`
`
`
`Exhibit 1023
`
`P.J. Watts et al., Encapsulation of 5-
`aminosalicylic Acid into Eudragit RS
`Microspheres and Modulation of Their
`Release Characteristics by Use of
`Surfactants, 16 J. CONTROLLED RELEASE
`
`311 (1991) (“Watts”)  
`
`

`
`Journal of Controlled Release, 16 ( 199 1) 3 1 l-3 18
`0 1991 Elsevier Science Publishers B.V. 0168-3659/91/$03.50
`ADONIS016836599100072M
`
`COREL 00605
`
`311
`
`acid into Eudragit RS
`Encapsulation of 5aminosalicylic
`microspheres and modulation of their release characteristics by
`use of surfactants
`P.J. Watts, M.C. Davies and C.D. Melia
`
`Department ofPharmaceutical Sciences, University ofNottingham, Nottingham, U.K.
`(Received October 11, 1990; accepted
`in revised form November 29, 1990)
`
`An emulsification-solvent evaporation technique has been used to produce microspheres containing 5-
`aminosalicylic acid from the acrylate-methacrylate copolymer, EudragitTM RS. Two emulsifiers were
`used in the preparation procedure, either sodium dodecyl sulphate (SDS) or Tween 20, at a range of
`concentrations. Microspheres could be produced with both surfactants, although the yield using SDS
`was very low. The rate of drug release was found to be dependent on the concentration of emulsifier
`used to produce the microspheres. For example, microspheres produced using 0.25% w/v SDS released
`their encapsulated drug instantaneously on dissolution testing, whereas microspheres produced with-
`out surfactant provided prolonged release of drug ( tsO% > 360 min). Low temperature scanning elec-
`tron microscopy revealed for these two systems that the differences in release resulted from different
`degrees of porosity in the microsphere drug-polymer matrix.
`Keywords: Microencapsulation; Eudragit TM RS; Colon delivery; 5Aminosalicylic acid; Porosity
`
`Introduction
`Microencapsulation can be employed for the
`production
`of multiparticulate
`sustained or
`modified-release drug delivery systems. Several
`publications on the formulation and use of drug-
`containing microspheres
`have utilised
`the
`Eudragit TM (Rohm Pharma) series of polymers
`as the encapsulating materials
`[ l-5 1. The Eu-
`dragits are a family of polymers based on acrylic
`and methacrylic acids suitable for use in orally-
`administered drug delivery systems. They are
`available in a number of different grades pos-
`
`Correspondence to: M.C. Davies and C.D. Melia, Dept. of
`Pharmaceutical
`Sciences, University of Nottingham, Not-
`tingham NG7 2RD, U.K.
`
`sessing a range of physicochemical properties
`[ 6 1. Some rapidly dissolve at clearly defined pH
`values whereas two grades, Eudragits RL and RS,
`are insoluble in aqueous media but permeable
`and as such have been shown to be suitable for
`use in sustained-release microencapsulated dos-
`age forms [ l-41. The permeability of Eudragits
`RL and RS to aqueous solutions is due to the
`presence of quaternary ammonium groups in
`their structure; RL has the greater proportion of
`these groups and as such is more permeable than
`RS [7].
`As part of a research programme investigating
`the controlled delivery of drugs to the colon, we
`have produced Eudragit RS microspheres con-
`taining 5-aminosalicylic acid ( 5-ASA), an agent
`active against inflammatory bowel disease [ 8 1.
`
`

`
`312
`The colon-specific delivery of 5-ASA is currently
`receiving considerable research interest. Since 5-
`ASA largely is absorbed from the upper intes-
`tine, selective delivery into the colon is required
`for it to be therapeutically effective [ 81. A num-
`ber of approaches have been used to achieve this;
`for example, tablets coated with polymers which
`dissolve at colonic pH levels [ 93 and sustained
`release pellets whose release rate is maximal at
`colonic pH [ lo]. Other methods of achieving
`colon-specific delivery exploit the metabolising
`properties of colonic bacteria. Thus delivery sys-
`tems activated by bacterial cleavage of azo- bonds
`are under investigation: the azo- bonds have been
`incorporated
`into both polymers
`[ 11,12 ] and
`prodrugs
`[ 8 1. Prodrugs containing glycosidic
`linkages also have the potential to provide site-
`specific release of drugs into the colon [ 13 1. This
`paper describes the preparation, characterisa-
`tion and release properties of 5-ASA-loaded Eu-
`dragit RS microspheres, a potential sustained-re-
`lease colonic drug-delivery system, and describes
`how the concentration and type of surfactant
`used for fabrication can modulate microsphere
`porosity and subsequent drug release. These
`microspheres do not have intrinsic colon-spe-
`cific release characteristics and thus to maximise
`their therapeutic use they will require delivery
`within a colon-targeted device.
`Materials and Methods
`
`Materials
`Eudragit RS (Dumas, U.K., Tunbridge Wells),
`5-aminosalicylic acid (95-98% purity) (Sigma,
`Poole, U.K. ), sodium dodecyl sulphate (Sigma),
`Tween 20 (Sigma), methylene chloride (GPR
`grade) (Rhone-Poulenc, Dagenham, U.K. ) . All
`reagents were used as received.
`Methods
`Microsphere preparation
`Microspheres were produced using an emulsi-
`location-solvent evaporation procedure
`[ 14 1.
`Eudragit RS was dissolved in methylene chloride
`to produce a 10% w/v solution. 5-ASA was finely
`
`ground using a pestle and mortar to produce par-
`ticles less than 20 pm (as determined by light
`microscopy) and added to the polymer solution
`to form a 5% w/v suspension. The dispersion of
`drug into the polymer solution was aided by son-
`ication for 20-30 min. When necessary, extra
`cold water was added to the sonic bath to pre-
`vent excessive warming of the drug-polymer so-
`lution mixture. The drug-polymer-solvent phase
`was poured into an aqueous solution of either so-
`dium dodecyl sulphate (SDS) or Tween 20 with
`agitation using an overhead paddle stirrer (200
`rpm ). A range of surfactant concentrations were
`employed: 0, 0.05, 0.075, 0.10 and 0.25% w/v
`for SDS and 0.025, 0.05, 0.25, 0.5, and 1% w/v
`for Tween 20. Each microsphere formulation was
`produced at least in duplicate. Stirring was con-
`tinued at room temperature until evaporation of
`the methylene chloride was complete (typically
`4-5 h). In all cases, drug-polymer agglomerates
`were formed at the liquid surface as the solvent
`evaporation proceeded. This material was de-
`canted from the sunken microspheres at the end
`of solvent evaporation. The resulting micro-
`spheres were collected by filtration through a 20
`pm filter, washed with 200 ml of water and dried
`overnight in a freeze-drier. A 250-500 ,um size
`fraction of the dried microspheres was collected
`by sieving.
`Determination of drug content
`Microspheres were finely ground using a pes-
`tle and mortar and accurately weighed quantities
`were transferred to 100 ml flasks. 100 ml of pH
`7 phosphate buffer (0.05 M) was added to each
`and the solution was sonicated to ensure disper-
`sion of the powdered microspheres. After several
`hours of regular shaking, samples were with-
`drawn, passed through a 1 pm membrane filter
`and the UV absorbance measured at 330 nm. The
`drug concentration was calculated with refer-
`ence to a calibration curve of 5-ASA in pH 7
`phosphate buffer.
`Drug release studies
`Profiles of drug release from the microspheres
`were obtained using a USP (Apparatus 2) dis-
`solution tester. Microsphere samples were placed
`
`

`
`313
`range of Tween 20 and SDS examined. In the
`complete absence of surfactant microspheres
`were produced, but the yield was low (see Table
`1): at the end of solvent evaporation 20 to 30%
`of the added mass of drug and polymer formed
`into microspheres, the remaining material hav-
`ing formed agglomerates. With SDS, yields were
`little different, varying between 20 and 40% and
`were not related to surfactant concentration.
`Likewise for Tween 20, yields were similar at all
`surfactant concentrations
`although
`the effi-
`ciency of the process was considerably higher
`with between 70 and 80% of the drug and poly-
`mer formed into microspheres. The stirring speed
`was chosen
`to maximise
`the proportion of
`microspheres between 250 and 500 pm in diam-
`eter. Typically at least 30% by weight of the
`microspheres
`fell within this size range. The
`emulsification procedure was clearly consider-
`ably more efticient with Tween 20 as surfactant.
`The reason for the marked difference in yield
`seen between the two surfactants is unclear and
`is currently under investigation.
`Drug encapsulation efficiency was high (the-
`oretical maximum loading 33% w/w) and the fi-
`nal drug content of the microspheres did not ap-
`
`Yield
`(%)
`
`20-30
`
`in 500 ml of pH 7 phosphate buffer, containing
`0.02% w/v Tween 20 to aid wetting, and agita-
`ted at 100 rpm. 10 ml samples were withdrawn
`from the dissolution flasks at regular intervals,
`passed through a 1 pm membrane filter and the
`UV absorbance at 330 nm measured. The drug
`concentration was calculated from the calibra-
`tion curve of pure drug in pH 7 phosphate buffer.
`Any microspheres retained on the filter were re-
`turned to the dissolution vessel by washing with
`10 ml of fresh buffer. Into each dissolution vessel
`was placed approximately 120- 140 mg of micro-
`spheres containing 40-45 mg of 5-ASA. We esti-
`mated the solubility of 5-ASA to be at least 2 mg/
`ml at pH 7 and 37 ‘C, so the amount of drug in
`each dissolution vessel was well below saturation
`level. Dissolution profiles for equivalent concen-
`trations of unencapsulated drug were obtained in
`the same way. UV scans of 5-ASA between 200
`and 400 nm remained unchanged over the pe-
`riod of the release study. Two dissolution runs
`were
`carried
`out
`for
`each microsphere
`formulation.
`Low temperature scanning electron microscopy
`Electron micrographs were obtained of two
`samples; those produced using no surfactant and
`those produced using 0.25% w/v SDS. The elec-
`tron microscope
`stage was maintained
`at
`- 180’ C with liquid nitrogen in order to min-
`imise damage to the samples from electron beam
`irradiation
`[ 15,16 1. Samples were mounted on
`a holder using carbon cement and then plunged
`into nitrogen slush. For fracturing, the holder was
`placed into the microscope preparation chamber
`and a cold knife run through the sample. Sam-
`ples were then moved onto the microscope stage
`and examined at a low kV, while the temperature
`was raised to - 80” C to allow sublimation of any
`surface water. The sample was then moved back
`into the preparation chamber and sputter coated
`with gold, prior to return to the stage for imaging.
`Results and Discussion
`Microsphere production
`Microspheres containing 5-ASA could be pro-
`duced successfully over the entire concentration
`
`29.2+ 1.5
`30.1fl.5
`30.7f 1.0
`30.7* 1.4
`
`29.5 f 1.3
`30.1 f 1.4
`28.9+ 1.4
`29.2f0.4
`29.6f2.0
`
`Mean microsphere
`5-ASA content I
`w/v (n=3)
`29.3 + 1.3
`
`TABLE 1
`Drug content and yield of 5-ASA: Eudrogit RS microsphere
`formulations
`Surfactant concentration
`used to produce
`microspheres
`(% w/v)
`No surfactant
`SDS
`0.05
`0.075
`0.10
`0.25
`Tween 20
`0.025
`0.05
`0.25
`0.5
`1.0
`
`1
`
`I 20-40
`
`I
`
`* 70-80
`
`

`
`314
`of
`the concentration
`to depend upon
`pear
`surfactant (Table 1).
`An important issue to be considered when us-
`ing solvent evaporation
`techniques for produc-
`tion of microspheres is the potential problem of
`residual solvent. Since many of the solvents used,
`including methylene chloride, are toxic, it is im-
`portant
`that
`levels remaining
`in the micro-
`spheres at the end of production are minimal. It
`has recently been reported that methylene chlo-
`ride levels in 50: 50 Eudragit RS : RL micro-
`spheres produced by solvent evaporation and
`dried in air at 37°C were less than 0.1% [ 171.
`Although we did not measure the residual sol-
`vent levels in our microspheres,
`it would seem
`reasonable to assume that they are of a similar
`order of magnitude.
`
`Drug release characteristics
`The drug release profiles from microspheres
`prepared using SDS and Tween 20 as surfactants
`are shown in Figs. 1 and 2 respectively, and the
`times for 50% of the encapsulated drug to be re-
`leased ( t5,,%) are given in Table 2. For both SDS
`and Tween 20, the rates of drug release were
`found to be dependent upon the concentration
`of surfactant used in the production procedure,
`with a progressive enhancement
`in the rate of
`drug release with increasing surfactant concen-
`tration. The microspheres made without surfac-
`tant showed the greatest retardation
`in drug
`availability with a t5,,% for release greater than 360
`
`so
`
`60
`
`40
`
`20
`
`-----
`r
`-
`-
`-
`-
`-09b
`
`dmp&C
`
`*asI
`0.26%
`0.1%
`
`o.om?h
`O.MI
`
`0
`0
`
`60 120 180 240 300 260
`Time (mind
`Fig. 1. Effect of concentration
`of sodium dodecylsulphate
`(SDS) on the release
`rate of 5-ASA from Eudragit RS
`microspheres.
`
`0
`
`a0
`
`120 180 240 200 260
`mnc (mills)
`Fig. 2. Effect of concentration
`of Tween 20 on the release
`rate of 5-ASA from Eudragit RS microspheres.
`
`TABLE 2
`
`Time for 50% of encapsulated 5-ASA to be released ( tSosc from
`Eudragit RS microsphere
`formulations
`Surfactant
`type
`t5% for drug
`release
`and concentration
`(min)
`(0~ w/v)
`SDS
`0
`0.05
`0.075
`0.10
`0.25
`Tween 20
`0
`0.025
`0.05
`0.25
`0.5
`1.0
`
`> 360
`300
`160
`140
`<30
`> 360
`300
`120
`100
`30
`<30
`
`min. In contrast, microspheres made with 0.25%
`w/v SDS and 1% w/v Tween 20 showed little or
`no retardation
`in availability over unencapsu-
`lated 5-ASA. Such dramatic changes in drug re-
`lease rates with changing SDS and Tween 20
`concentrations suggested that these surfactants
`may have a significant influence on the structure
`of the microsphere drug-polymer matrix.
`Low temperature SEM
`In order to investigate the influence of surfac-
`tant on the structure of the microspheres and
`hence the drug release kinetics, we used low tem-
`perature scanning electron microscopy to exam-
`
`

`
`Fig. 3. Electron micrographs of microspheres prepared without surfactant. Top left, overall view of microsphere population
`(magnification
`x SO). Top right, detail of microsphere surface (X460). Bottom left, freeze-fractured section of microsphere
`( x260). Bottom right, high magnification view of bottom left x2600).
`ine two microsphere formulations that exhibited
`mated these pores to be up to 2 pm in diameter.
`extreme drug release profiles i.e. a rapidly releas-
`When cryogenically fractured, more dramatic
`ing system produced using 0.25% w/v SDS and
`structural differences were revealed between the
`a sustained-release
`system produced without
`two systems. Fig. 3c shows a sectioned view of a
`surfactant.
`representative microsphere from the surfactant-
`The surfactant-free system produced micro-
`free system. The drug-polymer matrix appeared
`spheres that were smooth and spheroidal in ap-
`generally dense with a number of large holes
`pearance (Fig. 3a). Higher magnification micro-
`present. In a magnified view of this section (Fig.
`graphs emphasised the smoothness and integrity
`3d), there appear to be drug crystals visible. In
`of the surface with only a few small surface pores
`contrast,
`the internal structure of the micro-
`being visible (Fig. 3b). In comparison,
`the
`spheres produced using 0.25% w/v SDS re-
`microspheres produced with surfactant were
`fleeted the surface topography. The drug-poly-
`roughly spheroidal in shape with an uneven outer
`mer matrix was extremely open and porous
`surface (Fig. 4a). The surface was seen to con-
`resulting in a sponge-like appearance (Fig. 4~).
`tain areas of high porosity (Fig. 4b). We esti-
`Fig. 4d shows a crystal of 5-ASA suspended in
`
`

`
`316
`
`prepared with 0.25% SDS as surfactant. Top left, overall view of microsphere
`Fig. 4. Electron micrographs of microspheres
`population
`( X 50). Top right, detail of microsphere surface ( X 330). Bottom left, freeze-fractured section of microsphere
`( x 340).
`Bottom right, high magnification view of bottom
`left ( x 3200 ).
`
`this open framework of polymer. There are fewer
`crystals visible in the micrographs of the 0.25%
`w/v SDS system because, although the w/w drug
`loading is equivalent to the surfactant-free sys-
`tem, the bulk density is considerably lower due
`to their highly porous nature.
`The SEM work demonstrates
`that the ex-
`tremely rapid release of drug from the 0.25%
`w/v SDS system is a consequence of a highly po-
`rous surface and internal structure, which allows
`rapid entry of dissolution medium into and dis-
`solved drug out of the microspheres. Conversely,
`in the surfactant-free
`system
`the 5-ASA is
`
`embedded in a dense, low porosity matrix. As a
`result, diffusion of aqueous medium
`into and
`dissolved drug out of the microspheres will be
`slow and to a greater extent through the hydrated
`polymer
`itself, rather than primarily
`through
`pores as in the case of the rapidly releasing
`system.
`
`Conclusions
`We have demonstrated
`that G-ISA can be suc-
`cessfully encapsulated into Eudragit RS to pro-
`duce microspheres
`for potential sustained-re-
`
`

`
`lease oral drug-delivery applications. Moreover,
`the rate of drug release can be controlled by the
`concentration and type of surfactant used in the
`manufacturing process. For SDS, the enhance-
`ment in release was seen to be due to surfactant-
`induced changes in the drug-polymer matrix re-
`sulting in increased microsphere porosity. Fur-
`ther studies are examining
`the mechanism by
`which pores are formed
`in these systems, al-
`though they probably arise from the deposition
`of the drug-polymer matrix around droplets of
`aqueous phase which have entered the forming
`microspheres
`as a result of the action of
`surfactant.
`There were also marked differences
`in the
`emulsification efficiency between the two sur-
`factants, with SDS producing microsphere yields
`as low as 20%, compared to 70% with Tween 20.
`Clearly to be considered as an economically via-
`ble process, microsphere yields as low as 20%
`would be unacceptable. However, in this work we
`have made no attempt to optimise the micros-
`phere yield by other means. As one example, by
`including baffles in the mixing vessel and thus
`reducing vortex formation,
`the yield of micro-
`spheres in a solvent evaporation process could
`be increased from 50 to 90% [ 18 1.
`There are several parameters that can be used
`to produce microsphere
`systems with a pro-
`grammed rate of drug delivery, for example mi-
`crosphere size or the level of drug-loading, but if
`there are fixed requirements
`for these, control
`over release needs to be exercised through addi-
`tional parameters such as the nature of the en-
`capsulating polymer or the inclusion of additives
`in the microsphere formulation e.g. plasticisers
`[ 141. We have shown, in addition, that the con-
`centration and type of surfactant can be used as
`a controlling factor to modulate the rate of drug
`release from Eudragit RS microspheres pro-
`duced by the emulsification-solvent evaporation
`process.
`
`Acknowledgements
`
`We would like to thank Dr. J. Sargent for assis-
`tance with the electron microscopy work and
`
`317
`and Colman Pharmaceuticals
`SERC/Reckitt
`(Dr. G. Parr) for funding PJW.
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