Polymeric Drug Delivery II Polymeric Matrices and ... - ACS Publications

Chapter 21

Downloaded by STANFORD UNIV GREEN LIBR on July 6, 2012 | http://pubs.acs.org Publication Date: March 9, 2006 | doi: 10.1021/bk-2006-0924.ch022

Preparation of Nanostructured Particles of Poorly Water Soluble Drugs via a Novel Ultrarapid Freezing Technology 1

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J. C. Evans , B. D. Scherzer, C. D. Tocco , G. B. Kupperblatt , J. N. Becker , D. L. Wilson , S. Saghir , and E. J. Elder 1

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Dowpharma and ToxicoIogy and Environmental Research and Consulting, The Dow Chemical Company, Midland,MI48674 Current address: Mylan Technologies, 110 Lake Street, St. Albans, V T 05478 3

Many drugs are rejected or have less-than-optimal performance because of poor water solubility. This research evaluated the dissolution rate and relative bioavailability of the poorly water soluble drug ketoeonazole following processing via an ultra-rapid-freezing, particle-engineering technology. Dissolution of ketoeonazole USP powder reached approximately 62% dissolved in 30 minutes. By comparison, the modified powders were 96% dissolved within 2 minutes. Improved bioavailability was demonstrated by a 4- to 7-fold increase in A U C with a corresponding 2- to 3.5-fold increase in Cmax.

Introduction Over 40% of potential drugs are rejected because of poor water solubility (1). O f the drugs currently on the market, approximately 17% have less-thanoptimal performance because of poor solubility and low bioavailability. Increasing the rate of dissolution may favorably impact the performance of poorly water soluble drugs (2). © 2006 American Chemical Society

In Polymeric Drug Delivery II; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction The precipitation of proteins in the presence of nonionic water-soluble polymers has been described by many investigators in the scientific literature. The interaction of dextran with proteins such as fibrinogen and other proteins was described in 1952 (1,2). For example, poly(vinylpyrolidone), (PVP), was used to precipitate Factor VIII and fibrinogen (3). Polyethylene glycol), (PEG), has been extensively studied as a precipitating agent for proteins (4). These publications focused on the conditions and mechanisms of the straightforward precipitation of proteins in aqueous solutions. However, this process of combining proteins and water-soluble polymers such as P V P or P E G typically resulted in the formation of an amorphous precipitate of no defined shape or size (Figure 1). In contrast to these amorphous precipitates, research in our labora tory has shown that we can reproducibly form discrete, monodisperse, protein microspheres by controlling the ionic strength, pH, polymer concentration, protein concentration and temperature of the aqueous protein polymer mixture. Previously, we have shown that the P R O M A X X process formed protein microspheres with very narrow particle size ranges (5). These protein micro spheres can be fabricated for sustained or immediate release (5,6). In this article, we describe the formulation of P R O M A X X insulin microspheres in the 1 to 2 micron particle size range. This particle size range was chosen in order to allow deep lung delivery of the therapeutic insulin molecule. We characterize the insu lin microspheres by particle size, aerodynamic performance, long term stability and bioactivity in vivo.

Figure 1. SEM of an amorphous mass of insulin, precipitated in the presence of PEG in aqueous solution.


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Materials and Methods Figure 2 shows crystalline recombinant human zinc insulin (Calbiochem, LaJolla, C A ) being formulated into P R O M A X X microspheres. The insulin crystals were suspended in deionized water. P E G 3350 polymer (Spectrum Pharmaceuticals, Gardena, C A ) was dissolved at p H 5.6 in a controlled ionic strength, aqueous solution in a glass, temperature controlled, water-jacketed liquid chromatography column. The insulin suspension was added to and dissolved in the P E G solution. The insulin/polymer solution was then cooled, which subsequently resulted in the formation of a turbid suspension. The turbid suspension was diafiltered to remove the P E G and lyophilized to dryness (Virtis, Gardiner, N Y ) .

Figure 2. Transformation of zinc insulin crystals into PROMAXX microspheres is shown by first dissolving the insulin in a solution of PEG 3350 and then cooling the solution over 70 seconds. The microspheres are virtually all insulin.

The lyophilized microspheres were analyzed by scanning electron micro scopy ( A M R A Y 1000, Bedford, M A ) , light scattering particle size analysis (Beckman Coulter LS230, Miami, FL), aerodynamic particle size determination (TSI Aerosizer 3225, St. Paul, M N ) and the USP assay for insulin (7). Insulin microsphere powder was also incorporated into Capsugel Vcaps #3CS capsules (Capsugel, Morris Plains, NJ) for testing, using a dry powder inhaler (DPI). Cascade impactor particle size distribution studies were conducted using a Thermo Andersen eight stage non-viable cascade impactor, series 20-800 Mark



332 II, in a 60-liter per minute configuration (Smyrna, GA). A i r flow and actuation time were controlled using a Copley Instruments Critical Flow Controller (Copley Scientific Limited, Nottingham, U K ) . The microspheres were intro duced into the cascade impactor using a Cyclohaler DPI (Pharmachemie, Haarlem, The Netherlands) as shown in Figure 3. Biological activity was shown by the administration of P R O M A X X insulin powder to non-diabetic beagle dogs. The presence of deamidated insulin, insulin dimers and oligomers as well as related compounds was determined by H P L C (Waters, Milford, M A ) in the lyophilized insulin starting material and compared to lyophilized insulin micro spheres. The comparison was conducted at 25 and 37 °C over a 12-month period. Residual P E G was determined by size exclusion chromatography (SEC) and evaporative light scattering detection (ELSD).

Figure 3. Cyclohaler Dry Powder Inhaler used in the Andersen Cascade Impactor Studies.

Results and Discussion In Vitro Studies The particle size of the insulin microspheres was determined by several methods. The S E M image in Figure 2 clearly illustrates that the particle size of the insulin microspheres was approximately 1 micron in diameter and that the insulin precipitates are spherical in shape.


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Figure 4 quantifies the particle diameter by two additional analytical methods. Light scattering particle size analysis using a Beckman Coulter LS230 showed a remarkable superimposition of the percent number, percent surface area and percent volume particle size determinations for the P R O M A X X microspheres. This observation is indicative of a monodisperse distribution of particles in the P R O M A X X insulin microsphere preparation. These studies revealed that 95% of the microspheres were between 0.95 and 1.2 microns in diameter. Furthermore, the TSI Aerosizer device, which uses a time-of-flight method to determine particle aerodynamic diameter, showed the microspheres to be 1.47 microns in diameter.

Figure 4. Light scattering and time-of-flight quantitative distribution of insulin microspheres of approximately 1 to 2 microns in size. The cascade impactor studies were conducted to show the actual performance of the P R O M A X X insulin microspheres when delivered from the Cyclohaler DPI. 10 mg of P R O M A X X insulin was loaded into the capsule and then placed into the Cyclohaler DPI. A flow rate of 60 liters per minute was used in this study. The Cyclohaler was activated to release the insulin for collection onto the impactor's stages. The cascade impactor stages were disassembled, the powder on each stage was collected and submitted for H P L C


334 analysis. Figure 5 shows that greater than 75% of the insulin microspheres deposited on the Andersen stages, corresponding to a particle size of 1.1 to 3.3 microns. Particles in this size would be predicted to deposit in the alveoli of the lung and be available for systemic absorption (8). 45

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Figure 5. Andersen Cascade Impactor Results for PROMAXX insulin delivered from the Cyclohaler DPI.

In Vivo Studies A single dose forced-maneuver inhalation and subcutaneous study in five female beagle dogs was conducted to evaluate the bioactivity of the P R O M A X X insulin. The average weight of the beagle dogs was 9.5±0.5 kg. Dry powder insulin was administered via inhalation at two dose levels (1.5 and 0.5 mg) and Humulin R insulin (Eli Lilly, Indianapolis, IL) was administered via sub cutaneous injection at a dose of 0.1 mg per animal (0.35 IU/kg body weight). A l l five dogs received the 3 doses of insulin in repeat dose fashion with at least a three day wash-out period between doses. Figure 6 shows a significant decrease in blood glucose within 10 minutes after administration. Glucose levels remained depressed for approximately 200 minutes. Inhaled insulin was absorbed significantly more rapidly than the subcutaneous dose of insulin. The serum glucose concentrations of the inhaled insulin groups decreased faster than the subcutaneous group of animals. Serum glucose concentrations remained depressed below normal for 95 minutes for the


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0.5 mg inhaled dose and for approximately 180 minutes for the 1.5 mg inhaled dose. The 0.1 mg subcutaneous dose remained depressed for 100 minutes after insulin administration.

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Figure 6. Efficacy of PROMAXX insulin is shown for the 0.5 (M) and 1.5 mg (+) inhaled dosage forms. The initial serum glucose was measured 5 minutes before dosing ("-5 minutes '). The insulin dose was administered at 0 minutes. The inhaled dosage forms reduced the serum glucose more rapidly than the 0.1 mg subcutaneous dose (A). 1

Stability Indicating Studies The stability-indicating assays for the insulin samples, designed to show the generation of insulin dimers, desamido-insulin and total insulin-related compounds, were conducted using the size exclusion chromatography or reverse phase H P L C as specified in USP 25 official monographs for human insulin (7). Figure 7 shows that at 37°C, the P R O M A X X microspheres demonstrated a significantly lower total percentage of dimer formation than the insulin starting material used to form these microspheres.



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Figure 7. Dimer formation of PROMAXX insulin compared to lyophilized insulin starting material (stored at 3 7°C). The PROMAXX insulin showed approximately one half the dimer formation after one year.

Figure 8 shows the formation o f desamido-insulin in the insulin starting material and the P R O M A X X insulin microcapsules. The deamindated insulin was significantly reduced in the P R O M A X X insulin microsphere group compared to the insulin starting material stored at 37°C.

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Figure 8. Desamido-insulin formation of PROMAXX insulin compared to lyophilized insulin starting material (stored at 3 7°C). The PROMAXX insulin showed approximately one half the desamido-insulin compoundformation after one year.


337 The total insulin related compounds is shown in Figure 9. Again the data clearly indicates that higher molecular weight insulin related compounds are reduced in the P R O M A X X insulin group compared to the lyophilized starting insulin.


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Figure 9. Total insulin related compoundformation of PROMAXX insulin is compared to lyophilized insulin starting material (stored at 37°C). The PROMAXX insulin showed approximately one half the total insulin related compoundformation after one year.

The higher stability of P R O M A X X insulin with respect to insulin dimers, desamido-insulin and total high molecular weight insulin related compounds compared to lyophilized starting material was repeated for several lots of insulin. Corresponding results, i.e., significantly less deamidated insulin, fewer insulin dimers and less total related compounds, were found for samples stored at 25°C. Additional studies in our laboratory indicated that commercial insulin formu lations generated up to 15-times more high molecular weight insulin related compounds compared to P R O M A X X insulin microspheres stored at 37°C. Further analysis indicated that the weight percent insulin of the P R O M A X X microspheres is higher than 96% by weight zinc insulin. Size exclusion chromatography indicated that the residual weight percent P E G in the microspheres was less than 0.2% in these early formulations. The remainder of the microsphere is residual water.


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Conclusions P R O M A X X insulin microspheres were produced with a novel and important advance, which may be related to the process of protein precipitation in the presence of water-soluble polymers. However, unlike the volume exclusion process of protein precipitation, the P R O M A X X process resulted in the formation of discrete spherical particles 1 to 2 microns in diameter. These microspheres were shown to be comprised of virtually all insulin. Analyses of these microspheres exhibited a narrow particle size distribution by S E M , light scattering, time of flight measurements and by die Andersen Cascade Impactor. Additional testing of the P R O M A X X insulin powder delivered from the Cyclo haler DPI showed that the formulation was potentially suitable for systemic pulmonary delivery of proteins (9). These lyophilized insulin microspheres also showed enhanced stability over a 12-month time period under accelerated storage conditions, compared to starting material in the absence of stabilizing agents, added to the formulation. The P R O M A X X process must, therefore, be precipitating the insulin in a manner and form which inhibits the formation of insulin dimers and desamido-insulin. The preservation of the insulin's biological activity was shown in a glucose depression assay in beagle dogs.

Acknowledgements This work was funded by Baxter Healthcare Corporation's wholly owned subsidiary, Epic Therapeutics, Inc. The authors acknowledge the assistance of William Fowle of Northeastern University Biology Department for S E M analysis. The contributions of Roxanne Lau, Karen Kuzmich and Kathleen Boutin are also very much appreciated.

References 1. 2.

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Rickets, C.R. Interaction of dextran with fibrinogen. Nature 1952, 169, 970. Laurent, T.C. The interaction between polysaccharides and macromolecules 5. The solubility of proteins in the presence of dextran. Biochem. J. 1963, 89, 253-257. Casillas, G . ; Simonetti, C. Polyvinylpyrrolidone (PVP): A new precipi tating agent for human and bovine factor VIII and fibrinogen. British J. Haemal 1982, 50, 665-672.


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Atha D.H.; Ingham K . C . Mechanism of precipitation of proteins by poly ethylene glycols. Analysis in terms of excluded volume. J. Biol. Chem. 1981, 256, 12108-12117. Brown, L . ; Jarpe, M . ; McGeehan, J.; Qin, Y.; Moore, E.; Hogeland, K . P R O M A X X microsphere characterization. Resp. Drug. Del. 2004, IX, 477480. Rashba-Step, J.; Brown, L.; Hogeland, K . ; McGeehan, J.; Proos, R.; Rulon, P.; Sullivan, A . ; Scott, T. Albumin microspheres as a drug delivery vehicle for multiple routes of administration. Proc. Int. Symp. ControlRel.Bioact. Mater. 2001, 28, 1001-1002. USP 25, Official Monographs 2002, p. 911. Byron, P.R. Prediction of drug residence times in regions of the human respiratory tract following aerosol inhalation. J. Pharm. Sci. 1986, 75, 433438. Skyler, J.S.; Cefalu, W.T.; Kourides, I.A.; Landschulz, W . H . ; Balagtas C.C.; Cheng, S.; Gelfand, R.A. Efficacy of inhaled human insulin in type 1 diabetes mellitus: A randomized proof-of-concept study. The Lancet 2001, 357, 331-335.


Polymeric Drug Delivery II Polymeric Matrices and ... - ACS Publications

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