had poor solubility in Tricaprylin
`C80 with 8 carbons per hydrocarbon
`chain compared
`to the short chain triacylglycerols such as tributyrin C40
`and tricaproin C60 Triacetin C20 resulted in a high
`solubility of paclitaxel 75 mgmL while mineral oil
`led to
`structure The
`poor solubility because of its hydrocarbon
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`8
`
`ISRN Pharmacology
`
`TABLE 1 Solubility of paclitaxel
`permission from Kan et al 61
`
`in various oils reproduced with
`
`Oil
`
`Tributyrin
`
`Tricaproin
`
`Tricaprylin
`
`Corn oil
`
`Soybean oil
`
`Cotton seed oil
`
`Mineral oil
`
`Solubility of paclitaxel mgg
`962
`
`903
`
`119
`
`023
`
`018
`
`014
`
`Not detected
`
`1H
`
`08 H
`
`06
`
`04
`
`E
`
`cn
`
`02 H
`
`OH
`
`0
`
`30
`
`40
`
`50
`
`60
`
`Time day
`
`FIGURE
`
`11 Survival
`
`ratio of ascitic
`
`tumor bearing mice
`represents the group treated with
`control
`represents untreated
`60 mgkg paclitaxel emulsion and
`represents the group treated
`with same quantity of paclitaxel
`free emulsion vehicle reproduced
`with permission from Kan et al 61
`
`El
`
`1
`
`4111111111
`
`1
`
`08
`
`06
`
`E
`
`04 H
`
`02 H
`
`OH
`
`01
`
`10
`
`100
`
`1000
`
`FIGURE
`
`Paciltaxel nM
`rate of HeLa S3 tumor cells exposing
`10 Survival
`emulsions and Diluent 12 with or without paclitaxel
`the formulated emulsion with paclitaxel
`represents Diluent 12
`in 1 mL 50 ethanol
`and 50 Cre
`containing 6 mg paclitaxel
`mophor EL respectively 0 represents emulsion without paclitaxel
`and El
`reproduced with
`represents Diluent 12 without paclitaxel
`permission from Kan et al 61
`
`to
`
`represents
`
`emulsions were prepared by dissolving egg phosphatidyl
`choline EPC Tween 80 and paclitaxel
`in the oil phase
`and mixing it with the aqueous phase containing 225 wv
`and extrusion through a
`of glycerol followed by sonication
`022 µm filter The concentration of paclitaxel
`in emulsion for
`50 inhibition IC50 of HeLa cells is approximately 30 nM
`in Diluent 12 6 mg of paclitaxel
`which is similar to paclitaxel
`in 1 mL of 50 50 ethanol Cremophor EL The survival of
`HeLa S3 cells exposed to the test emulsions and Diluent
`is shown in Figure 10 61
`12 with or without paclitaxel
`Ascitictumorbearing mice were injected with the paclitaxel
`emulsion 60 mgkg and the vehicle
`the vehicle did not
`increase the life span of the tumor bearing mice but
`the
`span significantly Figure 11
`emulsion increased the life
`61 Even though the IC50 values are similar the paclitaxel
`lifespan than the Diluent 12
`emulsion resulted in a longer
`formulation when given in equivalent
`doses of
`30 mgkg Figure 12 61
`
`paclitaxel
`
`0
`
`
`
`1010
`
`20
`
`30
`
`40
`
`50
`
`60
`
`Time day
`
`FIGURE 12 Comparison oftreatment of Diluent 12 and emulsion on
`represents untreated control A
`the ascitic tumor bearing mice El
`represents group treated with 30 mgkg emulsion and
`represents
`group treated with Diluent 12 reproduced with permission from
`Kan et al 611
`
`Constantinides
`
`et al developed an injectable emulsion
`formulation containing paclitaxel 810 mgmL using Vita
`min E as the oil phase 25 The mean droplet diameter
`and 99 cumulative particle size distribution was less than
`02 µm therefore the emulsion could be filter sterilized A
`in PEG
`preemulsion was prepared by adding octocopherol
`the surfactants TGPS and Pluronic F127
`400 containing
`with or without paclitaxel
`to degassed water for injection It
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`ISRN Pharmacology
`
`9
`
`20
`
`18
`
`16
`
`60 14
`
`2 12
`
`10
`
`8
`
`8
`
`A 6
`
`4
`
`5
`
`10
`
`15
`
`t h
`
`120
`
`100
`
`e
`
`80
`
`60
`
`40
`
`v
`
`20
`
`CL7
`
`0
`
`0
`
`f5
`
`10
`
`100
`
`1000
`
`10000
`
`Concentration nM
`
`48 v 72
`FIGURE 14 Inhibition of HeLa cell proliferation by paclitaxel oleate
`in lipid emulsions Incubation times were 24
`and 96 h The effect of paclitaxel
`in Cremophor ELethanol at
`48 h A is shown as comparison Control growth = 100 Values are
`mean ± SD n = 4 reproduced with permission from Lundberg et
`al 64
`
`350
`
`14 300
`
`250
`
`t
`
`200
`
`2
`
`150
`
`100
`
`2
`
`50
`
`10
`
`20
`
`30
`
`40
`
`50
`
`Time hours
`
`FIGURE 15 Drug concentration
`versus time curves of 3Hpaclitaxel
`in Cremophor EL ethanol 1 1
`vv v and 3Hpaclitaxel
`in lipid emulsion
`3Hpaclitaxel
`oleate in lipid emulsion
`single iv bolus Values are mean ± SD n = 3 P < 005 versus
`from Lundberg et al 64
`
`paclitaxel
`
`in Cremophor ELethanol
`
`reproduced with permission
`
`following
`
`reconstituted with sterile water 63 In
`be successfully
`research the group incorporated a paclitaxel
`subsequent
`derivative which consisted of a paclitaxel oleate in a nanosize
`lipid drug carrier emulsion and evaluated its pharmacoki
`netic profile 64 The dose
`response curve in Figure 14
`shows that paclitaxel oleate is cytotoxic and the cytotoxic
`activity increases with an increase in incubation time 64
`The comparative pharmacokinetic
`profiles of paclitaxel
`lipid
`formulation and paclitaxel
`emulsion Cremophorethanol
`in an oleate emulsion are shown in Figure 15 64 It was
`of paclitaxel
`the plasma concentration
`suggested
`oleate emulsion was increased as a result of interaction with
`
`in the
`
`that
`
`lipoproteins
`
`FIGURE 13 Mean plasma concentration
`istration of paclitaxel
`injection
`
`0 in rats n = 5 reproduced with permission from He et al
`
`of paclitaxel after IV admin
`and paclitaxel microemulsion
`
`9 w
`
`as mixed vigorously at 45°C followed by homogenization
`and sterile filtration through a 02 pm Posidyne filter Drug
`from this emulsion was slower compared to Taxol
`both in the presence or absence of human serum albumin
`tolerated and more
`The tocopherol
`emulsion was better
`in the melanoma tumor model mice
`efficacious than Taxol
`
`release
`
`25
`He et al prepared and evaluated a microemulsion formu
`lation of paclitaxel with lower levels of solubilizers anticipat
`ing less hypersensitivity reactions 9 The hypersensitivity
`
`tests were done on guinea pigs While the Taxol vehicle led
`to frequent nose scratch tremble sneeze erect hair twitch
`or dyspnea with a grade of hypersensitivity equal to 3 the
`microemulsion vehicle resulted in erect hair and less trem
`bling with a grade of 1 The plasma concentration
`profile
`after a single dose of paclitaxel microemulsion injection is
`the microe
`that
`mulsion was able to increase the circulation time of pacli
`in rats while maintaining a relatively higher plasma
`concentration 9
`Simamora et al prepared two parenteral emulsions using
`benzyl benzoate and tributyrin as oil phase and Tween
`80
`as emulsifiers with 5 dextrose
`and sorbitan monolaureate
`phase These formulations had minimal
`side effects with no sign of phlebitis in rabbits 62
`Lundberg
`a lipid emulsion with triolein as oil
`prepared
`and dipalmitylphosphatidylcholine
`as the primary
`emulsifying agent 63 Tween
`80 and PEGdipalmitoyl
`phosphatidylethanolamine were used as stabilizing agents
`The emulsion resulted in prolonged
`circulation time for
`paclitaxel while the in vitro cytotoxicity experiments indi
`its IC50 value was approximately 35 nM which
`cated that
`to Taxol Lyophilization of
`with 5 dextrose resulted in a stable product which could
`
`given in Figure 13 9 The data suggests
`
`taxel
`
`as the continuous
`
`phase
`
`is comparable
`
`this emulsion
`
`Abraxis EX2054
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`
`

`

`10
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`4
`
`8
`
`12
`Time hours
`
`16
`
`20
`
`24
`
`FIGURE 16 Time courses of paclitaxel
`levels in tumor of murine B16
`melanoma induced mice after iv administration of 50 mgkg dose
`
`of GenexolPM + and 20 mgkg dose of Taxol v Each point
`represents the mean ± SD of four mice per time point reproduced
`with permission from Kim et al 3
`
`7 Micelles
`
`The process of entrapping a hydrophobic drug in a bio
`degradable drug carrier micelle without covalent bonding
`to the micelle is called micellisation A polymeric micelle
`consists of an aggregate of hydrophobic block copolymers
`with a diameter of 2050 nm with a densely packed core in
`which the hydrophobic drugs are incorporated by hydropho
`bic interactions Among the block copolymers the diblock
`copolymers are suitable for the formation of micelles because
`
`biodegradable
`
`of their simple structure 3 Micelles are convenient
`for use
`with anticancer drugs because they are structurally strong
`and avoid being captured by the MPS due to their particle
`size 65 66
`Kim et al prepared paclitaxel containing biodegradable
`polymeric micelles using a low molecular weight nontoxic
`called mon
`amphiphilic diblock copolymer
`omethoxy polyethylene glycol block poly D Llactide
`mPEGPDLLA and evaluated their pharmacokinetics The
`polymeric micelles were prepared by the solid dispersion
`technique The plasma concentration
`in the
`of paclitaxel
`polymeric micelles following IV administration in a murine
`is shown in Figure 16 3 The
`B16 melanoma mice model
`in vivo antitumor efficacy was evaluated in human ovarian
`tumor and human breast tumor xenografted nude mice In
`received the polymeric micelles a three fold
`the mice that
`response was apparent compared to the
`higher antitumor
`
`Taxol formulation 3
`
`formulations of
`et al also prepared micellar
`Zhang
`in the diblock copoly
`paclitaxel by incorporating paclitaxel
`mer poly dllactideblockmethoxy
`polyethylene
`glycol
`PDLLAMePEG The survival
`time of the animals treated
`
`ISRN Pharmacology
`
`with paclitaxel micelles was increased and the lipoprotein
`distribution and plasma concentration were similar to that
`a five fold increase in the maximum
`of Taxol There was
`tolerated dose when the paclitaxel micelles were administered
`intraparenterally in P388 leukemia tumor bearing mice The
`formulations in vitro