,,.
`
`Volume 8 •
`
`.~''.
`
`PHARMACY LIBRARY
`UNIVERSITY OF WISCONSIN
`JUN 0 6 2001
`
`Madison, WI 53705
`
`Editors~in-Chief
`-~Affred Stracher • -rony L. Whateley
`
`IPR2015-00410
`Petitioners' Ex. 1019
`Page 1
`
`

`

`DRUG
`DELIVERY
`
`THE JOURNAL OF DELIVERY AND TARGETING OF THERAPEUTIC AGENTS
`
`J I
`
`EDITORS-IN-CHIEF
`Alfred Stracher
`Department of Biochemistry, The State University of
`New York, Health Science Center at Brooklyn, 450
`Clarkson Avenue, Brooklyn, NY 11023, (718) 270-1256
`phone; (718) 270-3316 fax; strachea@hscbklyn.edu
`
`Tony L. Whateley
`Department of Pharmaceutical Sciences, University of
`Strathclyde, SIBS, 27 Taylor Street, Glasgow 04 ONR,
`Scotland, +44-141-5524400phone; +44-141-5526443 fax;
`t.l.whateley@strath.ac.uk
`
`ADVISORY EDITORS
`Carl Alving Walter Reed Army Institute of Research
`W. French Anderson
`USC Norris Cancer Center
`Sydney Brenner Scripps Clinic Research Institute
`Robert S. Langer Massachusetts Institute of Technology
`Richard A. Lerner Scripps Clinic Research Institute
`George Poste SmithKline Beecham Pharmaceutical
`Glynn Wilson
`Tacora Corporation
`
`EDITORS
`Theresa M. Allen University of Alberta; David R.
`Bard Strangeways Research Laboratory; Brian W.
`Barry University of Bradford; John H. Collett
`University of Manchester; Elazer R. Edelman
`Massachusetts Institute of Technology; Robert F.
`Furchgott State University of New York, Brooklyn;
`Colin R. Gat·dner Merck Sharp and Dohme Research
`Laboratory; Yoshita Ikada Kyoto University;
`Rakesh K. Jain Harvard Medical School; Leo Kesner
`State University of New York, Brooklyn; Jindrich
`Kopecek University of Utah; Rimona Margalit Tel
`Aviv University; Claude F. Meares University of
`California, Davis; Dirk K.F. Meijer University of
`Groningen; Michel Monsigny University of Orleans;
`Marc Ostro The Liposome Company; Demetrios
`Paphadjopoulos University of California, San
`Francisco; William M. Pardridge University of
`California School of Medicine, Los Angeles; Colin W.
`Ponton University of Bath; Murray Saffran Medical
`College of Ohio; Peter Senter Bristol-Myers Squibb
`Pharmaceutical Research Institute; David Shepro
`Boston University; John W. Weinstein National
`Institutes of Health; Clive G. Wilson University of
`Strathclyde; Richard J. Youle National Institutes of
`Health
`
`Abstracted and/or Indexed in: Biochemistry & Biophysics Citation Index, Chemical Abstracts, EMBASE, Index Medicus, and
`MEDLINE, Research Alert, and SciSearch.
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`@The paper in this publication meets the requirements of the ANSI Standard Z39.48-1984 (Permanence of
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`
`IPR2015-00410
`Petitioners' Ex. 1019
`Page 2
`
`

`

`

`

`94
`
`Q. YANG ET AL.
`
`2000). To date, there are three Food and Drug Administra(cid:173)
`tion (FDA) approved drugs for the treatment of Alzheimer's
`disease-tacrine, donepezil and rivastigmine-and several oth(cid:173)
`ers in clinical trials (Alzheimer's Association 2000). Tacrine,
`an acetylcholinesterase inhibitor, was approved by the FDA
`in 1993 for treatment of Alzheimer's disease. Tacrine is indi(cid:173)
`cated for patients with mild
`to moderate Alzheimer's
`disease, and its clinical effects have been limited due to as(cid:173)
`sociated cholinergic, hepatic, and gastrointestinal adverse reac(cid:173)
`tions (Abramowicz 1993; Qizilbash et al. 2000). One recent
`study showed that gastrointestinal side effects, such as diar(cid:173)
`rhea, anorexia, dyspepsia, and abdominal pain, and raised serum
`liver enzymes were the major reason for withdrawal (Qizilbash
`et al. 2000).
`Controlled drug delivery systems offer an advantage over the
`conventional approach because the polymer that is applied in
`the design of such systems controls the rate at which the drug
`is released. Microencapsulated tacrine could be delivered sys(cid:173)
`temically for a prolonged period of time at controlled lower
`concentrations to reduce the hepatic adverse reactions and by(cid:173)
`pass the gastrointestinal route. Encapsulation of water-soluble
`drugs by the use of nonaqueous systems such as oil-in-oil emul(cid:173)
`sion technique generally results in high encapsulation efficiency
`(0 'Donnell and McGinity 1998; Marinina et al. 2000). However,
`processing the microparticles could be quite tedious if vegetable
`and mineral oils are involved. Our paper describes the formula(cid:173)
`tion and characterization of a controlled release tacrine delivery
`system prepared by a simple oil-in-water emulsion technique
`with a high encapsulation efficiency, intended for the treatment
`of Alzheimer's disease.
`
`MATERIALS AND METHODS
`
`Materials
`Poly(D,L-lactide-co-glycolide) (PLG) (50/50) of different
`molecular weight grades were supplied by Birmingham
`Polymers (Birmingham, AL, USA). Polyvinyl alcohol (PVA)
`(M.W. 30,000-70,000) and tacrine were obtained from Sigma
`Chemical Company (St. Louis, MO, USA). Methylene chloride
`and methanol were supplied by Fisher Scientific (Norcross, GA,
`USA). Distilled de-ionized water was used. All materials were
`used as supplied.
`
`Preparation of Microspheres
`Microspheres were prepared by the emulsion solvent evapo(cid:173)
`ration procedure. PVA was dissolved in water to prepare
`1% w/v solution. Then 500 mg of PLG and 300 mg of tacrine
`were dissolved in a mixed organic solvent of methylene chloride
`and methanol. Afterward, the drug-polymer solution was added
`to 25 mL ofPVA solution that was being stirred at 500 rpm with
`aLightninMixer (General Signal, NY, USA). The emulsion was
`stirred overnight to remove the organic solvents. The microparti(cid:173)
`cles were collected by filtration, washed with water, and dried in
`
`air for 2 days. All products were sieve-sized using a combination
`of U.S. staqdard sieve numbers 40, 60, 120, and 400. Fractions
`collected b~tween 40/60 (250-425 ttm), 60/120 (125-250 f.tln),
`and 120/400 (37-125 ttm) were used for further studies. The
`effects of polymer molecular weight, stir speed during prepa(cid:173)
`ration, and drug loading on the encapsulation efficiency, and
`kinetics of tacrine release were investigated. The experiments
`were conducted in triplicates.
`
`Morphology of Microspheres
`The surface morphology, shape and size of tacrine micro(cid:173)
`spheres, was obtained using an Electroscan Model E-3 scanning
`electron microscope. The environmental scanning electron mi(cid:173)
`croscope allowed samples to be examined in their natural state
`without modification or preparation.
`
`Determination of Tacrine Encapsulation Efficiency (EEF)
`Fully 10 mg of tacrine microspheres were dispersed in
`10 mL of methylene chloride that dissolved the polymer but not
`the drug. Tacrine was extracted five times with a total of 100 ml of
`water and analyzed spectrophotometrically. The amount of drug
`in the microspheres was determined using an UV-1201 spec(cid:173)
`trophotometer (Schimadzu Scientific Instruments, MD, USA)
`at 323 nm. The EEF was determined as the ratio of the amount
`analyzed to the initial amount of the drug added during prepa(cid:173)
`ration.
`
`In Vitro Release of Tacrine from Microspheres
`The equivalent of 10 mg of microspheres or free drug was
`used in all studies. The microspheres or the free drug was placed
`in 900 mL of distilled deionized water in dissolution beakers
`(USP Apparatus 2), and stirred at 100 rpm using a VK 7000 dis(cid:173)
`solution testing station (VanKel Technology Group~NC, USA).
`The VK 7000 was interfaced with the ADS 2000 sampling sta(cid:173)
`tion and an UV-120 1 spectrophotometer. The dissolution system
`was programmed to collect samples automatically through full
`flow filters at specified time intervals from the various beakers
`and circulated through the spectrophotometer for automatic
`recording of absorbances on a computer. Each sample was au(cid:173)
`tomatically returned to its original beaker after each measure(cid:173)
`ment. The volume of the dissolution medium in each beaker
`was maintained at 900 mL throughout the dissolution studies.
`Absorbances of dissolved iacrine were measured at 323 nm and
`concentrations of the drug were calculated from a standard curve.
`Release data for three batches of microspheres were combined
`in one plot.
`In another release study conducted for more than 1 month
`in an incubator, microspheres were suspended in 200 ml of
`pH 7.4 phosphate buffer in closed containers and agitated at
`50 rpm. Samples of dissolved tacrine were removed at specified
`time intervals for spectrophotometric analysis. Fresh buffer was
`added each time to replace withdrawn samples to maintain the
`volume at 200 ml.
`
`IPR2015-00410
`Petitioners' Ex. 1019
`Page 4
`
`

`

`

`

`96
`
`Q. YANG ET AL.
`
`TABLE2
`Encapsulation efficiency and particle size distribution of microspheres.prepared with poly(D,L-lactide-co-glycolide)
`of molecular weight 155,000 as a function of stitJ/speed during preparation
`
`EEF (%)
`
`Particle size range (%)
`
`Stir speed (RPM)
`
`250-425 f.-till
`
`125-250 p,m
`
`250-425 p,m
`
`125-250 f.-till
`
`37-125 p,m
`
`400
`500
`800
`
`84.9 (0.6)
`85.0 (1.1)
`46.8 (2.0)
`
`51.0 (0.3)
`33.0 (1.1)
`43.3 (2.1)
`
`73.2 (2.2)
`71.7 (2.1)
`52.8 (0.4)
`
`26.5 (2.2)
`27.9 (2.0)
`39.4 (1.0)
`
`0.3 (0.1)
`0.4 (0.2)
`7.8 (1.4)
`
`Standard error of the mean shown in parenthesis.
`
`of stir speed during preparation is the particle size distribution
`of microspheres collected in different size ranges (Table 2). The
`higher the stit· speed, the higher the shearing force provided to
`break down the oil phase into smaller microdroplets. About 50%
`of microspheres prepared with stir speed of 800 rpm were under
`250 p,m compared with 30% for microspheres prepared with a
`stir speed of 500 rpm. However, no significant difference could
`be seen between microspheres prepared at 400 and 500 rpm. A
`number of studies involving emulsion solvent evaporation tech(cid:173)
`nique clearly show that the limiting factor determining particle
`size and particle size distribution of microparticles is the stir
`speed used during preparation (Jeffrey et al. 1991; Sandrap and
`Moes 1993; Cleland et al. 1997). The effect of stir speed during
`preparation on the release profiles of tacrine from microspheres
`of 250-425 p,m can be seen in Figure 3. Although the EEF for
`microspheres prepared with stir speed 800 was about half of
`those prepared with stir speed 400 and 500 rpm, no significant
`difference in release profiles could be seen. This suggests that
`within the range of stir speed studied, stir speed did not have an
`effect on the integrity of the microspheres.
`
`100
`
`The amount of drug to be incorporated into microparticles
`during preparation may play a role in the EEF and release of
`drug from the microparticles. For microspheres of250-425 f.-tin,
`increasing the drug loading from 16.7% to 37.5% w/w did not
`have a significant effect on the EEF (Table 3). However, fur(cid:173)
`ther increase from 37.5% to 50% w/w resulted in a significant
`decrease in EEF. Although this phenomenon is not clearly un(cid:173)
`derstood, it could be attributed to the integrity of the micro(cid:173)
`spheres formed when the drug loading was increased to 50%
`w/w during preparation. Continuous stirring overnight during
`preparation of microspheres to remove organic solvents could
`result in a significant loss of a water-soluble drug if very leachy
`microspheres are formed. This assumption is supported by the
`fact that drug loading did not have a significant effect on the
`release profiles of microspheres of 16.7%, 28.6%, and 37.5%
`w/w drug loading, respectively (Figure 4). Less than 5% of the
`drug was released from these microspheres in 24 hr. In sharp
`contrast, about five times as much drug was released from mi(cid:173)
`cropheres of 50% w/w drug loading during the same period of
`time.
`To find out if these microspheres would be suitable as a
`prolonged release dosage form, we conducted a prolonged re(cid:173)
`lease study for microspheres prepared at 500 rpm and 37.5%
`w/w drug loading. A 35-day continuous release study can be
`seen in Figure 5 for microspheres of size range 125-250 p,m
`
`TABLE3
`Encapsulation efficiency of microspheres prepared with
`poly(D,L-lactide-co-glycolide) of molecular weight 155,000 as
`a function of drug loading
`
`EEF (%)
`
`Drug loading(%)
`
`250-425 p,m
`
`125-250 p,m
`
`50
`37.5
`28.6
`16.7
`
`71.5 (2.4)
`85.0 (1.1)
`88.7 (2.5)
`89.0 (2.5)
`
`40.0 (1.1)
`33.0 (1.1)
`59.0 (1.1)
`70.0 (1.1)
`
`Standard error of the mean shown in parenthesis.
`
`Q)
`
`Q)
`
`'0. 80
`g)
`60 &
`
`Q)
`t:
`'I:
`u
`~
`'#.
`
`40
`
`20
`
`0
`
`0
`
`......,.._ 400 rpm
`-11- 500 rpm
`__....... 800 rpm
`-+- Free drug
`
`4
`
`8
`
`12
`Time (h)
`
`16
`
`-·
`
`24
`
`20
`
`FIG. 3. The effect of stir speed (rpm) during preparation at a constant PYA
`concentration of 1% w/v and a volume fraction of 22% on the release profiles
`of tacrine microspheres. (A.) 400, C•) 500, (e) 800. The dissolution of the free
`drug is shown for comparison C•).
`
`IPR2015-00410
`Petitioners' Ex. 1019
`Page 6
`
`

`

`

`

`98
`
`Q. YANG ET AL.
`
`Sansdrasp, P., and Moes, A. J. 1993. Influence of manufacturing parameters on
`the size characteristics and the release profiles of nifedipine from poly(DL(cid:173)
`lactide-co-glycolide) microspheres. Int. J. Pharm. 98:157-164.
`Simonson, W. 1998. Promising agents for treatment of Alzheimer's disease. Am.
`J. Health Syst. Pharm. 55(Suppl2):S11-S16.
`Sramek, J. J., and Cutler, N. R. 1999. Recent developments in the drug treatment
`of Alzheimer's disease. Drugs Aging 14:359-373.
`
`Yang, Q., Reams, R., and Owusu-Ababio, G. 1999. Effect of solvent composition
`during prep,aration on the characteristics of enoxacin microspheres. J. Ph arm.
`Pharmacd/.51:659-665.
`You an, B. B. C., Benoit, M. A., and Gillard, J. 1999. Protein-loaded poly( epsilon(cid:173)
`caprolactone) microparticles. I. Optimization of the preparation by (water(cid:173)
`in-oil) in water emulsion solvent evaporation. J. Microencap. 16:587-
`599.
`
`IPR2015-00410
`Petitioners' Ex. 1019
`Page 8
`
`