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Article pubs.acs.org/molecularpharmaceutics

Role of Molecular Interactions for Synergistic Precipitation Inhibition of Poorly Soluble Drug in Supersaturated Drug−Polymer−Polymer Ternary Solution Dev Prasad,† Harsh Chauhan,‡ and Eman Atef*,§ †

School of Pharmacy, MCPHS University-Boston 179 Longwood Avenue, Boston Massachusetts 02115, United States School of Pharmacy & Health Professions, Creighton University, Omaha, Nebraska 68178, United States § College of Pharmacy, California Northstate University, Elk Grove, California 95757, United States ‡

ABSTRACT: We are reporting a synergistic effect of combined Eudragit E100 and PVP K90 in precipitation inhibition of indomethacin (IND) in solutions at low polymer concentration, a phenomenon that has significant implications on the usefulness of developing novel ternary solid dispersion of poorly soluble drugs. The IND supersaturation was created by cosolvent technique, and the precipitation studies were performed in the absence and the presence of individual and combined PVP K90 and Eudragit E100. The studies were also done with PEG 8000 as a noninteracting control polymer. A continuous UV recording of the IND absorption was used to observe changes in the drug concentration over time. The polymorphic form and morphology of precipitated IND were characterized by Raman spectroscopy and scanning electron microscopy. The change in the chemical shift in solution 1 H NMR was used as novel approach to probe IND−polymer interactions. Molecular modeling was used for calculating binding energy between IND−polymer as another indication of IND−polymer interaction. Spontaneous IND precipitation was observed in the absence of polymers. Eudragit E100 showed significant inhibitory effect on nuclei formation due to stronger interaction as reflected in higher binding energy and greater change in chemical shift by NMR. PVP K90 led to significant crystal growth inhibition due to adsorption on growing IND crystals as confirmed by modified crystal habit of precipitate in the presence of PVP K90. Combination of polymers resulted in a synergistic precipitation inhibition and extended supersaturation. The NMR confirmed interaction between IND−Eudragit E100 and IND−PVP K90 in solution. The combination of polymers showed similar peak shift albeit using lower polymer concentration indicating stronger interactions. The results established the significant synergistic precipitation inhibition effect upon combining Eudragit E100 and PVP K90 due to drug−polymer interaction. KEYWORDS: supersaturation, NMR indomethacin, ternary solid dispersion, solubility, poorly soluble drug, drug−polymer interaction

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INTRODUCTION Conversion of crystalline drugs to an amorphous form is one successful approach for enhancing the solubility of poorly soluble active pharmaceutical ingredients (APIs).1−4 Many researchers have extensively studied the mechanism of the solubility advantage of formulating drugs in an amorphous form.5−9 The drawback of using a metastable amorphous or any other supersaturating dosage form is its tendency to precipitate out from supersaturated solution during dissolution. Therefore, the acquired solubility advantage can be compromised upon erratic precipitation during dissolution. The drug precipitation inhibition in solution determines the success of a given amorphous or other supersaturating dosage form. Precipitation of a drug from solution involves two critical phases: nucleation and crystal growth. If one or both phases can be delayed or inhibited, supersaturation may be maintained for a long enough physiologically relevant time period to create a significant enhancement in bioavailability. Supersaturation maintenance © XXXX American Chemical Society

and amorphous form stabilization are the keys to the successful solid dispersions formulations. It has been shown that polymers can successfully inhibit the precipitation of drugs from solutions once the higher apparent solubility is achieved.10−12 Although precipitation inhibition of drugs from aqueous supersaturated solutions by polymers has been documented, the drug−polymer interactions have not been widely studied. Such interactions may promote or inhibit precipitation (nucleation and crystal growth) in an unpredictable manner, which in turn has a significant impact on the extent and duration of supersaturation and overall bioavailability.13−15 Received: August 25, 2015 Revised: February 5, 2016 Accepted: February 11, 2016

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DOI: 10.1021/acs.molpharmaceut.5b00655 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

and the method was run for 8 min. The method was validated for precision and linearity from 1−100 μg/mL.16 Precipitation Studies of IND in the Absence and Presence of Individual and Combined Polymers. For precipitation studies, supersaturation was generated by adding 1 mL of IND solution (2 mg/mL in methanol) to 200 mL of media (0.01 N HCl, pH 2) at a rate of 0.1 mL/min using a syringe pump with continuous stirring. Real-time absorbance of IND in the solution phase was determined at a wavelength (λ) of 272 nm using a Cary 50 UV−vis continuous spectrophotometer (Varian Analytical Instrument, Palo Alto, California) equipped with a fiber optic probe. For IND precipitation inhibition studies, PVP K90, Eudragit E100, and PEG 8000 were studied at a molar ratio of 1:1 (IND/polymer) in binary mixtures and 2:1:1 (IND/polymer/polymer) in ternary systems. Polymers were predissolved in the precipitation media to study the effect, while precipitation media without polymer was used as a control. The initial and final pH and viscosity were measured using an Orion 3 Star pH meter (Thermo Scientific, Beverly, MA) and a Brookfield DV 2 Viscometer (Middleboro, MA), respectively. Once the precipitation experiment was completed, the precipitate was filtered using 0.22 μm filters and kept for overnight drying under vacuum. Solid-state characterization of IND precipitate was carried out using Raman spectroscopy, whereas morphology of the precipitate was studied using scanning electron microscopy (SEM). The n = 3 samples were run for each study. Calculation of the Precipitation Time of IND. The IND precipitation time in the absence and presence of polymers was calculated by determining the changes in their respective absorbance curves. The decrease in the absorbance was measured continuously at 272 nm for concentration change over a period of time. Precipitation curves were used to calculate t1% and t10%. The t1% and t10% in our experiments are defined as the time required for a 1% and 10% concentration drop of IND from its original concentration, respectively. During precipitation, the IND concentration in solution decreased with time, which resulted in observable changes in the absorbance curves. A correction was applied to account for the first 10 min used in adding the IND solution to the precipitation medium. Characterization of IND Precipitate. Raman Spectroscopy. The Raman spectra were collected to characterize the crystalline polymorphic form of the precipitate. The Raman spectra were collected using a RamanRxn System Model no. RXN1−785, Kaiser Optical Systems equipped with an excitation laser operating at 785 nm with a laser power setting of 400 mW. Data acquisition was done using an exposure time of 10 s for 10 accumulations. Scanning Electron Microscopy. Morphology of the precipitate was studied by SEM operating between 5 and 24 kV. The specimens were mounted on a metal stub (with double-sided adhesive tape) and coated under vacuum with gold in an argon atmosphere. Solution 1H NMR. Solution 1H NMR spectra were recorded on a Varian solution NMR operating at 500.13 MHz using a 5 mm multinuclear probe with π/2 pulses of 6.0 μs. The measurements were performed at room temperature in deuterated chloroform (CDCl3) using tetramethylsilane (TMS) as an internal standard.17,18 All analyses were carried out in triplicate. The NMR spectrum was collected for pure IND in solution and with various ratios with polymer(s). For binary systems, 70:30, 50:50, and 30:70 w/w of IND/polymer

The current manuscript investigates the precipitation inhibition mechanism to maintain supersaturation by utilizing very low polymer concentrations. The manuscript highlights an important modification of current solid dispersions to get high drug loading by creating an efficient ternary, that is, drug− polymer−polymer system. Although there is some literature that studied the precipitation inhibition phenomenon in the presence of a single polymer, there is a growing need to understand drug precipitation in the presence of two polymers due to increase in the synthesis of poorly soluble drugs where two polymers system can be used to enhance apparent solubility. The effects of individual and combined polymers on the precipitation of indomethacin (IND) in solution were determined through precipitation studies in absence and presence of polymers. The morphology and polymorphic characterization of the collected drug precipitates were carried out as part of the precipitation inhibition mechanism, and finally proton NMR was used to probe interaction between drug and polymer. Another advantage of studying the mechanism of precipitation inhibition of individual polymers is the ability to predict and identify polymer combinations that could result in synergistic effects. These drug−polymer interactions can result in significant changes in solution properties, which may result in a favorable or unfavorable effect on the inhibition of precipitation. In this study, the effect of polymer and polymer combinations on nucleation and crystal growth inhibition was investigated.

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MATERIALS AND METHODS Materials. IND was purchased from Sigma-Aldrich, St. Louis, MO. Eudragit E100 was a gift from Degussa (Parsippany, NJ). PVP K90 and PEG 800 were purchased from Sigma (St. Louis, MO). HPLC solvents (acetonitrile, methanol) were purchased from J.T. Baker (Phillipsburg, NJ). Hydrochloric acid, 10 N, ACS grade (lot # SN0543), potassium phosphate monobasic crystals, lot no. VQ 0785, and sodium hydroxide, lot no. QX0299, were purchased from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Solubility Determination of IND. Solubility studies of IND in 0.01 N hydrochloric acid (pH = 2) and in precipitation media (0.01 N HCl and methanol mixture) were carried out. An excess amount of IND (50 mg) was added to a 20 mL vial containing 10 mL of either 0.01 N HCl or precipitation media. The vials were shaken for 24 h at 25 °C. After centrifugation and filtration through 0.22 μm filters, the samples were analyzed using HPLC. Supersaturation was calculated based on the following equation: S=

⎛C ⎞ ⎜ ⎟ ⎝ C* ⎠

(1)

where S = supersaturation, C = concentration of IND in the media, and C* = equilibrium solubility of IND in the same media. HPLC Assay of IND. An HPLC method (Hewlett-Packard 1100 series HPLC system, HP ChemStation software, A Hypersil ODS C18, 150 × 5.4 mm2 column) was used to quantify IND. The mobile phase consisted of acetonitrile and 0.1 M glacial acetic acid (60:40 v/v) at 0.8 mL/min; the UV detection was done at λ = 272 nm. The IND eluted at 4.56 min, B


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Molecular Pharmaceutics were studied. In the case of ternary system, 70:15:15, 50:25:25, and 30:35:35 w/w/w IND/PVP K90/Eudragit E100 were studied. Molecular Modeling. The Jaguar (Schrodinger LLC, 2012) program was used to calculate the optimized geometries of the IND−polymer dimers using the B3LYP/6-31G**. Calculations were performed with gradient-corrected density functionals and default convergence criteria for geometry optimization. Intermolecular energy values, Ebind, were computed by using Jaguar’s hydrogen bond function to solve the following equation: E bind = [aΔE bind(cc − pvqz) − bΔE bind(cc − pvtz)] /(a − b)

where Ebind(cc − pvqz) and Ebind(cc − pvtz) are the cc-pVQZ (-g) and cc-pVTZ (-f) counterpoise corrected binding energies, respectively. The values of the parameters a and b are determined by fitting to a “training set” of molecular dimers, for which benchmark results have been obtained using conventional MP2 methods and ultra large basis sets.19,20 For Eudragit E100, individual monomer units were used for calculations. Statistical Analysis. One-way analysis of variance (ANOVA) and Tukey’s test were applied to compare precipitation time, t1%, and t10%. A value of P < 0.05 was denoted as significant throughout the study.

Figure 1. t1% of IND in the presence of polymer (or polymer combination). A significant increase in t1% was observed in the presence of Eudragit E100 and Eudragit E100/PVP K90. ∗, Statistically different (p < 0.05) from the reference group, IND alone.

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RESULTS Precipitation Studies. Precipitation Inhibition of IND in the Absence of Polymers. The equilibrium solubility (Cmax) of IND in 0.01 N hydrochloric acid (pH 2) was found to be 3.15 μg/mL at 25 °C. The initial supersaturated concentration was generated by adding 1 mL of 2 mg/mL of methanolic solution of IND in 200 mL of precipitation medium (IND concentration = 10 μg/mL). Thus, by using eq 1, the supersaturation (S) was found to be 3.17 during precipitation inhibition studies. In the absence of polymers, the t1% and t10% values for IND were 8.2 ± 3.2 and 13.8 ± 3.1 min, respectively, suggesting rapid nucleation (Figures 1 and 2). No change in the viscosity or pH was observed during the study. The average Δt (Δt = t10% − t1%) was calculated as 5.7 min. The precipitate generated was an α polymorphic form of IND, as confirmed by Raman (Figure 3), while the morphology of the IND crystals was found to be acicular or long needle-shaped (Figure 4A). Precipitation Inhibition of IND in the Presence of Single Polymers. The addition of polymer(s) to the precipitation media may change the solubility of IND; therefore, solubility studies were performed in the precipitation media containing an equal amount of polymer(s), as used in these studies. No significant changes in the solubility of IND were observed in the precipitation media composed of 0.01 N HCl and polymer(s), confirming that the same supersaturation levels of 3.17 were maintained throughout the studies. For IND/PVP K90, a 1:1 molar ratio is equivalent to 3.16 μg/mL of PVP K90 in the precipitating media. At this molar ratio, no change in the t1% was observed compared to precipitation of IND in the absence of polymers (Figure 1). On the other hand, a significant increase in the t10% was observed. The latter increased from 13.8 ± 3.1 min without polymer to 46.2 ± 3.0 min in the presence of PVP K90 (Figure 2). The Δt ≈ 32 min was significantly higher in the presence of

Figure 2. t10% of IND in the presence of polymer (or polymer combination). ∗, Statistically different (p < 0.05) from the reference group, IND alone.

polymer than in its absence. For IND/Eudragit E100, a 1:1 molar ratio is equivalent to 16 μg/mL of Eudragit E100 in the precipitating media. In presence of Eudragit E100, a significant increase in both t1% and t10% was observed. The t1% in the presence of polymers increased to 20.8 ± 2.0 min, while t10% increased to 32.2 ± 2.2 min, as compared to the precipitation studies in the absence of polymers (Figures 1 and 2). However, an increase in Δt was not observed, and it was found to be about 12 min. Among both the polymers, the Δt was highest in the presence of PVP K90. The precipitate formed in the presence of PVP K90 or Eudragit E100 was the α polymorphic form of IND (Figure 3). In the presence of PVP K90, IND crystals with small columnar habits were obtained (Figure 4C), while for the IND/Eudragit E100 system, the morphology of crystals was same in the absence and presence of the polymer (i.e., acicular or needleshaped) (Figure 4B). Precipitation Inhibition of IND in the Presence of PVP K90/ Eudragit E100 Combination. The 1:1 ratio in ternary systems is equivalent to 1.58 μg/mL of PVP K90 and 8 μg/mL of Eudragit E100 in precipitation media. The amount of individual C


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Figure 3. Raman spectra of IND precipitate obtained from precipitation studies, which confirm the α IND forms in all precipitates.

microscope showed that IND precipitated as small columnar crystals (Figure 4D), similar to the crystal habit observed in the presence of PVP K90. Precipitation Inhibition of IND in the Presence of PEG 8000. These studies were performed with PEG 8000, which has previously shown no molecular interaction with IND in binary mixtures.21 The purpose of this study was to establish the hypothesis that drug−polymer interaction is responsible for precipitation inhibition, and a synergistic effect is possible only when both the polymers interact with IND. In the presence of PEG 8000, no significant changes in the solubility of IND were observed in the precipitation media, thus the supersaturation of 3.17 was maintained. In the presence of PEG 8000, the t1% and t10% were found to be 10.9 ± 2.1 min and 19.8 ± 2.8 min, respectively (Figures 1 and 2), showing no significant change in the t1% or t10% compared to the studies with no polymer. As shown in Figure 3, the precipitate was the α polymorphic form of IND. SEM showed that IND precipitated as acicular or long needle-shaped crystals (Figure 5A), the same as crystal habit obtained in case of IND without any polymers. Precipitation Inhibition of IND in the Presence of PEG 8000/PVP K90 and PEG 8000/Eudragit E100 Combination. In the presence of PEG 8000/PVP K90, no change in the t1% was observed, and it was found to be 9.2 ± 2.9 min (Figure 1). However, t10% was significantly increased to 37.2 ± 7.8 min (Figure 2). Similar results were obtained in the presence of PEG 8000/Eudragit E100, which showed no significant change in t1% while a significant increase in t10% was observed. As no effect was seen on IND crystal nucleation or crystal growth in the presence of PEG 8000 alone, this crystal growth rate inhibition can be attributed to the presence of PVP K90 and Eudragit E100. The PEG 8000/Eudragit E100 system showed inhibitory effect on indomethacin crystal growth, although individually none of the polymers showed any effect. This discrepancy cannot be explained in the current study and needs further exploration. However, it can be assumed that polymer combination might be interacting with each other resulting in some inhibitory effect on IND crystal growth.

Figure 4. SEM images of IND precipitate obtained from precipitation inhibition studies (A) without polymer−acicular or needle-shaped crystals in the polymer free media, (B) in the presence of Eudragit E100 acicular or needle-shaped crystals, (C) in the presence of PVP K90 small columnar-shaped crystals, and (D) in the presence of Eudragit E100/PVP K90 small columnar-shaped crystals.

polymer in these ternary systems was lower compared to individual polymers used for precipitation studies. No significant change in the solubility of IND was observed in this media (S = 3.17). By using the combination of polymers, t1% and t10% were significantly increased to 27.5 ± 2.7 min and 56.5 ± 3.1 min, respectively (Figures 1 and 2). The Δt was calculated as 29 min. These values were significantly higher than the studies carried out in the presence of one polymer or no polymers. Similar to previous studies, the precipitate was the α polymorphic form of IND (Figure 3). A scanning electron D


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Figure 5. SEM images of IND precipitate obtained from precipitation studies (A) in the presence of PEG 8000 acicular or needle-shaped crystals, (B) in the presence of PEG 8000/Eudragit E100 small columnar as well as acicular crystals, and (C) in the presence of PEG 8000/PVP K90 small columnar crystals.

Figure 6. Solution 1H NMR spectrum and structure of IND with 1H chemical shift expressed in ppm.

The precipitate was found to be the α polymorph form of IND (Figure 3). The morphology of the precipitated IND was found to be small columnar crystals in the presence of PEG 8000/PVP K90 (Figure 5B). In the presence of PEG 8000/ Eudragit E100, combinations of small and large crystals were obtained (Figure 5C). The change in crystal habit was more pronounced in PEG 8000/PVP K90 systems compared to PEG 8000/Eudragit E100. Solution 1H NMR. Solution 1H NMR spectroscopic studies were performed to study the drug−polymer site of interaction in solution and to correlate the results obtained in the solution precipitation studies. Figures 6, 7, and 8 show the proton 1H NMR spectra of IND, PVP K90, and Eudragit E100, respectively. Sharp peaks are visible in the IND NMR spectrum. The assignments of proton chemical shifts (δ) of IND were based on the available literature in which IND was extensively characterized by proton NMR.22 For polymers, the lack of symmetry due to its random structure resulted in proton spectra with several strong peaks and some unresolved broad peaks. As a consequence, only a few protons can be successfully assigned to the polymers. To study the interaction between IND and polymer, the focus is kept on the proton present on carbon in the −COOH group assigned as carbon 2 (C2) and shown in Figure 9. The chemical

shift for proton at C2 in IND occurs at 3.70 ppm. Any molecular interactions should be reflected in the chemical shift variations since the electron density at the interacting atoms will be changed. Binary System (IND/PVP K90 and IND/Eudragit E100). 1H NMR spectra of IND, PVP K90, and their various ratios are shown in Figure 9. The chemical shift due to the proton at C2 shifted upfield to a lower value. This suggests the shielding of the proton at C2 in the presence of PVP K90. Further, a concentration-dependent change in this chemical shift (Δδ) was observed. In the presence of 20% PVP K90, Δδ was 0.014 ppm, while in the presence of 70% PVP K90, it increased to 0.053 ppm (Table 1). The 1H NMR spectra of IND, Eudragit E100, and their various ratios are shown in Figure 9. Similar to IND and PVP K90 systems, the Δδ observed in the presence of Eudragit E100 was concentration-dependent. However, the shift due to the presence of Eudragit E100 was more significant compared to PVP K90. The Δδ was 0.040 ppm in the presence of 20% Eudragit E100, which increased to 0.110 ppm in the presence of 70% Eudragit E100. This suggests that Eudragit E100 had more shielding effect compared to PVP K90. Ternary System (IND/PVP K90/Eudragit E100). A concentration-dependent upfield shift was observed, confirming the shielding of the proton in the presence of PVP K90 and E


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Figure 7. Structure and solution 1H NMR spectrum of PVP K90.

Figure 8. Structure and solution 1H NMR spectrum of Eudragit E100.

Eudragit E100 (Figure 9). The Δδ of 0.045 ppm was observed in the presence of 20% polymers (10% PVP K90 and 10% Eudragit E100), which increased to 0.100 ppm in the presence of 70% of these polymers (35% PVP K90 and 35% Eudragit E100). In ternary systems, higher peak shifts were observed compared to individual polymers. In the presence of 20% PVP K90 and Eudragit E100 in a binary system, Δδ of 0.014 and 0.040 ppm, respectively, were observed. The combination of polymers in ternary system showed a significantly higher Δδ of

0.070 ppm (Table 1), which suggests a synergistic effect on Δδ. These shielding effects in binary and ternary systems suggest a change in electron density as a result of interaction between IND and polymers. Molecular Modeling. The MP2 binding energy of IND with various polymers calculated using molecular modeling is given in Table 2. Molecular modeling calculations determine the binding energy and possible interactions between drug and monomer units of the polymers. The simulations undergo F


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Figure 9. Chemical shift of proton at C2 of IND in the presence of PVP K90, Eudragit E100, and PVP K90/Eudragit E100 at various ratios.

Table 1. Change in the Chemical Shift of C2 in the Presence of Polymers at Various Drug Loads IND/PVP K90

change in chemical shift (Δδ)

IND/E100


IND/PVP K90/E100


100:0 80:20 60:40 30:70

0.000 0.014 0.038 0.053

100:0 80:20 60:40 30:70

0.000 0.040 0.080 0.110

100:0:0 80:10:10 60:20:20 30:35:35

0.000 0.045 0.070 0.100

mine.24 One of the limitations of the current work is that it does not consider the interaction between additives and nuclei/ crystal surfaces, which had previously been known as an important mechanisms of nucleation/crystallization inhibition or morphology modification. These future studies coupled with the NMR and morphology changes observed in the current work can provide a more comprehensive understanding of drug−polymer systems.

Table 2. MP2 Binding Energies of Indomethacin with Polymers second molecule

MP2 binding energy (kcal/mol)

PEG PVP methyl methacrylate butyl methacrylate (2-dimethylamino ethyl) methacrylate

−4.87 −9.25 −8.14 −7.41 −13.89

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iterations until lowest energy state of drug−monomer complex had been established. The results indicated that IND has highest binding energy with Eudragit E100 followed by PVP K90 and PEG 8000 (least binding energy). The higher binding energy corresponds to the possibility of stronger interaction between IND and Eudragit E100 followed by IND and PVP K90. The results are in accordance with NMR data, which suggested similar rank ordering of IND interaction with these two polymers. In our study, these basic calculations correlated well with the experimental results providing valuable insights in the drug−polymer molecular interactions and their role in precipitation inhibition. However, caution should be exercised due to many unaccountable variables and assumption used in molecular modeling. These basic interaction energy calculations in conjunction with experimental results had been previously used to rank order polymers inhibitory effect on curcumin precipitation,20 correlate the improvement of benznidazole solubility with its interaction with PVP,23 and understand the intermolecular associations between celecoxib and meglu-

DISCUSSION

The precipitation of drugs from supersaturated solution is a topic of immense importance in the pharmaceutical industry. The mechanism of precipitation inhibition from high supersaturated solution in the presence of two polymers is still poorly understood, so more work is needed to understand this complex phenomenon. It can be concluded from the published literature that the addition of a polymer can have both thermodynamic and kinetic effects. Thermodynamically, polymers can change the solubility of the system and thus can reduce the supersaturation level, making the solution less susceptible to precipitation.25,26 In terms of kinetics, polymers reduce the activity coefficient and suppress the nucleation/ crystal growth rate by adsorption on the crystal faces. Further, drug−excipient interactions have been known to affect both the thermodynamics and kinetics of precipitation.21,26−28 In our experiments, no significant change in solubility was observed with any of the added polymers; this confirms that the supersaturation was maintained at the same level in all. This G


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Molecular Pharmaceutics rules out the possibility of modification in supersaturation levels as the mechanism of precipitation inhibition by these polymers. Further, in the presence of the noninteracting polymer PEG 8000, which results in the increase in entropy of the system, no effect was observed on nucleation and crystal growth. These results suggest that both the polymers Eudragit E100 and PVP K90 significantly interfere with the kinetics of precipitation via drug−polymer interactions. Precipitation is a two-step phenomenon involving formation of stable nuclei (nucleation) and then growth of the crystals. The nucleation process involves three steps: the diffusion of molecules through the bulk of the solution, collision with each other, and formation of nuclei of a critical size. Once the critical size is reached, crystal growth takes place.29,30 Many times, after initial nucleation, both steps can take place simultaneously. We utilized the t1% and t10% values calculated from absorbance curves as descriptive measures of precipitation. The true nucleation time, tn, is defined as the time that elapses from the creation of supersaturation in solution until critical nuclei form. This lag phase occurs as a consequence of the achievement of a steady-state distribution of sizes of the nuclei that are already present in solution or newly formed owing to the supersaturation that is created. However, it is not possible to directly observe the critical nuclei as they form in solution because they can only be observed after they have grown to a critical size. By utilizing the available techniques, it is not possible to directly measure the nucleation time. Therefore, in our study t1% is selected as a representation of nucleation as we were able to quantitatively distinguish the initial precipitation behavior of drug in the presence of polymers at this time. Below t1%, a lot of variability was observed, whereas above this value, crystal growth rate might predominate. Since we know that nucleation usually starts first followed by crystal growth, we adopted this simple method to distinguish the effect of polymers on indomethacin precipitation. In our study, t1% corresponds to the nucleation step of precipitation, whereas t10% provides information about the crystal growth. Further, the smaller the time difference (Δt) from t1% to t10%, the faster is the crystal growth rate. Any effect on t1% would signify the effect on the nucleation step, while changes in Δt (Δt = t10% − t1%) would signify the effect on crystal growth. In precipitation studies, spontaneous nucleation (t1% ≈ 8 min) and crystal growth (Δt ≈ 5 min) were observed for IND when no polymer was added. This indicates that the supersaturated solution was in an unstable labile zone (Figure 10). The degree of supersaturation involved in the system was high, so the poorly soluble drug in solution precipitated out rapidly.31 In the absence of polymers, once the nucleation is initiated at these high supersaturation levels, the crystal growth rate of IND was high. However, in the presence of Eudragit E100, the onset of nucleation was delayed (t1% ≈ 20 min), while the crystal growth rate was unaffected as confirmed by the Δt of about 12 min in precipitation experiments. In the presence of PVP K90, no effect was observed on nucleation, whereas the crystal growth rate was drastically decreased as suggested by increase in Δt to 32 min. The nucleation inhibition mechanism in the presence of Eudragit E100 can be explained on the basis of IND−Eudragit E100 interaction. IND showed stronger interaction with Eudragit E100 and higher binding energy compared to PVP K90 based on proton NMR studies and binding energy calculations. For nucleation to occur, IND molecules should overcome these drug−polymer interactions to diffuse and form

Figure 10. Various solubility zones in a concentration versus temperature curve. C* is the equilibrium solubility at temperature T*. If the concentration is in labile zone, spontaneous precipitation is expected from a system.

a critical nucleus. The drug−polymer interactions inhibit or retard the formation of nuclei through collision as well. Further, the strength of the drug−polymer interactions determines the time required for nucleation to take place; more energy is needed to break stronger interactions.31−34 The lower binding energy between IND−PVP K90 was not strong enough to have an effect on diffusion of IND molecules to form nuclei. Therefore, nucleation inhibition was observed in the presence of Eudragit E100, whereas no effect was observed when PVP K90 was present. Once the critical nuclei are formed, the IND crystals begin to grow. The crystal growth process involves transport of growth units (i.e., the molecule) from the bulk solution to the crystal surface, diffusion to the growth site, and incorporation into the crystal surface. Any of the above processes can be the rate-limiting step for crystal growth.30,35 The adsorption of PVP K90 might be the reason behind the decrease in the crystal growth rate of IND. The present results show that the growth inhibition and habit modification are achieved by the presence of PVP K90. As the solution approaches the crystal surface, the PVP K90 molecules get adsorbed onto the crystal surface. This leads to retardation in the growth of IND crystals and modification of its crystal morphology. Similar results were reported by Patel et al. where they found that PVP significantly inhibited the crystal growth of indomethacin due to the change in the indomethacin crystal growth mechanism that also resulted in a change in the rate limiting step from bulk diffusion to surface integration at high supersaturation.36,37 Thus, it seems that two important factors play significant roles in the process. First is the drug−polymer interaction, and second is the adsorption of polymer on the crystal growth sites. Eudragit E100 showed a higher extent of interaction with IND leading to a decrease in nucleation. However, Eudragit E100 was not adsorbed during the process, thus showing no effect on the IND crystal growth rate and crystal habit. On the other hand, the interaction of PVP K90 with IND was not strong enough to have an effect on nucleation activation energy, leading to no change in nucleation. However, the adsorption of PVP K90 on the IND crystal surface leads to crystal growth inhibition and habit modification. The ineffectiveness of PEG 8000 on the nucleation and crystal growth rate of IND further suggests that the drug−polymer interaction plays an important role in precipitation inhibition. H


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By using the combination of polymers, a significant increase in both t1% and Δt was observed. The change in morphology also confirmed the effect on crystal growth. These results imply that combining the two polymers showing effect on nucleation and crystal growth separately obtains both IND nucleation and crystal growth inhibition. Further, these significant effects observed by the combination of polymers in the ternary system were at half of the concentration used in the binary system. This clearly suggests the synergistic potential of using a combination of polymers showing drug−polymer interaction for precipitation inhibition. The synergistic effect was further confirmed by the 1H NMR studies, in which the ternary system produced Δδ similar to binary system at half the polymer concentration. In our previous publication on ternary solid dispersions, the comparison between the binary and ternary solid dispersions showed that the ternary system leads to a significant improvement in solid state stability and the dissolution rate of IND.38 Thus, overall our results show that the combination of two polymers with drug−polymer interaction can significantly enhance the stability in solution as well as in solid state due to a synergistic effect.

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CONCLUSION A combination of PVP K90 and Eudragit E100, two polymers with drug−polymer interaction, resulted in significant precipitation inhibition of the poorly soluble drug IND. This enhanced precipitation inhibition efficiency can be correlated to the synergistic effect of PVP K90 and Eudragit E100, as one significantly decreases crystal nucleation, and the other showed high crystal growth inhibition. The study highlights the importance of utilizing drug−polymer interaction by combining polymers with different crystallization inhibition mechanisms for precipitation inhibition and dissolution enhancement of poorly soluble drugs.

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AUTHOR INFORMATION

Corresponding Author

*Address: College of Pharmacy, California Northstate University, 10811 International Drive, Rancho Cordova, California 95670, United States. Phone: 617-832-5105. E-mail: eatef@ cnsu.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author wishes to acknowledge MCPHS University, Boston for funding and the use of instruments used in this project. The authors also extend their gratefulness to Jonathan Bernick at Creighton University for molecular modelling results.

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REFERENCES

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