Article pubs.acs.org/crystal
Understanding Polymer Properties Important for Crystal Growth InhibitionImpact of Chemically Diverse Polymers on Solution Crystal Growth of Ritonavir Grace A. Ilevbare,† Haoyu Liu,‡ Kevin J. Edgar,‡ and Lynne S. Taylor*,† †
Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States ‡ Department of Sustainable Biomaterials, College of Natural Resources and Environment, Virginia Tech, 230 Cheatham Hall, Blacksburg, Virginia 24061, United States S Supporting Information *
ABSTRACT: The use of supersaturating dosage forms, such amorphous dispersions, is an increasingly common approach for improving delivery of poorly water-soluble drugs. Crystallization must be prevented to maintain supersaturation, and so, the presence of an effective crystal growth inhibitor in solution is desirable to prolong supersaturation. In this study, the effectiveness of a group of chemically diverse polymers, including a number of novel cellulose derivatives, at inhibiting the crystal growth of ritonavir from solution was quantified, enabling key polymer properties important for crystal growth inhibition of ritonavir to be elucidated. In general, the greater effectiveness of the cellulose derivatives relative to the synthetic polymers was ascribed to a moderate level of hydrophobicity, the semirigid structure of the cellulose polymers, and their amphiphilicity. Interestingly, some of the novel cellulose polymers were found to be more effective crystal growth inhibitors than commercially available cellulose derivatives. Orthogonal partial least-squares analysis further pointed to the importance of polymer hydrophobicity. These properties of the cellulose-based polymers are likely to promote adsorption onto the crystallizing drug surface. Given the diversity of impact of polymers on crystal growth inhibition, it is clearly important to consider this factor when choosing a polymer for a supersaturating dosage form.
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INTRODUCTION Solubility is one of the key physicochemical parameters of a new molecule that needs to be assessed and understood very early on in the drug development process;1 adequate aqueous solubility is a prerequisite for oral therapeutic activity. Unfortunately, high-throughput screening methods have generated new drug candidates that tend to be hydrophobic and poorly water-soluble. Consequently, the use of an amorphous form of a poorly water-soluble active pharmaceutical ingredient (API) to generate supersaturated solutions is an increasingly common approach for improving drug delivery.2,3 Using this approach, an increase in bioavailability can be achieved if the enhanced concentrations are maintained long enough to enhance absorption. However, this formulation strategy can fail due to the strong driving force for crystallization from supersaturated solutions. The rate at which drug crystallization occurs thus determines the success of a given supersaturating dosage form. Crystallization of a drug from solution involves two processes, nucleation and growth. If one or both processes can be retarded or inhibited, supersaturation may be maintained for a physiologically relevant time period, leading to enhanced absorption. The presence of a polymeric additive, even at very low concentrations, can have a substantial effect on © 2012 American Chemical Society
nucleation and/or crystal growth rates, as well as on crystal morphology.4−7 Some of the commercially available polymeric crystallization inhibitors that have been explored in this context include polyvinyl pyrrolidone (PVP),8−13 polyethylene glycol (PEG), 1 4 , 1 5 and hydroxypropyl met hyl cellulo se (HPMC).9,10,13−16 Although a significant amount of research has been directed toward identification of polymers that stabilize supersaturated solutions of hydrophobic drugs, much less work has been performed to investigate and quantify the impact of these additives on the nucleation and growth stages of crystallization, key steps for understanding and predicting the inhibitory effect of polymeric additives on crystallization from the supersaturating dosage form. While it is virtually impossible to generate a general mechanism to explain the effects of additives on crystal growth and crystal morphology, there is a general consensus that the adsorption of additive molecules on the surface of crystal is a required step for the stabilization to occur.17 For example, Hasegawa et al. concluded that the adsorption of carboxymethylethyl cellulose (CMEC) on the surface of nifedipine Received: March 8, 2012 Revised: April 26, 2012 Published: April 27, 2012 3133
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literature; yet, they have outstanding promise as drug delivery polymers, particularly in amorphous matrix formulations.29
crystals was the most important factor in inhibiting crystallization from supersaturated solutions.18 The adsorption of polymers onto solids is important for controlling a number of interfacial processes.19 The polymer inhibits the introduction of drug molecules from solution into the crystal lattice by occupying growth sites and thereby acting as a mechanical barrier.18 The interactions responsible for adsorption can be either physical or chemical in nature.19 Physical adsorption is usually reversible, and van der Waals forces and electrostatic forces are primarily responsible, while chemical adsorption occurs through covalent bonding and is usually strong and irreversible. Several factors, such as hydrophobicity,20,21 electrostatic interaction,22,23 and hydrogen bonding between the adsorbate and the interfacial species16,19,24 have been found to contribute to the adsorption process. An understanding of the mechanism of adsorption is thus essential for understanding the principal factors responsible for inhibition of crystallization by polymeric additives. Adsorption of polymeric additives onto crystal surfaces is heterogeneous as a result of the anisotropic nature of most molecular crystals.25,26 Consequently changes in morphology may result if there is a high level of adsorption and inhibition of fast growing faces.5−7 For example, hydroxypropyl cellulose (HPC) and HPMC had a significant effect on the morphology of siramesine hydrochloride.5 HPC and HPMC interacted differently with the growing crystal surface, whereby the morphology of crystals was dominated by the crystal faces on which adsorption occurred. The objective of this study was to quantify the effectiveness of a group of chemically diverse polymers in inhibiting crystal growth from highly supersaturated solutions and to attempt to elucidate polymer properties important for inhibition. This was achieved by adding seed crystals to solutions of known supersaturation and monitoring bulk crystal growth rates in the presence and absence of predissolved polymer. It was hypothesized that only polymers with a certain level of hydrophobicity would be effective for the hydrophobic model compound, ritonavir (Figure 1), investigated in this study.
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MATERIALS AND METHODS
Materials. Ritonavir was purchased from Attix Corporation (Toronto, Ontario, Canada). Methanol was purchased from Macron Chemicals (Phillipsburg, NJ). The commercially available polymers were purchased from various sources: poly(vinyl pyrrolidone) K29/32, poly(acrylic acid), and cellulose acetate phthalate (Sigma-Aldrich Co., St. Louis, MO); poly(vinyl pyrrolidone vinyl acetate) K28 (BASF, Germany); HMPC 606 grade and HMPC acetate succinate AS-MF grade (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan); poly(allylamine), poly(N-methylvinylamine), poly(ethylenimine), poly(4-vinylphenol), poly(N-iso-propylacrylamide), and poly(4-vinylpyridine N-oxide) (Polysciences, Inc., Warrington, PA); poly(N,N-dimethyl acrylamide), poly(vinylacetate), poly(vinyl alcohol), poly(4-vinylpyridine), and poly(acrylamide) (Scientific Polymer Products, Inc., Ontario, NY); Eudragit L100 (Degussa, Rohm GmbH & Co. KG, Germany); HPC (Hercules Polymer and Chemicals, Inc., Florida); and carboxymethyl cellulose acetate butyrate (Eastman Chemical Company, Kingsport, TN). The abbreviations used in this report for the commercially available polymers are presented in Table 1. The abbreviations used in this report for the novel cellulose derivatives (Table 2) and some of their properties are presented in Tables 3 and 4. The general synthetic scheme for the novel cellulose esters polymers is presented in Scheme 1; a detailed description of the synthesis is outside the scope of this study and is discussed elsewhere.30 Adipate and other ω-carboxyalkanoate (e.g., suberate and sebacate) esters of the renewable natural polysaccharide cellulose were designed specifically for effectiveness in amorphous solid dispersions. The tetramethylene chain of the adipate group, in addition to the alkyl portions of other ester substituents, imparts hydrophobic character to the cellulose ester to enhance affinity for hydrophobic drugs. The pendent carboxyl group introduced by adipate substitution provides a mechanism for drug release via carboxylate ionization in the neutral pH of the small intestine, resulting in swelling or dissolution of the polysaccharide matrix. In addition, the carboxylate moieties can provide specific interactions with functional groups such as amines on the drug molecule. An important descriptor of all cellulose esters is the degree of substitution (DS) of each ester substituent. The DS value may range from 0 to 3 [because of the three hydroxyl groups per anhydroglucose unit (AGU) of cellulose], and the DS value is controlled by the conditions of synthesis. The nature and DS of substituents have profound effects on the properties of cellulose ester polymers.31 The novel cellulose esters had varying DS and type of substituents, with a total DS ranging from 2.16 to 2.98. One aspect that could be important in amorphous solid dispersions, for the reasons elucidated above, is the DS of carboxyl-containing substituents [DS (CO2H)]. The DS (CO2H) values of the new cellulose esters are listed and ranked in Table 3. Methods. Solubility Studies. The equilibrium solubility of ritonavir was determined in the absence and presence of selected polymers, at a polymer concentration of 5 μg/mL. An excess amount of ritonavir was equilibrated in sodium phosphate buffer, pH 6.8, at 37 °C for 48 h. The supernatant was separated from excess solid in solution by ultracentrifugation at 40000 rpm (equivalent of 274,356 × g) in an Optima L-100 XP ultracentrifuge equipped with Swinging-Bucket Rotor SW 41 Ti (Beckman Coulter, Inc., Brea, CA). Subsequently, the supernatant was diluted, and the solution concentration was determined using an Agilent 1100 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA). The chromatographic separation was performed with a Zobrax SB-C18 analytical column (150 mm × 2.1 mm i.d., 5 μm, 100 Å) (Agilent Technologies). Ritonavir was detected by ultraviolet (UV) absorbance detection at a wavelength of 240 nm. The mobile phase used consisted of 10 mM sodium phosphate buffer, pH 6.8, and acetonitrile, and the mobile phase flow was maintained at 0.2 mL/min. The total analytical run time was 20 min. The injection volume was 20 μL.
Figure 1. Molecular structure of ritonavir.
Ritonavir is a poorly water-soluble drug with a relatively slow crystallization tendency.27 To test this hypothesis, a range of novel cellulose ester derivatives with different hydrophobic substituents were synthesized (Figure 2) and evaluated together with commercially available cellulose derivatives and a variety of synthetic polymers (Table 1) chosen for their diverse chemical and physical properties. Cellulosic polymers are amenable to chemical modification and are biologically compatible, and particular derivatives have been shown to improve the amount of drug solubilized through stabilizing the amorphous form.28 Cellulose adipates have not been widely reported in the 3134
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Figure 2. Molecular structure of the novel synthesized cellulose derivative and the substitution groups. These cellulose derivatives are not regioselectivity substituted, and particular positions of substitution are shown only for convenience of depiction. Abbreviations are presented in Table 2. Characterization of Seed Crystals. Ritonavir was used as received from the manufacturer. The seeds were sieved to a size below 250 μm. The size and shape of ritonavir crystals were characterized using crosspolarized light microscopy (PLM) and scanning electron microscopy (SEM). Sample slides for PLM were prepared by placing a small
Table 2. Abbreviation for Novel Synthesized Cellulose Derivatives polymer cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose cellulose
Table 1. Abbreviations for Commercially Available Polymers Used in This Study polymer
abbreviation
poly(vinylpyrrolidone) (K 29/32) poly(vinylpyrrolidone vinyl acetate) (K 28) poly(allylamine) poly(N-methylvinylamine) poly(ethylenimine) poly(N,N-dimethyl acrylamide) poly(vinylacetate) poly(vinyl alcohol), 99.7% hydrolyzed poly(4-vinylpyridine) poly(acrylamide) poly(N-iso-propylacrylamide) poly(acrylic acid) poly(4-vinylphenol) poly(4-vinylpyridine N-oxide) Eudragit L100 hydroxypropyl cellulose cellulose acetate phthalate hydroxypropyl methyl cellulose (606 grade) hydroxypropyl methyl cellulose acetate succinate (AS-MF) carboxymethyl cellulose acetate butyrate
PVP PVPVA PAlAmn Pn-MVAmn PEE Pnn-DMAAmd PVAc PVAh PVPd PAcAmd Pn-IPAAmd PAA PVPh PVPdn-O EUD L100 HPC CAPh HPMC HPMCAS CMCAB
propionate adipate acetate 320S adipate acetate propionate 504-0.2 adipate 0.85 acetate 398-30 adipate acetate butyrate 553-0.4 adipate 0.81 acetate propionate 504-0.2 adipate 0.33 acetate propionate sebacate 0.67 acetate propionate 482-20 adipate acetate propionate suberate acetate butyrate 381-20 adipate acetate butyrate 553-0.4 adipate 0.25 acetate propionate sebacate 0.24 acetate butyrate suberate acetate butyrate sebacate
abbreviation CP Adp CA 320S Adp CAP Adp 0.85 CA 398-30 Adp CAB Adp 0.81 CAP Adp 0.33 CAP Seb 0.67 CAP 482-20 Adp CAP Sub CAB 381-20 Adp CAB Adp 0.25 CAP Seb 0.24 CAB Sub CAB Seb
aliquot of ritonavir seed crystal suspension between a glass slide and a coverslip. The cross-polarized optical microscope used was a Nikon Eclipse E600 Pol microscope, with NIS-Elements version 2.3 software package (Nikon Co., Tokyo, Japan). Seed crystals were prepared for SEM by dispersing them in water and allowing the suspension equilibrate for at least 24 h. Approximately 20 μL of the solution was placed on a glass slide and allowed to dry overnight in a vacuum oven at room temperature. Cover slides were mounted using double sticky copper tape and sputter-coated with Pt for 60 s prior to imaging. Subsequently, samples were imaged with a FEI NOVA nanoSEM field emission SEM using the Everhart−Thornley (ET) detector and through-the-lens detector (TLD). Parameters were 5 kV accelerating 3135
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28.44° 2θ. The X-ray tube consists of a target material made of copper, which emits a Kα radiation with a power rating of 2200 W and an accelerating potential of 60 kV. Experiments were performed using an accelerating potential of 40 kV and current of 30 mA. The divergence and scattering slits were set at 1.0°, and the receiving slit was set at 0.3 mm. The experiment was conducted with a scan range from 5 to 35° 2θ, while the scanning speed was 4°/min. Thermal transitions were measured using TA Q2000 DSC (TA Instruments, New Castle, DE) attached to a refrigerated cooling accessory (RCS) (TA Instruments). Both the DSC and the RCS were purged with nitrogen gas. Tin was used for temperature calibration, while cell constant and enthalpy calibrations were performed using indium. Baseline calibration was performed by heating the empty cell from −50 to 300 at 20 °C/min. The reference and sample pans were identical. The sample was sealed in an aluminum pan with a pinhole in the lid. The thermogram was obtained by heating the sample at a rate of 5 and 20 °C/min for melting point and glass transition temperature determinations, respectively. The temperature range used was 25−150 °C. Thermal transitions were viewed and analyzed using the analysis software Universal Analysis 2000 for Windows 2000/XP provided with the instrument. Characterization of Novel Cellulose Derivatives. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to determine the DS of the novel polymers. 1H NMR spectra were acquired on an INOVA 400 spectrometer operating at 400 MHz. The sample tube size was 5 mm, and the sample concentrations were ca. 10 mg/mL in CDCl3 or DMSO-d6. The DS values of the products were calculated from the 1H NMR spectra using the ratios of the integrals for appropriate acyl protons to the backbone AGU protons. Size-exclusion chromatography (SEC) and DSC were used to determine the number-average molecular weights (Mn) and the glass transition temperatures (Tg) of the novel polymers, respectively. SEC was performed in HPLC grade THF at 40 °C at a flow rate of 1 mL/ min using a Waters size exclusion chromatograph equipped with an autosampler, three in-line 5 μm PLgel Mixed-C columns, and a Waters 410 refractive index (RI) detector operating at 880 nm, which was programmed to a polystyrene calibration curve. All reported molecular weights are relative to polystyrene standards. Polymer DSC analyses were performed using the DSC instrument described above. Samples (approximately 3−6 mg) were sealed in aluminum pans with a pinhole in the lid. Individual samples were first equilibrated at −25 °C, heated to 200 °C at a heating rate of 20 °C/ min, cooled down to −10 at 50 °C/min, and heated up to 200 at 20 °C/min; glass transition values are reported from the second heating scan. Solubility parameter (SP) calculations were used to compare the relative hydrophobicities of the novel polymers. The method proposed by Fedors32 was used to estimate the SP. This method requires only knowledge of the structural formula of the compound. It is based on group additive constants, and the contribution of a large number of functional groups was evaluated. SP can be evaluated using:
Table 3. DS and SP Values for Novel Synthesized Cellulose Derivates and Their Ranking polymer abbreviation
DS (CO2H)
DS (other)a
DS (total)
DS (CO2H) rank
SP (MPa1/2)
SP rank
CP Adp CA 320S Adp CAP Adp 0.85
0.48 0.67 0.85
2.16 2.49 2.98
4 3 1
23.28 22.95 21.27
1 2 3
CA 398-30 Adp CAB Adp 0.81
0.21
Pr 1.68 Ac 1.82 Ac 0.04; Pr 2.09 Ac 2.47
2.68
10
20.91
4
2.84
2
20.86
5
CAP Adp 0.33
0.33
2.46
5
20.56
6
CAP Seb 0.67
0.67
2.80
3
20.41
7
CAP 482-20 Adp CAP Sub
0.19
Ac 0.14; Bu 1.99 Ac 0.04; Pr 2.09 Ac 0.04; Pr 2.09 Ac 0.10; Pr 2.50 Ac 0.04; Pr 2.09 Ac 1.0; Bu 1.70 Ac 0.14; Bu 1.99 Ac 0.04; Pr 2.09 Ac 0.14; Bu 1.99 Ac 0.14; Bu 1.99
2.79
11
20.29
8
2.39
6
20.19
9
2.89
10
20.11
10
2.38
7
20.05
11
2.37
8
19.94
12
2.38
7
19.84
13
2.35
9
19.62
14
0.81
0.26
CAB 381-20 Adp CAB Adp 0.25
0.19
CAP Seb 0.24
0.24
CAB Sub
0.25
CAB Seb
0.22
0.25
a
Additional abbreviations: Ac, acetate; Pr, propionate; and Bu, butyrate.
Table 4. Number-Average Molecular Weight (Mn) and Glass Transition (Tg) Values for Novel Synthesized Cellulose Derivates
a
polymer abbreviation
Mna (g/mol)
Tg (°C)
CP Adp CA 320S Adp CAP Adp 0.85 CA 398-30 Adp CAB Adp 0.81 CAP Adp 0.33 CAP Seb 0.67 CAP 482-20 Adp CAP Sub CAB 381-20 Adp CAB Adp 0.25 CAP Seb 0.24 CAB Sub CAB Seb
3850 20500 9700 26800 9500 12000 19100 58400 16700 61000 18300 18800 20900 22600
90 134 110 131 82 125 74 125 114 115 94 116 90 83
δ=
∑i Δei ∑i Δvi
=
ΔEv V
(1)
where the Δei and Δvi are the additive atomic and group contribution for the energy of vaporization and molar volume, respectively. The contributions applicable at a temperature of 25 °C are presented in ref 32. For high molecular weight polymers that have a glass transition greater than 25 °C, there is a deviation between the experimentally measured ΔEv and V and the estimated values. A small correction factor was introduced to take into account the divergence in the V values:
Number-average molecular weight in polystyrene equivalents.
voltage, ∼4−5 mm working distance, beam spot size of 3, and 30 μm aperture. Magnifications were 100−10,000×. The SEM images were analyzed using ImageJ, processing and analysis in Java (National Institute of Health). A total of 50 seed crystals were counted so as to ensure a representative sample. To further evaluate the crystal form of the seed crystals, powder Xray diffraction (PXRD) and differential scanning calorimeter (DSC) experiments were performed. PXRD patterns were obtained using Shimadzu XRD 6000 diffractometer (Shimadzu Scientific Instruments, Columbia, MD). The geometry of the X-ray diffractometer is the Bragg−Brentano parafocusing geometry. The equipment was calibrated using a silicon standard, which has a characteristic peak at
Δvi = 4n ,
n 1) are considered effective. Some of the commercially available polymers were effective in inhibiting crystal growth from solution; however, the majority of the polymers were ineffective. Out of the 20 commercially available polymers that were investigated, only five of them were effective: Pn-IPAAmd > HPMCAS > HPMC > CAPh > CMCAB. Pn-IPAAmd (Figure 6) was the most effective crystal growth inhibitor; it had an effectiveness ratio of 3.25. Five out of the 14 novel synthesized cellulose derivatives were effective
Table 5. Equilibrium Solubility of Ritonavir in the Presence of Polymer at a Concentration of 5 μg/mL polymer PVP PVPVA HPMC HPMCAS CAP Adp 0.85 CAB Adp 0.81 CMCAB Pn-IPAAmd
solubilitya (μg/mL) 1.2 1.3 1.5 1.4 1.3 1.3 1.6 1.3
± ± ± ± ± ± ± ±
0.05 0.03 0.02 0.09 0.02 0.01 0.01 0.01
The solubility of ritonavir at pH 6.8 and 37 °C in the absence of polymer is 1.3 ± 0.10 μg/mL.
a
MPa1/2. The hydrophobicity ranking is presented in Table 3, where the least hydrophobic polymer, CP Adp, is ranked #1, and #14 represents the most hydrophobic polymer, CAB Seb. Molecular weight and glass transition temperature values of the novel polymers are summarized in Table 4. The Mn values ranged from 3850 to 61000 g mol−1, where CP Adp had the smallest Mn and CAB 381-20 Adp had the largest Mn. The Tg values ranged from 82 to 134 °C; CAB Adp 0.81 had the lowest Tg, while CA 320S Adp had the highest Tg. Characterization of Seed Crystals. The physical form of ritonavir seed crystals before crystal growth was confirmed to be the most stable form II polymorph. The PXRD pattern (Figure S1 in the Supporting Information) had distinctive peaks at 9.51, 9.88, and 22.2 2θ, which are characteristic of the form II polymorph, while the characteristic combination diffraction peaks (3.32 and 6.75 2θ) for metastable form I polymorph of ritonavir were missing.35 In addition, the melting point and glass transition temperatures (measured for the cooled melt) of the seed crystals were 121.0 and 50.4 °C, respectively. These values are consistent with those reported by Law et al.36 for crystalline form II ritonavir and glassy ritonavir, respectively. Representative micrographs of ritonavir seed crystals from PLM and SEM before and after crystal growth in the absence and presence of polymer are shown in Figure 4a−d. The PLM
Figure 3. Molecular structure of synthetic amide polymers: (a) PnnDMAAmd, (b) Pn-IPAAmd, and (c) PAcAmd.
micrograph (Figure 4a) qualitatively confirmed the crystalline form of ritonavir seed crystals before crystal growth; the seed crystals were needlelike, which is the characteristic crystal habit of form II ritonavir.37 SEM analysis was used to quantitatively characterize the seed crystals (Figure 4b). The predicted crystal morphology of the ritonavir crystal form II (CSD ref code: YIGPIO01), obtained from using the BFDH morphology prediction tool in the Mercury (Mercury CSD 2.4.6, CCDC, Cambridge, United Kingdom) (Figure 4e) is a rodlike shape, while the actual experimental material used is more needlelike. The morphology predictions suggest that the fast growth direction is along the a-axis with four fast growing faces [(−10−1), (−1−10), (−101), (−110)] perpendicular to this 3138
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Figure 4. (a) PLM micrograph of ritonavir crystal seeds before the growth experiment. The SEM micrograph of ritonavir seeds (b) before growth experiment, (c) after growth experiment in the absence of polymer, and (d) after growth experiment in the presence of Pn-IPAAmd. (e) Predicted crystal morphology of ritonavir form II (rodlike habit, orthorhombic stable, P212121, CSD ref code: YIGPIO01) showing the crystal faces. The axes of the crystal are also shown.
cellulose-based polymers, both commercially available and novel cellulose-based polymers, were effective crystal growth inhibitors. Evaluation of Influence of Hydrophobicity of Novel Synthesized Cellulose Derivatives on Crystal Growth Inhibition. The crystal growth rate results for the novel polymers arranged in order of hydrophobicity (least to most hydrophobic, L to R, based on SP values) are presented in Figure 7. There is a notable trend, with the exception of CA 398-30 Adp, which is highlighted with a yellow column: the polymers with a SP ranging from 20.56 to 23.28 MPa1/2 inhibited crystal growth, while more hydrophobic polymers with SP < 20.56 MPa1/2 were ineffective. Evaluation of Influence of DS of Ionic Groups [DS (CO2H)] of Novel Synthesized Cellulose Derivatives on Crystal Growth Inhibition. Even though some of the novel cellulose derivatives share the same type of substituents, the DS of those substituents (in cases where this is known and/or controlled) and the position of these substituents (attachment to the 2-, 3-, or 6-hydroxyl, or some combination thereof) strongly impact their properties, conformation, and dynamics.38 Consequently, it is anticipated that the substituent DS may also influence the inhibitory ability of the various polymers. The crystal growth rate result for the novel polymers, arranged in order of decreasing DS for the CO2H-containing substituent [highest to lowest DS (CO2H)], is presented in Figure 8. There is a trend between DS and inhibitory effect with a higher DS giving a better extent of inhibition, with the exception of CAP Seb 0.67 (highlighted with a yellow column). Evaluation of Influence of Cellulose-Based Polymer Properties on Crystal Growth Inhibition. In an attempt to identify the physicochemical parameters important for inhibition of crystal growth, an OPLS-DA was performed
Figure 5. Desupersaturation of ritonavir with seed crystals in the absence of polymer (black square) and the presence of 5 μg/mL polymer (CAP Adp 0.85, red circle; CAP Adp 0.33, blue upward triangle; and CAB Adp 0.25, pink downward triangle) at an initial ritonavir concentration of 10 μg/mL. All experiments were performed in triplicate, and errors indicate one standard deviation.
crystal growth inhibitors to varying extents. CAP Adp 0.85 and CAB Adp 0.81 were more effective in inhibiting the crystal growth of ritonavir as compared to HPMCAS, HPMC, CAPh, and CMCAB. Summarizing, in general, the synthetic polymers (with the exception of Pn-IPAAmd) and the very hydrophobic novel cellulose derivatives were ineffective in inhibiting crystal growth of ritonavir, while the moderately hydrophobic 3139
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Figure 6. Crystal growth rate ratio of ritonavir at an initial ritonavir concentration of 10 μg/mL. Crystal growth rate experiments were performed in triplicate. Each column is an average of the effectiveness ratio, and error bars indicate one standard deviation. The y-axis is a ratio of the growth rate of ritonavir in the absence of polymer to growth rate of ritonair in the presence of polymer (5 μg/mL). Polymers with a ratio >1 are considered effective crystal growth inhibitors. The blue columns represent the commercially available cellulose-based polymers, the white columns are the commercially available synthetic polymers, and the red columns represent the novel synthesized cellulose derivatives.
Figure 7. Crystal growth rate ratio of ritonavir in the absence of polymer to the presence of novel synthesized cellulose derivative polymers (5 μg/mL) at an initial ritonavir concentration of 10 μg/mL. The data are arranged in order of hydrophobicity: least hydrophobic to most hydrophobic (left to right). The yellow column represents the outlier, CA 398-30 Adp.
Figure 8. Crystal growth rate ratio of ritonavir in the absence of polymer to the presence of novel synthesized cellulose derivative polymers (5 μg/mL) at an initial ritonavir concentration of 10 μg/mL. The polymers are arranged in order of decreasing DS (CO2H): high to low DS (CO2H). The yellow column represents the outlier, CAB Seb 0.67.
using various structure-based (SP, DP, HBA, HBD, polymer Mn, and monomer Mw) and thermal (Tg) input parameters for the set of 19 cellulose-based polymers. The model had a total nine variables, seven were X variables, and two were Y class discriminant variables. The polymers were divided into two classes based on their ability to inhibit crystal growth, as described above. One predictive component and three orthogonal components were calculated in this model with a goodness of fit (R2Ycum) of 0.713 and goodness of prediction (Q2Y) of 0.512. A large Q2Y (Q2Y > 0.5) indicates a good prediction. The modeled variation of X, using all predictive components and orthogonal components in X (R2Xcum), was
0.91. The R2Xcum value is a measure of fit, that is, how well the model fits the X data. Figure 9a shows the score plot for the predictive and first orthogonal components. Each data point represents a polymer, which was colored according to its class, where the red observations are the ineffective cellulose-based polymers and the black observations are the effective cellulosebased polymers. The plot shows the possible presence of outliers, groups, and other patterns in the data. Observations that lie close to each other are more similar than observations that lie relatively distant from each other. An OPLS-DA model reveals the between class variation in the first t1 predictive component (horizontal direction), while the within class 3140
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DISCUSSION
Supersaturating dosage forms are attractive for improving the delivery of poorly water-soluble drugs since they result in a solution with a higher thermodynamic activity, which may enhance the absorption of a drug relative to a saturated solution.39 However, to maintain supersaturation, crystallization must be prevented; hence, the presence of an effective crystal growth inhibitor in solution is desirable to prolong supersaturation. While polymers are often used to fulfill this role, there is little mechanistic understanding of the properties of a polymer that make it a good inhibitor of crystal growth for a given compound. Ritonavir was chosen as the model compound for this study based on intrinsic properties of drug molecules that are expected to contribute toward their insolubility in water. The insolubility of pharmaceuticals results primarily from high log P (lipophilicity) and/or high melting point, Tm (representing lattice energy). Ritonavir is a highly lipophilic compound with a log P value of 5.9827 and melting point of 121 °C, yielding an equilibrium solubility of 1.3 ± 0.10 μg/mL, at pH 6.8 (ritonavir is un-ionized at this pH) and 37 °C. Importantly, at the low polymer concentrations tested in this study (5 μg/mL), the equilibrium solubility of ritonavir does not change; therefore, any impact of the polymer on crystal growth rates is not due to its altering the degree of supersaturation. In addition, it is reasonable to assume that the viscosities of the polymer solutions are essentially the same as for water at these very low concentrations, thus eliminating bulk mass transport effects. Therefore, it is possible to seek correlations between the polymer properties and the extent of crystal growth rate inhibition without the analysis being complicated by bulk thermodynamic and mass transport effects. On the basis of the data presented in Figure 6, it is clear that polymer hydrophobicity appears to be a key parameter in determining the impact of a given polymer upon ritonavir crystal growth. The hydrophobicity of the polymer is likely to affect the extent of adsorption of polymer to the crystal surface, which in turn influences the effectiveness of the polymer as a crystal growth inhibitor. Polymer adsorption affects crystal growth by blocking the sites for incorporation of new growth units. Thus, crystal growth is inhibited, and crystals of reduced size are formed.20 If a polymer is very hydrophilic, it would be expected to interact more favorably with the solvent molecules while if the polymer is too hydrophobic, it is likely to interact more favorably with other monomer units and form a more condensed globule, which either may not adsorb or, if adsorbed, may not have a high surface coverage. On the basis of this reasoning, it is apparent that a polymer should have an optimal level of hydrophobicity to be an effective crystal growth inhibitor; that is, have a certain hydrophilic/hydrophobic balance to drive adsorption to a crystal surface. At the same time, a sufficient amount of the polymer must dissolve in aqueous media for it to be effective. These expectations are, in general, supported by the results of this study, in particular for the cellulose derivatives. With the exception of a few polymers (Pnn-DMAAmd, EUD L100, CA 398-30 Adp, and PAlAmn), the moderately hydrophobic polymers located in middle portion of Figure 6, with SP values ranging from 20.56 to 25.98 MPa1/2, were effective crystal growth inhibitors, albeit to varying extents. Interestingly, with the exception of Pn-IPAAmd, the synthetic polymers were ineffective in inhibiting crystal growth, even when they had SP values similar to an effective cellulose
Figure 9. (a) Score plot of predictive (t1) and orthogonal (t01) components of the OPLS-DA model, colored according to the discriminate y-class. The black squares represent the effective cellulosebased polymers, while the red circles represent the ineffective cellulose-based polymers. The Hotelling's T2 line (ellipse) represents the boundary 95% confidence limit of the model. (b) Loadings of the predictive component of the OPLS-DA model constructed from the data set.
variation is revealed in the first to1 orthogonal component (vertical direction). It is apparent from Figure 9a that the ineffective cellulose-based polymers tend to populate the left side (negative X direction) of the plot, while the effective cellulose-based polymers dominate the right side (positive X direction) of the plot, with the exception of CAB Adp 0.81 and CAP 504-0.2 Adp (CAP Adp 0.33). There is less within-class variation within the ineffective polymer class; the polymers all lie very close to one another along the vertical direction, with the exception of CAB 381-20 Adp and CAP 482-20 Adp. On the other hand, the effective polymers are more spread out along the vertical direction. There is a distinct within-class variation between the effective commercially available and the novel cellulose-based polymers. This variation reveals the structural dissimilarity between the novel and the commercially available polymers, such as number of HBD and HBA, SP, and Mn. HPC is outside the 95% confidence limit of the model. Although HPC inhibits growth by a small extent, it does not fit any class in the model. The loadings plot (Figure 9b) can be used to understand what variables are important in the predictive component of the model. SP (i.e., polymer hydrophobicity) was the most important variable. No clear correlations with other properties, such as Mn, could be determined. 3141
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in inhibiting crystal growth of the hydrophobic drug ritonavir. There was no significant correlation observed between polymer Mn and monomer Mw, DP, HBA, or Tg and the ability of a polymer to inhibit crystal growth from solution. These properties do not appear to be the most important considerations when selecting a polymer that will inhibit ritonavir solution crystal growth. The effect of hydrophobicity on polymer adsorption alone does not adequately explain why Pn-IPAAmd, a synthetic and moderately hydrophobic polymer, is the most effective in inhibiting crystal growth of ritonavir. It is speculated that PnIPAAmd adsorbs to one or more of the fast growing faces of ritonavir crystals through the formation of specific intermolecular interactions. To gain a better insight into the impact of Pn-IPAAmd upon ritonavir crystallization, the crystals extracted after crystal growth in the absence and presence of the predissolved polymer were analyzed using SEM and PXRD. The aspect ratio of ritonavir crystals after growth in the absence of polymer increased (from 7.7 to 8.8, before and after growth, respectively) since the crystals grew length-wise and became more elongated along the a-axis. There are four faces shown by the BFDH morphology predictor (Figure 4e) that grow approximately perpendicular to the a-axis and are the fastest growing faces [(−10−1), (−1−10), (−101), (−110)]. These planes expose functional groups containing electronegative atoms such as carbonyl oxygen and nitrogen atoms from the thiazole functional group of ritonavir, which (assuming these are also the fast growing faces in the experimental system) can potentially interact with the amide group of Pn-IPAAmd. In the presence of predissolved Pn-IPAAmd, the crystals appeared to be less elongated, suggesting adsorption of Pn-IPAAmd to the fast growing faces, resulting in an increase in width (from 5.7 ± 1.4 to 9.3 ± 2.5 μm, in the absence and presence of predissolved Pn-IPAAmd, respectively) and in turn, a reduced average aspect ratio of the crystals. Therefore, the strong crystal growth inhibitory ability of Pn-IPAAmd can likely be attributed to a combination of the moderate hydrophobicity of the polymer and fortuitous intermolecular hydrogen bonding to the fast-growing faces at the solid−liquid interface. The other amide polymers that were investigated (Pnn-DMAAmd and PAcAmd) were probably ineffective because they were either too hydrophilic or have no hydrogen bond donor groups (Figure 3).
derivative. For example, Pnn-DMAAmd, EUD L100, and PAlAmn are ineffective crystal growth inhibitors, even though their respective SP values are between 20.56 and 25.98 MPa1/2. The ineffectiveness of the synthetic polymers most likely can be attributed to their flexible structures as compared to the relatively rigid structure of the cellulose-based polymers. The usual description of conformations at an adsorbing interface is in terms of three types of subchains: trains, which have all of their segments in constant contact with the substrate; loops, which have no contact with the surface and connect two trains; and tails, which are nonadsorbed chain ends. A balance of these three populations gives the adsorbed layer its unique properties.40 The more flexible synthetic polymers are more likely to form loops and thus have limited contact with the crystallizing surface, while polymers with a more rigid backbone, such as the cellulose polymers,41 will be more planar and hence have more contact with the substrate. Kramarenko et al., using molecular dynamics simulations, studied the adsorption of polymer chains and concluded that stiffer chains adsorb more easily, while the average length of the adsorbed section of the polymer (train) strongly depends on the chain stiffness and adsorption energy.42 The amphiphilic nature of the novel cellulose polymers appears to further enhance their ability to inhibit crystal growth, with the polymers possessing higher DS (CO2H) proving to be more effective inhibitors (Figure 8). The pKa of the adipate substituent is approximately 4.43,43 so the degree of ionization of the CO2H-containing substituents (adipate, suberate, and sebacate) will be nearly complete (∼100%) at pH 6.8.44 The presence of an ionized carboxylic acid group will render the polymers more amphiphilic, where the ionized carboxylic acid group is the “water-loving” part and may influence the conformation of the adsorbed polymer. Using atomic force microscopy measurements (AFM), Roiter and Minko found that extended molecules of poly(2-vinylpyridine) at the solid− liquid interface go through a coil-to-globule phase transition as the degree of protonation decreases.45 The only exception to the trend of increasing inhibition with DS (CO2H) is the very hydrophobic CAB Seb 0.67. Interestingly, PAA, which has a large number of ionized carboxylic acid groups, was completely ineffective, supporting the conjecture that the correct balance of multiple chemical and/or structural features is needed for effective inhibition. So far, the discussion has emphasized the importance of hydrophobicity, polymer conformation (rigidity), and the amphiphilic nature of the novel cellulose-based polymers on crystal growth inhibition. These key factors appear to be jointly responsible for the stabilizing ability of the cellulose-based polymers; they are interrelated and influence one another. From Figures 7 and 8, it is apparent that CA 398-30 Adp and CAB Seb 0.67 are outliers. Even though CA 398-30 Adp has the right level of hydrophobicity (20.56 < SP < 25.98 MPa1/2), it has a low number of ionizable groups [DS (CO2H) = 0.21]. As a result, it is less amphiphilic as compared to the effective polymers, CAP Adp 0.85 and CAB Adp 0.81 (Figure 7). In the same way, CAB Seb 0.67 is an outlier when the polymers are ranked based on their DS (CO2H) (Figure 8) because, although it has a relatively highly amphiphilic nature [DS (CO2H) = 0.67], it is too hydrophobic (SP < 20. 56 MPa1/2). The importance of hydrophobicity can be further inferred from the OPLS-DA analysis. The predictive component of the OPLS-DA model shows that level of hydrophobicity is an important polymer characteristic that influences its effectiveness
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CONCLUSIONS In this study, the effect of 34 polymers, including a series of novel cellulose derivatives, on the solution crystal growth of ritonavir was quantified enabling key polymer properties important for crystal growth rate inhibition of ritonavir to be elucidated. The general effectiveness of the cellulose derivatives (in particular the novel polymers) relative to the synthetic polymers was attributed to the following: (1) hydrophobicity the effective polymers had a moderate level of hydrophobicity; (2) rigidity of polymer structurethe semirigid cellulose polymers were more effective than the semiflexible synthetic polymers of similar hydrophobicity; and (3) amphiphilicity of the novel cellulose-based polymersthe cellulose polymers containing more ionizable carboxylic acids were better inhibitors relative to neutral or less ionized cellulose polymers. These factors are likely to promote adsorption onto ritonavir crystal surfaces. Multivariate analysis indicated that hydrophobicity was the most important polymer property, impacting its ability to inhibit crystal growth. No significant correlation 3142
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was found between polymer Mn, monomer Mw, DP, HBA, or Tg values and their ability to inhibit crystal growth from solution. This study enhances our understanding of the important structural factors necessary for polymer inhibition of crystal growth, enhancing our ability to design new polymers for this purpose.
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ASSOCIATED CONTENT
S Supporting Information *
PXRD predicted and experimental patterns of ritonavir crystals (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1-765-496-6614. Fax: +1-765-494-6545. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Eastman Chemical Company for their kind donation of CMCAB. Support of the National Science Foundation through Grant DMR-0804609 is gratefully acknowledged. Pfizer Inc. is acknowledged for providing a fellowship for G.A.I. We also thank Xuanhao Sun of the Life Sciences Microscopy Facility of Purdue University for performing SEM analyses.
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