ELSEVER
`
`Journal of Controlled Release
`
`75 2001 307
`
`315
`
`journal of
`controlled
`release
`
`www.elsevier.com/locate/jconrel
`
`Preparation characterization and in vivo evaluation of 120-day
`
`polyDL-lactide leuprolide microspheres
`
`Byung Wo Janusz
`
`Kostanski Sisay Gebrekidanc Bhas
`B.C Thanoo
`DeLuca
`Patrick
`800 Rose Street Lexington KY 40536 USA
`Faculty of Phormoceutical Sciences College of Phormocy University of Kentucky
`Institute One Squibb Drive P0 Box 191 New Brunswick NJ 08903 USA
`Reseorch
`bBristolMyers Squibb Phormoceuticol
`Esterbrook Lone Cherry Hill NJ 08002 USA
`Wyeth-Ayerst Loborotories
`d0oood Loborotories 7670 First Ploce
`Ookwood OH 44146 USA
`Suite
`
`Dania
`
`Received
`
`12 February 2001 accepted
`
`21 May 2001
`
`Abstract
`
`luteinizing hormone-releasing hormone LHRH
`120-day polyDL-lactide PLA microsphere delivery system for
`analogue leuprolide was prepared and evaluated Leuprolide microspheres were prepared with PLA mw 11 000 Da by
`characterized for drug load by HPLC particle
`extraction-evaporation method and
`dispersion/solvent
`size by laser
`and surface morphology by scanning electron microscopy
`release and polymer degradation
`In vitro peptide
`diffractometry
`modified dialysis method Serum peptide and testosterone
`were studied using
`levels were analyzed after subcutaneous
`rat model Spherical microspheres with
`mean diameter of 52 pm containing 13.4% peptide released
`administration using
`10% of the peptide within 24
`linear release for 150 days Serum leuprolide levels increased
`followed by
`immediately
`after administration of the microspheres to 45.6 ng/ml
`at 15 days and
`at 30 days
`to 4.3 ng/ml
`2.0 ng/ml
`where they remained for 120 days The testosterone
`to 15 ng/ml and then decreased to below 0.5
`increased initially
`where they remained for 120 days In conclusion
`ng/ml by day
`120-day microsphere formulation of leuprolide was
`2001 Elsevier Science BY All
`developed with excellent controlled peptide release characteristics and in vivo efficacy
`rights reserved
`
`but
`
`then fell
`
`levels
`
`Keywords Leuprolide Luteinizing
`
`hormone-releasing hormone LHRH analogue PolyoL-lactide Microsphercs
`
`Testosterone suppres
`
`sion
`
`Introduction
`
`Controlled release dosage forms have been investi
`
`Conesponding author Tel
`323-0242
`
`1-859-257-1831
`
`fax
`
`1-859-
`
`moil oddress ppde1u1pop.uky.edu
`
`PP DeLuca
`
`gated to improve the efficacy of peptide drugs and
`frequent administration Bio
`eliminate the need for
`degradable microsphcres were shown to improve the
`hioavailahility of peptides by protecting them from
`and proteolysis in body fluids
`PolyDL-lactide PLA and polyuL-lactide-co-gly
`colide PLGA are the most widely used and well-
`
`physical degradation
`
`0168-3659101/8
`see front matter
`P11 SOl 68-365901 00403-5
`
`2001 Elsevier Science BY All
`
`rights reserved
`
`WIT
`
`DATES
`
`LLUERRMRCRR
`
`ALKERMES Exh. 2026
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`308
`
`EN Woo ci 0/
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`Journol
`
`Controlled Releose
`
`75 2001 307 315
`
`characterized materials for the preparation of bio
`
`Materials and methods
`
`potent
`
`luteinizing hormone-releasing
`
`degradable microspheres
`Leuprolide the peptide drug used in this study is
`agonist of
`hormone LHRH currently used for the treatment of
`endometriosis and precocious pu
`prostatic cancer
`in-water drying method
`Utilizing an
`berty
`based on the w/o/w emulsion technique 1- 3- and
`4-month release leuprolide microcapsules Lupron
`16 In the in-water-drying
`have been developed
`method leuprolide acetate was dissolved in aqueous
`polymer solution
`gelatin or water dispersed
`into
`which was then emulsified into an external aqueous
`phase to form droplets The microcapsules solidified
`removal and were freeze-dried Several
`after solvent
`publications have described in vivo pharmacological
`and pharmacokinetic evaluations of these microcap
`sules
`
`In most
`
`in
`
`literature reports of in vitro release the
`certain volume of
`microspheres were incubated
`release medium generally phosphate buffered saline
`PBS with
`preservative using glass or plastic test
`tubes at 37C with optional agitation
`In these
`methods the pH of the release medium consistently
`dropped due to the formation and accumulation of
`Con
`acidic polymer degradation products
`tinuous removal of the acidic polymer
`fragments and
`released
`be desirable to
`drug by dialysis might
`the release medium
`the pH drop of
`an LHRH
`release method
`antagonist omtide loaded PLGA microspheres has
`small dialysis unit and 0.1
`been developed by using
`The in vitro release
`acetate buffer pH 4.0
`profile obtained with the dialysis method permitted
`better correlation with in vivo release However only
`information is available on in vitro degra
`in vitro and
`dation kinetics and correlation betweeii
`
`prevent
`
`dialysis
`
`in vitro
`
`for
`
`little
`
`in vivo release of long-term release peptide loaded
`PLA microspheres
`The goal of this study was to prepare and char
`4-month formulation of leuprolide micro-
`acterize
`dispersion method
`and to study in
`spheres using
`vitro drug release and in vivo efficacy Also an
`modified dialysis method for
`to develop
`in vitro release from biodegradable micro-
`testing
`spheres which would exhibit
`good in vitro in vivo
`correlation was undertaken
`
`attempt
`
`2.1 Materials
`
`Leuprolide
`
`ethylamide was obtained
`
`Pro9-LHRH
`o-Leu6
`from Bachem Torrance
`CA USA PolyDL-lactide mw 11 000 Da
`Resomer R202H PLA was supplied by Boehringer
`Ingelheim Ingelheim Germany Polyvinyl alcohol
`mw 30 000 70 000 PVA was supplied by Sigma
`St Louis MO USA Dialysis
`tubes Tube-O
`Dialyzer were purchased
`from Research Products
`Co Mount
`IL and
`Prospect
`International
`Spectrapor CE dialysis membranes MWCO 7000
`300 000 Da were supplied by Spectrum Medical
`Industries Houston TX All other chemicals were
`obtained commercially as analytical grade reagents
`
`2.2 Preparation of microspheres
`
`solution of
`
`drug into
`
`Leuprolide PLA microspheres were prepared by
`solution of polymer and
`dispersing the homogeneous
`PVA solution followed
`by
`solvent
`de
`extraction evaporation dilution
`as previously
`leuprolide in methanol
`scribed
`34% w/w solution of polymer
`was added into
`in
`methylene chloride to form the clear solution The
`resulting solution was
`then
`slowly injected into
`0.35% aqueous PVA solution while mixed with
`Silverson L4R mixer Silverson Machines MA
`USA at 7000 rpm The solvents were removed by
`stirring and continuous
`phase replacement at 38
`40C for
`The
`solidified microspheres were
`by filtration and dried under
`vacuum at
`recovered
`room temperature for 48
`
`2.3 Particle characterization
`
`2.3.1 Particle size distribution
`Particles were sized by laser diffractometry using
`Malvern 2600 laser sizer Malvem 2600c Particle
`Sizer Malvem UK The average particle size was
`the volume mean diameter Vmd
`
`expressed
`microns
`
`as
`
`in
`
`2.3.2 Surface morphology
`The surface morphology was examined by scan-
`
`

`

`RH Woo el ol
`
`.Joornol of Controlled Releose
`
`75 2001 307 315
`dry weight Wa The mass remaining MR and the
`degree of hydration DH were calculated
`MR%-/ 100
`DHWW-Wd/Wd
`
`309
`
`as follows
`
`ning electron microscopy Hitachi Model
`800
`Japan after palladium/gold
`of
`the micro-
`coating
`sphere sample on an aluminum stub
`
`solved
`
`2.3.3 Peptide content
`the microspheres was dis
`10-mg amount of
`The peptide
`in 2.0 ml methylene chloride
`from the polymer solution by addition
`was extracted
`acetate buffer pH 4.0 followed
`of 10 ml of 0.1
`The peptide was assayed in the
`by agitation for
`aqueous phase by HPLC method Bondclone 10 C18
`column 150X3.Q mm Phenomenex Torrance CA
`USA mobile phase 27% v/v acetonitrile
`0.1%
`acid flow rate 1.0 ml/min UV de
`trifluoroacetic
`tection at 215 nm
`
`2.4 In vitro release study
`
`in 0.1
`
`large
`
`PBS at
`In vitro release was carried out
`37C using
`which
`modified dialysis method
`allowed control of the pH of the release medium and
`complete sample recovery Dialysis membranes with
`various MWCOs
`and dialysis tubing with
`used
`area were
`surface
`the release
`to optimize
`method by assessing the permeability of the peptide
`through the membranes and the recovery of
`the
`50-mg amount of microspheres was
`peptide
`suspended in ml 0.1
`phosphate buffered saline
`pH 7.4 PBS containing 0.02% sodium azide in
`cm
`tube 1.5 cm i.d.X5.5
`7-ml Tube-O-Dialyzer
`length The
`tubes were
`capped with
`dialysis
`dialysis membrane-windowed screw cap molecular
`weight cut off 300 000 placed in
`4-1 dialysis tank
`filled with the same release medium and incubated at
`37C with continuous
`agitation At each
`time point
`remaining microspheres were recovered
`by filtration
`The amount of
`through
`0.8-jim filter
`leuprolide
`remaining in the microspheres was determined by
`HPLC as described previously
`
`2.5 In vitro polymer degradation
`
`2.51 Mass loss and hydration
`At
`time point
`each
`recovered
`weighed accurately wet weight
`under vacuum at
`room temperature and reweighed
`
`wet mass was
`dried for 48
`
`gel
`
`permeation
`
`2.52 Molecular weight Mw of polymer
`The molecular weight distribution of PLA was
`determined
`by
`chromatography
`GPC as described previously
`Waters M-45
`Waters 990 Photo-
`solvent delivery system with
`diode Array Detector was used Two Ultrastyragel
`300 mm each one
`columns connected in series 7.8
`with 104-A pores and one with 103-A pores were
`used Samples mg/ml were eluted with tetrahy
`drofUran at ml/min The weight average molecular
`weight Mw of each
`sample was calculated
`using
`standards molecular
`
`monodisperse
`polystyrene
`weight 100050 000
`
`2.6 In vivo study
`
`at
`
`weighing 300
`Male Sprague Dawley rats
`were used to evaluate
`in vivo performance of
`The microspheres were
`leuprolide microspheres
`the back of the neck 12
`injected subcutaneously
`mg leuprolide/kg after reconstitution in
`suitable
`vehicle 1% carboxymethylcellulose
`and 2% man
`nitol w/v Blood samples were collected
`from the
`time points The samples were
`vein at specific
`centrifuged in Microtainer
`tubes Becton Dickin
`son Franklin Lakes NJ and serum was collected
`20C
`Serum samples were frozen and stored at
`
`tail
`
`using
`
`until analysis
`in rats were
`Serum leuprolide levels
`radioimmunoassay RIA method as de
`The lower detection
`scribed previously
`pg/ml The intra- and interassay
`of the assay was
`and 9% respectively
`coefficients of variation were
`levels were
`Serum testosterone
`assayed
`using
`TM Testosterone RIA DSL-4000 kits Diagnos
`tic Systems Webster TX The
`lower
`of
`limit
`for this assay was 0.08 ng/ml
`detection
`intra- and interassay
`coefficients of variation were 10
`and 9% respectively
`The cross-reactivity of
`testosterone antiserum was less than 6%
`
`assessed
`
`limit
`
`and the
`
`the
`
`

`

`310
`
`Bk Woo ci ol
`
`.Joorno/ of Coo/rolled Releose
`
`75 2001 3073 15
`
`Results and discussion
`
`at
`
`the bottom of
`
`the tube
`
`that
`
`could
`
`affect
`
`the
`
`Microsphere characterization
`
`Leuprolide microspheres were
`spherical with
`relatively non-porous surface Fig
`The aver
`age particle size was 51.7 sm which is suitable for
`injections The target
`intramuscular or subcutaneous
`load was 16.5% and the actual peptide content was
`determined to be 13.4% Leuprolide encapsulation
`efficiency was 81.2% The major difference between
`and the
`the microspheres prepared
`in this study
`microcapsules of 4-month Lupron depot
`is the drug
`in the polymer matrix Lupron mi
`distribution
`water-in-oil-in-
`are prepared
`crocapsules
`using
`water w/o/w emulsion technique
`and the particles
`of drug hence
`contain
`discrete
`intemal
`pockets
`called microcapsules The microspheres in this study
`from clear homogeneous
`solution of
`are prepared
`polymer and drug and the drug is believed to be
`molecularly distributed in the PLA matrix
`
`3.2 In vitro release
`
`that membranes with higher
`Table
`indicates
`MWCOs showed higher rates of peptide permeabili
`ty faster equilibrium between the dialysis cells and
`the media and good peptide
`recovery The dialysis
`large surface area showed
`tubing with
`complete
`equilibrium after 48
`it showed
`lower
`however
`to the dialysis cells due
`peptide recovery compared
`the peptide
`to higher adsorption of
`area Based
`on these
`membrane MWCO 300 000 was selected for the in
`vitro release study The dialysis method used in this
`the conven
`study showed
`over
`several advantages
`tionally used test
`tube methods This method elimi
`nated
`
`surface
`
`on the large
`
`results
`
`dialysis
`
`the undesired loss of microspheres during
`pH changes of
`sample preparation and handling
`the release medium and
`shear stresses due to the
`centrifugation for sample recovery Use of higher
`and frequent
`buffer
`the
`replacement of
`capacity
`release medium in the conventional
`tube method
`
`pH change However centrifu
`could also prevent
`gation is necessary to remove the supematant with
`loss of microspheres for
`the conventional
`out
`tube
`method This will
`
`cause the packing of microspheres
`
`of the microspheres
`physical characteristics
`The in vitro release study of
`spheres is shown in Fig
`15 days
`In the initial
`29% peptide released Thereafter release was nearly
`linear through day 150 with 0.5% released per day
`is 10%
`during this period Release in the first 24
`probably due to the fast diffusion of
`associated peptide The initial
`24-h release is illus
`5% initial
`trated in the small panel
`decrease to 2%
`observed in 30 mm followed by
`Then
`
`leuprolide micro-
`
`the surface-
`
`burst was
`
`in the next
`
`linear
`
`release
`
`ensued
`
`thereafter The initial
`
`decrease in release in the first
`
`few hours
`
`has been
`
`typical
`leuprolide microspheres and the only reason which
`
`observation with
`
`can be offered is that adsorption of released peptide
`occurs to the hydrated polymer surface in the early
`phase of release Once the binding sites are saturated
`release proceeds
`
`then the diffusional
`
`3.3 In vitro polymer degradation
`
`the initial
`
`and blank PLA micro-
`Both the peptide
`loaded
`spheres showed slow mass loss in the first 50 days
`followed by linear profiles for 150 days Fig
`The
`was only 2.0% confirm
`initial mass loss within 24
`release was caused by diffusion of
`ing that
`on the surface of
`located
`the peptide
`the micro-
`spheres Blank microspheres showed lower mass loss
`than drug loaded microspheres The peptide loaded
`and blank microspheres were slowly hydrated within
`50 days Fig
`between
`Hydration rate increased
`50 and 90 days with the peptide
`loaded micro-
`higher degree of hydration The
`spheres showing
`loading of PLA microspheres led to higher
`drug
`into the polymer matrices and
`uptake of water
`faster mass loss The molecular weight of residual
`and blank micro-
`polymers from both drug loaded
`spheres gradually decreased from 11 000 to 7000 Da
`There was no significant
`after 90 days Table
`difference in polymer degradation rate between drug
`and blank microspheres Blank and drug
`loaded
`loaded microspheres showed relatively smooth sur
`the beginning of
`the
`faces with spherical shape at
`study Fig
`The drug loaded micro-
`very porous interior
`spheres possessed
`while the blank microspheres showed an extremely
`shrunken shape after 35 days After 70 days the drug
`
`degradation
`
`structures
`
`

`

`RH Woo
`
`et ol
`
`Journol of Conarolled Releose
`
`75 2001 307
`
`315
`
`311
`
`Scanning electron microscopy of
`Fig
`Ieuprolide microsphere
`PLA blank microspheres
`PLA blank microspheres incubated
`
`and
`
`leuprolide microsphere incubated
`in PBS at 37C for 35 days
`
`in PBS at 37C for 35 days
`
`

`

`112
`
`RH 11
`
`fl
`
`1W
`
`.UUU I/CC
`
`It
`
`/1/C
`
`2001
`
`/1
`
`Table
`Equilibrium of leuprolide thiough dialysis membranes and peptide
`
`recovery
`
`MWCO
`Da
`
`8000
`
`14000
`
`50000
`
`300 000
`
`300 000
`
`Dialysis
`
`unit
`
`Equilibrium
`
`Dialysis cell
`
`Dialysis cell
`
`Dialysis cell
`
`Dialysis cell
`
`Dialysis tubing
`
`66.62.1
`
`67.33.4
`
`964
`1.8
`97.3 1.8
`102.52.2
`
`Peptide
`
`recovety
`
`88.8l.2
`
`88.30.5
`92.3 0.2
`91.90.6
`
`77.02.4
`
`deformed cake-like
`
`loaded microspheres showed
`shape compared to the lumpy shape of blank micro-
`spheres This suggests that
`the presence of peptide
`the morphological
`in the polymer
`changes
`matrix during in vitro release The overall
`
`affects
`
`in vitro
`
`release of
`
`the peptide
`
`correlated well with the
`
`polymer degradation profile
`
`3.4 In vivo leuprolide level
`
`Fig 5A shows serum leuprolide levels in rats after
`the subcutaneous administration of the microspheres
`Serum leuprolide levels increased immediately after
`administration to 45.6 ng/ml due to the initial burst
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Cu
`
`4-
`
`Cu
`
`30
`
`60
`90
`Time day
`
`120
`
`150
`
`Mass
`Fig
`PBS at 37C
`
`loss of peptide loaded
`
`and blank microspheres in
`
`release
`
`the
`
`initial
`
`fell
`
`at day
`
`15
`
`from the microspheres After
`elevation the levels
`to 4.3 ng/ml
`plateau level around
`ng/ml or less
`second dose of the microspheres at
`
`followed by
`over 120 days
`
`100
`
`Cu
`
`60
`
`Cu
`
`40
`
`20
`
`Drug loaded
`Blank
`
`18
`
`16
`
`14
`
`12
`
`1-I
`Cu
`
`24
`
`12
`
`18
`
`Time hr
`
`30
`
`60
`90
`Time day
`
`120
`
`150
`
`30
`
`60
`90
`Time day
`
`120
`
`150
`
`In vitro release of leuprolide loaded PLA microspheres in
`Fig
`PBS at 37C for 150 days large panel and initial
`release within
`small panel
`24
`
`Fig
`Hydration of peptide loaded
`PBS at 37C
`
`and blank microspheres in
`
`

`

`fiji
`
`Woo
`
`0/
`
`Joiwool of Cooov/leci Release
`
`2001 307
`
`315
`
`313
`
`20
`
`c16
`
`E12
`
`-J
`
`Cl
`
`CO
`
`CO
`
`2nd Dose
`
`30
`
`60
`
`90
`
`120
`
`150
`
`180
`
`30
`
`00
`
`00
`
`120 150
`
`ieo
`
`Time tdeyt
`
`Dose
`
`30
`
`60
`90
`120
`Time day
`
`150
`
`180
`
`AUC
`In vitro release
`
`120
`
`-100
`
`CO
`
`Ct
`
`80
`
`40
`
`30
`
`60
`90
`Time day
`
`120
`
`150
`
`Fig
`Comparison of in vitro leuprolide release profile with in
`vivo release profile the latter plotted as cumulative area under
`serum peptide curve nonrialized as percent of the total area large
`
`panel and in vitro in vivo correlation plot small panel
`
`for
`
`The testosterone levels were suppressed to 0.5 ng/ml
`and the levels remained near
`at day
`this level
`50 days before falling below 0.5 ngfml
`through day
`120 The challenges
`of 100 p.g of aqueous
`leup
`at 40 64 89 105 and 120 days did not
`rolide/kg
`levels which suggests that
`elevate the testosterone
`the pituitary LHRH receptors were occupied by the
`analogue and down-regulated Leuprolide challenges
`135 and 160
`the
`caused an elevation
`of
`
`Fig
`
`trations
`
`in
`
`In vivo serum leuprolide
`after single administration
`dose 12 mg/rat n6 small panel and
`panel Arrows
`indicate challenges of 100 sg/kg
`
`testosterone concen
`and
`of PLA mierospheres
`second
`dose large
`
`leuprolide
`
`at
`
`days
`
`123 days resulted in
`the first dose
`
`serum peptide level similar to
`
`3.5 In vivo testosterone
`
`suppression
`
`The small panel of Fig SB shows
`single subcutaneous administra
`levels in rats after
`the peptide-containing PLA microspheres
`
`testosterone
`
`tion of
`
`testosterone
`
`level
`
`the microspheres
`suggesting that
`for 120 days The large panel of
`were only effective
`Fig SB shows that
`elevation of
`following the initial
`testosterone the levels were suppressed below 0.5
`ngl ml through 123 days and
`second dose did not
`similar initial elevation of testosterone
`
`cause
`
`Fig
`
`compares
`
`profiles plotted as cumulative area Tinder
`
`the in vitro and in vivo release
`semm
`
`leuprolide normalized as percent of
`between days
`and 123 total area under
`AUC0 23d447 ng/day per ml The overall
`vitro release rate was slower
`vivo release However
`the in vitro release of leup
`rolide obtained with the modified dialysis method
`
`than the estimated
`
`in
`
`in
`
`the total area
`
`the curve
`
`

`

`314
`
`Table
`
`B.H Woo
`
`ci 0/
`
`.Jnnrnc/ of Control/ed Re/cave
`
`75 21W 217
`
`2/5
`
`Molecular weight changes of the residual polymer of leuprolide loaded
`
`and blank PLA microspheres
`
`Time
`
`day
`
`14
`
`24
`
`35
`
`50
`
`70
`
`Drug loaded
`
`10100
`
`9800
`
`9710
`
`8870
`
`7910
`
`7830
`
`6780
`
`S850
`
`5350
`
`5510
`
`4920
`
`5050
`
`4720
`
`PD
`
`1.49
`
`1.68
`
`1.81
`
`1.61
`
`1.61
`
`1.55
`
`1.56
`
`Blank
`
`Al
`
`11 000
`
`10700
`
`9830
`
`8780
`
`8530
`
`8540
`
`7610
`
`Sf
`
`6830
`
`6420
`
`5950
`
`5210
`
`6450
`
`5120
`
`5070
`
`PD
`
`1.61
`
`1.67
`
`1.65
`
`1.69
`
`1.32
`
`1.67
`
`1.50
`
`90
`
`7340
`
`6910
`
`4480
`
`1.54
`
`7060
`
`4640
`
`1.52
`
`average of molecular weight with three-significant
`Weight
`Number average of tnolecular weight
`
`Polydispersity PD MM
`
`figures
`
`correlated rather well with the estimated
`release r0.995 as shown in Fig
`
`small panel
`
`in vivo
`
`Conclusions
`
`controlled release 4-month formulation of leup
`from PLA
`rolide microspheres was
`fabricated
`dialysis method for measurement of in vitro release
`and successfully applied In vitro
`was developed
`release of
`leuprolide conelated well with polymer
`as with in vivo release The
`as well
`degradation
`leuprolide PLA microspheres are potentially useful
`formulation for testosterone
`suppres
`120-day
`
`as
`
`sion
`
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`
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`
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`
`active
`
`luteinizing
`
`between
`
`the
`
`for
`
`highly
`
`and relation
`
`that of
`
`

`

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