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`Enhancing Initial Release of Peptide from PoIydI
`lactide-co-glycolide PLGA Microspheres by
`Addition of Porosigen and Increasing Drug Load
`
`Harish
`
`Ravivarapu Heeyong Lee
`
`Patrick
`
`DeLuca
`
`DeLuca 2000 Enhancing
`To cite this article Harish
`Ravivarapu Heeyong Lee
`Patrick
`PLGA Microspheres by Addition
`Initial Release of Peptide from Polydl-lactide-co-glycolide
`of Porosigen and Increasing Drug Load Pharmaceutical Development and Technology 52
`287-296 DOl 10.1081/PDT-1 001 00543
`
`To link to this article http//dx.doi.org/10.1081
`
`/PDT-1 001 00543
`
`Published online 19 Apr 2000
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`Pharmaceutical Development and Technology 52 287
`
`296 2000
`
`RESEARCH ARTICLE
`
`Enhancing Initial Release of Peptide from
`Polyd/-lactide-co-glycolide PLGA
`Microspheres by Addition of
`Porosigen
`and Increasing Drug Load
`
`Harish
`
`Ravivarapu1 Heeyong Lee2 and Patrick
`
`DeLuca3
`
`1Atrix Laboratories Inc 2579 Midpoint Drive Ft Collins Colorado 80525
`2Hundai Pharm lnd Co Ltd 213 Sosa Bon 1-Dong Sosa-Gu
`Bucheon-Si Kyoungki-Do Korea
`of Kentucky College of Pharmacy Rose Street Lexington Kentucky
`
`40536-0082
`
`.0
`
`Received May 14 1999 Accepted October 12 1999
`
`ABSTRACT
`
`The objective of this study was to evaluate formulation variables such as drug load and addition of
`release ofpeptidefrompolyd1 lactide-co-glycolide PLGA
`porosigen in achieving an increased initial
`by altering carrier characteristics Leuprolide acetate-loaded PLGA microspheres were
`microspheres
`and were characterized
`for their drug load
`prepared by
`solvent-extraction evaporation
`process
`HPLC assay bulk density tapping method size distribution dynamic light scattering specfic sur
`face area BrunauerEmmett--Teller
`analysis surface morphology scanning electron micros
`copy in vitro drug release at 37C and in vivo efficacy suppression of rat serum testosterone
`Increasing the drug load and adding various amounts of calcium chloride to organic and aqueous
`phases of the emulsion during processing yielded particles with increased porosity lower bulk density
`these formula
`higher specf Ic surface area and accordingly higher initial
`release In an animal model
`tions showed
`
`compared
`to microspheres without higher drug
`faster onset of testosterone suppression
`load or calcium chloride The approaches employed in this study were found to be effective in avoiding
`lag phase usually observed with microencapsulated macromolecular drugs
`the therapeutic
`KEY WORDS Drug load In vitro characteristics
`PLGA microspheres Porosigen
`
`In vivo efficacy Leuprolide acetate Peptide
`
`DeLuca University of Kentucky College of Phamiacy Rose Street Room 3270 Lexington
`to Patrick
`Address correspondence
`KY 40536-0082 Fax 606 323-0242 E-mail ppdelul@pop.uky.edu
`
`Copyright
`
`2000 by Marcel Dekker
`
`Inc
`
`www.dekker.com
`
`287
`
`

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`
`INTRODUCTION
`
`There is
`
`painful
`
`cancer
`
`sional
`
`and have been
`
`in
`
`need for developing sustained-release dos
`and proteins because these mac
`age forms for peptides
`romolecular drugs pose unique difficulties
`that
`include
`low oral bioavailability instability
`in the gastrointestinal
`frequent and
`tract dosage form design and subsequent
`injections 12 Recent
`shown that
`reports havc
`in achieving sus
`polymer-based systems can be useful
`tained and controlled delivery with less frequent paren
`teral administration
`Injectable systems of leutein
`izing hormone releasing hormone LHRH analogues
`based on polyd.1-lactide-co-glycolide PLGM were re
`ported to be therapeutically advantageous
`in hormonal
`successfi.illy commercialized
`treatment of
`11 An advantage with these systems is that
`their biodegradation and drug release can be controlled
`by modifying polymer characteristics such as molecular
`comonomer
`and
`weight hydrophilicity
`lactide/gly
`colide ratio 12 15 In particular compared to implants
`and
`and
`nonbioerodible matrices
`biodegradable
`jectable microspheres obviate the need for surgical proce
`removal
`dures during administration and subsequent
`Release of conventional
`low molecular weight drug
`molecules from polymer systems is primarily via difth
`sional process 16 Macromolecular
`drugs such as pro
`teins and peptides release via both diffusion and matrix
`degradation processes 1719 Often the initial diffu
`release of proteins and peptidcs is not sufficient
`because
`to elicit
`
`sustained
`
`pharmacological
`response
`diffusion of macromolecules through the polymer struc
`ture may be limited This results in an unwanted
`the onset of polymer erosion and
`peutic lag phase until
`drug release In addition to drug
`subsequent
`and polymer characteristics diffusional
`release of drugs
`is controlled by carrier-related factors such as structure
`size surface area porosity and drug load 16172021
`ap
`These
`factors can be
`by formulation
`proaches
`in altering the drug release
`to avoid the undesired therapeutic lag phase
`The purpose of this study was to evaluate preparation
`variables such as drug load and addition of
`porosigen
`in achieving modified and in particular increased initial
`peptide from PLGA micro
`release of
`spheres L1-IRH-analogue-loaded nuicrospheres were pre
`and
`solvent-extraction
`
`thera
`
`accelerated
`
`controlled
`
`and can be useful
`
`difflisional
`
`pared by
`
`evaporation process
`characteristics of microspheres were
`physicochemical
`correlated with in vitro release profiles of peptide These
`formulations were also compared for their efficacy in
`suppressing serum testosterone levels in rats The peptide
`for these studies was the LHRH superagonist
`
`selected
`
`leuprolide Polymer selected was 5050 PLGA with un
`capped end groups to increase hydration Calcium chlo
`ride was chosen as
`porosigen to increase the porosity
`of peptide-loaded mierospheres
`
`MATERIALS AND METHODS
`
`Materials
`
`PLGA 5050 polymer Resome RG5O3H MW
`28032 was obtained
`from Boehringer
`Ingelheim Wall
`ingford CT LHRH analogue
`and leuprolide as an ace
`tate salt was purchased
`from Bachem Inc Torrance
`CA Calcium chloride dihydrate was obtained
`from
`Sigma Chemical Co St Louis MO All other ehemi
`eals used were of analytical
`
`reagent grade
`
`Preparation of Microspheres
`
`in
`
`typical batch size of
`
`by
`
`solvent-extraction
`
`evaporation
`
`1.5
`
`follows
`
`contained
`
`mixed with
`The typical
`
`Microsphere formulations
`were prepared
`method 22 Briefly the preparation procedure was as
`methylene chloride CH2CI2 solution that
`16% w/w of polymer was
`approximately
`methanolie solution CH3OH of peptide
`ratio of CH3OH to CH2CI2 was 0.24 The
`resulting mixture dispersed phase DP was then slowly
`added to 500 ml of 0.35% w/v polyvinylalcohol PVA
`aqueous solution continuous phase CP maintained at
`25C with
`water jacket CP and DP were emulsified for
`homogenizer Silverson L4R Silverson
`mm
`using
`Instruments Corp MA at 7000 rpm The stirring rate
`was decreased
`to 500 rpm and the temperature of the
`emulsion was increased to 40C to extract and evaporate
`hr The system was cooled
`the organic phase over
`25C and particles were recovered
`on
`pore size
`solvent-resistant membrane filters
`and then dried over
`night under vacuum at room temperature
`The target drug load for the formulations was 12.5%
`ww except
`for higher drug-loaded formulation
`load of 20% slightly higher polymer
`which had
`set
`concentration 18% w/w in CT-12C1
`and 0.27 ratio of
`CH3OH/CHCl2
`were employed for the higher drug load
`tormulation For calcium-chloride containing
`formula
`various amounts Table
`of salt were also
`tions
`dissolved in the CP and the CH3OH component of the
`DP
`
`to
`
`Process Characterization
`
`The mierosphere preparation process was character
`ized by determining mierosphere yield encapsulation ef
`
`271 Madison Avenue New York New York 1001
`
`\1.ro.EL DEkKER INC 11\
`
`

`

`nhaneing Initial
`
`kelease of Peptide
`
`289
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`Table
`
`Manitfoc liiiiog Parameters of Pep/ide-Loaded RGSO3H Microsplierec
`
`Addition qf Porosigen
`
`Calciun Chloride Molar Concentration
`
`Formulation
`
`ID
`
`Methanol
`
`CP
`
`Ratio Methanol/CP
`
`0.268
`
`0.268
`
`0.155
`
`0.117
`
`0.117
`
`0.100
`
`0.060
`
`0.060
`
`0.060
`
`0.045
`
`2.68
`
`4.46
`
`2.58
`
`1.95
`
`2.60
`
`Target drug load for
`
`is 12.5% w/w and
`
`for
`
`Ia 20% w/w
`
`it
`
`total mi
`ficiency and mass balance of peptide The
`yield was
`gravimetrically on the
`calculated
`crosphere
`basis of polymer/drug recovery For the incorporation ef
`in microspheres was ex
`ficiency peptide encapsulated
`load The
`percentage of theoretical
`pressed as
`target
`mass balance of the peptide was obtained as the sum of
`the peptide remaining in the CP unincorporated plus the
`amount incorporated in microspheres
`
`Characterization of Microspheres
`
`Microspheres formulated as above were characterized
`for their drug content bulk density specific surface area
`mean sizes surface morphology in vitro drug release
`and in vivo efficacy
`
`Drug Content
`
`nyvale CA and an autosampler Thenno Separation
`Products Fremont CA The mobile phase was an iso
`cratic mixture of 1-IPLC grade water and acetonitrile in
`the ratio of 68 32 adjusted to pH 4.0 by adding approxi
`mately 0.1% trifluoroaeetie acid The flow rate was set
`at 1.1 mI/mm
`
`Bulk Density
`
`to
`
`The thy mierospheres were quantitatively transferred
`tube The test
`tube was subsequently
`graduated
`test
`tapped 20 times from vertical distance of approximately
`volume was recorded The tap
`0.5 in and the occupied
`ping process was repeated until
`the volume occupied
`by
`particles remained unchanged The final volume was re
`corded as bulk volume
`and the tapped bulk density
`g/cm3 was calculated
`as rn/yb where in was the weight
`of microspheres employed
`
`Specific Surface Area
`
`area was determined using an
`The specific surface
`ASAP 2000 surface
`area analyzer Micromeritics Nor-
`cross GA by BrunauerEmmett Teller BET transfor
`isotherms of Kr on
`mation of the adsorptiondesorption
`The area values were
`the surface of the microspheres
`normalized to the sample weight The sample weight typ
`ically was in the range of 250300 mg
`
`Size Distribution
`
`by shaking
`
`for
`
`hr on wrist-
`
`re
`
`Drug-loaded microspheres were quantitatively dis
`into ace
`solved in CH2CI2 and the peptide was extracted
`tate buffer pH 0.1
`action shaker Burrell Pittsburgh PA The
`aqueous
`and ex
`buffer phase was
`by centrifhgation
`separated
`tracted peptide was quantitated by modification of
`ported reversed-phase HPLC method 23 This extrac
`tion procedure was repeated with fresh buffer and the
`combined peptide amounts from the two extractions were
`reported as the drug content expressed as percent w/w
`of microspheres Triplicate samples were used to deter
`mine the drug content and mean values HPLC analytical
`conditions were as follows ehromatographie separation
`was achieved
`C18 pBondapak column 3.9
`on
`300
`mm Waters by using
`variable-wavelength detector
`at
`220 nm gradient pump both from Dionex Corp Sun-
`
`laser diffraction
`
`particle
`
`Particle size distribution was determined by using
`sizer Malvern Instruments
`
`Southborough MA The microspheres were suspended in
`63-mm for
`0.1% aqueous Tween 80 solution and either
`
`\1.cdusonAveiiue
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`290
`
`Ra\ varapu Lee and DeLuca
`
`100 mm for
`118 jim or
`size range of 0.5
`size range
`188 pm focal
`length lens was employed to deter
`of 1.9
`mine particle size while the sample was stirred at about
`100 rpm in the sample cell using magnetic stirrer bar
`
`calculated
`
`ited stability in the releasing medium Released drug was
`as the difference between initially loaded drug
`and that remaining in the microspheres and expressed as
`loaded amount
`percent of initially
`
`Surface Morphology
`
`In Vivo Evaluation
`
`Particle surface morphology was examined by scan
`ning electron microscopy model S800 Hitachi Japan
`the microsphere sample was
`coated with gold
`after
`palladium on an aluminum stub
`
`In Vitro Drug Release
`
`test
`
`Approximately 10mg of peptide-loaded microspheres
`was incubated with 10 ml of 0.033
`buffer
`phosphate
`pH at 37C in
`temperature-controlled oven Separate
`tubes with equal amounts of microspheres were
`maintained for each time point of the release study Ag
`gregation and settling of microspheres were noticed dur
`tubes were shaken
`twice
`Test
`ing the release
`study
`weekly and ml of supernatant was replaced with fresh
`buffer weekly to maintain sink conditions At the sam
`pling time microspheres were separated by centrifuga
`tion To minimize the loss of microspheres only ml of
`the supernatant was removed and analyzed by HPLC for
`the drug content The total peptide remaining in the mi
`that was left behind
`ml
`and supematant
`crospheres
`was extracted into acetate
`and quantitated by
`HPLC On the basis of the supematant analysis peptide
`in the remaining ml of supernatant was calculated
`and
`accounted
`for in the final calculations of peptide that re
`mained in the microspheres Compared to standards no
`additional peaks were noted in the chromatographs ob
`tained from the analysis of test samples
`release was determined on the basis of the
`Peptide
`drug remaining in the microspheres rather than on the
`released amount of peptide because the peptide has lim
`
`buffer
`
`Male Sprague Dawley rats Harlan Sprague Dawley
`Inc Chicago IL were employed
`in evaluating
`the formulation efficacy in suppressing serum testoster
`one Animals were maintained as per
`the guidelines set
`forth in reference 24 All
`formulations were suspended
`and 2% man
`in mixture of 1% carboxymethylcellulose
`nitol and injected into rats subcutaneously just below the
`based on
`neck region at
`drug dose of 100 jig/kg/day
`literature reports 25
`single injection was given to
`each animal
`immediately after an initial
`sample was col
`lected from the tail vein Additional
`samples were col
`15 25 32 33 42 and 43 days
`On days 32 and 42 all
`after dose administration
`the
`groups were challenged with 100 jig/kg of leuprolide ac
`
`lected at 0.25
`
`etate Following regular sampling additional
`samples
`were taken at
`and 24 hr after the challenge Samples
`were assayed in duplicate for testosterone analysis by ra
`dioimmunoassay using standard commercial kits
`
`RESULTS AND DISCUSSION
`
`Process Characterization
`
`The adapted manufacturing procedure resulted in
`high microsphere yield 8892% high encapsulation ef
`ficiency 7288% and close to 100% peptide mass bal
`ance Leuprolide acetate is highly water soluble and was
`to have low microencapsulation
`expected
`efficiency by
`using the conventional oil/water o/w solvent evapora
`tion method due to rapid partitioning
`into the aqueous
`
`.0
`
`-C
`
`.0
`
`CO
`
`CO
`
`Ct
`
`CO
`
`C-
`
`-c
`
`-o
`
`CO
`
`Characteristics of Peptide-Loaded Microspheres
`
`Effect of Drug Load and Calcium Chloride
`
`Formulation
`
`ID
`
`Drug Content
`%w/w
`
`Encapsulation
`
`Efficiency
`
`Surface Area
`m2/g
`
`Size
`jim 0.5
`
`Bulk Density
`g/cmr
`
`Table
`
`11.9
`
`16.3
`
`9.79
`
`9.08
`
`10.1
`
`10.9
`
`11.0
`
`95.2
`
`81.5
`
`78.3
`
`72.6
`
`80.8
`
`87.2
`
`88.0
`
`0.387
`
`7.278
`
`1.249
`
`1.480
`
`1.023
`
`0.913
`
`0.778
`
`18.0
`
`27.0
`
`24.7
`
`24.2
`
`20.5
`
`28.5
`
`24.9
`
`0.54
`
`0.29
`
`0.48
`
`0.42
`
`0.57
`
`0.56
`
`0.64
`
`Si7c distributi on on
`
`volume bans
`
`tsr
`
`RcEL DoucHe
`ulproii Ar enue New York New rI
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`Enhancing Initial
`
`Release of Pcptidc
`
`291
`
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`Figure
`
`Scanning electron micrographs of RG5O3H microsphercs with different drug loads
`
`w/w
`
`11.9
`
`16.3 at 1500X
`
`phase of the emulsion during the manufacturing process
`26 Instead of using the suggested
`and reported multi
`
`ple-emulsion water/oil/water
`
`cated that
`
`approaches
`
`to
`
`achieve high encapsulation efficiency 727 cosolvent
`system of CH1OH and CH2CI2 was employed as the or
`ganic phase of u/w emulsion The results obtained mdi
`high percentage of initially
`added peptide
`formu
`could be associated with the microspheres for all
`lations produced irrespective of variables Although the
`mechanism of peptidepolymer association is not clear
`it appears that as CH3OH rapidly partitions into the aque
`ous phase or evaporates drug precipitates and remains
`trapped in the polymeric matrix and is inhibited from par
`titioning into the continuous phase by the rapidly forming
`polymeric wall Possible interaction between peptide and
`polymer however was not evaluated
`in this study This
`for scaleup and is suitable for en
`is amenable
`procedure
`into microspheres with high
`capsulating similar peptides
`
`efficiency
`
`In Vitro Characteriation of Peptide
`Loaded Microspheres
`
`Effect of Drug Load
`
`Two microsphere batches with different drug target
`12.5%
`20.0% were prepared
`loads
`to evaluate
`the effect of drug load on microsphere characteristics
`The physicochemical properties of these formulations are
`listed in Table
`The results indicate that
`loading the
`microspheres with higher amounts of peptide
`tially increased the specific surface area and decreased
`These micro
`the bulk density of
`the microspheres
`spheres examined under scanning
`electron microscope
`
`substan
`
`at
`
`tained
`
`magnification of l500X Fig
`possessed higher
`porosity relative to lower drug-loaded micro-
`surface
`spheres When the microsphere field was examined at
`lower magnifications 40X and 200x data not shown
`con
`the higher drug-loaded microsphere formulation
`certain amuunt of norispherical and amurphous
`like material which could be polymeric debris and/or
`prematurely formed polymeric particles Coupled with
`feature of higher
`increased surface porosity this process
`drug-loaded microspheres possibly contributed to the ob
`served increase in specific surface area Another contrib
`uting factor could be an increase in internal microsphere
`porosity No attempt to fracture and examine the internal
`structure of these microspheres was made because of
`their small size Table
`
`120
`
`iR
`
`.4
`
`20
`
`aIS 3%
`ali 9%A
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`lime day
`
`In vitro peptide release from RG5O3H micro-
`Figure
`spheres Effect of drug load
`
`C-
`
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`
`

`

`292
`
`Ravivarapu
`
`Lee and DeLuca
`
`IIll
`ORDER jjj
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`The assumption that higher drug-loaded microspheres
`space was supported by
`increased internal porosity/void
`the bulk density data presented
`in Table
`Smaller bulk
`density values of high drug-loaded microspberes suggest
`higher number of hollow or more po
`the formation of
`rous mierospheres Another possible contribution
`less-than-optimum packing of nonspherical particles
`area porosity and de
`Because
`increased
`surface
`bulk density should increase
`drug release
`rate of peptide release was expected
`from higher
`higher
`drug-loaded mierospheres With higher drug load more
`of the drug is available at
`the surface of mierospheres
`release The cu
`which should also lead to higher initial
`rnulative release profiles of these two formulations
`shown in Fig
`discernible difference in the released
`amount of peptide was observed
`release study Increased drug load provides interconnect-
`
`creased
`
`is
`
`are
`
`at each time point of the
`
`greater poros
`ne channels in the particle structure and
`ity for penetration of water into and peptide leaching out
`of the matrix Complete release of drug was obtained by
`49 days for both formulations
`
`Effect of Calcium Chloride
`
`release of pep-
`Another approach to increase the initial
`tide from the mierospheres is to increase the porosity
`and internal by addition of porosigenie
`both surface
`materials such as water-soluble calcium chloride and so
`dium chloride salts Juni et al observed
`increased poros
`and bleomycin release from polylaetie acid micro-
`esters 28
`due to the addition of
`spheres
`fatty acid
`Recently Herrmann and Bodmeier studied the effects of
`adding salts calcium or sodium chloride to the internal
`and external aqueous phases of w/o/w emulsion on so-
`
`ity
`
`Figure
`
`Scanning electron micrographs of RGSO3H microspheres
`phases were
`none
`methanol/continuous
`0.268/0.1
`0.268/0.06
`
`1500
`
`Effect of calcium chloride Molar salt concentrations
`0.117/0.045 Magnification
`
`0.155/0.06
`
`0.117/0.06
`
`in
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`Enhancing Initial Release of Peptidc
`
`293
`
`rnatostatin release
`
`125
`
`100
`
`lime days
`
`Figure
`
`In vitro peptide release from RG503H micro-
`spheres Effect of calcium chloride Molar salt concentrations
`phases were
`0.155/0.06
`
`in methanol/continuous
`
`0.268/0.06
`
`none
`
`0.117/0.06
`
`0.268/0.1
`0.117/0.045
`
`from polylactic
`acid microspheres
`29 and showed that varied particle structures and modi
`fied drug release could be obtained through variations in
`In comparison to the Herrmann
`the salt concentrations
`and Bodmeier study the present study assessed the addi
`tion of salt to both aqueous and organic phases and the
`particles were prepared from PLGA by an o/w solvent-
`extraction evaporation method in contrast
`to prepara
`tion of polylactic
`w/o/w emulsion
`system
`The physicochemical
`properties of the microsphere
`formulations prepared in the absence and presence of salt
`at various concentrations are shown in Table
`Calcium
`salt was
`
`acid particles using
`
`chloride
`
`readily
`
`soluble in methanol
`
`and
`
`in methy
`methanol/methylene
`chloride mixture but not
`lene chloride alone As an initial
`attempt salt 0.46
`was added only to the CH3OH phase of the DP However
`the drug content of these microspheres was very low at
`1.5% Because of the unacceptable
`low drug load these
`
`15
`
`13
`
`11
`
`Formulation
`
`Formulation
`
`high drug load
`
`Baseline
`
`C3
`
`59
`
`15
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`10
`
`15
`
`20
`
`25
`
`30
`
`40
`
`45
`
`Time days
`
`Figure
`
`In vivo testosterone suppression with RG5O3H microspheres Effeci of drug load The inset shows the data in the extended
`range and the upward pointing arrows along the x-axis represent the challenge
`
`MAnUeL DuKes INC
`270 Muiisoo Avenue New York New York 1001
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`Ii
`
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`

`294
`
`Ravivarapu
`
`Lee and DeLuea
`
`ORDER
`
`REPRINTS
`
`These
`
`trations were varied in the CP and DP Table
`increases are consistent with the scanning electron photo
`which illustrate increased porosity
`mierographs in Fig
`and surface texture of formulations prepared in the pres
`For the calcium chloride
`ence of salt
`versus
`formulations as the microspheres became more porous
`and ex
`the surface area increased drug load decreased
`rate of initial
`drug
`
`trapolating this correlation
`
`higher
`
`release occurred
`
`Figure
`
`shows the in vitro cumulative release of pep-
`tide from these fomniulations The formulations seemed
`
`for
`
`the initial
`
`re
`to have similar release profiles except
`lease This particular characteristic may be advantageous
`because it
`release alone and not
`implies that diffusional
`be controlled by this formulation
`release can
`erosional
`approach As the porosity and surface area increase due
`to an osmotic gradient
`in favor of the organic phase the
`peptide release also increases Note that
`in Table
`the concentration ratio of salt in CH3OH and the CP
`was similar for some of the fonnulations
`however
`the
`
`initial
`
`characteristics including in vitro release profiles were
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`niicrospheres were not characterized further Apparently
`the osmotic gradient created with salt in the organic phase
`promoted water influx from the outer aqueous phase re
`sulting in very porous microspheres with low drug load
`As
`formulations were modified to
`
`result subsequent
`have
`lower osmotic gradient favoring the organic phase
`by incorporating salt in the CP also These modifications
`led to an increase in the drug loads which were close to
`that of formulations without calcium chloride The effect
`
`of salt gradient could be correlated with the drug content
`values For example at
`fixed salt concentration in the
`CP as the concentration in the DP decreased from 0.268
`to 0.117
`and
`Tables
`as for formulations
`an increase in the drug load was achieved
`and
`No major changes in the particle size and bulk density
`values were observed even when salt concentrations were
`varied Mean particle size of these microspheres was in
`range of 18 25 jim suitable size for convenient
`injections However notice
`subcutaneous/intramuscular
`areas were ob
`able differences in the specific surface
`because salt was added and salt concen
`served Table
`
`11
`
`Formulation
`
`.Formulation
`
`nFormulation
`
`Baseline
`
`01
`
`01
`
`In
`
`01
`
`Cl
`
`1J
`
`10
`
`15
`
`20
`
`25
`
`30
`
`40
`
`45
`
`Time days
`
`Figure
`
`In vivo testosterone suppression with calcium chloride formulations The inset shows the data in the extended range and
`
`the upward pointing arrows along the x-axis represent
`
`the challenge
`
`so
`
`270
`
`tadisoll As eoue Ness York
`
`York 11016
`
`MAISCELDEI
`
`hER
`
`

`

`Enhancina Initial
`
`Release of Peptvle
`
`29
`
`lHl
`
`ORDER
`
`REPRINTS
`
`jJJ
`
`very dissimilar From this observation it appears that
`the physical amount of cal
`addition to osmotic gradient
`cium chloride present
`in the aqueous and organic phases
`plays an important
`role in modifying mierosphere proper
`the higher the amount
`the higher the ini
`ties In general
`
`in
`
`tial
`
`release
`
`In Vivo Evaluation
`
`Figures
`
`and
`
`show serum testosterone profiles after
`hr val
`formulation administration
`The average of the
`ues was considered
`to be the baseline Only descriptive
`statistics mean SD were employed in comparing the
`efficacy Leuprolide acetate being an LHRH super ago
`flare-up as expected The
`nist caused initial
`with all of the formu
`levels however declined by day
`
`testosterone
`
`lations
`
`the effect of drug load on efficacy
`Figure
`compares
`Even though the levels were similar by day 15 suppres
`sion earlier to that was faster for the higher drug-loaded
`formulation
`This possibly is due to the higher release
`that was seen
`in vitro assuming similar trends in vivo
`the calcium chloride formulations
`
`Figure
`
`were chosen based on in vitro
`As discussed earlier the in vitro
`
`compares
`Formulations
`and
`release profiles Fig
`release profiles of these formulations were similar except
`for the initial burst Of the three considered formulations
`formulation
`had the lowest burst Accordingly testos
`terone suppression with this formulation was the slowest
`leuprolide re
`However
`no direct correlation between
`lease in vitro and in vivo could be made because leupro
`lide serum levels were not measured When the testoster
`one levels were measured
`
`as
`
`indicator
`
`pharmacological activity thus minimizing the undesired
`lag phase often seen with these macromolecules
`
`REFERENCES
`
`Lee Biopharmaeeutieal properties and pharmaeoki
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`Amidon eds American
`DrugDesign
`Taylor and
`Chemical Society Washington DC 1995 pp 69 97
`Sanders Dmg delivery systems and routes of ad
`ministration of peptide and protein drugs Eur
`Drug
`152 95
`102 1990
`Metab Pharmacokinct
`Hora
`Rana
`Tafarn
`
`Nunherg
`
`Gilley and
`
`Hudson Develop
`
`Tice
`ment of
`controlled release microspheres formulation of
`interleukin-2 Proc mt Symp Controlled Release Bio
`act Mater 16 509510 1989
`The pharmaceutics and delivery of thera
`Talmadge
`and proteins Adv Drug Del Rev
`
`peutic polypeptides
`10 247299 1993
`Okada
`and
`Toguchi Biodegradable microspheres
`in drug delivery Crit Re Ther Drug Carder Sys
`121 199 1995
`Ogawa and
`Heya
`Okada
`Toguehi
`In vitro
`and
`releasing hor
`in vivo evaluation of thyrotrophin
`mone release from copolydl-Iactic/glycolic acid micro
`Pharm Sci 835 636
`640 1994
`spheres
`Cohen
`Hwang and
`Yoshioka
`Lucarelli
`Langer Controlled delivery systems for proteins based
`on polylactic/glycolic acid microspheres Pharm Res
`86 713
`720 1991
`Mchta
`
`Burton and
`
`Thanoo
`Calis
`Jeyanthi
`DeLuca Biodegradable microspheres
`as depot system for parenteral delivery of peptide drugs
`Controlled Release 29 375384 1994
`Okada
`Doken
`Ogawa and
`Toguehi Prepa
`ration of three month depot
`injectable microspheres of
`Icuprorelin acetate using biodegradable polymers Pharm
`Res 118 1143
`1147 1994
`Asch
`Bartke
`Rojas
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`Sehally
`
`Tiee
`
`drol
`
`and
`
`Kiemeke
`Bray and
`Silerkhodr
`Hogan Prolonged suppression of plasma LH levels
`single injection of an LHRH agonist
`in male rats after
`An
`in polydl-lactide-co-glycolide microeapsules
`8388 1985
`Walker
`Turkes
`Buck
`cock
`Peeling and
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`slow-
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`formulation
`of
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`118630 Zoladex
`Endocrinol
`Cutwright
`Perez
`Beasley
`Larson
`Posey Degradation of polylactic
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`Zwink
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`Bea
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`Griffiths Treatment
`
`tCl
`
`10
`
`11
`
`12
`
`270 \tadison
`
`MACSOLDErER tNt
`0016
`Avenac New York NewYori
`
`.0
`
`.0
`
`to
`
`C-
`
`pharmacological
`of leuprolide released in vivo the levels did not differen
`tiate the efficacy of the calcium chloride formulations
`There were however notable differences in in vitro drug
`release More importantly as compared to formulation
`Fig
`formulations with calcium chloride showed
`suppression and met the objec
`faster rate of testosterone
`tive of providing increased
`release of peptide
`similar observation could be made with the higher drug-
`loaded formulation An unstated objective of this study
`was to maintain the testosterone
`suppression for at
`least
`1-month period This objective was met with both
`higher drug and calcium chloride formulations
`In conclusion increasing drug load and adding poro
`sigen modified the carrier characteristics by increasing
`porosity and specific surface area of PLOA mierospheres
`This enhanced initial
`drug release due to increased/rapid
`hydration of carrier particles and provided faster onset of
`
`initial
`
`

`

`lIl
`
`ORDER
`
`e_
`
`REPRINTS
`
`jJJ
`
`Ravivarapu
`
`Lee and DeLuea
`
`Kanke
`DeLuca
`Schroeder
`Sato and
`Porous mierospheres for drug delivery and methods for
`making same U.S Patent 4818 542 1990
`Ogawa
`Yamamoto
`Okada
`Yashiki and
`Shimamoto
`new technique to efficiently
`entrap lee
`acid or
`into mierospheres of polylactic
`acid Chem Pharm Bull
`
`prolide acetate
`
`296
`
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`
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`
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`
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`
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`
`18
`
`19
`
`20
`
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`Park Degradation of polydl-lactic acid micro
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`173 1994
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`Brieh
`Laneranjan
`Bantle
`Vit Parenteral dcpot systems on thc basis of
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`biodegradable polyesters
`
`Kissel
`
`fall
`
`and
`
`42 199
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`Traeehslin Factors influ
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`gradable parcntcral dcpot systcms
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`rcvicw tnt
`
`critical
`
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`sys
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`tcms
`Sah
`Chicn Thc
`Toddywala and
`influence
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`trolled iclease of
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`Pitt PoIyglyeolie aeid-eo
`dI-tactie acid Diffusion or degradation controlled drug
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`Hadgraft and
`from polymers in Polymers in Controlled Drug Delivery
`Davis eds Wright Bristol 1987
`IlIum and
`pp 99I 16
`Heya
`Ogawa
`Toguehi Effects of counterion of TRI-1 and loading
`amount on control of TRH release from eopolydl-lactie/
`glyeolie acid mierospheres prepared by an in-water dry
`ing method mt
`75 1991
`Pharm 69 69
`Cohen
`Alanso
`Park
`Gupta
`Siber and
`Langer Determinants of release rate of teta
`from polyester mierospheres Pharm Res
`953 1993
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`
`protein
`
`Okada
`
`Tanigawara
`
`and
`
`nus vaccine
`
`107 945
`
`22
`
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`
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`
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`27
`
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`
`29
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`eopolylaetic/glyeolic
`1103 1988
`1095
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`DHEW Pub No 78-23 revised National
`Institutes of
`Health
`Okada
`
`lnoue
`
`Ueno
`
`Heya
`Ogawa and
`Toguehi
`Pharmacokineties
`of once-a-month
`jeetable mierospheres of leuprolide acetate Pharm Res
`86 787791 1991
`Bodmeier and
`
`in
`
`MeGinity Polylactie acid micro-
`
`spheres containing quinidine base and quinidine sulfate
`prepared by the solvent evaporation technique Methods
`279288 1987
`and morphology Mieroeneap
`MeGinity Preparation of multiphase
`iwata and
`
`mierospheres of polydl-laetie
`
`eo-glyeolie acid containing
`
`acid and polydl
`w/o emulsion by multiple
`
`lactic
`
`Mieroeneap
`
`emulsion solvent evaporation technique
`201214 1992
`Nakano
`Juni
`Matsui
`Kubota and
`Ogata
`Control of release rate of bleomycin from polylaetie acid
`mierospheres by additives Chem Pharm Bull 334
`16091614 1985
`Herrmann
`and
`
`Bodmeier The effect of particle
`on the somatostatin release
`from poly
`mierostrueture
`laetide mierospheres prepared by w/o/w solvent evap
`36 63
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`Controlled Release
`71
`1995
`
`no
`
`270 \l.idton Avenue
`
`MARLO DEI.KEs INt
`New York \..rn Yrk Dolt
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