ELSEVIER
`
`European Journal of Pharni.u.caucs
`
`and Biopharmiceutics 50 2000 263 270
`
`Jjjj_ieJnjj
`
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`Pjjsujjaj
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`wu w.elsevier.coni/locate/ejphabio
`
`Polymer and microsphere blending to alter the release of
`PLGA microspheres
`
`peptide from
`
`Research paper
`
`Harish
`
`Ravivarapu\ Kevin Burtonb Patrick
`
`DeLucac
`
`Dvnavas Technologies Coiporotion Berkley CA USA
`hPrhe Phaimo L.P Ardale NY USA
`College of Pharnaci
`Faculty of Pharmaceutical Sciences University of Kentucky
`
`I.e sington KY USA
`
`Received
`
`31 January 2000 accepted
`
`April 2000
`
`Abstract
`
`solvent-extraction/evaporation
`
`microspheres
`
`The objective of this study was to evaluate the effect of polymer and microsphere blending in achieving both
`sufficient
`release and
`loaded hydrophilic 5050
`peptide from polyDL-lactide-co glycolide microsphcres
`desired continuous
`release of
`acetate
`Leoprolide
`PLGA microspheres were prepared by
`process and were characterized for their drug load bulk density size
`distribution surface area surface morphology in vitro drug release and in vivo efficacy Combining PLGA polymers that varied in their
`molecular weights in various ratios yielded microspheres with varied drug release profiles commensurate with the hydration tendencies of the
`polymers Increasing the component of lower molecular weight SflSO hydrophilic PLCIA polymer
`kfla increased the initial drug release
`similar microsphere formulation prepared instead with blending microspheres from individual polymers showed
`animal model microspheres obtained from polymer or microsphere blends attained
`faster onset of testosterone suppression as compared to
`from higher molecular weight 5050 hydrophilic PLGA polymer 28.3 kDa alone These
`blending polymers or microspheres of varied characteristics
`in achieving modified drug release In particular the increased initial
`lag phase usually observed with microencapsulated macromolecules
`2000 Elsevier Science
`the peptide could help avoid the therapeutic
`B.V All
`rights reserved
`
`initial
`
`similar increase In an
`
`studies illustrated the feasibility
`
`of
`
`release of
`
`Keyworda PLGA microspheres Leuprolide Peptide release Polymer and miciosphere
`
`blend
`
`Introduction
`
`and
`
`low molecular weight mole
`Compared with conventional
`cules proteins and peptides have unique requirements and
`limitations for delivery These agents in general have short
`plasma half-lives are unstable in the gastrointestinal GI
`tract and also have low bioavailabilities due to their large
`molecular weights
`high
`aqueous
`solubility
`injections might be acceptable in cases of acute
`Frequent
`situations but not in chronic conditions Thus development
`of sustained release dosage forms for peptide/protein deliv
`to realize their full potential
`ery will help these agents
`drugs while enhancing patient compliance and convenience
`Microspheres prepared from polyoL lactic-co-glyculic
`acid PLGA polymers have been studied extensively in the
`last two decades
`as sustained release dosage forms and hase
`
`as
`
`author University of Kentucky college of Pharmacy
`Corresponding
`Rose Street Rm 3270 Lexington KY 40536 0082 USA Tel
`1-606-
`257-1831 fax
`606-323-0242
`H.B Ravivarapu kevin
`E-mail addresses Hravivarapu@dvax.com
`Burton ppdelu @pop.uky.edu
`P.P DeLuea
`
`burton@pharma.com
`
`shown improved patient compliance and/or
`therapeutic effi
`cacy of contraceptive steroids narcotic antagonists antima
`and
`Recent
`anticancer
`studies
`agents
`larials
`hormone
`especially with
`luteinizing hormone-releasing
`LHR-I analogues
`have shown these systems to be effec
`tive in the sustained delivery of macroniolecules
`In
`degradation rates of PLGA
`addition to being biocompatable
`and the accompanying release of encapsulated drug can be
`by the polymers physical
`controlled
`properties such
`as
`and the ratio of lactide
`molecular weight hydrophilicity
`Thus it
`is possible to obtain the desired
`to glycolide
`drug release from PLGA microspheres by altering the pol
`An extension to this approach
`mers characteristics
`is to
`optimally combine microsphetes
`prepared from different
`polymers with known drug release or to blend polymers
`The effect of mixed
`prior to preparing the microspheres
`populations of controlled release particles on the resulting
`release pattern has been cited in some early literature reports
`14 but there has been little experimental
`follow-up to
`show the feasibility and practical application of blending
`The purpose of this study was to pre
`are and evaluate
`
`0930641
`see front matter
`P11 S0939 64110000099
`
`11001$
`
`2000 Elsevier Science B.V All
`
`rights reseived
`
`EXHIBIT _________
`WIT________
`cL11
`DATE
`DAWN HILLIER RMR CRR
`
`ALKERMES Exh. 2024
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`264
`
`H.B Ravivarapu
`
`ci at
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`European Jaui 170/ of Phannuc eutics and Biophannaceutics
`
`50 2000 263 270
`
`28.3 kDa polymet
`fottuulatiotis
`Total polymer concentration was adjusted so that
`chloride
`viscosities of polymer/niethylene
`
`as detailed in Table
`
`solutions as
`
`the
`
`Brooksfield
`
`viscometer were comparable
`formulations was
`
`for all
`
`the
`
`measured by
`The
`loading
`di ug
`target
`12.5% w/w Microsphetes
`prepared from individual poly
`in 31 28.3 kDaJ8.6 kDa drug
`niers were mixed physically
`content
`ratio to obtain formulation
`of
`these microspheres were compared with those of micro-
`31 polymer mixture formulation
`spheres prepared from
`
`Characteristics
`
`2.3 Characterization
`
`of rnicrospheres
`
`The microspheres were characterized for drug content
`surface area mean particle
`bulk density specific
`size
`surface morphology and in vitro drug release and in vivo
`
`efficacy
`
`0.1
`
`dissolved
`
`and
`
`2.3.1 Drug content
`Drug loaded microspheres were quantitatively
`in methylene chloride and the peptide was extracted into
`acetate buffer pH
`by shaking the mixture for
`wrist action shaker Burrell Pittsburgh PA The
`on
`aqueous buffer phase was separated by centrifugation
`extracted peptide was quantitated by
`reverse phase-HPLC
`after some modifications The extraction was
`method
`combined
`and
`repeated with
`amount
`
`fresh buffer
`
`the
`
`peptide
`and
`the drug content
`values were reported as
`w/w of microspheres Triplicate samples
`the drug content and mean
`were used
`for determining
`values were reported HPLC analytical
`conditions were
`separation was achieved
`on
`as follows
`chromatograph
`300 mm Waters using
`C18 p.Bondapak column 3.9
`variable wavelength detector at 220 nm gradient pump
`both from Dionex Corp Sunnyvale CA and an autosam
`pIer Thermo Separation Products Fremont CA The
`6832 isocratic mixture of HPLC
`mobile phase was
`grade water and acetonitrile which was adjusted to pH
`acid The flow rate was
`4.0 with 0.1% trifluoracetic
`1.1
`mI/mm
`
`expressed as
`
`peptide-loaded microspheres trom various blends of
`PLGA polymers with
`commercial
`different molecular
`weights as an alternative to modifying the polymer charac
`
`two
`
`initial drug release Further
`teristics in achieving enhanced
`formulations obtained by physically blending microspheres
`that were prepared from individual polymers were evaluated
`to making microspheres from
`as an alternative
`approach
`blended polymers LHRH superagonist analogue leuprolide
`and peptide loaded microspheres from
`acetate was selected
`hydrophilic PLGA 5050 polymers were prepared by
`method Physico-chemical
`solvent extraction/evaporation
`characteristics of
`the microspheres were correlated with
`the in vitro peptide release and the formulation efficiency
`in suppressing serum testosterone levels for sustained pen
`ods was evaluated in an animal model
`
`Materials aad methods
`
`2.1 Materials
`
`PLGA 5050 polymers Resomer RGSO3H MW 28
`032 and RG5O2H MW 8631 were obtained from Boeh
`ringer Ingelheim Ingelheim Germany LHRH analogue
`from Bachem
`salt was purchased
`leuprolide as an acetate
`Inc Torrance CA All other chemicals
`reagent grade
`
`used were of
`
`analytical
`
`2.2 Preparation of microspheres
`
`.5
`
`typical batch size of
`Microsphere formulations in
`method
`were prepared by
`solvent-extraction/evaporation
`combination of PLGA polymers
`PLGA polymer or
`itt methylene chloride was mixed with methanolic solution
`of the peptide The resulting mixture dispersed phase DP
`0.35% w/w polyvinylalcohol PVA MW 30
`was added to
`phase CP
`70 k.Da Sigma aqueous
`solution continuous
`while stirring at 7000 rev./min using
`homogenizer Silver
`son L4R Silverson Instruments Corp MA After
`mm at
`25C the stirring rate was decreased
`to 500 rev/mm
`and the
`temperature raised to 40C to slowly extract and evaporate
`After cooling to 25C particles
`the organic phase over
`were recovered by filtration
`under
`and dried overnight
`vacuum at room temperature
`To evaluate
`the effect of polymer blends the 8.6 kDa
`combined
`polymer was
`in various
`with
`
`proportions
`
`Table
`
`Manufacturing
`
`parametets of peptide loaded microapheie
`
`2.3.2 Bulk density
`The dry microspheres were quantitatively
`tube The test
`tube was subsequently tapped
`
`graduated test
`
`transferred to
`
`Formulation ID
`
`Polymers
`
`Ratio
`
`wiw
`
`of polymer in DPb
`Ratio of CFI3OH/CH2CI
`
`in DP
`
`16.3
`
`0.24
`
`38.0
`
`0.22
`
`31
`
`24.0
`
`0.20
`
`41
`
`21.1
`
`0.20
`
`28.3 kDa
`
`8.6 kDa
`
`28.3 kDa/8.6 kDa
`
`28.3 kDa/8.6 kDa
`
`28.3 kDa/8.6
`51
`
`kDa
`
`19.6
`
`0.20
`
`Formulation
`was
`physical
`DP dispersed phase containing
`
`combination
`
`of
`
`and
`
`peptide and polymer in methanol
`
`and methylene
`
`chloride
`
`

`

`HR Rawvai apa ci
`
`European
`
`Journal of Phaniaeeuncs
`
`and Biaphannaceuan 50 2000 263 270
`
`265
`
`vertical
`
`20 times from
`
`distance of approximately 0.5
`volume
`recorded The tapping
`inches and the occupied
`the volume occupied by particles
`process was repeated until
`remained unchanged The final volume was recorded as
`and the tapped bulk density g/cc
`bulk volume
`was
`calculated as iiilv where in was the weight of nñcrospheres
`
`employed
`
`2.3.3 Specific surface circa
`The specific surface area was determined using an ASAP
`2000 surface area analyzer Micromeritics Norcross GA
`by BET
`adsorption desorption
`The area
`isotherms of Kr on the surface of microspheres
`values were normalized to the sample weight which was
`typically in the range 0f 250300 mg
`
`of
`
`the
`
`transformation
`
`2.3.4 Sic distribution
`Particle size distribution was determined using Malvern
`
`suspended
`
`2600c
`
`Laser Diffraction Particle Sizer Malvern Instru
`
`ments Southborough MA The microspheres
`were
`in pre-filtered 0.1% aqueous Tween 80 solution
`63 mm for
`size range of 0.5118 p.m or
`and either
`100 mm for
`size range of 1.9188 p.m focal
`length lens
`to determine particle size Mean diameter
`was employed
`based on volume was determined
`
`2.3.5 Surface morphology
`The surface morphology was examined by scanning elec
`tron microscopy Hitachi Model S800 Japan after coating
`the microsphere sample with gold-palladium on an alumi
`num stub
`
`2.3.6 In vitro drug release
`Approximately 10 mg of peptide loaded microspheres
`tubes and incubated
`were quantitatively
`transferred to test
`at 37C in
`phosphate buffer pH
`with 10 ml of 0.033
`samples were main
`temperature controlled oven Separate
`tained for each time point The tubes were shaken
`twice
`weekly and
`ml of supernatant were replaced with fresh
`sink conditions After
`days to maintain
`
`buffer every
`
`sampling niicrospheres were separated by centrifugation
`To minimize the loss of microspheres only 80% of
`supernatant was removed Correction
`for peptide in the
`remaining ml of supernatant was made in the final calcu
`
`the
`
`Table
`
`characteristics of peptide loaded microspheresa
`
`The drug
`in microspheres
`lations of peptide remaining
`in the microspheres was quantitated as described
`content
`earlier by HPLC
`Peptide release was based on the peptide remaining in the
`microspheres rather than on the released amount of peptide
`as the released peptide has limited stability
`unpublished
`laboratory studies in the in vitro releasing medium under
`Released drug was calculated
`conditions
`the experimental
`loaded drug and that
`initially
`of
`and expressed as
`
`as
`
`the difference between
`
`remaining in the microspheres
`loaded amount
`
`initially
`
`2.3.7 In vivo evaluation
`Male SpragueDawley rats Harlan Sprague Dawley
`least 12 weeks old weighing 200
`Inc Chicago IL at
`250
`were employed
`formulation to assess
`per
`serum testosterone levels Animals were maintained as per
`forth in Guide for the Care and Use of
`the guidelines set
`Laboratory Animals DHEW Pub No NIH 78-23
`revised The microspheres were suspended
`in mixture
`7LFPH USP Aqualon
`of 1% carboxymethylcellulose
`Delaware NJ and 2% mannitol USP/EP and injected
`into rats subcutaneously just below the neck
`region at
`mg leuprolide/kg
`drug dose of
`body weight based
`on
`single injection was given to
`
`literature reports
`
`each animal
`
`an initial
`
`the
`
`Following the
`
`immediately after collecting
`sample
`from the tail vein Further samples were collected at 0.25
`15 25 32 33 42 and 43 days after dose administra
`tion On days 32 and 42 animals were challenged with
`100 .tg/kg of leuprolide acetate to investigate whether
`LH receptors were still
`down-regulated
`and
`samples were taken
`challenge doses additional
`at
`The lack of an elevation in testosterone levels above
`0.5 ng/ml would indicate that
`the receptors were still occu
`
`24
`
`pied Samples were assayed
`
`levels by radioimmunoassay
`using
`kit Active Testosterone Diagnostic
`tories Webster TX
`
`in duplicate for testosterone
`standard commercial
`
`Systems Labora
`
`Results and discussion
`
`3. In vitro characterization
`
`The physico-chemical
`
`characteristics of peptide loaded
`
`Formulation ID polymer-ratio
`
`Drop content %w/w
`
`Surface area m2/g
`
`Size1 p.m
`
`i/nIb density p/cc
`
`28.3 kDa
`86 kDa
`31
`28 3/8.6
`28.3/8.6 41
`51
`
`28.3/8.6
`
`II
`
`88
`
`11.34
`
`9.87
`
`9.48
`
`9.75
`
`Formulation wi
`11 combination of
`physical
`Mean diameter based on volume
`
`and
`
`0.387
`
`1.540
`
`0.584
`
`0.420
`
`0.602
`
`18.0
`
`21.0
`
`28.0
`
`28.5
`
`20.0
`
`0.54
`
`0.30
`
`52
`
`0.55
`
`0.56
`
`

`

`266
`
`jIB Roiivarapu et al lEuropean Journal of Phunnaceunn and Biopharnaceutic3
`
`502000 263 270
`
`Fig
`
`Scanning electron micrographs
`
`of microspheres
`
`from polymer combinations
`
`at 1500
`
`28.3 kDa/8.6 kDa
`
`10
`
`01
`
`3.1
`
`41
`
`51
`
`PLGA microspheres are tabulated in Table
`and scanning
`of representative microspheres
`electron photomicrographs
`from each
`formulation at
`1500
`magnifications
`shown in Fig
`formulations except
`All
`the
`8.6 kDa microspheres had
`similar surface morphology
`In general microspheres were spherical smooth and non
`the 8.6 kDa microspheres were very
`In contrast
`poruus
`porous with rough surfaces
`and were expected to have
`surface
`and
`
`are
`
`for
`
`the
`
`drug
`
`faster
`
`ities
`
`gauge needle
`w/w values for 28.6 and 8.6 kDa micro
`Drug content
`spheres were similar however niicrospheres from the poly
`mer combinations
`have
`decreased
`loading
`The encapsulation efficiency
`Table
`formulations calculated against
`the target drug loading of
`12.5% w/w ranged from 78 to 95% Slightly higher amounts
`
`values
`
`for all
`
`the
`
`efficiency
`
`specific
`
`area
`
`release
`
`high
`The
`formulations had
`similar size distribution with
`mean diameters in the range of 1829 l.Lm As the viscosity
`of polymer solution can influence the microsphere charac
`teristics including size distribution similar polymer viscos
`in the organic phases were maintained by modifying
`polymer concentrations Table
`In the case of the lower
`the viscosity was maintained by
`molecular weight polymer
`The microspheres
`increasing the polymer concentration
`used in this study were in an injectable range for convenient
`subcutaneous
`and intramuscular
`21- or 23-
`injections via
`
`dual
`
`of methylene chloride were used in preparing the polymer
`combination batches
`resi
`and possibly due to this higher
`levels of methylene chloride in the range of 132 vs
`20 ppm with single polymer
`formulations data
`not
`shown were observed It was possible that as methylene
`chloride was extracted at an apparent slower rate
`amount of peptide was lost
`phase through
`into the aqueous
`the polymer wall
`remained soft and permeable for
`
`that
`
`higher
`
`100
`
`80
`
`is
`
`20
`
`30
`
`40
`
`50
`
`TIme days
`
`Fig
`
`In vitro peptide release from microapheres
`01
`283 kDa/8.6 kDa
`10
`combinations
`
`prepared from polymer
`41
`i1
`31
`
`

`

`1-i
`
`Rovivarcipiv
`
`et of
`
`uropean
`
`Journal
`
`Pliai
`
`rnoceuric.s
`
`and Biophai nuuiuai
`
`50 2000 26
`
`270
`
`267
`
`surface
`
`can also be correlated with specific surface area values
`formulations with lower bulk density having higher specific
`In this study 8.6 kDa
`areas-internal plus external
`microspheres with the lowest bulk density had the highest
`total specific surface area Formation of porous or hollow
`microspheres with high specific surface area in general trans
`No major differences in
`faster drug release
`lates into
`bulk density were observed among 28.3 kDa and polymer
`from
`combination microspheres which were predominantly
`the 28.3 kDa polymer The specific surface areas with formu
`were higher as compared to that of formulation
`lations
`however not as high as that was seen with 8.3 kDa poly
`mer formulation
`
`show the in vitro cumulative release of
`and
`Figs
`peptide As expected
`drug release from 8.6 kDa micro-
`spheres was very rapid with approximately 55% of encap
`sulated drug being released within 24
`This high initial
`release can be attributed to the more rapid hydration of
`lower molecular weight polymer as well as higher specific
`surface area The high initial
`release was
`followed
`
`by
`
`slower uniform release until exhaustion after 30 days In
`kDa microspheres was
`drug release from 28.3
`contrast
`very slow and gradual up to 14 days at which time the
`and subsequently
`polymer apparently
`started dissolving
`clear modi
`polymer erosion controlled the drug release
`
`10
`
`Is
`
`20
`
`25
`
`00
`
`35
`
`40
`
`45
`
`00
`
`Time days
`
`In vitro peptide release from mierospheres prepared from polymer
`Fig
`blends
`and mierosphere
`
`time in case of combination formulations
`
`relatively longer
`leading to lower drug content values
`Microspheres prepared froni 8.6 kDa polymer in this study
`had the lowest bulk density which correlated well with the
`observed porous surface morphology Fig lB Low bulk
`qualitative indicator of the formation
`density value can be
`of hollow microspheres and/or lack of optimum packing of
`irregular non spherical microparticles Bulk density values
`
`10
`
`15
`
`20
`
`25
`
`30
`
`45
`
`Time days
`
`t32
`
`He
`
`In vivo testosterone suppression SEM with individual polymers
`show the data in
`formulations The inserts
`and
`and nsierosphere blend
`axis while mean baseline value from 180 rats is shown by the
`ngJrnl Mean zero time values for the groups are shown on the
`the extended range above
`
`and
`
`dotted line
`
`indicates
`
`the challenge
`
`

`

`268
`
`H.B Rca ivoropu ci at
`
`European Journal qi Pharmaceutics
`
`and Biaphornaceuncs
`
`50 2000 263 270
`
`and
`
`and
`
`as
`
`As
`
`release of
`
`no major
`between
`
`differ
`
`favoring
`
`the release
`
`profiles
`
`the extent of
`
`difference
`
`in the
`
`formulations
`
`and
`
`initial
`
`appeared
`
`after the initial
`
`fication in the peptide release particularl\
`during the initial
`period was obsei
`ccl with the polymer combination formu
`lations versus
`the single polymer formulations
`vs
`well as within polymer combinations
`8.6 kDa polymer content was increased the initial
`peptide was increased formulation
`Although
`release was observed
`initial
`20 and 16% respectively
`ences were noted after approximately
`10 days of study
`with the higher 8.6 Wa component
`formulation
`release In general
`slightly higher
`seemed
`to be similar with the exception of
`release Preparations
`from polymer combinations
`to have
`14 days of drug release
`lag phase
`release This lag is believed to be due to the
`predominance of the 28.3 kDa polymer Previous studies by
`Li et al
`that gelling and solidification of
`suggested
`homogeneous
`the polymers occurred separately even in
`mixture of the two polymers In such
`case it could be
`the more hydrated domains of 8.6 kDa
`hypothesized that
`polymer within the microsphere matrix accounted
`for the
`days of the study and the
`release during the first
`28.3 Wa domain dictated the subsequent
`more prominent
`release in the later period
`Fig
`shows
`the comparative
`peptide from twn formulations
`and mierosphere mixing
`
`initial
`
`polymer
`
`cumulative
`
`release of
`
`and
`
`obtained from
`
`As the
`
`drug
`
`content of 7X
`
`and 8.6 kDa microsphcrcs was very siniiiai
`31 ratio of drug content
`Table
`rather than polymer
`content was used in obtaining formulation
`The profiles
`were very close except for some differences on the first day
`and during the 1449 day period PLGA polymers degrade
`hydrolytically giving rise to an acidic microenvironment
`in
`which enhances polymer degrada
`the particle structure
`tion and mass loss An acidic microenvironment
`is attained
`the 8.6 kDa PLGA as this polymer
`in the case of
`hydrates faster owing to its higher number of carboxylic
`from the
`endgroups
`acid
`mierospheres
`Additionally
`lower MW polymer had
`more porous internal structure
`which would also facilitate hydration Thus microspheres
`that contain 8.6 kDa PLGA as
`combination in their struc
`
`faster
`
`and release drug faster
`ture are expected
`to degrade
`blended as
`compared to microspheres that are physically
`the hydration of the 8.6 kDa polymer will also hydrate the
`closely associated 28.3 kDa polymer This may explain the
`higher drug release seen with polymer combination formu
`later time points However
`as the noted difference
`in the drug release from polymer or microsphere blends is
`
`lations at
`
`as
`
`not substantial obtaining microspheres by physical mixing
`to be
`suitable formulation alternative
`appears
`These studies also showed that
`
`release
`
`the experimental
`
`closely approximated the theoretical calculated proportio
`nately from profiles of
`individual polymer formulations
`release values Fig
`Thus it
`is possible to determine an
`
`03
`
`Cl
`
`Fig
`
`In vivo testosterone suppression tn
`
`with potymer blend formulations
`
`and
`
`The inseris
`
`and
`
`show the data points above
`
`ng/mt and
`
`indicates
`
`the chattenge
`
`Time days
`
`

`

`Rat/va i/Ill
`
`CI 0/
`
`European
`
`lout /111/ of Pharnaceanrs and B/op/ta Inaceutics 50 2000 26
`
`270
`
`269
`
`optimum combination of microspheres without performing
`high number of experiments
`
`3.2 In viva evaluation
`
`maintained
`
`30 days of
`the suppression for at
`study meeting the study objective of rapidly achieving and
`testos
`testosterone levels In contrast
`sustaining suppressed
`terone levels were seen to be elevated with the 8.6 kDa
`
`least
`
`the
`
`and
`
`causes an
`
`show the testosterone suppression obtained
`Fig
`with formulations
`the
`testosterone
`depicts
`suppression obtained with 28.3 kDa
`8.6 kDa
`31 28.3
`kDa
`kDa/8.6
`mixture of microspheres
`LHRH superagonist
`Leuprolide acetate being
`in the serum testosterone levels Insert
`shows the elevated levels of 25.3 and 12.2 ng/rnl at
`and
`the testoster
`respectively For Formulation
`one peak occurred prior to
`and by that time had already
`rise to 4.79 ngml Within
`days
`descended
`from the initial
`levels were below baseline As expected from the in vitro
`the suppression with 8.6 kDa micro-
`release profiles Fig
`spheres was rapid as compared to 28.3 kDa formulation The
`kDa
`effect of substituting
`component microspheres
`28.3
`with 8.6 kDa at 25% w/w 31 28.3 kDa/8.6
`kDa was
`as the suppression with the combination
`clearly evident
`kDa micro
`was
`than with 28.3
`microspheres
`than with 8.6 kDa microspheres alone
`spheres but slower
`correlates with the higher amount of drug
`This effect
`released in vitro In addition to achieving faster onset of
`combination microspheres
`
`Figs
`
`and
`
`initial elevation
`
`for
`
`all
`
`faster
`
`testosterone
`
`suppression
`
`the
`
`microspheres by 25 days due to faster depletion of the drug
`Fig
`shows
`the testosterone profiles obtained with the
`As
`combinations
`from polymer
`microspheres
`compared with the 28.3 kDa polymer microspheres alone
`the three polymer combinations yielded faster testoster
`and
`one suppression Figs
`suggesting an initial higher
`release of peptide in vivo as was the case in vitro Fig
`Challenging animals with leuprolide solution at days 32 and
`42 showed the formulations to be effective at
`least until 30
`days Compared to the elevation observed on day 32 the
`elevation on second challenge was very high indicating the
`exhaustion of drug levels to completely occupy the recep
`tors The suppression profiles obtained with formulations
`though the 8.6 kDa
`did not differ much even
`and
`formulations varied from 16 to 25%
`component
`vs
`w/w and in vitro difference in drug release
`and
`was noticed
`This lack of pharmacological
`be explained as the testosterone suppression deter
`indicator of the
`niined in this study is
`pharmacological
`drug release in vivo Once the gonadotropin LHRH recep
`tors are down regulated
`the serum levels of
`leuprolide
`are known to be very
`needed
`to maintain desensitization
`
`all
`
`in these
`
`cannot
`
`difference
`
`31
`
`26
`
`21
`
`16
`
`11
`
`025
`
`41
`
`42
`
`43
`
`44
`
`in
`
`Baseline
`
`oC28.3 kd/8.6 kd 31
`aFAB 31
`
`Fig
`points shove
`
`tn vivo testosterone
`
`suppression
`
`ng/ml and
`
`indicates
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`Time days
`
`t32
`
`with potynler hend formulation
`the chattenge
`
`and microsphere
`
`blend formulation
`
`The inserts
`
`and
`
`show the data
`
`

`

`270
`
`1-1.8 Rat
`
`varapu ci 0/
`
`European Journal of Phannaceutics
`
`and Biopharnacentics 50 2000 263270
`
`low
`
`and it
`
`is possible that variatinnc
`in drug release
`from these formulations in vivo were not significant enough
`to affect varied suppression profiles Since serum leuprolide
`levels nearly nondetectable after the testosterone levels are
`
`reduced
`
`direct assessment of in vivo
`
`to below 0.5 ng/ml
`drug release were not determined in this study
`compares the efficacy of formulations
`Fig
`physical blend of 28.3 and 8.6 kDa microspheres
`that was prepared from 31
`kDaI8.6 kDa polymer
`28.3
`blend The testosterone suppression profiles obtained with
`from the
`these formulations were very similar as expected
`Thus physically mixing
`similar in vitro drug release Fig
`from single polymers whose drug release
`microspheres
`are known
`to be an attractive approach
`to making microspheres from
`and can be an alternative
`custom made or blended polymers
`this study had shown the feasibility
`In conclusion
`
`profiles
`
`appears
`
`31
`
`and
`
`of
`
`110
`
`1141
`
`utilizing blends of polymers or microspheres
`to prepare
`formulations that will provide the desired release of peptides
`testosterone as well as
`and effect earlier suppression of
`continued
`
`30
`
`These
`
`for
`
`at
`
`least
`
`days
`suppression
`offer two practical alternatives to the expensive
`approaches
`and laborious process of customizing the polymer properties
`for the desired drug release
`
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`
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`
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