Pliarrnscile cit9
`AAPS PharmSciTech
`2000
`http//ph arm sc itech.com
`Evaluation of Orntide Microspheres in Rat Animal Model and
`Correlation to In Vitro Release Profiles
`
`article 27
`
`Submitted April
`
`Janusz
`
`Patrick
`
`Kostanski
`DeLuea
`
`2000 Accepted July 19 2000
`Dani
`
`Bhas
`
`George-Ann Reynolds Cyril
`
`Bowers
`
`and
`
`Sciences University of Kentucky College of Pharmacy Lexington KY 40536
`1Faculty of Pharmaceutical
`School of Medicine Tulane University New Orleans LA 70112
`
`Key Words LHRH antagonist Orntide
`acetate
`delivery PLGA microspheres
`
`controlled
`
`Peptide
`
`Prostate cancer
`INTRODUCTION
`
`as
`
`prostatic
`continuous
`
`Since the discovery
`luteinizing hormonereleasing
`numerous
`have
`been
`analogues
`synthesized
`studied for potential application in gynecology and
`oncology as antigonadotropic agents for ovarian and
`suppression
`acetate
`testicular
`Leuprolide
`developed and marketed by TAP Pharmaceuticals
`as Lupron Depot
`Inc Deerfield Ill
`is one of
`the most widely used LHRH superagonists
`treatment of sex hormone-dependent
`tumors such
`in men
`carcinoma
`
`and structural elucidation
`
`of
`
`hormone LHRH
`
`and
`
`in the
`
`During
`
`Leuprolide
`administration
`initially
`and release of testosterona
`stimulates production
`LHRH
`an
`but
`prolonged
`the
`to
`exposure
`superagonist eventually causes down-regulation of
`LHRH receptors
`and
`of
`inhibition
`hormone LH release which
`Due
`chemical
`this
`castration
`to
`initial
`in humans
`testosterone elevation which may last
`days approximately 11% of all
`from to
`and
`experience
`patients
`painftil
`dangerous flare-ups of disease
`
`luteinizing
`
`finally
`
`leads
`
`to
`
`treated
`
`potentially
`
`Patrick
`
`corresponding Author
`DeLuca
`Faculty of Pharmaceutical
`Sciences University of
`exington KY
`Kentucky College of Pharmacy
`40536 telephone 606 257-1831 facsimile 606
`323-0242 e-mail ppdeluI@pop.uky.edu
`
`EXHIBIT
`
`DATE
`DAWN HILLIER RMR CRR
`
`novel
`
`luteinizing
`
`ABSTRACT
`Omtide acetate
`hormone LHRH antagonist was
`hormone-releasing
`prepared and evaluated in vivo in 30-day and 120-day
`rat animal
`delivery formulations using
`sustained
`ide PLGA
`model Omtide polydl- lactide-co-glycol
`lactide PLA microspheres were
`and polydl-
`prepared by
`dispersion method and administered
`liquid vehicle to rats at 2.2 mg
`subcutaneously in
`Orntide/kg of body weight 30-day forms or 8.8 mg
`Orntide/kg 120-day forms Serum levels of Omtide
`monitored
`and
`
`testosterone
`
`were
`
`by
`
`radioimmunoassays and
`dose-response study at
`1.75 mg Orntide/kg
`was
`doses
`2.25 1.5 and
`to determine the effective dose of Orntide
`
`conducted
`
`3.9 and
`Microspheres with diameters between
`14
`were prepared The onset and duration of testosterone
`varied
`
`microsphere
`for
`different
`suppression
`formulations and were influenced both by polymer
`and
`characteristics
`
`properties
`
`by microsphere
`5050
`
`with
`
`and
`
`7525
`
`Microspheres
`prepared
`copolymers effectively sustained peptide release for 14
`to 28 days whereas an 8515 copolymer and the PLA
`microspheres extended the pharmacological
`response
`for more than 120 days Increase in drug load generally
`from the microspheres
`accelerated
`release
`serum levels of Orntide and
`resulting in higher
`shorter duration of the release In general apparent
`release was faster in vivo than under in vitro conditions
`Omtide
`
`peptide
`
`initial
`
`microspheres
`effectively
`suppressed
`testosterone in rats providing rapid onset of release and
`extended periods of chemical castration Testosterone
`suppression occurred immediately after microsphere
`administration without
`elevation seen with
`the initial
`LHRH superagonists
`
`ALKERMES Exh. 2025
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`HYPOTHALAMUS
`
`LHRH
`
`Jfln
`
`____
`
`PflJtTARY
`
`____
`
`H_LH
`
`_______
`
`_______
`
`11
`
`TESTOSTEROP
`
`Normal physiology of
`
`the pituitary-gonadal
`
`that
`
`cleave
`
`the
`
`glycine-leucine
`
`linkage
`
`between
`
`Scheme
`
`axis
`
`Scheme
`
`normal physiology of
`axis LI-IRH peptide
`hypothalamo-pituitary-gonadal
`hormone synthesized in the arcuate
`nucleus of the
`released
`
`illustrates
`
`the
`
`is
`
`in
`
`to
`
`low
`
`hypothalamus
`response
`testosterone levels and is transported to the pituitary
`known
`gland via capillaries
`the hypothalamic-
`as
`pituitary portal system where it binds to the surface
`of
`
`the
`
`containing
`
`follicle-stimulating
`
`that consist
`
`of
`
`subunits
`
`stimulates
`
`development
`
`of
`
`the
`
`maintains
`
`spermatogenesis
`
`the
`
`and
`
`the
`
`receptors
`
`cells
`
`or
`
`gonadotropin-producing
`LHRH
`Once
`gonadotropes
`are
`receptors
`stimulated the gonadotropes synthesize and secrete
`by exocytosis of storage granules
`gonadotropins
`LH and
`hormone
`FSH Both hormones are glycoproteins
`tx and
`FSH
`seminiferous
`tubules
`and LH stimulates
`to produce and secrete
`Leydig cells in the testes
`testosterone Under normal physiologic conditions
`LI-IRH is rapidly removed from the site by enzymes
`
`positions
`
`and
`
`released in
`
`In this way the gonadotropins
`fashion which
`regular pulsatile
`necessary to maintain normal ftmnctions of the pituitary
`gonadotropes and gonads
`
`are
`
`is
`
`shows
`
`the
`
`effect
`
`of
`
`continuous
`
`the
`
`to
`
`Scheme
`administration of an LI-IRH superagonist
`on
`pituitary-testicular axis Initially
`transient phase of
`testosterone elevation caused
`by stimulation of the
`pituitary LI-IRH receptors and increased release of LH
`is observed Soon after however
`the gonadotrophic
`receptors become desensitized and unresponsive
`The
`fUrther
`stimulation
`receptor down-regulation
`process includes reduction in the number of surface
`of
`from
`the
`
`uncoupling
`receptors
`receptors
`intracellular mediators and postreceptor
`active LH
`Finally instead of producing
`gonadotrophs start to produce and release an inactive
`as lh that
`form of LH shown in Scheme
`unable to stimulate
`production
`
`inactivation
`
`of
`
`testosterone
`
`is
`
`in
`
`testes
`
`

`

`1r
`
`IAgeiist
`
`AgnIst
`
`Th Th
`
`Aprnst
`
`AgoSt
`
`Agnkt
`
`Agonist
`\.__J
`
`c\
`\J
`
`Agonist
`
`-H_H_
`
`Li
`
`JLJ
`
`ii
`
`PITUITARY
`
`ri
`
`____
`
`Hi
`
`Agnist
`
`PITUITARY
`
`____
`
`l_r
`
`STES
`
`TESTO STEROME
`
`lb
`
`LHRH Receptor Desensitization
`.1 number of receptors
`uncoupling from mediators
`postreceptor
`inactivation
`
`Scheme
`
`The effect of continuous
`
`exposure to
`superagonist on the pituitary-gonadal axis physiology
`
`LHRH
`
`It
`
`has
`
`been
`immediate
`and
`complete
`reported that
`testosterone suppression may be achieved with LHRH
`These compounds show much higher
`antagonists
`affinity to the pituitary LHRI-I binding sites than does
`native LHRH they also compete with endogenous
`the LI-IRH receptor sites Blockade of the
`LI-IRH at
`pituitary LHRH receptors prevents stimulatory effects
`of native LHRH and results in rapid inhibition of the
`pituitary- gonadal axis thus testosterone suppression is
`immediate without
`the characteristic stimulatory phase
`for LHRH superagonists Scheme
`hi addition it has
`been reported that L1- antagonists directly inhibit
`tumors
`growth of numerous
`including mammary
`and
`tumors
`by
`binding
`to
`
`pancreatic
`prostatic
`receptors localized on tumor cells
`
`More
`
`than
`
`3000
`
`L1-IRH
`
`and
`
`studied
`
`analogues
`Whereas
`
`have
`
`been
`
`the LI-IRH
`
`synthesized
`agonists have already found clinical application in the
`therapy of gonadal
`steroidassociated
`diseases the
`LHRH antagonists have not reached this stage Animal
`studies with early LHRH antagonists revealed that due
`from mast cells these
`release
`to high histamine
`compounds
`caused
`
`severe
`
`adverse effects
`
`including
`
`anaphylactoid reactions hypotension
`formation of edema
`During the past
`decades
`developers of safer LHRH antagonists focused
`potency and minimizing
`increasing antigonadotropic
`histamine-releasing properties As
`result of enormous
`series of modern
`efforts by several
`research groups
`and more potent LI- antagonists has been
`Orntide acetate synthesized by
`synthesized
`Bowers Karl Folkers and Anna Janecka
`Tulane School of Medicine New Orleans LA is one
`of the most promising LHRH antagonists developed
`date Ti.lle1_e1
`
`skin lesions and
`
`on
`
`at
`
`to
`
`safer
`
`Cyril
`
`

`

`itt\ An
`4t 4h AM in Mt
`/M\
`crxwt k1 gaki kg.a nit gnst et emii
`
`pabt
`
`óath
`
`4\
`
`1tI
`
`LURtI
`
`ltuttkt
`
`R1H1
`
`TESTES
`
`TESTOSTERONE
`
`Scheme
`
`antagonist
`
`The effect of continuous
`
`exposure to
`on the pituitary-gonadal axis physiology
`
`LHRH
`
`Table
`
`Leading LHRH antagonists Modified from
`Bowers Karl Folkers and Anna Janecka
`Cyril
`United States Patent US5480969 1996
`
`Note
`
`The most
`
`preferred
`
`LHRH analogues
`high
`for histamine ED50
`high effective dose level
`activity and also
`antiovulatory
`dose releasing 50% of total histamine from mast cells for native LHRH
`EDsollO g/mL
`
`of
`
`the table
`
`have
`
`this
`
`is
`
`potency and
`
`It combines both high antigonadotropic
`safety At
`undergoing
`analogue
`present
`preclinical evaluation in animals and soon will enter the
`first phase of clinical
`trials Therefore sustained-release
`formulations of Omtide acetate are urgently needed to
`make the clinical use more feasible Such preparations
`may in addition help to reduce the therapeutic dose
`required for continuous
`suppression of pituitary and
`functions and may cause fewer adverse effects
`
`gonadal
`
`To enable
`
`further
`
`studies
`
`and
`
`clinical
`
`trials with
`
`Omtide this analogue must
`
`be
`
`formulated
`
`as
`
`prepare
`
`using
`
`biodegradable
`
`therapeutic system that provides the highest efficiency
`and safety of the treatment and preferably good patient
`compliance Therefore the purpose of this work was to
`controlled-release microparticulate Omtide
`low-molecular-
`formulations
`lactide PLA and
`weight
`polylactides polydl-
`polyd.l- lactide-co-glycolide PLGA and to evaluate
`formulations
`for
`their efficacy
`in suppressing
`rat animal model
`
`these
`
`testosterone
`
`in
`
`

`

`MATERIALS AND METHODS
`
`Preparation of Orntide Microspheres
`
`Materials
`
`were prepared
`Orntide microsphere batches 0.9-2.7
`dispersion method followed by solvent extraction
`by
`solution of peptide in methanol
`and evaporation
`solution of PLGA or PLA in
`was combined with
`methylene chloride and stirred until clear The solution
`reactor with baffles
`was then slowly injected into
`Inc Vineland NJ
`Ace Glass
`phase CP 0.35%
`continuous
`PVA pH 7.2 and
`Silverson
`
`stirred at
`
`5500
`
`rpm with
`
`containing
`solution of
`
`the
`
`L4R homogenizer Silverson Machines Ltd Waterside
`The
`of
`reactor was
`England
`the
`temperature
`maintained initially at 25C for 30 minutes and then at
`40C for 60 minutes Once microspheres were formed
`and
`of
`reactor were
`hardened
`the
`contents
`the
`
`filtration
`
`transferred to
`equipped with
`apparatus
`0.8 membrane filter Gelman Sciences Ann Arbor
`Ml and the recovered product was washed with water
`and dried under reduced pressure for 48 hours at room
`
`temperature
`
`Microsphere Characterization
`
`in microspheres was determined by
`Peptide content
`HPLC after dissolving the microspheres in chloroform
`0.1 mol/L acetate
`and extracting the peptide with
`buffer AB with
`pH of 4.0 Total product yield was
`assessed gravimetrically on the basis of polymer/drug
`bulk density was determined
`recovery Microsphere
`with the tapping method using approximately 100 taps
`10 mL graduated
`and
`tube Particle size
`glass test
`distribution was determined using
`laser diffraction
`technique Malvem 2600c Particle Sizer Malvem
`UK
`In Vitro Release Study
`
`previously described dialysis technique was used to
`determine
`from Omtide
`the
`vitro
`release
`
`in
`
`microspheres a7 Approximately 20 mg of
`the
`mL
`microspheres were quantitatively transferred to
`dialysis tube Tube-O-Dilalyzer MWCO 300 000 Da
`containing 5.0 mL of AB which in turn was placed in
`50 mL tube containing 40 mL of the same release
`medium The
`of
`tube were
`the
`contents
`larger
`magnetic stirrer At various
`continuously stirred with
`times 1.0 mL of the supematant was removed from the
`
`in
`
`from
`
`Boehringer
`
`Ingeiheim
`
`Inc
`
`D3PaI
`Orntide acetate
`DpClPhe
`LHRH
`Ilys DAIa 10
`PicLys D6AnicOrn
`was supplied
`by California Peptide Research
`Inc
`Napa CA Orntide 30-day and
`120-day PLGA
`PLA microspheres were
`and
`developed
`collaboration with Oakwood
`LLC
`Laboratories
`Oakwood OH
`PLGA
`5050
`Three
`copolymers MW 10 777 31 281 with different
`degrees of hydrophilicity 7525 PLGA MW 11
`PLA homopolymer MW 9489 were
`161 and
`obtained
`Ingeiheim Germany and 8515 PLGA MW 17
`903 was obtained from Birmingham Polymers Inc
`Birmingham AL The hydrophilic
`resomers
`groups
`carboxyl
`ones were
`capped with
`alkyl alcohols Polyvinyl alcohol PVA average
`MW 30 000-70 000 was obtained
`from Sigma
`Chemical Co St Louis MO The solvents
`and
`other excipients were analytical grade and were
`purchased from commercial sources dialysis tubes
`Tube-O-Dialyzer were from Research Products
`IL and
`International Corp Mount Prospect
`CE dialysis membrane molecular
`Spectra/Por
`weight cut-off or MWCO 300 000 Da was from
`Inc Houston TX
`Spectrum Medical
`Industries
`Injections of free Orntide acetate were prepared by
`to reach the desired
`dissolving the peptide in water
`concentration Male Sprague Dawley rats weighing
`were purchased from Harlan
`approximately 300
`Indianapolis IN The studies were conducted
`at
`the University of Kentucky College of Pharmacy
`Animal Research Facility
`
`had
`
`free
`
`hydrophobic
`
`end
`
`whereas
`
`the
`
`long-chain
`
`Methods
`
`High-Performance Liquid
`Chromatography HPLC Method for
`Peptide Assay
`The peptide was analyzed by reverse-phase HPLC
`3.90 mm using an
`Bondclone 10 C18 column 150
`elution phase of 34% vollvol acetonitrile and 0.1%
`in water Ultraviolet
`trifluoroacetic
`acid
`vol/vol
`detection was at 215 nm
`
`

`

`50 rnL tube and 1.0 mL of fresh buffer was added The
`sample was analyzed for peptide
`concentration using
`the HPLC method
`
`Animal Study
`
`approximately
`
`Male
`
`Sprague
`
`Dawley
`weighing
`rats
`were used to study testosterone
`300
`liquid Omtide
`treatment with
`suppression
`during
`The
`and Omtide microspheres
`formulations
`acetate
`were injected subcutaneously at the lower back section
`liquid vehicle 1%
`of the neck after reconstitution in
`and 2%
`carboxymethylcellulose
`wt/volJ
`mannitol Blood samples were collected from the tail
`vein at specific times The samples were centriftiged in
`Microtainer
`etubes Becton Dickinson Franklin Lakes NJ and
`serum was collected Serum samples were frozen and
`stored at -20C until analysis
`Serum Omtide levels in rats were assessed using
`
`free-
`
`j.t
`
`RESULTS AND DISCUSSION
`flowing powder
`fonned
`Dried microspheres
`with the average particle size varying from 3.9 to 19
`Table
`Average peptide incorporation efficiency
`was 84% and drug load varied from 6.63% to 17.2%
`As shown in
`single adniinistration of liquid
`Omtide to rats at 0.1 mg/kg resulted in serum Orntide
`hours later observed Cm 35 ng/mL At the
`peak
`same time mean serum testosterone
`from the initial 3.4 ng/mL to below 0.1 ng/mL
`
`level decreased
`
`Table
`
`Physical
`
`characteristics
`
`of
`
`Orntide
`
`microspheres
`
`6.63
`
`112
`
`14.6
`
`8.91
`
`136
`
`108
`
`61
`
`86
`
`81
`
`81
`
`91
`
`83
`
`68
`
`77
`
`78
`
`78
`
`70
`
`70
`
`048
`
`0.49
`066
`
`0.63
`
`0.51
`
`29
`
`36
`
`19
`
`27
`
`28
`
`29
`
`10
`
`14
`
`5.9
`
`11
`
`10
`
`11
`
`6.6
`
`2.3
`
`2.0
`
`1.7
`
`1.6
`
`2.3
`
`17
`
`labeled
`
`at Tulane School of
`radioimmunoassay
`developed
`Medicine New Orleans LA Tyrl -Omtide was
`radioiodinated by the lactoperoxidase method and the
`ligand was purified by HPLC Omtide was
`carbodiimide method and
`an
`by
`the
`conjugated
`antibody to Omtide was produced in rabbits The lower
`limit of the assay was 0.008 ng/mL The
`detection
`intra- and interassay coefficients of variation were 6%
`and 9% respectively
`levels were assayed using Active
`Serum testosterone
`Testosterone RIA DSL-4000 kits Diagnostic Systems
`Inc Webster TX The lower
`limit of detection for this
`assay was 0.08 ng/mL and the intra- and interassay
`9%
`10% and
`of
`were
`variation
`coefficients
`The
`cross-reactivity of the testosterone
`antiserum was less than 6%
`Statistical Evaluation of Data
`as means standard deviation For
`Data are presented
`the limit was
`values below the assay detection limit
`used for calculations To evaluate the effectiveness of
`
`respectively
`
`continuous
`
`treatment with Omtide microspheres to
`in rats the areas under
`
`testosterone level
`
`suppress
`serum Omtide and serum testosterone curves were
`calculated using GraphPad Prism GraphPad Software
`The San Diego CA The results were analyzed by
`one-way analysis of variance at a-level of 0.05
`
`Ts1D
`
`202111
`
`202112
`
`202113
`
`..3A85
`
`P1
`PLA
`
`PLA
`
`9489
`
`9489
`
`9489
`
`142
`
`980
`
`15.1
`
`172
`
`74
`
`80
`
`86
`
`89
`
`88
`
`94
`
`82
`
`26
`
`29
`
`23
`
`46
`
`9.8
`39
`
`34
`
`15
`33
`
`Average molecular weight
`
`Serum Testosterone
`
`-S Serum Omtide
`
`Time
`
`03
`CO
`
`fjgpre
`
`Serum Omfide and testosterone
`liquid Omde 0.1 mg/kg
`level of testosterone
`
`administrafion of
`
`indicates
`
`castration
`
`50
`
`40
`
`-30
`
`20
`
`10
`
`-J
`
`03
`
`CO
`
`levels in rats after
`
`single
`
`Doffed
`
`line at 0.5 ng/mL
`
`

`

`40
`
`30
`
`-J
`
`20
`
`10
`
`20
`
`TIme
`
`30
`
`40
`
`.502H
`
`.503H
`
`503H
`
`s--7525N
`
`Serum Orntide levels in rats after administration of 30-day
`9gpre
`Orntide PLGA microspheres 2.2 mg Orntide/kg
`
`These
`
`results
`
`indicate that Orntide
`
`is
`
`acetate
`an
`rapid onset of
`agent with
`effective antigonadotropic
`action The testosterone suppression was reversed 24
`hours after Orntide
`
`that
`
`administration suggesting
`in animals
`
`Orntide
`
`acetate
`
`was
`
`rapidly degraded
`result of poor
`probably as
`resis tance to peptidases
`pH 29I
`and physical
`at physiological
`Short biological half- lives of LHRH analogues have
`been one of the reasons
`for frequent administrations
`and poor patient compliance during chronic treatment
`
`instability
`
`ggeshows serum Omtide levels in rats after
`administration of 30-day Omtide microspheres 2.2 mg
`resomers of PLGA
`Omtide/kg prepared with different
`The 502H microspheres resulted in
`serum
`high initial
`Omtide level observed Cnnx of 69 nglmL at
`hours but
`serum Orntide levels beyond
`did not sustain elevated
`15 days The
`had the highest
`release Cm of 147 ng/mL at
`hours and
`initial
`maintained serum Omtide levels above
`ng/mL for
`more than 22 days
`
`microspheres
`
`7525
`
`microspheres produced
`
`concentration
`
`at
`
`highest
`
`The 503H
`sharp Omtide
`hours 27 ng/mL and Cm of 29 ngmL was
`peak at
`the mean serum level of Orntide
`observed on day
`ng/mL for approximately 20 days
`remained above
`The 503H
`microspheres with
`higher drug load
`14.6% showed
`serum Omtide
`the
`hours Cmof 25 ng/mL and
`the mean
`second
`smaller peak occurred on day
`serum Omtide level
`remained above
`ng/mL for 36
`days These serum Orntide profiles indicate that drug
`load had an effect on the in vivo release rate from the
`503H microspheres
`increase
`in drug load shifted the
`maximum release from the microspheres from to
`days
`
`Serum testosterone levels in rats treated with 30-day
`Omtide PLGA microspheres and in
`control group are
`shown injjgjje The 503
`and
`hydrophobic
`microspheres did not suppress testosterone in rats with
`the only exception of the 6-hour data point when the
`levels decreased to below 0.5 ng/mL castration level
`
`10
`
`20
`
`Time
`
`30
`
`40
`
`---502H
`
`503H1
`5032 e-- 7525H
`
`--5031
`
`503FU2
`
`Control
`
`The lack of
`503
`suppression
`testosterone
`microspheres was expected because very little release
`from these microspheres was detected in vitro
`
`for
`
`the
`
`Figure
`
`Testosterone suppression in rats after administration of 30-
`2.2 mg Orntide/kg
`day Orntide microspheres
`ng/mL indicates castration level of testosterone
`
`Dotted line at 0.5
`
`

`

`The initial
`
`suppression of testosterone in these two
`be ascribed to the
`release of
`could
`free
`
`testosterone for
`showed
`better
`503H
`and maintained
`microspheres
`the
`testosterone level below 0.1 ng/mL for more than 22
`The
`days Figure
`difference
`in suppression
`duration between the two 503H microsphere batches
`illustrates the effect of drug load on the release rate
`from PLGA delivery
`of
`systems Incorporation
`hydrophilic drugs into polymeric matrices increases
`water
`the microspheres
`thereby
`by
`uptake
`accelerating hydrolytic degradation of the polymer
`is also true and
`high load of
`the opposite
`drug may delay polymer degradation
`
`initial
`
`testosterone suppression than
`
`10
`
`Control
`
`20
`
`llmed
`
`mg/Kg
`
`30
`
`40
`
`225 mg/Kg
`
`M---- 1.5 mg/Kg
`
`.--- 0.75 mg/Kg
`
`Dose-response
`Figure
`study
`PLGA503H microspheres Dotted
`castration level of testosterone
`
`in
`
`rats
`
`with
`
`30-day Orntide
`
`line
`
`at
`
`0.5
`
`ng/mL
`
`indicates
`
`Timed
`
`300
`
`-2
`
`200
`
`1100
`
`-J20
`
`10
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`160
`
`Time fd
`
`PLA2O2H
`
`P1.4202
`
`PLA2O2H
`
`PLGA8SI5H
`
`fjjge5 Serum Orntide levels in rats after administration of Orntide
`120-day microspheres
`
`hydrophobic
`
`microspheres
`in rats was
`
`Dose-response study with the 503H
`showed that
`suppression
`testosterone
`dose-dependent and could be enhanced by increasing
`the initial dose of Orntide microspheres Figure
`mg/kg extended
`Increase of the Orntide dose to
`testosterone suppression by an additional week and
`sustained-release Omtide formulation
`
`provided
`effective in vivo for 30 days
`
`Serum Orntide levels in rats after administration of
`120-day Orntide microspheres 8.8 mg Orntide/kg
`are shown in jgure
`all microspheres
`Initially
`produced high levels of Orntide in serum ranging
`from 52 ng/mL 85I5H microspheres to 212 ng/mL
`hours The 202H
`202H
`microspheres at
`with
`low drug load resulted in lower Orntide
`hours the mean
`levels and after the initial
`peak at
`Omtide concentration decreased to 6.5 ng/mL on day
`and remained below that
`for the remainder of the
`
`study This serum Orntide level was not sufficient to
`suppress testosterone and maintain castration in rats
`for an extended period of time jgje6
`
`groups
`Orntide adsorbed to the microspheres
`ascribed to stress during administration stress also
`affected serum levels in the animals in the control
`testosterone suppression were
`group Rapid onsets.of
`seen in all animal groups treated with hydrophilic
`503H
`Orntide microspheres
`but
`the
`only
`of
`maintained
`
`surface or
`
`levels
`
`microspheres
`
`microspheres
`
`castration
`weeks The 503H
`
`

`

`-J
`
`03
`
`Table
`
`Response areas under serum Orntide and
`testosterone curves in rats treated with Orntide
`
`microspheres
`Formulation Omtide mgkg mL ngcurve Mean area under
`
`Omtide serum
`testosterone
`underarm Mean ngd/mL
`
`curve
`
`Control 0.0
`02H microspheres 2.2
`
`503H
`
`503H
`
`microspheres 2.2
`
`microspheres 2.2
`
`503 1rnicrospheres22
`5032 microspheres 2.2
`
`75251-1 microspheres
`
`20-Day Control 00
`202111 microspheres 8.8
`2021-12 microspheres 88
`20203 microspheres
`851 51-I microspheres 88
`
`14
`
`3754
`
`04
`
`TO
`
`ID
`
`36
`
`59
`
`49
`
`31134
`
`05
`128
`59 12
`
`27
`
`44
`
`11833
`
`.115
`
`545
`l4
`
`.5
`
`71
`
`13
`
`12
`
`34
`13325
`243
`
`L4
`
`0.8
`
`10
`
`5.8
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`140
`
`160
`
`burst release followed by an extended
`steady release
`
`phase of
`
`Time
`
`Table
`
`shows mean areas under serum Orntide and
`
`PLA2O2I-1
`
`--PLA2O2H
`
`PLA2O2H
`
`e-PLGAB515H
`
`Control group
`
`Figure
`
`Testosterone
`
`suppression
`120-day Orntide microspheres Dotted line at 0.5 ng/mL indicates
`castration level of testosterone
`
`in rats after administration
`
`of
`
`the 202H
`In contrast
`serum Omtide level
`
`microspheres maintained
`that was twice as high as that
`weeks
`produced by the previous batch during the first
`and suppressed testosterone in rats to below 0.5 ng/mL
`sma 11 elevation to
`for approximately 130 days with
`Even more rapid castration was
`0.8 ng/mL on day
`seen in rats treated with the 202H
`and 8515H
`microspheres mean testosterone levels decreased
`hours and remained below
`below 0.5 ng/mL within
`0.1 ng/rnL for months Fig6 Interestingly these
`batches of microspheres maintained higher serum
`Omtide levels than did the
`previous batches 202H
`and
`for most of
`only during the first
`10 days
`the study the Omtide levels were below
`ng/mL This
`indicates
`that
`to achieve
`prolonged
`rapid
`to maintain higher serum
`is crucial
`castration in rats it
`Omtide levels initially above
`10 nglmL for the first
`10-14 days and it seems that the subsequent
`levels as
`low as
`ng/mL are sufficient
`to suppress testosterone
`for several months This may be an advantage
`because
`the biodegradable PLGA and PLA microspheres cause
`
`to
`
`and
`
`in
`
`at
`
`microspheres
`
`testosterone curves
`
`various Orntide
`
`batches
`
`testosterone
`
`in
`
`4.4
`
`the
`
`resulted
`
`in
`
`of
`in rats after administration
`formulations All microsphere
`decrease
`of
`resulted
`significant
`levels compared with control groups
`503H
`503H
`and
`the
`Statistically
`microspheres were most effective among the 30-day
`formulations area under the curve
`T036d 4.5
`4.5 ng dImL respectively whereas
`3.1 and 5.5
`the 202H3 microspheres
`showed
`the
`best
`4-month period
`over
`testosterone
`suppression
`AUC T0115
`0.8 ng d/mL On average
`30-day formulations produced an area under serum
`Orntide curve Orntide AUC of 273 ng d.mL and
`the 4-month Orntide microspheres
`mean AUC of 581 ng d/mL
`Cumulative Orntide AUCs were compared with in
`vitro release profiles previously obtained with
`dialysis method
`Assuming that Orntide acetate
`has
`and that
`very short biological half-
`the
`life
`does not stimulate an antibody reaction
`peptide
`then the cumulative Orntide.AUC should reflect the
`vivo
`from sustained-release Orntide
`release
`formulations Figure 7A shows the Orntide AUC
`curve and in vitro release profile obtained with
`dialysis method in 0.1 mol/L acetate buffer pH 4.0
`PLGA 503H
`5050
`370
`for
`the
`
`

`

`Auc
`
`100
`
`tao
`
`SR
`
`60
`
`40
`
`20
`
`-100
`
`80
`
`60
`
`40
`
`20
`
`00
`
`10
`
`20
`
`30
`
`40
`
`071x
`
`21 265
`
`0783
`
`100
`
`60
`
`60
`
`40
`
`20
`
`100i
`
`in
`JL.J
`
`80
`
`60
`
`40
`
`20
`
`04
`
`Auc
`
`In v/kr
`
`release
`
`10
`
`20
`
`Tmed
`
`30
`
`40
`
`18.391
`2.0414c
`R0.7913
`
`20
`
`40
`
`60
`
`80
`
`100
`
`in Vitro Release 14
`
`20
`
`40
`60
`In Vitro Release 14
`
`80
`
`100
`
`fjge 5050 PLGA microspheres
`serum Orntide curve vs in vitro release profile and
`
`Mean area under
`
`in vivo-in vitro
`
`Figure
`
`7525 PLGA microspheres 7525H
`serum Orntide curve vs in vitro release profile and
`
`Mean area under
`
`in vivo-in
`
`correlation
`
`vitro correlation
`
`the in vivo
`
`The Orntide AUC curve shows that
`release started immediately after administration and
`15 days 77% release
`was rapid during the first
`During the same period only 24% of the initial drug
`load was released from the microspheres
`in the in
`vitro study 50% release was reached after T50 of 23
`days After the initial 25%
`days in vivo T50 was
`release the in vivo and in vitro release rates became
`similar although the in vitro release curve remained
`weeks for
`delayed by approximately
`the entire
`in vivo release was seen for the
`study Even faster
`7525H Orntide microspheres where
`the initial
`day and was over 93%
`release reached 24% after
`after 15 days Figure 8A
`
`In contrast only 7.3% of the drug was released
`within
`and the
`day during the in vitro study
`microspheres continued to release with
`steady rate
`50% after
`of 1.7% per day reaching
`28 days
`in vivo release was seen for
`Similarly higher initial
`the 120-day forms Figure 9A shows
`typical 5-
`cumulative
`in vitro release profile and
`shaped
`Orntide AUC for the 851 5H microspheres
`
`in vitro
`
`The release rate was much slower under
`conditions during the first month 14% than in
`similar release occurred within
`vivo where
`days The PLA 202H
`released in
`microspheres
`vivo more than 60% of the drug within
`weeks and
`continued to release 0.37% per day figje19j
`In contrast only 16% release occurred in vitro in 14
`days but subsequently
`the release rate stabilized at
`0.3 8% per day
`
`

`

`100
`
`80
`
`60
`
`40
`
`to
`
`Vi
`
`20
`
`120
`
`100
`
`60
`
`if
`
`60
`
`40
`
`20
`
`AUC
`
`In v/Ire
`
`release
`
`20
`
`40
`
`80
`
`Time
`
`80
`
`100
`
`120
`
`0.964x
`
`25.307
`
`R2
`
`08011
`
`20
`
`40
`
`60
`
`80
`
`100
`
`In I/lire Release
`
`8515 PLGA microspheres 8515H
`Figure
`serum Orntide curve vs in vitro release profile and
`
`Mean area under
`
`in vivo-in vitro
`
`correlation
`
`CONCLUSIONS
`
`The comparison between in vitro and in vivo release
`in vivo release is
`
`that
`the initial
`profiles indicates
`times higher than under
`in vitro conditions
`several
`The biggest difference in release rates was seen
`14 days for the 30-day forms and
`during the first
`during the first month for the 120-day microspheres
`The higher
`in vivo release rate resulting in poor
`in vivoin vitro correlation Figures 7B and
`overall
`lOB could be ascribed to several
`factors including
`higher peptide solubility in serum than in
`presence of hydrolytic
`enzymes
`of
`acidic microenvironment
`within
`
`system
`formation
`
`buffer
`
`100
`
`E1
`
`80
`
`60
`
`40
`
`to
`
`Vt
`
`20
`
`100
`
`80
`
`leo
`
`20
`
`if
`
`law/rn
`
`release
`
`20
`
`40
`
`60
`Tme
`
`80
`
`100
`
`120
`
`yal 9498x18.106
`0.8264
`
`20
`
`40
`
`60
`
`80
`
`100
`
`In Vitro Release
`
`Figure 10 PLA microspheres 1202H3l
`Orntide curve vs in vitro release profile and
`
`Mean area under serum
`
`in vivo-
`
`in vitro
`
`correlation
`
`recently published study showed
`
`that solubility of
`was
`somatostatin
`analogue
`pamoate
`vapreotide
`in protein-containing media such as
`greatly enhanced
`bovine serum albumin BSA solution and
`was
`serum and
`on
`dependent
`protein
`The increased peptide solubility in
`the release rate from biodegradable
`serum could affect
`higher dissolution rate of
`microspheres because of
`free fraction of the peptide trapped inside the matrix
`
`aqueous
`human
`
`concentration
`
`pores
`
`or
`
`adsorbed
`
`to
`
`the
`
`it
`
`microspheres
`seems unlikely that
`surface.However
`the higher
`solubility in serum could play major role in release
`period of several months because the rate of
`over
`peptide release from biodegradable microspheres is in
`most cases limited by the rate of hydrolytic degradation
`of
`
`degrading microspheres and
`effect of plasma
`proteins on degradation of polyesters
`
`the polymeric matrix erosion-controlled release
`The hydrolytic activity of esterolytic enzymes
`
`

`

`could
`
`also accelerate
`
`the
`
`in vivo
`
`release from
`it seems more likely that
`biodegradable systems but
`the catalytic effect of these enzymes would be more
`pronounced during the latter part of treatment when
`is more accessible
`the highly hydrophobic
`polymer
`greater contact area and
`to the enzymes because of
`large number of hydropltilic
`formation of
`the
`
`oligomers
`
`recent study performed with the application of
`resonance EPR showed the
`electron paramagnetic
`formation
`of
`highly
`
`acidic
`
`rapid
`microenvironment
`
`in
`
`within
`biodegradable
`Decrease in pH within polyester
`microspheres
`could
`microspheres to values below
`result
`accelerated polymer erosion caused by the catalytic
`of
`and
`on
`
`effect
`
`lactic
`
`acids
`
`ester
`
`glycolic
`hydrolysis and hence could increase the release rate
`The microenvironment
`effect may play
`more
`important role in vivo than under in vitro conditions
`the presence of plasma proteins
`because of
`and
`on
`polymer
`degradation
`properties
`
`their.effect
`
`the
`
`at
`
`Serum
`and
`albumin
`y-globulins
`fibrinogen were shown to form adsorption layers on
`PLA
`of
`surface
`microspheres
`through
`the
`in such
`way that
`hydrophobic interactions
`outer part of the adsorbed protein layer is rich in
`positively charged groups and the inner part
`is rich
`groups which
`causes
`in negatively charged
`variation in the zeta potential of plasma protein-
`coated microspheres The potential
`increase on the
`polymer-protein interface causes an increase in
`interface which may result in
`concentration
`that
`an acceleration of the hydrolytic degradation of the
`microspheres Also the presence of plasma proteins
`increase the solubility of PLA causing the
`can
`polylactide to exist in an expanded form and hence
`and
`increase
`for
`
`its
`
`availability
`hydrolytic
`enzymatic processes Therefore the formation of
`microenvironment
`acidic
`inside
`
`highly
`
`from
`
`an
`
`systems
`biodegradable
`resulting
`products and
`accumulation of polymer degradation
`concentration on the polymer-plasma
`increase in
`protein interface may indeed be
`for
`responsible
`in vivo releases than under
`higher
`initial
`conditions
`
`in vitro
`
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`LHRH and Its Analogs
`Vicker
`JJ Jr.
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`Weinbauer OF Nieschlang
`LH-Ri
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`the art and future perspectives Recent Results Cancer Res
`1992 13-136
`Campen CA Lai MT Kraft
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`Potent pituitary-gonadal
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`
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`Comparison of LH-RH analogue 1-month depot and
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`LM Trials with Leuprolide In Vickery BH Nestor
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`
`Kienle
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`Eflicac
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`for prostate cancer The German Leuprorelin Study Group
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`1990 1446 1479-1480
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`LHRH antagonist
`adniinistration with
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`prostate
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`
`concurrent
`
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`treatment of prostate
`
`Costa
`
`Louis
`
`17-22
`
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