III Di Iii
`
`1110
`
`IO
`
`IIII
`
`Oil Oil DIV iii
`010 IlO II0 110
`US 20070122487A1
`
`II0I
`
`III
`
`IlII
`
`19 United States
`12 Patent Application Publication
`Deluca et al
`
`10 Pub No Us 2007/0122487 Al
`May 31 2007
`43 Pub Date
`
`Related U.S Application lata
`
`60 Provisional application No 60/455590
`19 2003
`
`filed on Mar
`
`Publication Classification
`
`51 lot Cl
`AOJK
`38/22
`A6JK 38/28
`A6IK 38/18
`A61K 38/21
`A6IK 9/14
`A61K 9/SO
`52 U.S Cl
`
`2006.01
`2006 01
`2006.01
`2006.01
`2006.01
`
`2006.01
`424/490 514/2 514/3 514/15
`424/85.4 424/46
`
`57
`
`ABSTRACT
`
`54 POLYACRYLOYL-HYIROXYETHYL
`STARCH-PLGA COMPOSITION
`MICROSPHERES
`
`75 Inventors Patrick Deluca Lexington KY US
`Ge Jiang Durham NC US Byung
`IL US
`Woo Schaumburg
`
`Correspondence Address
`BUCHANAN INGERSOLL
`POST OFFICE BOX 1404
`ALEXANDRIA VA 22313-1404 US
`
`ROONEY PC
`
`73 Assignee UNIVERSITY
`KENTUCKY
`OF
`RESEARCH FOUNDATION Lexing
`ton KY US
`
`21 Appl No
`
`10/549760
`
`22
`
`86
`
`PCT Filed
`
`PCT No
`
`Mar 19 2004
`
`PCT/USO4/08399
`
`371c1
`Date
`
`Dec 21 2006
`
`invention
`
`relates to
`
`composite microsphere
`
`The present
`system comprising polyDL-lactide-co-glycolide PLGA
`starch AcHES and
`phar
`polyacryloyl hydroxyethyl
`biologically active com
`maceutically effective amount of
`pound Thc
`active compound may be for example
`an
`luteinizing hormone-releasing hor
`insulin an interferon
`mune LHRH analog
`somatostatin and/or derivatives
`parathyroid hormone PTH bone
`mnrphngenic protein BMP an erythropoietin EPO an
`epidennal growth factor EGF or
`growth hormone This
`also relates to methods of using the composite
`and methods of preparing same
`microspheres
`
`thereof
`
`calicitonin
`
`invention
`
`20301
`WIT bElACr
`
`DATE..2-
`
`DAWN HILLIER RMR CRR
`
`ALKERMES Exh. 2030
`Luye v. Alkermes
`IPR2016-1096
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 200710122487 Al
`
`Figure
`
`SEM micrcgraphs of insulin-loaded AcHES-PLGA composite
`
`microspheres
`
`interior structure of
`
`fractured microsphere
`
`arrows point to
`
`embedded AcHES microparticles and freeze dried AcHES hydrogel
`
`microparticles
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 2007/01 22487 Al
`
`Figure
`
`HPLC chromatogram of insulin sample isolated from composite
`
`microspheres by ACN extraction and intact
`
`insulin standard
`
`to
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 200710122487 Al
`
`Figure Characterization of insulin integrity in the composite microspehres
`
`SDS-PAGE with DII Lanel Molecular weight marker Lane
`
`Bovine insulin
`
`standard Lane
`
`insulin sample from 81021 and lane
`
`insulin sample from
`
`61022
`
`MALDI-TOF MS of insulin extracted from composite in comparison to
`
`intact standard
`
`4iIC
`
`AK
`
`4K
`
`1K
`
`M1fl734
`
`insutin
`
`standard
`
`Response
`%lnt
`
`insulin extracted
`From microspheres
`
`MU2
`
`2867
`
`Mass/charge
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 2007/01 22487 Al
`
`Figure
`
`In vitro release of insulin from composite microspheres in glycine buffer
`at 37C Sonication levels are indicated in
`
`100
`
`20
`
`-o
`
`-s-51022 3.3
`s- 61020 LI
`
`61021 3.0
`81016 vortex
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Time day
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 200710122487 Al
`
`Figure
`
`Serum glucose suppression in diabetic rats treated with insulin loaded
`
`composite microsphere batches B1021
`
`and 81022
`
`10
`
`15
`
`Timeday
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 2007/0122487 Al
`
`Figure
`
`Serum Insulin level of
`
`81021 and
`
`B1022 treated diabetic rats
`
`12
`
`10
`
`12
`
`10
`
`10
`
`15
`
`Time day
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 200710122487 Al
`
`Figure
`
`Blood glucose suppression of multiple dosing treatment of insulin
`
`loaded composfte microspheres nB Dose 80 lUIrat
`
`Boo
`
`700
`
`lime day
`
`

`

`Patent Application Publication May 31 2007 Sheet
`
`of
`
`US 2007/0122487 Al
`
`Figure
`
`Serum insulin level of multiple dosing treatment of insulin loaded
`
`composite microspheres
`
`Dose 80 lU/rat
`
`.3
`
`UI
`
`25
`
`30
`
`Tlm day
`
`

`

`US 2007/0 122487 Al
`
`May 31 2007
`
`POLYACRYLOYL-HYDROXYETHYL
`STARCH-PLGA COMPOSITION MICROSPHERES
`
`FIELD OF THE INVENTION
`
`sphere
`
`The present invention relates to
`system comprising
`polyDL-lactide-co-glycolide
`
`composite micro-
`
`PLGA polyacryloyl hydroxyethyl
`starch AcHES and
`effective amount of biologically active
`pharmaceutically
`The active compound may be for example an
`compound
`hormone-releasing hor
`insulin an interferon luteinizing
`mone LHRH analogs
`and
`somatostatin
`derivatives
`thereof calicitonin parathyroid hormone PTH bone mor
`phogenic protein BMP erythropoietin EPO epidermal
`growth factor EGF or growth hormone This invention also
`relates to methods of using the composite microspheres and
`methods of preparing same
`
`BACKGROUND OF THE INVENTION
`
`Biodegradable microspheres are effective as deliv
`ery systems for biologically active peptides and proteins
`Sustained release characteristics of microspheres reduce the
`and enhance patient com
`need for frequent administrations
`pliance by maintaining in vivo drug levels in the therapeutic
`of this drug delivery system
`range Additional advantages
`include
`controlled
`hiocompatibility
`
`biodegradability
`
`absorbability non-toxicity degradation products
`potential
`for sustained release and targeting and ease of administra
`tion
`
`associated with
`there are problems
`Nevertheless
`the use of currently known niicrosphere systems for protein
`has been observed
`delivery For example protein instability
`during the preparation of protein-loaded microspheres See
`Control Release 6841-51 2000 Usually an
`Li et at
`aqueous protein solution is dispersed in an organic polymer
`solution by using
`homogenizer or sonicator
`to create
`water-in-oil emulsion However
`the exposure of protein to
`organic solvent often has adverse effects on the stability of
`the proteins During drug release the absorption of protcin
`on the hydrophobic polymer matrices and
`low pH gener
`ated during the polymer degradation process
`could cause
`degradation of the entrapped protein
`
`second problem is presented by the initial burst
`The fast difib
`release of protein drugs from microspheres
`sion of protein drugs located on the surface of internal pores
`and channels
`formed by the evaporation of solvent and water
`during the microsphere preparation contributes to the burst
`release One approach to overcome this probleoi
`is the
`or physical blending of PLGA with
`chemical modification
`hydrophilic monomers and polymers such as polyethylene
`glycol PEG polyethylene-co-vinyl acetate
`and polyvi
`nyl alcohol PVA See Nam et al
`.J Microencapsul
`16625-637 1999 Another approach
`is physical encapsu
`Iatioo of protein-loaded hydmphilic particles or hydrogels
`PLGA matrix
`into
`
`However
`in the preparation of microspheres pro
`to large amounts of organic
`tein drugs are often exposed
`solvent and multiple freezing-thawing
`or heating-cooling
`processes during protein loading in the primary hydrophilic
`
`delivery of proteins and polypeptides
`Structural or con.for
`mational changes of proteins during microsphere manufac
`and release are known See Cleland et al
`turing storage
`Pharni Res 14 420-5 1997 and Crotts etal..J Microen
`capsuL 15 699-7 13 1998 For example during micro-
`is often
`protein solution
`an aqueous
`sphere preparation
`homog
`dispersed in an organic polymer solution by using
`water-in-oil emulsion The
`enizer or sonicator
`to create
`exposure of proteins to organic solvent and high sheer has
`on the integnty of the proteins See Nihani
`adverse effects
`et al. .1 Gollotd Interface Set 17355 65 1995 During
`and release protein unfolding and aggregation
`drug storage
`often occurs due to interaction of protein molecules with the
`the low pH gen
`surface Moreover
`hydrophobic polymeric
`erated duriog polymer erosion causes chemical degradation
`of entrapped proteins
`
`PLGA otierospheres are also considered to have an
`inconsistent
`high initial burst
`release profile For example
`plateau and
`effect within 24 hours followed
`by
`then
`culminating in incomplete release is often seen This initial
`burst
`because high initial
`drug release is not
`is undesirable
`suitable for therapeutic proteins due to the risk of side effects
`been made to
`from high serum levels Attempts
`have
`improve protein stability and release kinetics of the PLGA
`system by changing the physieo-ehemical
`properties of the
`polymer For example both chemical derivation and physi
`cal blending of PLGA with hydrophilie monomers
`and
`such
`polymers
`glycol polyethylene-eo
`as polyethylene
`vinyl acetate and polyvinyl alcohol have been reported See
`Péan et al Pharm Res 161294-1299 1999 However
`to
`date there is little understanding of the in vitro and in vivo
`behavior of composite microspheres and essentially no in
`evaluation
`of
`vivo pharmacological
`from these composite systems
`
`therapeutic proteins
`
`SUMMARY OF THE INVENTION
`An object of the present invention is to provide
`composite micmsphere
`system comprising polyD.L-lae
`tide-eo-glyeolide PLGA polyaeryloyl
`hydroxyethyl
`starch AcHES and
`effective amount
`phnrmaeeutieally
`of biologically active compound wherein the biologically
`active compound is
`polypeptide having molecular weight
`of about 200
`60000 Daltoos Preferably
`to about
`the
`biologically active compound is an insulin an interferon
`hormone LHRH analog
`luteinizing hormone-releasing
`somatostatin and/or somatostatin derivative
`ealicitonin
`parathyroid hormone PTH
`bone morphogenic protein
`BMP an erythropoietin EPO an epidermal growth factor
`EGF or
`growth honnone
`Another object of the present invention provides
`drug formulation comprising
`composite mierosphere sys
`polyDL-laetide-co glycolide PLGA
`tem comprising
`starch AcHES and
`phar
`polyaeryloyl hydroxyethyl
`biologically active com
`effective amount of
`maceutically
`pound wherein the biologically active compound is an
`luteinizing hormone-releasing hor
`insulin an interferon
`mone LHRH analog
`somatostatio and/or
`somatostatin
`parathyroid hormone PTH
`bone morphogeoie protein BMP an erythropoietin EPO
`an epidermal growth factor EGF or
`growth hormone and
`
`ealicitonin
`
`derivative
`
`particles
`
`pharmaceutically
`
`acceptable
`
`vehicle
`
`Despite success with small peptides PLGA micro-
`are known to have
`number of problems with
`
`spheres
`
`further object of the present invention provides
`method for the sustained release delivery of
`
`therapeutic
`
`

`

`US 2007/0 122487 Al
`
`May
`
`2007
`
`subject
`
`subject comprising administering
`compound to
`to the
`composite microsphere system comprising polyl
`L-lactidc-eo-glycolide PLGA polyacryloyl
`hydroxy
`ethyl starch AcHES and
`effective
`pharmaceutically
`biologically active compound
`amount of
`wherein the
`biologically active compound is an insulin an interferon
`hormone LHRH analog
`
`luteinizing hormone-releasing
`
`somatostatin and/or somatostatin derivative
`calicitonin
`parathyroid hormone PTH
`bone morphogenic
`BMP an erythropoietin EPO an epidermal growth factor
`EGF or
`growth hormone
`
`protein
`
`tin derivative
`
`calicitonin
`
`the subject is suffering from condition
`Preferably
`Which may be treated and/or cured by the administration of
`an insulin an interferon
`luteinizing hormone-releasing
`hormone LHRH analog
`somatostatin and/or somatosta
`parathyroid hormone PTH
`bone morphogenic protein BMP an erythropoietin EPO
`an epidermal growth factor EGF or
`growth hormone
`the subject
`vertebrate or an invertebrate
`Preferably
`is
`organism More preferably the subject
`an ovine
`primate an equine
`porcine
`camelid an avian
`bovine an amphibian
`murine organism Most preferably the primate is
`
`is
`
`canine
`
`feline
`
`caprine
`
`fish or
`human
`
`Preferably the microspheres are in
`pharmaceu
`vehicle and the microspheres are adniin
`tically acceptable
`istered topically Topical administration may include inha
`administration Also preferably
`lation
`nasal
`the
`or
`microspheres may be administered parenterally Preferably
`the microspheres may be administered intramuscularly
`
`parathyroid
`
`calicitonin
`
`protein
`
`further object of the present invention provides
`composite microsphere system com
`method of preparing
`selected
`biologically active ingredient
`prises incorporating
`from the group consisting of an insulin an interferon
`hormone LHRH analog
`luteinizing hormone-releasing
`somatostatin and/or somatostatin derivative
`hormone PTH bone morphogenic
`BMP an euythropoietin EPO an epidernial growth factor
`EGF or
`growth hormone into AcHES hydrogel micro-
`AcHES
`the
`particles encapsulating
`resulting
`hydrogel
`containing the biologically active ingredient
`microparticles
`into PLGA matrix Preferably the AcHES hydrogel micro-
`the biologically active ingredient
`are
`particles containing
`into the PLGA matrix using either solvent
`solvent evaporation
`spray drying freeze drying
`extraction
`combination thereof
`
`incorporated
`
`or
`
`FIG
`shows
`insulin from
`in vitro release of
`composite micmspheres in glycine buffer at 37
`Sonica
`tion levels are iodicatcd by
`
`FIG
`serum glucose suppression in dia
`betic rats treated with insulin loaded composite microsphere
`and
`
`shows
`
`batches
`
`FIG
`treated diabetic rats
`
`shows
`
`senun
`
`insulin level of
`
`and
`
`FIG
`shows blood glucose suppression of niul
`tiple dosing treatment of insulin loaded composite micro-
`spheres n8 Dose 80 lU/rat
`FIG shows serum insulin level of multiple dos
`ing treatment of
`insulin loaded composite microspheres
`n8 Dose 80 lU/rat
`
`DETAILED DESCRIPTION
`PRFFERRED EMBODIMENI
`INVENTION
`
`OF THE
`OF THE
`
`lefinitinns
`
`the tenns in the present application are
`In general
`used consistently with the manner in which those terms are
`understood in the art
`
`By micro is meant
`particle having
`from nanometers to micrometers
`
`diameter of
`
`By composite microsphere is meant
`sphere matrix formed of at
`two different polymeric
`least
`materials In the present invention
`the polymeric materials
`include PLGA particles and AcHES particles Acompusite
`is an aggregation of microspheres made as described herein
`bound by materials known to those skilled in the art for this
`
`micro-
`
`purpose
`
`vehicle is meant
`By pharmaceutically acceptable
`the vehicles in which the composite mierospheres are sus
`pended or distributed The vehicles may include excipients
`and auxiliaries which
`facilitate processing of the active
`compounds into preparations wluch can be used for delivery
`to the site of action such as oil adjuvants
`
`By effective amount or dose effective amount
`ortherapeutically eflective amount is meant an amount of
`an agent which modulates
`activity of the pro
`biological
`teins of the invention
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`II Introduction
`
`FIG shows SEM micrographs of insulin-loaded
`AcHES-PLGA
`interior struc
`
`composite microspheres
`the arrows indicate
`tare of
`fractured microsphere
`embedded AcHES microparticles and freeze dried AcHES
`hydrogel microparticles
`
`HPLC chromatogram of an insulin
`FIG shows
`by ACN
`sample isolated from composite microspheres
`extraction and the intact
`insulin standard
`
`FIG shows
`characterization of insulin integrity
`shows SDS-PAGE with
`in the composite microspheres
`DTT Lane
`shows the molecular weight marker Lane
`shows
`the bovine insulin standard Lanes
`shows an
`and
`shows MALDI-TOF MS of insulin
`insulin samples
`extracted from composite in comparison to intact standard
`
`sphere
`
`The present invention provides
`composite micro-
`pulylL-Iactide-eo-glyeolide
`system comprising
`PLGA pulyaeryloyl hydroxyethyl
`starch AcHES and
`effective amount of
`biologically active
`pharmaceutically
`compound Preferably
`the biologically active compound is
`molecular weight of about 200 to
`polypeptide having
`about 160000 Daltuns More preferably the active com
`luteinizing hormone-
`pound is an insulin an interlèron
`releasing hormone LHRH analogs
`somatostatin and/or
`parathyroid hor
`mone PTH bone morphogenic protein BMP an eryth
`ropoietin EPO an epidennal growth factor EGF or
`growth hormone This novel composite mierosphere system
`on PLGA and polyaeryloyl hydroxyethyl starch
`based
`AcHES was developed using bovine serum albumin and
`horseradish peroxidase HRP These
`composite micro-
`
`somatostatin derivative
`
`calicitonin
`
`

`

`US 2007/0122487 Al
`
`May 31 2007
`
`show more favorable and complete release than
`spheres
`conventional PLGA microspheres
`The present
`invention
`also provides methods of using and preparing the composite
`microsphere system
`
`Composite Microsphere System and Formulations
`
`invention
`
`comprises
`
`and/or
`
`somatnstatin
`
`calicitonin
`
`parathyroid
`
`The composite microsphere system of the present
`polyDL-lactide-co-glycolide
`PLGA pnlyacryloyl hydroxyethyl
`starch AcHES and
`effective amount of biologically active
`pharmaceutically
`compound The biologically active compound may include
`is not limited to an insulin an interferon
`but
`luteinizing
`hormone-releasing hormone LHRH analog
`somatostatin derivative
`bone morphogenic protein BMP an
`hormone PTH
`erythropoietin EPO epidermal growth factor ECiF or
`growth hormone Types of insulin appropriate for use with
`limited to human
`the present invention include but are not
`insulin and bovine insulin Types of interferons appropriate
`for use with the present invention include but are not limited
`beta-interferon and gamma-interferon
`to alpha-interferon
`Types of calcitonin
`appropriate for use with the present
`invention include eel human and salmon
`
`ing polyDL-lactide-co-glycolide
`
`hydroxyethyl
`amount of
`
`drug formu
`The present invention also provides
`composite niicrosphere system compris
`lation comprising
`PLGA polyacryloyl
`starch AcHES pharmaceutically effective
`and
`pharma
`biologically active
`compound
`vehicle Preferably
`the biologically
`ceutically
`acceptable
`active compound is an insulin an interferon
`hormone-releasing hormone LHRH analog
`and/or
`somatostatin derivative
`calicitnnin
`protein BMP an
`hormone PTII
`bone morphogenic
`erythropoietin EPO an epidermal growth factor EGF or
`growth hormone
`
`luteinizing
`
`snmatostatin
`
`parathyroid
`
`The
`
`vehicles may
`pharmaceutically
`acceptable
`limited to any excipients and auxiliaries
`include but are not
`which facilitate processing of the active compounds into
`preparations which can be used pharmaceutically
`for deliv
`ery to the site of action For example suitable formulations
`fur parenteral administration may include aqueous solutions
`of the active compounds in water-soluble form for example
`salts Optionally the suspension may also
`water-soluble
`contain stabilizers
`
`The composite microsphere system and formula
`tion are appropriate for parenteral or topical administration
`topical routes may include nasal administration
`types of composi
`or inhalation
`indicated
`the different
`formulations may be used simultaneously
`systemic administration of the active ingredient
`
`Specifically
`
`tions and
`
`achieve
`
`If
`
`to
`
`mone-releasing
`
`and/or somatostatin derivative
`
`calicitonin
`
`parathyroid
`
`the
`
`the
`
`conditions according to generally accepted medical practice
`The agents of the present
`invention can be provided alone
`in combination with other agents
`or
`that modulate
`par
`ticular pathological process As used herein two or more
`agents are said to be administered in combination when the
`or are admin
`two agents
`are administered simultaneously
`istered independently in
`act contemporaneously
`
`fashion such that
`
`the agents will
`
`Methods of Treatment Using the Composite Microsphere
`System
`
`method for
`
`compos
`
`polymeric
`
`The present
`invention also provides
`therapeutic compound to
`the sustained release delivery of
`subject comprising administering to the subject
`ite microsphere system comprising AcHES
`such as PLGA and
`material
`effective
`pharmaceutically
`amount of biologically active compound contained within
`the AcHES microsphere Preferably the biologically active
`luteinizing hor
`compound is an insulin an interferon
`hormone LHRH analog
`somatnstatin
`protein BMP an
`hormone PTH bone morphogenie
`erythropoietin EPO an epidermal growth factor EGF or
`growth hormone The subject may be
`vertebrate or an
`the subject may be
`invertebrate organism Specifically
`canine
`feline ass ovine
`primate an equine
`porcine
`bovine an amphibian
`camelid an avian
`fish
`caprine
`human
`or murine organism Preferably the primate is
`The amount of active biologically active com
`pound incorporated
`in the microsphere
`system delivery
`device varies widely depending on the particular agent
`desired effect and the time span over which it
`takes
`matrix to release the compound The upper and lower
`limits
`on the amount of the compnund to be incorporated into the
`device can be determined empirically by comparing micro
`range of compound The dosage admin
`spheres containing
`patient will be dependent also on the age health
`istered to
`and weight of the recipient
`if
`treatment
`type of concurrent
`any frequency of treatment and the nature and stage of
`disease and the nature of the effect desired The microsphere
`system of the methods of the present
`invention are prefer
`ably administered topically via inhalation or nasal admin
`istration or by parenteral administration
`as described above
`produced
`are
`Micrnspheres
`standard gauge needle
`small enough to be injected through
`under the skin or into the peritoneum for subsequent
`release
`of incorporated drug Adhesion of the microspheres to the
`peritnneum aids
`release of the incorporated
`in localizing
`drug Microspheres can also be implanted or injected intra
`for immunization or other
`and subcutaneously
`muscularly
`purposes where slower
`release into the bloodstream is
`desirable
`
`The composite microsphere system and formula
`tions of this invention may be used alone or in combination
`with each other or in combination with other therapeutic or
`diagnostic agents especially in eases where an insulin an
`hormone
`interferon
`luteinizing
`hormone-releasing
`LHRH analog
`somatostatin and/or somatostatin deriva
`hormone PHI
`bone
`morphogenic protein BMP an erythropoietin EPO an
`factor EGF or
`growth hormone is
`epidermal growth
`In certain preferred embodiments
`indicated for treatment
`the compounds of this invention may be co-administered
`along with other compounds typically prescribed for these
`
`tive
`
`calicitonin
`
`parathyroid
`
`The micrnspbere system of the present
`invention
`may be administered in order to target
`specific organ For
`example the lung may be targeted by administering
`the
`microsphere system via inhalation The lung may also be
`targeted by using intravenous IV administration so long as
`the micro
`the microsphere size is appropriate Specifically
`should be about 8-30 microns in size in order to
`
`spheres
`lodge in the capillaries of the lungs Microspheres as large
`as 130 microns may be administered
`and subcutaneous
`needle for intramuscular
`
`via
`
`hypodermic
`administration
`
`Microspheres may be suspended
`suitable phar
`using methods appro
`maceutical vehicle for administration
`
`in
`
`

`

`Us 2007/0122487 Al
`
`May 31 2007
`
`pnate for the vehicle and site of administration Vehicles
`may include phosphate buffer saline gelatin
`cellulosic
`surfactant or an oil adjuvant Suitable phammccu
`agent
`tical vehicles are known to those skilled in the art and
`
`and make ttìe subsequent
`release of the active ingredient
`on polymer hydration and mass loss As
`release dependant
`such as Tween 20
`surfactants
`an alteniate to sonication
`may be used to reduce the burst of the active ingredient
`
`commercially available
`
`used
`
`The diseases disorders and conditions which may
`be treated by the methods
`and systems of
`the present
`include diabetes The present
`invention may be
`uivention
`to deliver
`insulin to the body in the treatment of
`diabetes Patients suffering from diabetes often have to take
`one or more types of insulin for the rest of their lives The
`method of treatment of the presesit invention may be con
`venient and sate in delivering
`insulin to diabetic patients
`
`The present invention may also be used to deliver
`in need of same The
`human growth honisone to
`subject
`present invention may further be used to deliver
`interferon
`to the body in the treatment of diseases
`including but not
`and
`limited to cancer
`conditions
`related to
`Acquired Immune Deficiency Syndrome AIDS In addi
`tion the present invention may be used to deliver LHRH
`somatostatin and denvatives
`analogs
`PTH BMP EPO and EGP to
`The
`
`hepatitis
`
`thereof ealeitnnin
`in need thereof
`
`subject
`
`include but
`
`is not
`
`ingredient may
`active
`also
`biologically
`limited to melanocyte stimulating hor
`hormone TRH lutein
`mone MSH1 thyrotropin-releasing
`izing hormone LH follicle
`stimulating hormone FSH
`vasopressin oxytonin parathyroid hormone glucagon gas
`secretin pancreozymin
`
`laetogen human ehononie gonadotropin HCG enkepha
`
`eholecystokinin
`
`angiotensin
`
`EXAMPLE
`
`Preparation and Characterization of Mierospheres
`
`starch-based
`
`hydrngel particles con
`Hydrophilie
`taining model proteins were prepared by
`simple swelling
`procedure The protein-loaded hydrogel particles were then
`in PLGA mierospheres to form hydrogel
`encapsulated
`PLGA combined composite mierospheres
`solvent
`using
`extraction or evaporation method Bovine senim albumin
`BSA and horseradish peroxidase HRP were used
`as
`model protein drugs Physieoehemieal characteristics and in
`vitro protein release of mierospheres were studied to estab
`stareh-PLGA AcHES
`hydroxyethyl
`lish polyaerylol
`PLGA composite microspheres as
`protein delivery sys
`tem
`
`PolyD.L-laetide-eo-glyeolide PLGA copoly
`mer ratio of 5050 laetie/glycolie M\/
`and
`28000
`Resomer RG503H were supplied by Boehringer
`Ingel
`heim Ingelheim Germany Hydroxyethyl starch was
`obtained from Dupont Phannaceutieals Wilmington Del.
`Aenyloyl chloride was purchased from Aldrich Chemicals
`Company Inc Milwaukee Wis. BSA and polyvinyl alco
`hol PVA MW 30000-70000 were obtained from Sigma
`Chemical Co St Louis Mo HRP IStepTM Slow TIvIB
`ELISA and micro-BCA protein assay kit were obtained from
`Pierce Roekford Ill.
`
`Preparation of AcHES Hydrogel Particles
`
`distilled
`
`acryloyl
`
`An acrylic
`starch
`ester of hydroxyethyl
`acid
`of HES was dissolved in 60 mL
`CAcHES was prepared 20
`dimethyl acetamide and an appropriate amount 2-10 mL of
`chloride based on desired degree of
`derivatizatinn DD number of vinyl groups
`inirndueed on
`group in every unit of HES polymer
`the hydroxyethyl
`amount of triethylamine were
`chain and
`an equimolar
`added slowly to the HES solution The reaction mixture was
`precipitated by adding 200 inL of precooled acetone and the
`precipitate was dissolved in 50 mL deionized water The
`solution was transferred to
`dialysis tubing with molecular
`weight cutoff of 14000 and dialyzed against deionized water
`with frequent change of water for 48 hours The dialyzed
`The DD was determined
`solution was
`freeze-dried
`by
`proton-NMR spectroscopy 22
`AcHES polymer 73 mixture of DD0.14 and
`in 0.IM phosphate-buffered
`0.25 was dissolved
`saline
`PBS pH 7.4 to make
`30% w/v solution and arnrou
`nium peroxidisulfate
`was added to the solution to form
`dispersed phase The dispersed phase was added to 50 mL
`mineral nil containing 0.3% of Sorbitan Sesquinleate while
`stirring to form w/o emulsion NNNNtetramethyleth
`ylenediamine 300 t.LL was added to the emulsion to initiate
`the polymerization reaction followed by continuous stirring
`hour The suspension containing
`at room temperature for
`droplets was poured into preeooled hexane
`polymerized
`The AcHES hydrngel particles were col
`while sonicating
`at 1000 rpm for minutes washed
`lected by centrifugation
`twice with hexane and ethanol
`rinsed with deionized water
`several times and freeze-dried
`
`tn
`
`kyntorphin tuftsin thymopoietin thymosin
`lin endorphin
`factor TBP serum thymic
`thymosthymlin thymic humnral
`factor FTS tumor necrosis factors TNF colony stimu
`lating factors CSF motilin dynnrphin bombesin neuro
`asparaginase kal
`tensin eaerulein bradykinin urukinase
`likrein substance
`nerve growth factor blood coagulation
`factor VIII and IX lysozyme chloride polymyxin
`eolis
`tin gramieidin bacitraein
`synthesin-stimulating
`
`protein
`
`peptide
`
`invention
`
`ing incorporating
`
`luteinizing
`
`somatostatin
`
`parathyroid
`
`parathyroid
`
`hor
`
`gastric inh.ibitoD polypeptide GIP vasoactive
`intestinal polypeptide VIP platelet-derived
`growth factor
`PDGP growth hormone releasing factor GRF and bone
`morphagenetic protein BMIP
`Methods of Preparing the Composite Mierosphere System
`method for the
`The present
`provides
`composite mierosphere system of compris
`preparation of
`an insulin an interferon
`hormone-releasing hormone LHRH analog
`and/or somatostatin derivative
`ealicitonin
`protein BMP an
`honnone PTH
`bone morphogenic
`erythropoietin EPO an epidermal growth factor EGF or
`growth hormone into AcHES hydrogel micropartieles
`and
`encapsulating the resulting AcHES hydrogel micrnpartieles
`luteinizing hormone-
`containing an insulin an interferon
`releasing hormone LHRI-l analog
`somatostatin and/or
`somatostatin
`derivative
`ealicitonin
`mone PTH bone morphogenie protein BMP an eryth
`ropoietin PIN an epidennal growth
`factor EGF or
`growth hormone into PLGA matrix The AcHES hydrogel
`containing the active ingredient
`microparticles
`are prefer
`ably incorporated into the PLGA matrix using either solvent
`spray drying freeze drying
`solvent evaporation
`extraction
`combination
`thereof
`
`or
`
`more homogenous disn-ibution of hydrogel par
`the PLGA matrix will
`retard the initial
`ticles throughout
`
`

`

`US 2007/0 122487 Al
`
`May 31 2007
`
`Preparation of PLC1A-AcHES Composite Microsplieres
`
`The PLGA-AcHES
`composite and conventional
`PLGA microspheres were prepared by modified solvent
`extraction of ovaporation mcthod with 5-10% targot
`loading
`of BSA and 5% for HRP 25-50mg of BSA and 25 mg HRP
`were dissolved in 0.23 mL of 0.1
`PBS pH 7.4 The
`to AcHES particles 10% or
`protein solutions were added
`total polymer weight and the particles were allowed to
`mm with vortex mixing at
`room temperature
`swell
`for
`Twenty percent wlw PLGA 90% of total polymer weight
`to the swollen AcHES
`in methylene chloride was added
`particles and vortexed for minutes at room temperature to
`form protein in hydrogell.polymer in colvent dispersion
`This primary dispersion was then added to precooled 100
`mL 6% PVA solution
`Silverson mixer
`and stirred by
`Silverson Chesham Bucks England
`at 5000 rpm for
`minute The resulting secondary suspension was transferred
`deionized water and stirred gently for hours at room
`temperature to remove the organic solvent and solidify the
`polymer The microspheres were washed with water and
`the conventional PLGA microspheres
`freeze-dried For
`primary emulsion was prepared by mixing the protein solu
`tions with 20% PLGA solution and then the emulsion was
`to 6% PVA solution while stirring at 5000 rpm The
`added
`resulting suspension was transferred to
`deionized water
`and stirred gently for
`hours at room temperature to remove
`the organic solvent and fabricate the polymer The micro
`spheres were washed with water and freeze-dried
`
`to
`
`Particle Characterization and Morphology of Microspheres
`
`PLGA-AcHES microspheres 10 mg were dis
`persed in 10 mL 0.1% Tween 80 solution The particles were
`sized by laser difllactometry by using Malvern 2600 laser
`size The average particle size was expressed as the volume
`mean diameter
`in micrometers
`
`The surface morphology
`and intomal structure of
`fractured microspheres were examined by scanning electron
`microscopy SEM model S800 Hitachi Tokyo
`Japan
`coating of the microsphere samples on
`
`after palladium-gold
`aluminum stub
`
`Drug Loading Efficiency
`
`PLGA-AcHES
`Ten-milligram protein-loaded
`microspheres were hydrolyzed in mixture of 0.9 mL of
`NaOH and 0.1 mL PBS with vigorous shaking at room
`mL
`hour Protein standard solutions
`temperature for
`were also hydrolyzed after adding 0.9 mL
`NaOH using
`HCI was
`the same procedures After hydrolysis
`niL 0.9
`added to neutralize the samplesolutions Protein concentra
`tions were determined by micro-BCA total protein assay
`method The loading efficiency was calculated by the actual
`protein loading to the theoretical
`in
`loading of protein
`PLGA-AcHES microspheres
`based on the amount used in
`the microsphere preparation
`
`In Vitro Protein Release
`
`Microspheres were weighed and placed in 5-mL
`centrifuge tubes containing PBS with 0.02% sodium azide
`The tubes were incubated at 37
`as
`with
`preservative
`occasional shaking At designated times samples were col
`lected and the release medium was replaced with fresh PBS
`The samples were assayed by micro-BCA method or by
`fluorescence spectrophotometer model P2000 Hita
`using
`
`clii at excitation aod emission wavelengths of 280 and 350
`nm The two assay methods showed comparable results for
`in vitro release samples
`
`Protein Stability
`
`integrity of proteins extracted from
`The structural
`the microspheres was characterized by size exclusion chro
`matography SEC and sodium dodecyl sulfate polyacry
`lamide gel eleetrophoresis SDS PAGE Ten-mg of micro-
`spheres were dissolved in 0.1 mL CHC12 Proteins were
`extracted from the polymer solution by addition of ml 0.1
`PBS followed by agitation for
`hour
`
`SEC was performed by using Biosep SEC 52000
`column 4.6x300 mm Phenomenex Torrance Calif. The
`phosphate buffer pH 7.0 contain
`mobile phase was 0.1
`ing 0.02% sodium azide The flow rate was 0.5 mLimin and
`the detection wavelength was UV 280 nm The injection
`volume was 20 pL
`
`SDS-PAGE was carried out in the presence of 0.1%
`SDS using
`9% slab gel prepared by
`gel casting and
`eleetrophoresis unit Mini-Protean
`electrophoresis sys
`tem