Journal of Biomaterials Science Polymer Edition
`
`TaylorFrancis
`ThyIoFMc
`
`ISSN 0920-5063 Print 1568-5624 Online Journal homepage httpi//www.tandfonline.com/loi/tbsp2o
`
`Extended release peptide delivery systems
`through the use of PLGA microsphere
`combinations
`
`Burton
`
`Shameem
`
`Thanoo
`
`Deluca
`
`Deluca 2000 Extended
`Shameem
`Burton
`Thanoo
`To cite this article
`release peptide delivery systems through the use of PLGA microsphere combinations journal
`of Biomaterials Science Polymer Edition 117 71 5-729 DO 10.1163/156856200743977
`
`To link to this article http//dx.doi.org/10.1163/156856200743977
`
`Published online 02 Apr 2012
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`

`Biomater Sci PolymerEdn Vol 11 No
`VSP 2000
`
`pp 715729 2000
`
`Extended release peptide delivery systems through the use
`of PLGA microsphere combinations
`
`THANOO2 and
`SHAMEEM1
`BURTON
`Purdue Pharma L.P 444 Saw Mill River Road Ardsley NY 10502 USA
`Oakwood Laboratories 7670 First Place Oakwood OH 44146 USA
`University of Kentucky College of Pharmacy Rose Street Rm 327G Lexington KY 40535 0082
`
`DeLUCA3
`
`Received 26 July 1999 accepted
`
`February 2000
`
`AbstractThe purpose of this study was to evaluate the utility of combining polymer matrices to
`overcome extended lag periods or unacceptably
`short durations of action intrinsic in the individual
`polymer systems Leuprolide an LHRH superagonist was incorporated into variety of polylactide
`co-glycolide PLGA matrices using
`method The in vitro release
`solvent extraction/evaporation
`of Leuprolide from these matrices was evaluated at pH 7.0 and 37C in phosphate buffer The
`formulations were administered to an animal model at
`doses and serum testosterone
`or mg kg
`levels were followed using RIA method
`two-part system was made by combining microspheres
`25 acid terminated PLGA and microspheres made from 75
`made from 75
`25 ester terminated
`PLGA This combination elicited chemical
`castration from 10
`three-part combination
`100 days
`25 PLGA formulation an ester terminated 50 50 PLGA
`composed of an ester terminated 75
`formulation and an acid terminated 50 50 PLGA formulation also provided
`composite profile with
`duration of 100 days Additionally
`an onset of 10 days and
`single polymer system composed
`25 PLGA was employed to produce release over the
`of
`high molecular weight ester terminated 75
`desired 90-day release period This study demonstrates that microsphere combinations can potentially
`provide effective therapies over extended intervals when combined at the proper ratio
`
`-c
`
`Key words Peptide delivery sustained release microspheres polylactide-co-glycolide
`superagonist
`leuprolide
`
`LHRH
`
`INTRODUCTION
`
`Delivery of highly potent peptides and proteins pose some interesting challenges for
`the pharmaceutical scientist Low bioavailability and in vivo stability often preclude
`formulation For certain clinical applications
`conventional
`delivery system that
`peptide for longer than 30 days would provide
`can ensure continuous release of
`
`To whom correspondence should be addressed E-mail ppdelul @pop.uky.edu
`
`

`

`716
`
`WBurtonetal
`
`lEt
`
`is
`
`convenient and efficacious
`system for delivery of these compounds
`90-
`day duration provides for administration only four times per year providing better
`patient compliance Further from pharmacoeconomic
`90-day dosage
`standpoint
`form could provide
`less expensive alternative to daily or monthly injections
`Aliphatic polyesters such as polylactide-co-glycolide PLGA are biocompat
`able and biodegradable
`and are therefore good candidates for
`controlled delivery
`However because
`these polymers release higher molecular weight
`system
`drugs like peptides primarily by an erosional mechanism there exists the possibility
`lag phase between the diffusional controlled release and the erosional controlled
`of
`challenge for providing continuous release over the
`This presents
`release
`entire release period The lag phase of individual systems could be addressed by
`13 In this way the
`combining systems produced from different polymers
`desired extended continuous release periods could be achieved
`Leuprolide acetate when released in
`continuous manner significantly reduces
`serum testosterone levels which has implications in the treatment of several dis
`eases such as prostate cancer precocious puberty endometriosis and mammary
`nine amino acid peptide with molecular weight of -1200 Da
`cancer
`or no tertiary structure making it
`good candidate for incorpora
`having little
`tion into these PLGA systems Leuprolide is
`LHRH superagonist exploiting the
`pituitarytesticular axis to decrease testosterone levels Under the normal physiol
`burst of LHRH is released from the hypothalamus and travels to the pituitary
`ogy
`gonadotrope This stimulates the go
`where it binds
`receptor on the surface of
`nadotrope to release its store of gonadotropin LH FSH as
`burst and to begin
`LH binds
`synthesis of more gonadotropins
`receptor on the Leydig cells caus
`ing testosterone production and release When LHRH is released in
`continuous
`is down regulated
`saturate and the cell
`manner
`the receptors on the gonadotrope
`resulting in release of smaller amounts of less active gonadotropin This culminates
`in less stimulation of the Leydig cells and lower testosterone levels Superagonists
`like Leuprolide have greater serum stability and higher binding affinity for the go-
`nadotrope when compared to native LHRH
`The objective of this study was to evaluate the feasibility of combining mi
`90 day testosterone suppression profile using Le
`crosphere systems to produce
`uprolide incorporated into PLGA polymers of varying end groups monomer ratios
`and molecular weights In order to produce combinations capable of providing the
`desired 90 day efficacy many polymer systems were individually evaluated in vitro
`and in vivo Based on the data obtained from the individual systems several can
`didates having either prolonged lag phases or short durations were identified for
`further study in the form of microsphere combinations These microsphere combi
`nations were subsequently
`tested for in vivo suppression of testosterone
`
`

`

`Extended release pep tide delivery systems
`
`717
`
`MATERIALS AND METHODS
`
`Matrix selection
`
`The matrices selected for evaluation in the form of microsphere combinations were
`based on the screening of seventeen systems Each individual system was evaluated
`for in vitro release and in vivo efficacy Systems which showed shortened duration
`or extended lag which could be compensated by combining with other systems
`release and efficacy profiles Five candidate
`were identified based on the individual
`for combination to produce testosterone suppression
`
`formulations were selected
`through 90 days
`
`Polymer characterization
`Polymers obtained from Boerhinger Ingelhiem Ridgefield CT USA were used
`the Leuprolide loaded PLGA microspheres
`The PLGAs
`in the preparation of
`were characterized for acid number weight and number average molecular weight
`number of techniques
`polydispersity thermal transitions and cloud points using
`scanning calorimetry and
`including gel permeation chromatography differential
`The polymer properties are shown in Table
`
`titration
`
`Microsphere preparation
`Leuprolide acetate Bachem Inc Torrance CA USA was encapsulated
`18 The
`method
`solvent extraction/evaporation
`the various polymers by
`The
`polymer was dissolved in dichloromethane and the Leuprolide in methanol
`two solutions were then combined to provide the dispersed phase DP The DP
`was added to an aqueous continuous phase CP containing 0.10.35% polyvinyl
`alcohol under rapid stirring After the microspheres had partially solidified due
`the CP was increased to 40C for
`to solvent extraction the temperature of
`to remove the remaining solvent and to ensure
`low residual solvent content
`
`into
`
`i.e
`
`Table
`
`Polymer properties
`
`Polymer
`
`ID
`
`RG756
`RG752
`RG7525H
`RG5O3H
`RG502
`
`Ratio
`
`LAGA
`
`7525
`7525
`7525
`5050
`5050
`
`Molecular weight
`
`95285
`15577
`
`11161
`
`28022
`10754
`
`57373
`
`6541
`
`5062
`13233
`
`5014
`
`PD
`
`1.66
`
`2.38
`
`2.21
`
`2.12
`
`2.15
`
`Tga
`
`Acid
`
`number1
`
`Cloud
`
`pointC
`
`45.3
`
`42.4
`
`44.5
`
`46.2
`
`39.8
`
`0.3
`
`1.1
`
`14.3
`
`4.6
`
`0.9
`
`32.7
`
`57.6
`
`69.6
`
`28.0
`
`35.6
`
`2nd heating
`KOH/g PLGA
`bTitration
`CTitrationml CH3OH/g PLGA
`
`

`

`718
`
`Wfiurtonetal
`
`100 ppm The temperature of the CP was reduced to 25C and the microspheres
`using an 8-4um Miflipore SC filter
`The recovered
`were isolated by filtration
`vacuum oven at RT overnight and subsequently stored
`microspheres were dried in
`desiccator at 5C
`in
`
`Microsphere characterization
`
`HPLC method for determination of Leuprolide
`HPLC was used for the de
`Dionex A53500
`termination of Leuprolide The chromatography system was
`quaternary gradient pump an autosampler
`Chromatography system consisting of
`an Advanced Computer Interface
`Variable Wavelength Detector and the Ai-450
`Chromatography software all by Dionex Sunnyvale CA USA and
`Bondapak
`3.6 mm column with
`3.9 mm guard
`C18 300
`10 C18 Guard 30
`Bondclone
`column from Phenomenex Torrence CA USA Detection was at 220 nm Sam
`pie concentrations were determined relative to
`Leuprolide standard curve The
`mobile phase consisted of Miili-Q purified water Waters Milford MA USA and
`ratio of 6832 v/v with 0.1% Tn
`HPLC grade Acetonitrile Fisher Scientific at
`flouroacetic acid Fisher as an ion pairing agent The mobile phase was degassed
`by helium purge 10 mm and the reservoir was kept under slight pressure less than
`mm Hg with helium The flow rate was 1.1 ml mm
`.The injection volume was
`for the release media The total run
`30 4ul for the extraction samples and 100
`time was 8.0 mm
`
`Ten mg of the
`Drug content
`Drug content was accomplished by extraction
`tube The matrix
`microspheres were quantitatively transferred to
`12-ml glass test
`pH 4.0 acetate buffer
`was solubilized in ml of dichloromethane 10 ml of 0.1
`added and the tubes were either agitated by
`wrist action shaker or rotated on
`wheel
`Samples were centrifuged
`and the aqueous layer was analyzed
`for
`by HPLC
`second extraction with 10 ml of acetate buffer was made to ensure
`complete extraction and effect mass balance
`
`In vitro studies
`
`phosphate buffei pH 7.0
`In vitro release studies were conducted in 10 ml of 0.03
`incubated at 37C Individual samples of approximately 10 mg were transferred
`to screw capped glass tubes for each assay point and the tubes placed on
`tube
`rotator 18 rpm Because Leuprolide is unstable in the release media the drug
`release was based on extractable drug from the microspheres relative to time zero
`mm and
`To accomplish extraction the tubes were centrifuged
`at 3000 rpm for
`ml of release media were removed Extraction of the separated microspheres was
`accomplished by adding ml of dichioromethane to the release tubes and vortexing
`pH 4.0 acetate buffer were added and the
`to solubilize the matrix Eight ml of 0.1
`tubes were agitated on wrist action shaker for
`The samples were then briefly
`centrifuged ml of the aqueous layer was removed and 100
`were analyzed by
`
`

`

`Extended release peptide delivery systems
`
`719
`
`HPLC
`second extraction with ml of acetate buffer was made to ensure complete
`extraction The average of duplicate samples was plotted
`
`In vivo studies
`
`Experimental animals were SpragueDawley rats ob
`Animal experiments
`tained from Harlan Inc Indianapolis IN USA averaging 175200
`when pur
`chased The animals were allowed to acclimate for
`days prior to any treatment
`The animals were lightly anesthetized with anesthetic grade diethyl ether Fisher
`Scientific and
`baseline blood sample was taken from the tail
`prior to injection
`with the appropriate formulation The microspheres were suspended in
`1% w/w carboxymethyl cellulose Aqualon Wilmington DE USA
`containing
`and 2% w/w Mannitol Fisher Scientific and injected at
`body weight normal
`ized volume ml injection volume equal to
`in the dorsal medial
`kg body weight
`area of the animal The dose was equivalent
`to 3.0 mg Leuprolidekg
`body weight
`unless otherwise noted Subsequently samples were taken at specified intervals
`by lightly anesthetizing the animal with ether and removing 600 800 tl of blood
`from the tail Blood samples were collected in Microtainer brand serum separator
`tubes Fisher Scientific and allowed to clot These samples were then centrifuged
`at 10000 rpm for 35 mm and the serum decanted into 1.5 ml polypropylene tubes
`non-cycling freezer at 20C until analysis All formulation groups
`and stored in
`contained at least three animals
`
`vehicle
`
`cci
`
`Challenge injections of free Leuprolide
`
`In order to check the effectiveness of
`
`dose of 100 ptg kg was administered at
`the formulation Leuprolide at
`ml injection volume equaled
`weight normalized volume
`kg body weight at
`designated times during the study The Leuprolide injection vehicle was 0.1
`phosphate buffered saline The concentration was confirmed to be 100 pg ml
`HPLC
`
`body
`
`by
`
`com
`Serum testosterone concentration was measured by
`Testosterone assay
`from Diagnostic Systems
`mercially available coated-tube radioimmunoassay kit
`Laboratories Inc Webster TX USA Fifty jil of the serum samples standards
`and controls testosterone spiked serum were added in duplicate to the antibody
`anti-testosterone coated RIA tubes Next 500 /Ll of 251-labeled testosterone sup
`plied was added to each tube The tubes were gently mixed and incubated in
`37C water bath for 6070 mm The tubes were then aspirated and the radioac
`tivity of the tube bound 1251-labeled testosterone measured by MINIAXIa gamma
`counter Packard Downers Grove IL USA The serum testosterone levels were
`then calculated from standard curve produced at each assay The detection limit
`of the kit was 0.1 ngml
`
`

`

`720
`
`Burton et al
`
`RESULTS AND DISCUSSION
`
`Testosterone suppression in untreated animals
`
`The normal
`
`testosterone serum concentration from six untreated animals over the
`The dotted line represents the average zero
`course of 120 days is shown in Fig
`baseline reference The testosterone
`time values of 165 animals and is included as
`the baseline with means ranging from 0.8 to 3.5 ng ml
`about
`levels fluctuate
`The standard deviations were large as the animals showed significant variation in
`The arrows on the abscissa
`readings as one might expect
`individual
`indicate
`challenge injections with 100 ig Leuprolidekg
`Testosterone levels increase to
`1012 ng m11 by
`normal response to the challenge
`after challenge indicating
`
`Single part system
`
`The in vitro release of the peptide from the high molecular weight 75 25 ester
`system is shown in Fig
`Release occurred
`terminated polymer Polymer
`release
`14 days indicating substantial diffusional
`very rapidly through the first
`steady rate until 84 days when the final sample
`release continued at
`Thereafter
`revealed 70% release This is to be expected from this polymer system as the
`high molecular weight polymer degrades slowly over
`the period releasing the
`release of surface bound peptide
`peptide primarily by erosion with some diffusional
`occurring in the early stages of release
`In vivo release of the Leuprolide from polymer
`microspheres administered
`dose of 3.0 mgkg Fig 3a demonstrated suppression from to 20 days
`at
`corresponding to the in vitro diffusional period This was followed by
`period
`of marginal suppression from 20 to 50 days During this period it
`is likely that
`
`14
`
`12
`
`10
`
`c6
`
`24
`
`I-
`
`10
`
`20
`
`30
`
`50
`
`601
`
`70
`
`180
`
`Time Days
`
`Figure
`
`Testosterone levels of untreated controls Arrows represent challenge injections
`
`

`

`Extended release peptide delivery systems
`
`721
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`a240
`
`30
`
`20
`
`10
`
`14
`
`21
`
`28
`
`35
`
`42
`
`49
`
`56
`
`63
`
`70
`
`77
`
`84
`
`91
`
`98
`
`Time Days
`
`Figure
`
`In vitro release Polymer microspheres
`
`release was insufficient
`the erosionally controlled diffusional
`to produce effective
`castration but was enough to produce some attenuated suppression as indicated
`testosterone fluctuation below baseline testosterone levels From
`by the decreased
`50 to 100 days testosterone was again completely suppressed as peptide release
`The challenge at 100 days resulted in
`due to erosion of the polymer increased
`deflection indicating cessation of suppression as predicted by the in vitro release
`profile Fig 3a
`Based on the suppression profile of Polymer
`3.0 mg kg
`microspheres
`dose it was hypothesized that by simply increasing the dose the marginal release
`during the lag period from 20 to 50 days might be enough to prolong suppression
`the second phase of erosionally controlled release could begin The suppres
`until
`dose of 9.0 mg Leuprolide kg
`body weight
`is demonstrated
`sion profile after
`in Fig 3b The testosterone levels were suppressed below the 0.5 ng ml
`level
`Leuprolide at 41 63
`before day 10 until day 98 Challenges with 100 ptgkg
`76 and 90 days resulted in no increase in serum testosterone levels indicating full
`down regulation of the LHRH receptor sites Testosterone elevation following the
`100 day challenge occurred and was progressively higher at
`the 110 and 120 day
`normal response as more LHRH receptors
`challenges indicating
`progression to
`became available This dose allowed for continuous testosterone suppression for 90
`single polymer
`
`days using
`
`at
`
`Two-part systems
`
`and
`systems is shown in Fig
`The in vitro release from Polymer
`Polymer
`15 577 75 25 ester terminated PLGA shows an initial
`lower molecular weight
`release of 20% in
`day and about 25% within the first
`days This was followed
`
`

`

`722
`
`Burton et al
`
`3.5
`
`2.5
`
`1.5
`
`05
`go
`24
`
`C-
`
`9.0 mg/kg dose
`
`rd
`
`44
`
`.4
`
`10
`
`20
`
`30
`
`4d
`
`50
`
`60t
`
`70
`
`t80
`
`9t1
`
`idb ut
`
`io
`
`Time Days
`Testosterone levels Polymer microspheresa 3.0 mg kg doseb 9.0 mg kg
`
`dose
`
`Figure
`
`by an extended period 14 days of little or no release Between 21 and 80 days
`to the erosion of the polymer occurred
`the third phase of release corresponding
`between
`and 28
`To address the initial
`lag period demonstrated by polymer
`release system could be used One faster releasing candidate would
`days
`faster
`be microspheres composed of Polymer
`carboxylic acid terminated
`75 25 PLGA The in vitro release from the Polymer
`system Fig shows an ini
`day followed by gradual release through 28 days of 25%
`tial burst of 10% after
`After 28 days release increased in an exponential manner and at 63 days 80% re
`leased The more gradual
`release between
`and 28 days effectively eliminates the
`lag period exhibited in the in vitro release profile shown by polymer microspheres
`In order to combine the microspheres
`proper ratio to provide the desired
`efficacy the testosterone suppression profile of the individual microsphere systems
`is shown in
`in vivo suppression from Polymer
`were assessed
`microspheres
`testosterone spike immediately following injection
`After the expected initial
`
`Fig
`
`at
`
`

`

`Extended release peptide delivery systems
`
`723
`
`100
`
`90
`
`80
`
`70
`
`60
`
`Cs
`
`g40
`
`30
`
`20
`
`10
`
`14
`
`21
`
`28
`
`35
`
`42
`
`49
`
`56
`
`63
`
`70
`
`77
`
`84
`
`Time Days
`
`Figure
`
`In vitro release Polymer
`
`and Polymer microspheres
`
`4-
`
`3.5
`
`Cl
`.E 2.5
`
`0l
`
`ti
`
`1.5
`
`0.5
`
`00
`
`10
`
`20
`
`30
`
`40
`
`601
`Time Days
`
`70
`
`80
`
`sot
`
`100
`
`1110
`
`Figure
`
`Testosterone levels Polymer microspheres
`
`incomplete suppression was evidenced through -36 days However
`testosterone
`-P40 days and challenges at 51 and 64 days
`levels reached castration levels after
`indicating continuation of suppression and further suggesting that
`were uneventful
`the LHRH receptor sites were down regulated
`challenge at 92 days produced
`slight elevation indicating the early stages of cessation that was fully demonstrated
`by 107 days when
`normal response
`challenge resulted in
`significant gap in
`to the lag between diffusion and
`the suppression profile is observed corresponding
`erosion release from the Polymer matrix This suppression profile was predicted
`
`

`

`724
`
`Burton et al
`
`3.5
`
`12
`
`Figure
`
`Testosterone levels Polymer microspheres
`
`Time Days
`
`spike in the testosterone levels was caused by
`by the in vitro release The initial
`release of the peptide from the polymer However because
`the initial
`release was not sustained as evidenced by the lag period in the in vitro profile the
`recovered before depletion and normal
`testosterone variations were
`gonadotrope
`noted With the onset of erosional release the peptide began to release in more
`continuous manner resulting in testosterone suppression that was sustained through
`100 days While this profile would not provide full suppression through 100 days
`release formulation
`the possibility of combining these microspheres with
`faster
`may enable suppression through 100 days
`In vivo suppression profile after administration of Polymer
`microspheres
`remained
`days where it
`Suppression occurred within
`demonstrated by Fig
`through 71 days surviving challenges at 25 32 36 43 and 48 days The challenge
`However
`ng ml
`at 71 days produced
`in the
`slight elevation to nearly
`unchallenged state levels were 0.4 and 0.2 ng ml
`on 76 and 77 days respectively
`indicating some additional
`release The challenge at day 77 resulted in
`significant
`The 83-day sample remained elevated at which time the study was
`slightly longer releasing form
`discontinued This formulation in combination with
`such as Polymer microspheres could provide continuous suppression through 100
`
`this initial
`
`is
`
`elevation
`
`days
`As polymer
`microspheres suppressed from 50 to 90 days and polymer
`to 56 days
`suppressed from
`combination of the microspheres was
`formulated to produce suppression from to 90 days The in vivo testosterone
`suppression profile of the physical combination of microspheres administered at
`total dose of 9.0 mg Leuprolidekg is shown in Fig
`This combination of
`microspheres prepared with the hydrophilic and hydrophobic forms of 75 25 PLGA
`
`

`

`Extended release pep tide delivery systems
`
`725
`
`3.5
`
`2.5
`
`10
`
`20
`
`30
`
`50
`
`60
`
`130
`
`Time Days
`
`Figure
`
`Testosterone levels
`
`microsphere combination Polymer
`
`and Polymer
`
`showed suppression from about day 10 to day 98 surviving challenges at 41 63 76
`slight elevation occurred at 100 days which became progressively
`and 90 days
`larger at 110 and 120 days Based on this suppression profile it appears the lag
`system Fig
`has been effectively eliminated
`phase exhibited in the Polymer
`through combination with the Polymer
`system thereby providing continuous
`release through 100 days
`
`Three-part system
`
`second combination was explored in the form of 75 25 PLGA Polymer
`microspheres and 50 50
`microspheres 50 50 acid terminated PLGA Polymer
`PLGA Polymer
`The three-part combination offers greater
`microspheres
`in the formulation should minor alterations in the release or suppression
`flexibility
`profile be deemed necessary
`and Polymer
`The in vitro release of Leuprolide from Polymer
`microspheres
`The in vitro release profile of Polymer
`is shown in Fig
`microspheres
`microspheres 50 50 PLGA 30
`acid terminated exhibited
`smaller initial
`release of about 5% after the first
`days which was attributed to the diffusional
`release of surface bound accessible peptide From to 14 days only very gradual
`release was noted After 14 days the release accelerated
`as the Polymer
`began to
`erode marking the third and final phase of release which continued through 40 days
`burst of nearly 20% period of very gradual release through 21 days was
`After
`microspheres 50 50 PLGA 10
`noted during in vitro testing of Polymer
`ester terminated After 28 days the rate of Leuprolide release increased Release
`steady rate through 63 days before leveling at 70 days with 95%
`continued at
`
`released
`
`

`

`726
`
`Burton et al
`
`100
`
`90
`
`80
`
`70
`
`50
`
`40
`
`30
`
`20
`
`10
`
`14
`
`21
`
`28
`
`35
`
`42
`
`49
`
`56
`
`63
`
`70
`
`77
`
`Time Days
`
`Figure
`
`In vitro release Polymer
`
`and Polymer microspheres
`
`The in vivo testosterone
`suppression profile of the Polymer
`formulation is
`shown in Fig 9a After the customary
`and expected increase in testosterone
`immediately following administration levels fell below castration levels within 10
`days Levels remained suppressed through 38 days
`challenge at 32 days was
`indicating complete protection and full down regulation of the GnRH
`uneventful
`at 42 and 47 days elicited significant
`sites Subsequent
`increases in
`challenges
`testosterone levels indicating exhaustion of the depot and cessation of suppression
`Again close correlation with the in vitro release is evident
`An initial
`increase in the testosterone concentration
`in response to the initial
`release of Leuprolide from the microspheres
`is evident
`immediately following
`formulation Fig 9b From to 23 days
`in vivo administration of the Polymer
`remained below the zero time levels 2.8 ng ml
`but above
`the concentrations
`the target suppression level of 0.5 ng ml
`suggesting Leuprolide levels were not
`to cause castration By 28 days the animals had reached
`state of full
`sufficient
`castration exhibiting testosterone levels below 0.5 ng ml
`five times less than the
`average testosterone levels 2.8 ng m1 However
`the long lag phase between
`release 13 days and the onset of erosional release at 28
`
`completion of diffusional
`
`days is unacceptable
`Based on the individual
`
`release and suppression profile
`
`three-part microsphere
`
`combination was made Two parts 3.0 mg kg of the Polymer
`were used to produce release for the first 30 days One part 1.5 mgkg of the
`Polymer microspheres was used to produce suppression from 30 to 60 days only
`one part was deemed necessary due to the overlap with Polymer
`assumed to
`which begins to suppress
`reach exhaustion between 35 and 42 days and Polymer
`
`microspheres
`
`

`

`Extended release peptide delivery systems
`
`727
`
`3.5
`
`2.5
`
`1.5
`
`0.5
`
`2.5
`
`1.5
`
`0.5
`
`C-
`
`Cl
`
`I-
`
`10
`
`20
`
`30
`
`t40
`Time Days
`
`50
`
`60
`
`7Ot
`
`80
`
`90
`
`Figure
`
`Testosterone levels
`
`Polymer microspheresb Polymer microspheres
`
`tn
`
`Cr
`
`.4-
`
`-t
`
`-c
`-o
`
`-0
`Ct
`
`microspheres was
`
`at about 50 days Three parts 4.5 mg kg of the Polymer
`added to suppress from 50 to 100 days
`three microsphere batches showed good suppression over
`This combination of
`the 90-day period Fig 10 However
`the challenges at 41 and 63 days caused
`slight elevation to between 0.5 and 1.0 ngmt The challenges at 76 and 90
`days were uneventful The challenges at 41 and 63 days may indicate incomplete
`and Polymer
`and Polymer
`suppression during the overlap region of Polymer
`and Polymer
`This problem might be circumvented
`by simply increasing the
`amount of Polymer
`in the formulation It should be noted that the slight elevations
`at 63 and 100 days were caused by administration of high does of the superagonist
`Leuprolide It is reasonable to expect an uneventful change due to the normal release
`of the less potent native LHRH Challenging with Leuprolide is very aggressive and
`is questionable whether challenges should be with the superagonist Nevertheless
`
`it
`
`

`

`728
`
`WBurtonetal
`
`3.5
`
`02
`
`1.5
`
`Ii
`
`0.5
`
`riD
`
`-t
`
`-c
`
`Figure 10 Testosterone levels
`
`microsphere combination Polymer
`
`Polymer
`
`Polymer
`
`Time Days
`
`this microsphere formulation showed
`composite release as the early lag times
`and Polymer Fig 9b were eliminated by
`demonstrated by Polymer Fig
`combining these polymers with Polymer
`
`SUMMARY AND CONCLUSIONS
`
`This study showed that extended testosterone suppression profiles can be obtained
`by microspheres made from different polymers In two cases microsphere combi
`nations produced castration which persisted through the 90-day challenge dose By
`combining microspheres at the proper ratio effective therapies can be administered
`can be physically combined at
`variety of intervals Further microspheres
`over
`any ratio to manipulate the release by decreasing the onset time to suppression in
`creasing the duration of action or eliminating the lag phase to produce an effective
`similar approach can be followed to obtain
`delivery system through ninety days
`4- and 6-month dosage forms
`
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`TiceU.S Patent46751891987
`Shimamoto Chem Pharmacol Bull
`Okada and
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`

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