articles
`
`Design of Potent Amorphous Drug Nanoparticles for
`Rapid Generation of Highly Supersaturated Media
`
`Michal E. Matteucci,† Blair K. Brettmann,† True L. Rogers,‡ Edmund J. Elder,‡
`Robert O. Williams, III,§ and Keith P. Johnston*,†
`Department of Chemical Engineering, The UniVersity of Texas, Austin, Texas 78712,
`College of Pharmacy, The UniVersity of Texas, Austin, Texas 78712, and The Dow
`Chemical Company, Midland, Michigan 48640
`
`Received February 15, 2007; Revised Manuscript Received June 4, 2007; Accepted June 20, 2007
`
`Abstract: Controlled precipitation produced aqueous nanoparticle suspensions of a poorly water
`soluble drug, itraconazole (ITZ), in an amorphous state, despite unusually high potencies (drug weight/
`total weight) of up to 94%. Adsorption of the amphiphilic stabilizer hydroxypropylmethylcellulose
`(HPMC) at the particle–aqueous solution interface arrested particle growth, producing surface areas
`from 13 to 51 m2/g. Dissolution of the particles in acidic media yielded high plateau levels in
`supersaturation up to 90 times the equilibrium solubility. The degree of supersaturation increased
`with particle curvature, as characterized by the surface area and described qualitatively by the Kelvin
`equation. A thermodynamic analysis indicated HPMC maintained amorphous ITZ in the solid phase
`with a fugacity 90 times the crystalline value, while it did not influence the fugacity of ITZ in the
`aqueous phase. High surface areas led to more rapid and levels of supersaturation higher than
`those seen for low-surface area solid dispersions, which undergo crystallization during slow
`dissolution. The rapid generation of high levels of supersaturation with potent amorphous nanopar-
`ticles, containing small amounts of stabilizers oriented at the particle surface, offers new opportunities
`for improving bioavailability of poorly water soluble drugs.
`Keywords: Supersaturation; amorphous pharmaceutical; nanoparticle dissolution; poorly water
`soluble drug; metastable solubility; itraconazole
`
`increases the surface area, A, while high-energy polymorphs,
`including the amorphous state, increase the solubility, Csat,
`both leading to faster dissolution rates4–10 as predicted by
`the Noyes–Whitney equation
`
`1. Introduction
`For biopharmaceutical classification system (BCS) type
`II drugs with high permeability through biomembranes,
`bioavailability in oral delivery is limited by the dissolution
`rate.1–4 Particle size reduction of crystalline materials
`
`* To whom correspondence should be addressed: Department of
`Chemical Engineering, The University of Texas, 1 University
`Station, C0400, Austin, TX 78712. Telephone: (512) 471-
`4671. Fax: (512) 475-7824. E-mail: kpj@che.utexas.edu.
`† Department of Chemical Engineering, The University of Texas.
`‡ The Dow Chemical Co.
`§ College of Pharmacy, The University of Texas.
`(1) Amidon, G. L.; Lennernas, H.; Sha, V. P.; Crison, J. R. A
`theoretical basis for a biopharmaceutic drug classification: The
`correlation of in vitro drug product dissolution and in vivo
`bioavailability. Pharm. Res. 1995, 12, 413–420.
`(2) Horter, D.; Dressman, J. B. Influence of physicochemical proper-
`ties on dissolution of drugs in the gastrointestinal tract. AdV. Drug
`DeliVery ReV. 2001, 46, 75–87.
`
`782 MOLECULAR PHARMACEUTICS VOL. 4, NO. 5, 782–793
`
`(Csat
`
`- C)
`
`dm
`) DA
`dt
`h
`where D is the diffusion coefficient, h is the diffusional path
`length, and C is the concentration in solution. Precipitation
`of an organic drug solution with either water or a supercritical
`fluid antisolvent is commonly used to form high-surface area
`particles, both crystalline and amorphous. During precipita-
`tion, mixing energy11–13 and stabilization with polymers are
`
`(1)
`
`(3) Hancock, B. C.; Parks, M. What is the True Solubility Advantage
`for Amorphous Pharmaceuticals. Pharm. Res. 2000, 17 (4), 397–
`404.
`(4) Singhal, D.; Curatolo, W. Drug polymorphism and dosage form
`design: A practical perspective. AdV. Drug DeliVery ReV. 2004,
`56, 335–347.
`
`10.1021/mp0700211 CCC: $37.00  2007 American Chemical Society
`Published on Web 08/24/2007
`
`

`

`articles
`
`Design of Potent Amorphous Drug Nanoparticles for
`Rapid Generation of Highly Supersaturated Media
`
`Michal E. Matteucci,† Blair K. Brettmann,† True L. Rogers,‡ Edmund J. Elder,‡
`Robert O. Williams, III,§ and Keith P. Johnston*,†
`Department of Chemical Engineering, The UniVersity of Texas, Austin, Texas 78712,
`College of Pharmacy, The UniVersity of Texas, Austin, Texas 78712, and The Dow
`Chemical Company, Midland, Michigan 48640
`
`Received February 15, 2007; Revised Manuscript Received June 4, 2007; Accepted June 20, 2007
`
`Abstract: Controlled precipitation produced aqueous nanoparticle suspensions of a poorly water
`soluble drug, itraconazole (ITZ), in an amorphous state, despite unusually high potencies (drug weight/
`total weight) of up to 94%. Adsorption of the amphiphilic stabilizer hydroxypropylmethylcellulose
`(HPMC) at the particle–aqueous solution interface arrested particle growth, producing surface areas
`from 13 to 51 m2/g. Dissolution of the particles in acidic media yielded high plateau levels in
`supersaturation up to 90 times the equilibrium solubility. The degree of supersaturation increased
`with particle curvature, as characterized by the surface area and described qualitatively by the Kelvin
`equation. A thermodynamic analysis indicated HPMC maintained amorphous ITZ in the solid phase
`with a fugacity 90 times the crystalline value, while it did not influence the fugacity of ITZ in the
`aqueous phase. High surface areas led to more rapid and levels of supersaturation higher than
`those seen for low-surface area solid dispersions, which undergo crystallization during slow
`dissolution. The rapid generation of high levels of supersaturation with potent amorphous nanopar-
`ticles, containing small amounts of stabilizers oriented at the particle surface, offers new opportunities
`for improving bioavailability of poorly water soluble drugs.
`Keywords: Supersaturation; amorphous pharmaceutical; nanoparticle dissolution; poorly water
`soluble drug; metastable solubility; itraconazole
`
`increases the surface area, A, while high-energy polymorphs,
`including the amorphous state, increase the solubility, Csat,
`both leading to faster dissolution rates4–10 as predicted by
`the Noyes–Whitney equation
`
`1. Introduction
`For biopharmaceutical classification system (BCS) type
`II drugs with high permeability through biomembranes,
`bioavailability in oral delivery is limited by the dissolution
`rate.1–4 Particle size reduction of crystalline materials
`
`* To whom correspondence should be addressed: Department of
`Chemical Engineering, The University of Texas, 1 University
`Station, C0400, Austin, TX 78712. Telephone: (512) 471-
`4671. Fax: (512) 475-7824. E-mail: kpj@che.utexas.edu.
`† Department of Chemical Engineering, The University of Texas.
`‡ The Dow Chemical Co.
`§ College of Pharmacy, The University of Texas.
`(1) Amidon, G. L.; Lennernas, H.; Sha, V. P.; Crison, J. R. A
`theoretical basis for a biopharmaceutic drug classification: The
`correlation of in vitro drug product dissolution and in vivo
`bioavailability. Pharm. Res. 1995, 12, 413–420.
`(2) Horter, D.; Dressman, J. B. Influence of physicochemical proper-
`ties on dissolution of drugs in the gastrointestinal tract. AdV. Drug
`DeliVery ReV. 2001, 46, 75–87.
`
`782 MOLECULAR PHARMACEUTICS VOL. 4, NO. 5, 782–793
`
`(Csat
`
`- C)
`
`dm
`) DA
`dt
`h
`where D is the diffusion coefficient, h is the diffusional path
`length, and C is the concentration in solution. Precipitation
`of an organic drug solution with either water or a supercritical
`fluid antisolvent is commonly used to form high-surface area
`particles, both crystalline and amorphous. During precipita-
`tion, mixing energy11–13 and stabilization with polymers are
`
`(1)
`
`(3) Hancock, B. C.; Parks, M. What is the True Solubility Advantage
`for Amorphous Pharmaceuticals. Pharm. Res. 2000, 17 (4), 397–
`404.
`(4) Singhal, D.; Curatolo, W. Drug polymorphism and dosage form
`design: A practical perspective. AdV. Drug DeliVery ReV. 2004,
`56, 335–347.
`
`10.1021/mp0700211 CCC: $37.00  2007 American Chemical Society
`Published on Web 08/24/2007
`
`

`

`Potent Amorphous Drug Nanoparticles
`
`used to control particle size and morphology.14–19 Thera-
`peutic proteins are often formulated in the amorphous state
`with stabilizing excipients to ensure physical and chemical
`stability.20,21 Theoretical solubilities of amorphous pharma-
`ceuticals in aqueous media have been predicted with
`thermodynamic models to be 100-fold and up to 1600-fold
`greater than that of the crystalline form.3,19,22 Experimentally
`
`(5) Yamashita, K.; Nakate, T.; Okimoto, K.; Ohike, A.; Tokunaga,
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`maceutics 2004, 1 (6), 406–413.
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`articles
`
`measured solubilities, however, often reach only a fraction
`of the theoretical value, for example, 4.5 experimental versus
`∼100 theoretical for indomethacin.3,19 A high degree of
`supersaturation for amorphous formulations will increase Csat
`and thus the flux across biomembranes.2,23–27 In this case,
`drug molecules are in a metastable state and may precipitate
`by nucleation and growth to lower the free energy. Particle
`growth of embryo crystals may be inhibited by coating drug
`particles with polymeric stabilizers,25,28–31 particularly
`HPMC.5,23,25,29
`Storage stability of an amorphous solid is a great concern,
`as the high-energy solid state may relax to the lower-free
`energy crystalline form. Properties such as the glass transition
`(Tg), reduced crystallization temperature (Tc – Tg or Tm –
`Tg), and fragility of the amorphous drug can be used to
`describe the molecular mobility and characterize the stability
`at typical storage temperatures.3,19,32–34 Solid dispersions and
`solid solutions with high-Tg polymers are often utilized to
`
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`1144.
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`Moore, D. J. Solid-State Stabilization of R-Chymotrypsin and
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`Some Thermodynamic Relations of Glassy and R-Crystalline
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`1997, 86, 1–12.
`(25) Kumprakob, U.; Kawakami, J.; Adachi, I. Permeation Enhance-
`ment of Ketoprofen Using a Supersaturated System with Anti-
`nucleant Polymers. Biol. Pharm. Bull. 2005, 28 (9), 1684–1688.
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`Determination of Drug Solubility in Polymeric Matrices. J. Pharm.
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`cellulose polymers on supersaturation and in vitro membrane
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`(28) Gao, P.; Rush, B. D.; Pfund, W. P.; Huang, T.; Bauer, J. M.;
`Morozowich, W.; Kuo, M.-s.; Hageman, M. J. Development of a
`supersaturable SEDDS (S-SEDDS) formulation of paclitaxel with
`improved oral bioavailability. J. Pharm. Sci. 2003, 92 (12), 2386–
`2398.
`(29) Gao, P.; Guyton, M. E.; Huang, T.; Bauer, J. M.; Stefanski, K. J.;
`Lu, Q. Enhanced Oral Bioavailability of a Poorly Water Soluble
`Drug PNU-91325 by Supersaturatable Formulations. Drug DeV.
`Ind. Pharm. 2004, 30 (2), 221–229.
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`lization of hydrocortisone acetate: Influence of polymers. Int.
`J. Pharm. 2001, 212 (2), 213–221.
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`and Aqueous Solutions. Chem. ReV. 2002, 102, 2627–2650.
`
`VOL. 4, NO. 5 MOLECULAR PHARMACEUTICS 783
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`

`

`articles
`
`reduce the mobility of drug molecules, thereby preventing
`crystallization during both the particle formation step and
`storage.5,35,36
`Amorphous drug formulations, either solid dispersions or
`co-ground mixtures with polymers such as HPMC,5,36–41
`poly(vinylpyrolidone),42 polyethylene glycol,43 aminoalkyl
`methacrylate copolymer (Eudragit E 100),44 methacrylic acid-
`methacrylic acid methyl ester copolymer (Eudragit L),45
`carboxymethylethylcellulose,45 and microcrystalline cel-
`lulose,7 have been used to create supersaturated solutions.
`Suzuki et al. produced solid dispersions of nifedipine
`stabilized with HPMC and PVP at drug loadings (drug
`weight/total weight) from 11 to 33%, where supersaturation
`levels were up to 6 times the crystalline nifedipine
`solubility.38,40 A supersaturation of 45 in 2 h was achieved
`
`(33) Angell, C. A. Thermodynamic Aspects of the Glass Transition in
`Liquids and Plastic Crystals. Pure Appl. Chem. 1991, 63 (10),
`1387–1392.
`(34) Zhou, D.; Zhang, G. G. Z.; Law, D.; Grant, D. J. W.; Schmitt,
`E. A. Physical Stability of Amorphous Pharmaceuticals: Impor-
`tance of Configurational Thermodynamic Quantities and Molec-
`ular Mobility. J. Pharm. Sci. 2002, 91 (8), 1863–1872.
`(35) Hasegawa, A.; Kawamura, R.; Nakagawa, H.; Sugimoto, I.
`Physical Properties of Solid Dispersions of Poorly Water-Soluble
`Drugs with Enteric Coating Agents. Chem. Pharm. Bull. 1985,
`33 (8), 3429–3435.
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`Brewster, M. E. Characterization of solid dispersions of itracona-
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`Nakai, T. Dissolution mechanism and rate of solid dispersion
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`and Polyethylene Glycol as Carriers for Nifedipine Solid Disper-
`sion Systems. Chem. Pharm. Bull. 1997, 45 (10), 1688–1693.
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`the dissolution of nifedipine solid dispersions with combined
`carriers. Chem. Pharm. Bull. 1998, 46 (3), 482–487.
`(40) Suzuki, H.; Sunada, H. Some Factors Influencing the Dissolution
`of Solid Dispersions with Nicotinamide and Hydroxypropylm-
`ethylcellulose as Combined Carriers. Chem. Pharm. Bull. 1998,
`46 (6), 1015–1020.
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`K.; Mullens, J.; Benoist, L.; Thimon, M.; Meublat, L.; Verreck,
`G.; Peeters, J.; Brewster, M. E.; Van den Mooter, G. Characteriza-
`tion of Solid Dispersions of Itraconazole and Hydroxypropylm-
`ethylcellulose Prepared by Melt Extrusion. Part II. Pharm. Res.
`2003, 20 (7), 1047–1054.
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`with hydroxypropyl cellulose on the dissolution and particle size
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`and polyethylene glycol 2000: Dissolution properties and physico-
`chemical characterization. Eur. J. Pharm. Biopharm. 2005, 59
`(1), 107–118.
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`
`784 MOLECULAR PHARMACEUTICS VOL. 4, NO. 5
`
`Matteucci et al.
`
`in 0.1 N HCl for a solid dispersion of ITZ with HPMC
`formed by hot melt extrusion at a drug loading of 40%.36,41
`Amorphous solid dispersions of tacrolimus produced by
`solvent evaporation with PEG 6000, PVP, and HPMC at 50%
`drug loading were shown to supersaturate 0.1 N HCl up to
`25 in 2 h.5 In all of these studies, supersaturation levels were
`obtained only for drug loadings of e50%.
`Previous studies of supersaturation from amorphous drug
`particles have focused primarily on solid dispersions with
`stabilizers dispersed throughout the entire particle.5,36–45
`These formulations had relatively low surface areas (<1 m2/
`g) and required a large amount of stabilizer, typically 50%
`(weight) or more, to inhibit crystallization of drug domains
`and to achieve sufficient hydrophilicities on the particle
`surfaces for wetting. For high-dose drugs,
`it would be
`beneficial to produce amorphous particle formulations with
`larger drug loadings to achieve desired dosage sizes and to
`avoid side effects from excipients. The amount of am-
`phiphilic stabilizer may be reduced by forming the particles
`in the presence of water to orient the stabilizer preferentially
`at
`the interface between the drug particle and aqueous
`solution. This approach has been utilized to form crystalline
`particles in antisolvent processes16,46,47 and evaporative
`precipitation of organic48–50 and supercritical fluid51,52 solu-
`tions. Aqueous suspensions of sub-300 nm ITZ particles have
`been produced by CP with poloxamer 407 at drug loadings
`
`(45) Hasegawa, A.; Taguchi, M.; Suzuki, R.; Miyata, T.; Nakagawa,
`H.; Sugimoto, I. Supersaturation Mechanism of Drugs from Solid
`Dispersions with Enteric Coating Agents. Chem. Pharm. Bull.
`1988, 36 (12), 4941–4950.
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`precipitation of stabilized organic and block copolymer nanopar-
`ticles as unique composites. Polym. Mater. Sci. Eng. 2003, 89,
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`Theory, Experiment, and Use. Angew. Chem., Int. Ed. 2001, 40
`(23), 4330–4361.
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`dissolution of high potency itraconazole particles produced by
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`Potency Itraconazole Particles Formed by Evaporative Precipita-
`tion Into Aqueous Solution. Int. J. Pharm. 2005, 3.2 (1–2), 113–
`124.
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`Williams, R. O. I. Comparison of powder produced by evaporative
`precipitation into aqueous solution (EPAS) and spray freezing into
`liquid (SFL) technologies using novel Z-contrast STEM and
`complimentary techniques. Eur. J. Pharm. Biopharm. 2005, 60,
`81–89.
`(51) Reverchon, E. Supercritical antisolvent precipitation of micro- and
`nano-particles. J. Supercrit. Fluids 1999, 15 (1), 1–21.
`(52) Young, T. J.; Mawson, S. M.; Johnston, K. P. Rapid Expansion
`from Supercritical to Aqueous Solution to Produce Submicron
`Suspensions of Water-Insoluble Drugs. Biotechnol. Prog. 2000,
`16 (3), 402–407.
`
`

`

`Potent Amorphous Drug Nanoparticles
`
`up to 86%;53 however, the morphology and dissolution
`behavior were not investigated. To date, supersaturation of
`dissolution media from amorphous nanoparticles of poorly
`water soluble drugs has not been reported for particles with
`high drug loadings.
`The key objective of this study was to form amorphous
`ITZ nanoparticles with high drug loadings up to 80% to
`achieve unusually high supersaturation levels in acidic media.
`During particle formation, the orientation of the hydrophilic
`stabilizer HPMC near the surface of the particles may be
`expected to facilitate high drug loadings while still inhibiting
`particle growth and crystallization. In contrast, nearly all
`amorphous conventional solid dispersions, for example, ITZ
`and HPMC, are limited to 50% loading and surface areas of
`<2 m2/g. The rapid dissolution of high-surface area nano-
`particles has the potential to minimize the time window for
`solvent-mediated crystallization of the remaining solid phase
`and thereby favor higher supersaturation levels than for low-
`surface area solid dispersions.
`Itraconazole was chosen as a model poorly water soluble
`drug, given its extremely high lyophilicity, that is, octanol/
`water partition coefficient (log P) of 5.66, as reported by
`Janssen Pharmaceutica. The particles were characterized by
`modulated differential scanning calorimetry and X-ray dif-
`fraction to determine the crystallinity, scanning electron
`microscopy, Z-contrast transmission electron microscopy,
`X-ray photoelectron spectroscopy, BET surface area analysis,
`and contact angle measurements. The supersaturation be-
`havior of aqueous suspensions and dry powders containing
`ITZ was investigated in 0.1 N HCl acidic media. Contact
`angles with 0.1 N HCl were compared for the CP powders
`relative to solid dispersions formed by solvent evaporation
`to demonstrate preferential orientation of HPMC on the
`particle surface for the CP powders.
`The ITZ/HPMC suspensions were added to the dissolution
`media in increments to determine the maximum supersaturation
`level, which will be shown to approach the metastable solubility
`limit. High supersaturation values are facilitated by trapping
`ITZ in the amorphous state even for high drug loading. To place
`these results in perspective, the metastable solubility of a pure
`amorphous ITZ suspension was measured without any HPMC.
`A thermodynamic scheme was developed to examine the effect
`of HPMC on the fugacity of ITZ in the solid phase relative to
`the aqueous phase and to determine the ratio of amorphous and
`crystalline ITZ fugacity. The HPMC concentration influences
`the particle surface area during controlled precipitation and thus
`the supersaturation as predicted by the Kelvin equation. Mass
`transfer models were developed in an attempt to understand
`how the surface area and erosion of slowly dissolving HPMC
`influenced the dissolution rate. The exceptionally high surface
`area of the aggregated nanoparticles, with high wetted hydro-
`philic surface areas, will be shown to produce more rapid and
`
`(53) Matteucci, M. E.; Hotze, M. A.; Williams, R. O. I.; Johnston,
`K. P. Drug Nanoparticles by Antisolvent Precipitation: Mixing
`Energy Versus Surfactant Stabilization. Langmuir 2006, 22 (21),
`8951–8959.
`
`articles
`
`higher levels of supersaturation than in the case of lower-surface
`area solid dispersions (SD) with less hydrophilic surfaces.
`
`2. Materials and Methods
`
`2.1. Materials. BP grade ITZ was purchased from Hawk-
`ins, Inc. (Minneapolis, MN). HPMC E5 (viscosity of 5 cP
`in a 2% aqueous 25 °C solution) grade was a gift from The
`Dow Chemical Co. Poly(vinylpyrolidone) K15 (PVPK15)
`and poloxamer 407 (P407) NF grade were both obtained
`from Spectrum Chemical (Gardena, CA). Stabilized pa grade
`1,3-dioxolane was purchased from Acros Organics (Morris
`Plains, NJ). HPLC grade acetonitrile (ACN), ACS grade
`hydrochloric acid (HCl), and diethanolamine (DEA) were
`used as received from Fisher Chemicals (Fairlawn, NJ).
`2.2. Controlled Precipitation into Aqueous Solution.
`The method of CP developed by Rogers et al.16 was used to
`produce nanoparticle suspensions of itraconazole. Deionized
`water (120 g) containing an appropriate quantity of HPMC
`was used as the antisolvent phase into which 30 g of 1,3-
`dioxolane containing 3.3% (weight) ITZ was injected to form
`a fine precipitate. The organic phase was separated from the
`aqueous suspension via vacuum distillation. In some cases,
`the aqueous suspension was added dropwise to liquid
`nitrogen and lyophilized to form a powder using a Virtis
`Advantage Tray Lyophilizer (Virtis Co., Gardiner, NY) with
`primary drying at -35 °C for 24 h followed by secondary
`drying at 25 °C for 36 h. Dried powders were stored in a
`13% relative humidity environment.
`Solid
`Form a
`2.3. Solvent Evaporation To
`Dispersion (SD). Approximately 2 g of ITZ was added to
`20 mL of dichloromethane and the mixture agitated until
`the ITZ was completely dissolved. The ITZ solution was
`placed in a mortar, and an appropriate amount of HPMC
`was slowly added while the mixture was gently stirred with
`a pestle without any precipitation. The solution was stirred
`gently until approximately 90% of the dichloromethane
`volume was evaporated, leaving a clear viscous gel. The
`remaining dichloromethane was removed by heating to 50
`°C at a reduced pressure of ∼500 mtorr for 2 h. The resulting
`drug/polymer film was removed from the mortar and pestle
`with a straight razor blade and ground to a fine powder for
`30 min using a ceramic ball mill (1 cm bead size). The final
`powder was collected after filtration through a size 16 mesh
`sieve (<1190 µm pore size).
`2.4. Solubility Determination. To determine the solubility
`of crystalline ITZ at 37.2 °C, approximately 1.5 mg of bulk
`ITZ was placed in a glass vials containing 100 mL of 0.1 N
`HCl (pH 1.2). Two aliquots were removed from each vial
`after 18 h, immediately filtered with a 0.2 µm syringe filter,
`and diluted by one-half with ACN. Similarly, solubility was
`determined for solutions containing HPMC. Approximately
`50 mg of ITZ was added to 100 mL of HPMC dissolved in
`0.1 N HCl at concentrations of 0.5, 1, and 3.4 mg/mL. In all
`cases, drug concentrations were determined by high-
`performance liquid chromatography as described below with
`an n of at least 3.
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`Matteucci et al.
`
`2.5. Supersaturation Dissolution. Metastable solubility
`limits and rates of supersaturation were measured in 0.1 N HCl
`(pH 1.2) at 37.2 °C. A USP paddle method was adapted to
`accommodate smaller sample sizes using a VanKel VK6010
`Dissolution Tester with a Vanderkamp VK650A heater/circula-
`tor (VanKel, Cary, NC). Dissolution media (50 mL) were
`preheated in small 100 mL capacity dissolution vessels (Varian
`Inc., Cary, NC). Suspensions were added dropwise to the
`dissolution media at a rate of approximately 10 drops/min to
`reduce the amount of excess particle dosing after the solubility
`limit had been reached. The drops were no longer added when
`particles could barely be detected by the naked eye, minimizing
`heterogeneous sites for nucleation of the supersaturated solu-
`tions. In the case of powder dissolution, a sample weight (∼17.6
`mg of drug) equivalent to approximately 80 times the equilib-
`rium solubility (4.4 µg/mL, from the solubility study) of ITZ
`in 0.1 N HCl was added to the media. Sample aliquots (1.5
`mL) were taken at various time points. The aliquots were filtered
`immediately using a 0.2 µm syringe filter, and 0.8 mL of the
`filtrate was subsequently diluted with 0.8 mL of ACN. To
`investigate the possibility of sub-200 nm particles passing
`through the 0.2 µm filter, the experiment was also conducted
`using a 0.02 µm syringe filter. In all cases, the filtrate was
`completely clear upon visual inspection, and dynamic light
`scattering of the filtrate gave a count rate of less than 20K cps
`(too small for particle size analysis). For all samples, the drug
`concentration was quantified by high-performance liquid chro-
`matography as described below.
`2.6. High-Performance
`Liquid Chromatography
`(HPLC). ITZ concentrations were quantified using a Shi-
`madzu (Columbia, MD) LC-600 HPLC system. The mobile
`phase consisted of ACN, water, and DEA (70:30:0.05), and
`the flow rate was 1 mL/min. Using a detection wavelength
`of 263 nm, the ITZ peak eluted at 5.4 min. The standard
`curve linearity was verified from 500 to 1 µg/mL with an r2
`value of at least 0.999.
`2.7. Scanning Electron Microscopy (SEM). Aqueous
`suspensions were diluted by one-tenth using pure deionized
`water and then flash-frozen onto aluminum SEM stages.
`After lyophilization to remove all water, the remaining
`particles were gold-palladium sputter coated for 35 s.
`Micrographs were taken using a Hitachi S-4500 field
`emission scanning electron microscope with an accelerating
`voltage of 10 kV.
`2.8. X-ray Diffraction (XRD). Wide-angle X-ray scat-
`tering was employed to detect the crystallinity of dried drug
`powders using Cu KR1 radiation with a wavelength of
`1.54054 Å at 40 kV and 20 mA from a Philips PW 1720
`X-ray generator (Philips Analytical Inc., Natick, MA). A
`small amount of powder was pressed onto a glass slide to
`form a flat sample surface. The reflected intensity was
`measured at a 2θ angle between 10 and 30° with a step size
`of 0.05° and a dwell time of 9 s.
`2.9. Temperature-Modulated Differential Scanning
`Calorimetry (mDSC). Drug crystallinity was detected by a
`model 2920 modulated differential scanning calorimeter (TA
`Instruments, New Castle, DE) with a refrigerated cooling
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`
`xcryst
`
`system. The samples were purged with nitrogen at a flow
`rate of 150 mL/min. Samples were scanned after being placed
`in hermetically sealed aluminum pans. The amplitude used
`was 1 °C, the period 1 min, and the underlying heating rate
`5 °C/min. Integration of the area under the ITZ melting
`endotherm at 168 °C and crystallization peak at ap-
`proximately 120 °C estimated the percent crystallinity in the
`original sample (prior to DSC heating) by
`- ∆hcryst
`) ∆hmelt
`∆hmeltItz
`where xcryst is the percent crystallinity, ∆hmelt is the heat of
`melting, ∆hcryst is the heat of crystallization, and ∆hmeltItz is
`the heat of melting for pure crystalline ITZ. It was assumed
`that the heat necessary to melt 1 g of crystalline ITZ and
`that necessary to crystallize 1 g of amorphous ITZ are equal.
`For a purely crystalline material, the melting peak area will
`be equal to that of the bulk material and no crystallization
`peak will be observed, giving a value of 1 for xcryst. For a
`completely amorphous material, either no melting or crystal-
`lization peak will be observed or any crystallization peak
`area will be equal to the melting peak area, both giving an
`xcryst value