Pharmaceutical Research Vol 19 No
`
`April 2002
`
`2002
`
`In Vitro Characterization and in Vivo
`Testosterone Suppression of 6-Month
`Release PolyDL-Lactide
`Leuprolide Microspheres
`
`Byung Ho Woo1 Kyu-Heuin Na Bhas
`Thanoo2 and Patrick
`Ge Jiang5
`
`Dani
`DeLuca3
`
`Received December
`2001 accepted Deceribe
`14 2001
`KEY WORDS leuprolide LHRH analogue polyD.L-lactide mi
`ciuspheres peptide stability testustewjie suppression
`
`INTRODUCTION
`
`Controlled-release polymer delivery systems have been
`to achieve
`the efficacy of biologically active
`investigated
`and improve patient compliance by eliminating the
`need for frequent administration 15 Microsphere delivery
`
`agents
`
`lactide and glycolide
`systems fabricated from polyesters of
`were shown to improve the bioavailability
`of peptides pro
`teins and DNA by protecting them from physical degrada
`tion and proteolysis in body fluids before release 611
`
`uf
`
`luteinizing
`
`potent agonistic analogue
`Leuprolide
`hormone-releasing hormone inhibits
`the secretion of pitu
`itary gonadotropin when administered chronically
`in thera
`peutic doses 1213 Microsphere depot
`formulations of leu
`prolide were developed successfully and marketed for long-
`term testosterone suppression 1- 3- and 4-month release
`formulations of Lupron depot developed using
`water-in-
`oil-in-water w/o/w emulsion method currently are used for
`prostatie cancer endo
`the treatment of hormone-dependent
`metriosis and precocious puberty 1419
`4-month release polyDL-lactide PLA microsphere
`solvent extraction/evaporation
`delivery system using
`method have been developed recently
`The microspheres
`prepared with PLA molecular weight 11.000 provided sus
`tained release of leuprolide and suppression of serum testos
`for months in rats Compared to Lupron de
`terone level
`in which particles contain discrete internal pockets of
`pot
`the PLA microspheres were
`drug so-called microcapsules
`prepared from clear homogeneous solution of polymer and
`drug so that
`the drug was molecularly distributed throughout
`the PLA matrix
`The goal of this study was to prepare and characterize
`6-month leuprolide microsphere formulation using
`dispei
`sion/solvent extraction evaporation method Microspheres
`were prepared with PLA polymers of m.w 18000 28000 to
`obtain more convenient
`and effective microsphere formu
`lation for prostate cancer and endometriosis therapy
`
`EXHIBIT
`WIT DQJJrSc\
`ce9
`DATE
`DAWN HILLIER RMR CRR
`
`MATERIALS AND METHODS
`
`Materials
`
`Short Cooioinnicaoon
`
`D-Leu Pro9I-
`acetate
`Leuprolide
`luteinizing hormone releasing hormone ethlamide was ob
`tained from Bachem Torrance CA USA PLA polymers
`molecular weight of 18000 PLA 18k
`and 28000
`with
`PLA 28k were supplied by Birmingham Polymers Birming
`ham AL USA and Boehringer
`lngelheim Ingelheim Ger
`many tespectively
`alcohol molecular weight
`Polyvinyl
`30000 70000 PVA was obtained from Sigma Chemical Co
`St Louis MO USA All other chemicals were obtained
`commercially as analytical grade reagents
`
`Preparation of Microspheres
`
`ing
`
`Leuprolide PLA microspheres were prepared by dispers
`homogeneous solution containing polymer and drug into
`PVA solution followed by solvent extraction/evaporation
`solution of leuprolide in methanol was added to
`Briefly
`2129% w/w solution of PLA polymer in methylene chlo
`ride The resultant clear solution was then slowly dispersed in
`0.35% aqueous PVA solution while mixing with Silverson
`Inc East Longmeadow MA
`L4R mixer Silverson Machines
`USA at 7000 rpm using an in line mixer 20 The solvents
`removed by stirring air sweep and continuous phase
`were
`replacement at 40C for
`The solidified microspheres were
`recovered by filtration and dried under vacuum at room tem
`perature for 48
`
`Microsphere Characterization
`
`Microspheres were sized by laser diffractometry
`using
`Malvern 2600 laser sizer Malvern 2600c Particle Sizer Mal
`vern UK The average particle size was expressed
`as the
`volume mean diameter 1Imd in microns The morphology was
`electron microscopy Hitachi Model
`examined by scanning
`S800 Japan after palladiumlgold coating of the microsphere
`sample on an aluminum stub Peptide content was deter
`mined by high-performance liquid chromatography HPLC
`in mL
`as follows 10 mg of the microspheres was dissolved
`of methylene chloride The peptide was extracted from the
`polymer solution by addition of 10 mL 0.1
`acetate buffer
`pH 4.0 followed by agitation for
`The peptide concen
`tration was determined by HPLC using
`Bondclone 10 Cl8
`3.9 mm Phenomenex Torrance CA USA
`column 300
`Gradient elution was performed with 0.1% trifluoroacetic
`and 90% acetonitrile 0.1% trifluoroacetic
`acid
`acid
`from 10 to 60%
`and increasing the amount of the solution
`flow rate of 1.5 mL/min Leuprolide was
`over 12 mm
`at
`at 215 nm
`detected
`
`In Vitro Release Study
`
`Faculty of Pharmaceutical Sciences College of Pharmacy Univer
`90 Rose Street Lexington Kentucky 40536
`sity of Kentucky
`Oakwood Laboratories 7670 First Place-Suite
`Qakwood Ohio
`44146
`To whom correspondence should be addressed e-mail ppdelul@
`uk .edu
`
`Ten milligrams of the microspheres
`was incu
`bated in 10 mL 0.1
`saline PBS pH
`phosphate-buffered
`7.4 at 37C At each time point
`the residual microspheres
`and
`were recovered by centrifugation at 3000 rpm for 10 mm
`dissolved in mL of methylene chloride The peptide was
`extracted from the polymer solution li addition of 10 niL of
`
`0724-8741
`
`02/0001461
`
`002 Plenum Publishing Corporation
`
`546
`
`ALKERMES Exh. 2028
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`6-Month Release PLA Leuprolide Mierospheres
`
`547
`
`Table
`
`Characterization
`
`of Leoprolide PLA Microspheres
`
`\licrosphere
`
`Polymer
`
`Target drug load
`
`Drug content
`Drug incorporation
`
`Mean particle size sm
`
`efficiency
`
`PLA 28k
`low-drug load
`
`PLA 18k
`high-drug load
`
`PLA 18k
`
`PLA 28 000
`18.0
`
`PLA 28 000
`21.0
`
`PLA 18 000
`15.2
`
`16.3
`
`90.6
`
`22.0
`
`20.7
`
`98.6
`
`20.1
`
`15.0
`
`98.7
`
`7.2
`
`0.1
`
`acetate buffer pH 4.0 followed by agitation for
`The concentration of leuprolide was assayed by HPLC
`
`In Vitro Polymer Degradation
`
`was incu
`Thirty milligrams of the microspheres
`hated in 30 mL of 0.1
`PBS at 37C At each time pninr
`recovered wet microsphere was weighed accurately wet
`weight Wj dried for 48
`under vacuum at room tempera
`ture and reweighed dry weight Wd The mass remaining
`MR and the degree of hydration DH were calculated
`
`as
`
`follows
`
`MR
`
`Wd/WO
`
`100
`
`Where W0 is the initial mass at
`DH
`
`time zero
`
`WJ/Wd
`
`Peptide Stability
`
`0.1
`
`Peptide was extracted from the PLA microspheres incu
`PBS for 161 days using the methylene chloride/
`bated in 0.1
`acetate buffer extraction method described previously
`The stability was assessed by HPLC The peptide eluted at the
`same retention time of standard leuprolide was collected and
`analyzed using an lonSpec HiResMALDI Fourier Transform
`lonSpec Co Irvine CA USA
`mass spectrometer
`
`In Vivo Testosterone Suppression
`
`weighing 300
`Male Sprague Dawley rats
`were
`leuprolide micro-
`used to evaluate
`in vivo performance of
`spheres The microspheres were injected subcutaneously at
`the back of the neck 18 mg/kg
`as leuprolide after reconsti
`suitable vehicle 1% carboxymethylcellulose
`and
`tution in
`2% mannitol w/v Blood samples were collected from the
`
`tail vein at specific
`
`time points The blood samples were cen
`Co
`tubes Becton Dickinson
`trifuged in Microtainei
`Franklin Lakes NJ USA and serum was collected Seruni
`samples were frozen and stored at 20C until analysis
`Serum testosterone levels were assayed using Active0
`Testosterone RIA DSL-4000 kits Diagnostic
`Systems
`Inc.
`Webster TX USA The
`lower
`limit of detection for this
`assay was 0.08 ng/mL and the intra- and interassay coeffi
`cients of variation were 10 and 9% respectively
`The cross-
`reactivity of the testosterone antiserum was less than 6%
`
`RESULTS AND DISCUSSION
`
`Microsphere Characterization
`
`The PLA 28k
`with two different
`
`leuprolide microspheres were prepared
`target drug loads 18% and 21% The ac
`tual drug contents were determined to be 16.3% and 20.7%
`and 99% for
`and the drug incorporation efficiency was 91
`The
`the low and high target drug loads respectively Table
`mean particle size was 20 p.m which is suitable for intra
`injections The PLA mierospheres
`muscular or subcutaneous
`spherical shape Fig
`showed
`with some surface
`and
`pores and scratch marks lines of pores which could be from
`the shear stress during microsphere preparation Fig ib Mi
`crospheres of PLA 18k were prepared with 15.2% target drug
`load The actual drug content was found to be 15% and the
`efficiency was 99% The microspheres
`drug incorporation
`were spherical with relatively
`smooth surface morphology
`Fig ic The mean particle size was
`p.m The smaller
`particle size compared to the PLA 28K microspheres could
`be due to the lower viscosity of the polymer solution of the
`low molecular weight PLA polymer solution For the prepa
`ration of long-term release microsphere formulations maxi
`
`Fig
`
`leuprolide PLA 28k Leup 16.3% microspheres
`electron microscopy of
`Scanning
`leuprolide PLA 18k microspheres
`prolide PLA 28k Leup 20.7% and
`
`leu
`
`

`

`548
`
`Fig
`
`PLA 18k Leup 15.0%
`
`SA PLA 28k Leup 16.3%
`
`PLA 28k Leup 20.7%
`
`110
`
`100
`
`t90
`
`80
`
`CU
`
`cu
`
`40
`
`30
`
`10
`
`30
`
`60
`
`90
`120 150
`Time day
`In vitro release of leuprolide PLA microspheres in 0.1
`pH 7.4 at 37C
`
`180 210 240
`
`110
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`20
`
`10
`
`PBS
`
`Fig
`
`Mass loss of
`
`Woo et at
`
`SA PLA 28k Leup 16.3%
`PLA 28k Leup 20.7%
`PLA 18k Leup 15.0%
`
`30
`
`60
`
`90
`120 150 180 210 240
`Time day
`leuprolide PLA microspheres in 0.1
`7.4 at 173C
`
`PBS pH
`
`mum drug load into minimum amount of polymer is desirable
`to reduce the dose of synthetic polymer to patients while the
`microspheres contain the sufficient amount of peptide for
`long-term therapy With
`extraction
`dispersion/solvent
`evaporation method high drug incorporation
`at target peptide loads up to 21%
`achieved
`
`efficiency was
`
`In Vitro Release
`
`The PLA microspheres showed
`three-phase in
`vitro release Three to seven percent
`release peptide
`due to the fast diffusion of surface-associated
`was observed
`lag period of 3045 days until
`the poly
`peptide followed by
`mer hydrates and loses mass sufficient
`controlled release After the initial
`lag
`
`typical
`
`initial
`
`to initiate the erosion
`nearly linear and
`continuous release was observed over
`to months in vitro
`The low-drug load 16.3% PLA 28k microspheres showed
`3% initial
`release followed by
`very slow release until day
`45 The release increased
`after day 45 and
`continuous re
`
`20
`
`18
`
`16
`
`10
`
`14
`12
`88
`04
`
`Fig
`
`Hsdration of
`
`AA PLA28kLeup 16.3%
`PLA 28k Leup 20.7%
`sU PLA 18kLeup 15.0%
`
`180 210 240
`
`30
`
`60
`
`120 150
`90
`Time day
`leuprolide PLA microspheres in 0.1
`7.4 at 37C
`
`PBS pH
`
`days 60 and 210 Fig
`The
`lease was observed
`between
`high-drug load 20.7% PLA 28K niicrospheres
`showed
`faster and continuous release after 7% initial
`release The
`and subsequent
`faster release might be due to
`higher initial
`the highei drug load Peptide loading in the polymer matrix
`causes faster hydration and mass loss of polymer leading to
`PLA 18k micruspheres showed
`faster
`in vitro release
`release to the high-drug load PLA micro-
`similar in vi
`spheres The microspheres showed 3% initial
`release and
`release until day 30
`almost no further
`followed to 100% at day 210
`
`continuous release
`
`Hydration and Mass Loss
`
`low degree of hydration was observed with both PLA
`PBS at
`28k microspheres during the first
`45 days in 0.1
`37C Fig
`which
`correlated well with the slow in vitro
`for the first 45 days The hydration increased
`release observed
`maximum hydration at day
`slowly after day 45 and reached
`faster and higher hydration was observed with PLA
`150
`18k microspheres With PLA polymer the decrease of hydra
`the maximum hydration due to the
`tion was observed
`critical molecular weight and
`
`polymer degradation reaching
`
`after
`
`Table II Stability of Leuprolide Extracted from PLA Microspheres
`PBS pH 7.4 at 37C
`Incubated in 0.1
`
`Time days
`
`Stability
`
`PLA 28k
`Leap 20.7%
`
`14
`
`30
`
`45
`
`60
`
`90
`
`110
`
`161
`
`100
`
`99.4
`
`99.4
`
`99.4
`
`99.2
`
`99.5
`
`99.6
`
`97.5
`
`95.9
`
`91.9
`
`PLA 18k
`
`99.5
`
`99.5
`
`99.4
`
`99.4
`
`99.6
`
`99.2
`
`99.1
`
`97.6
`
`92.6
`
`88.1
`
`

`

`6-Month Release PLA Lenprolide Mierospheres
`
`549
`
`Lawo
`
`10
`
`LI
`
`AspJ.i.4.tvt
`
`4-
`
`4-
`Cl
`
`PLA 28k Leup 20.7%
`PLA 18k Leup 15.0%
`
`30
`
`60
`
`90
`
`120 150 180 210 240
`
`Time day
`In vivo serum testosterone concentrations after single admin
`Fig
`istration PLA microspheres dose
`18 mg/kg
`as leuprolide in rats
`The arrow indicates the challenge of 100 p.g/kg aqueous
`leuprolide at day 210 The zero time level
`represents the average
`serum testosterone level before the microsphere treatment
`18
`
`in rats
`
`Peptide Stability
`
`10
`
`15
`
`20
`
`lint n-in
`
`Fig
`
`HPLC chromatograms
`large panels and mass spectra small
`and the peptide extracted from the
`panels of standard leuprolide
`PLA 28k Leup 20.7% microspheres incubated in 0.IM PBS pH
`7.4 at 37
`for 161 days
`
`As shnwn in Table II over 99% of peptide remained as
`leuprolide in the PLA microspheres for 60 days before
`intact
`degrading slowly to 8892% after 161 days The large panel of
`shows the HPLC chromatograms of leuprolide
`Figure
`and the peptides extracted from the high-drug load PLA 28k
`The major degra
`microspheres incubated for 161 days
`dation product eluted prior to the intact
`leuprolide and minor
`degradation peaks were observed
`the leuprolide peak
`after
`The small panels are the mass spectra of leuprolide
`and
`the peptide from the PLA microspheres that eluted at
`same retention time of
`This confirms
`intact
`leuprolide
`the major peptide peak from the PLA microspheres is
`intact
`leuprolide
`
`that
`
`the
`
`loss of polymer matrix structure at which the capacity
`tain water
`is decreased
`
`to re
`
`Four
`initial mass loss was observed
`to seven percent
`at
`the first day with high drug load PLA 28k Leup 20.7% and
`PLA 18k microspheres whereas PLA 28k Leupl6.3% mi
`initial mass loss of 2% as shown
`showed
`lower
`crospheres
`Both PLA 28k microspheres showed
`slow mass
`in Figure
`loss for 45 days followed by
`nearly linear mass loss up to day
`150 The mass loss decreased
`between
`days 150 and 240 and
`the mass remaining was 5% at day 240 The low-drug load
`microspheres showed higher mass remaining values than the
`high-drug load microspheres due to the low initial mass loss
`However after
`the initial mass loss the mass loss profiles
`were similar The PLA 18k microspheres also showed
`slow
`mass loss for 30 days and then
`nearly linear mass loss up to
`slow mass loss between
`day 150 followed by
`day 150 and
`240 The mass loss profiles of the PLA microspheres corre
`lated well with the corresponding hydration and in vit ro pep-
`tide release profiles
`
`In Vivo Testosterone Suppression
`
`shows
`Figure
`the testosterone levels
`in rats after
`administration of the PLA leuprolide mi
`single subcutaneous
`crospheres dose
`as leuprolide Testosterone
`18 mg/kg
`levels increased immediately after administration to 13 ng/mL
`release of leuprolide from the PLA micro-
`due to the initial
`ng/mL
`spheres After the initial elevation the levels fell
`to
`ng/mL by day 14
`followed by
`at day
`increase to
`slight
`The testosterone levels were suppressed
`to below 0.5 ng/mL
`remained below 0.5 ng/mL through
`at 30 days and the levels
`180 days The testosterone level elevated above 0.5 ng/mL at
`day 210 and
`of 100 p.g/kg of aqueous
`challenge
`leuprolide
`caused
`testosterone This suggests that pituitary
`peak of
`LHRH receptors were occupied
`no longer than 180 days by
`leuprolide from the PLA microspheres
`single injection of
`the PLA microspheres inhibited the pituitary-gonadal
`system
`in rates for months and the in vivo efficacy correlated well
`with the duration of in vitro peptide release and the polymer
`degradation profiles
`
`

`

`550
`
`CONCLUSION
`
`Six-month release microspheres of leuprolide were pre
`pared successfully with PLA using
`dispersion/solvent evapo
`ration-extraction method The in vitro behavior correlated
`single injection of the micro-
`well with the in vivo results
`provided sustained testosterone suppression for
`spheres
`months in rats The leuprolide PLA microsphercs would be
`more convenient
`than 3- or month formulation and poten
`the patients who
`useful
`for
`improving compliance of
`need more reliable and effective hormone therapy
`
`tially
`
`ACKNOWLEDGMENTS
`
`The authors thank Charles Ritchie and Qui Wei
`nical assistance and Mr Henry
`Southgate Department of
`Entomology Agricultural Science University of Kentucky
`for SEM analysis of microsphere samples
`
`for tech
`
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`Li
`
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