Supersaturation Potential of Salt, Co-Crystal, and Amorphous Forms of

Article pubs.acs.org/crystal

Supersaturation Potential of Salt, Co-Crystal, and Amorphous Forms of a Model Weak Base Published as part of the Crystal Growth & Design Margaret C. (Peggy) Etter Memorial virtual special issue Luis Almeida e Sousa,† Susan M. Reutzel-Edens,‡ Gregory A. Stephenson,‡ and Lynne S. Taylor*,† †

Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States ‡ Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, United States S Supporting Information *

ABSTRACT: High energy solids, such as salts, co-crystals, or amorphous solid dispersions, have been widely used to generate supersaturated aqueous solutions and improve drug bioavailability. However, most research on solubility enhancing strategies has focused on the kinetics of dissolution, and there is relatively little comparison of the different degrees of supersaturation achieved by using different solid state forms of the same compound. Recent studies from our group have demonstrated that the maximum achievable supersaturation is dictated by the aqueous solubility of the amorphous form of the drug. Liquid−liquid phase separation (LLPS) occurs at concentrations above this value. Herein, it was hypothesized that the upper limit of supersaturation that can be achieved from dissolution of various high energy solids is also governed by the amorphous solubility. To test this hypothesis, the dissolution and supersaturation behavior of different solid forms of a model compound, CRH1, were investigated using a variety of techniques. With the exception of CRH1 crystalline free base, all solid forms generated supersaturated solutions. The extent of supersaturation, onset of crystallization time, and area under the curve increased significantly when a polymer with crystallization inhibitory properties was present in the dissolution medium or incorporated in the formulation (in the case of amorphous solid dispersions). In the presence of the polymeric crystallization inhibitor, several solid state forms, including the amorphous solid dispersion and the salts, dissolved to concentrations above the amorphous free form solubility and underwent LLPS, generating a drug-rich phase. Other solid state forms underwent crystallization prior to attaining the amorphous solubility and showed no evidence of LLPS (co-crystal and glass forms). These studies should aid in solid state form selection and formulation and help to understand how to achieve maximized supersaturation in vivo.

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permeability and flux across membranes as a result of lowered concentrations of free drug.2 On the other hand, supersaturating delivery systems generate concentrations higher than the thermodynamic solubility of the crystalline drug without compromising effective permeability across membranes. The main disadvantage of these systems is that supersaturated solutions are metastable and tend to fall back to equilibrium (via crystallization) unless crystallization inhibitors are added to the formulation. To generate and maintain supersaturation, two conditions must be met: the compounds must be administered as solid state forms that have a higher “apparent” or kinetic solubility than the thermodynamically stable form in the dissolution media, and the dissolution rate has to be higher than the rate of precipitation/crystallization of the lower energy

INTRODUCTION In recent years, the number of water-insoluble drugs has increased substantially, whereby it is estimated that more than 60% of newly discovered drug molecules currently have suboptimal aqueous solubility.1 This shift in paradigm is largely attributed to an increase in size and lipophilicity of new molecules targeting biologic membranes or membraneassociated proteins.1 Because poor aqueous solubility has a great impact on the biological exposure and bioavailability of drugs administered orally, it is of paramount importance to develop solubility enhancing strategies for these compounds. Two strategies have been widely used to address such solubility issues: solubilization techniques and supersaturating drug delivery systems. Solubilization strategies use cosolvents, surfactants, complexing agents (e.g., cyclodextrins) and/or lipids in the formulation to effectively increase the amount of drug dissolved or dispersed in water. Unfortunately, these systems sometimes lead to decreases in drug effective © XXXX American Chemical Society

Received: September 15, 2015 Revised: November 20, 2015

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DOI: 10.1021/acs.cgd.5b01341 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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amorphous in nature.15−17 This phenomenon is called liquid− liquid phase separation (LLPS)6,7 or glass−liquid phase separation (GLPS) depending if the new phase is a supercooled liquid or a glass.18 Co-crystals of poorly water-soluble drugs are an example of pharmaceutical solids that combine the stability advantages of crystalline solids with the solubility benefits of high energy solids. Although the mechanisms by which co-crystals enhance solubility are not yet fully understood, there is general agreement on the critical role of the coformer physicochemical ́ properties on the overall rate of dissolution. RodriguezHornedo found that the up to ∼150-fold solubility improvement of carbamazepine co-crystals is related to the coformer solubility and that the concentrations of each component at the eutectic are a direct measure of co-crystal equilibrium solubility.19,20 On the other hand, Nangia described the sudden generation of supersaturation in terms of the quick release of the co-former from the co-crystal lattice and the formation of supramolecular aggregates of randomly oriented molecules.21 These clusters apparently behave similarly to amorphous materials and, therefore, have improved dissolution and solubility properties. Despite generating high drug concentrations rapidly, co-crystals tend to dissociate readily in solution, converting to a more stable and less soluble crystalline form.22−24 These phase transformations are responsible for the decay in drug concentration that is often observed with time. Addition of excipients to the co-crystal formulation may retard the conversion kinetics.25 Another strategy that is commonly used to enhance the solubility of acidic and basic drugs is salt formation. Crystalline salts dissociate in water to form ionized species that are highly soluble compared to the conjugated un-ionized molecules. The bulk solution pH plays an important role in this solubility enhancement because it dictates the degree of ionization of the API molecules. Salts also dissolve significantly faster than free drugs, independently of bulk solution pH. 26,27 Rapid dissolution is explained by the higher drug concentrations at the unstirred diffusion layer which, in turn, result from differences in pH between the surface of the dissolving salt and the bulk of the solution. These high dissolution rates are responsible for the generation of supersaturation near pHmax, where the fraction of soluble ionized species is high enough to trigger a rapid increase in drug concentration.28 Supersaturated solutions have been generated from dissolution of salts and maintained using predissolved crystallization inhibitors such as polymers.29,30 Analysis of the solid phase in contact with those solutions showed that, in cases where supersaturation was maintained, an amorphous precipitate was present.30 Similar amorphous materials were obtained in experiments where supersaturation was generated by changing the bulk pH of solutions of slowly crystallizing drugs. In these studies, a clear relationship was found between the solution concentration in apparent equilibrium with the amorphous phase and the estimated amorphous solubility of drugs.31 Similar conclusions were obtained from dissolution of amorphous solid dispersions.14,15 These observations suggest that the maximum free drug concentration/supersaturation that is possible to obtain from the dissolution of salts also corresponds to the solubility of the amorphous free drug. The work presented herein aims to compare the supersaturation and dissolution behavior of different solid forms (crystals, co-crystals, amorphous, ASDs, and salts) of the corticotropin releasing hormone receptor 1 antagonist, CRH1

form. Metastable polymorphs, salts, co-crystals, amorphous drugs, and amorphous solid dispersions are some of the most relevant solid forms with the potential to form supersaturated solutions that are used in oral drug delivery. Metastable polymorphs have a crystal lattice in which the packing/conformation of the drug molecules precludes optimal interactions between adjacent molecules from occurring in three-dimensional space. Therefore, these solids have lower crystal lattice energy, stability, and higher solubility compared to the thermodynamically stable form. Despite the improved solubility of metastable polymorphs, solubility enhancement ratios are relatively moderate. Results from a study where the solubility ratio and dissolution rate of 55 compounds with different polymorphs were analyzed, showed that solubility ratios between 2 and 3 were typical for most compounds and were less than 5 for all but one compound.3 These ratios are much smaller compared to typical values obtained with amorphous forms. Similar to amorphous drugs, these polymorphs are metastable and will tend to convert to the most stable/less soluble form over time or after exposure to heat or humidity. Conversion of a crystalline solid to its amorphous form is an alternative approach that yields significantly higher solubility ratios.4,5 This high energy state has improved solubility over the crystalline counterpart because the crystal lattice is disrupted, which makes it energetically more favorable for the solid to dissolve. The maximum concentration obtained from dissolution of an amorphous drug is not a true equilibrium solubility because it refers to a thermodynamically metastable system. Nevertheless, because there is a well-defined difference in free energy between pure amorphous (supercooled liquids or glasses prepared under known conditions) and crystalline forms, it is possible to predict the “apparent” solubility of an amorphous drug (Camorphous) using the solubility of the crystal form (Ceq), the free energy difference between the crystal and amorphous form (ΔGc→a) and the activity of the amorphous solute saturated with water (exp[−I(a2)]):6,7 Camorphous = Ceq e[−I(a2)]eΔGc→ a / RT

(1)

where T is temperature and R is the ideal gas constant. Phase transformations are common when amorphous drugs are dissolved in aqueous media with crystallization occurring from solution and/or from the surface of the dissolving amorphous material.8 To stabilize the amorphous state, crystallization inhibitors, such as polymers, are often combined with the amorphous API to generate amorphous solid dispersions (ASDs). Usually, these have a relatively high glass transition temperature (Tg) compared to the pure amorphous drug and subsequently reduce API molecular mobility and recrystallization tendency.9 Hydrogen bonds formed between the drug and polymer can also hinder nucleation and growth processes. In addition to stability advantages, ASDs are used to enhance the dissolution rate of drugs.10,11 These two properties combined with the fact that amorphous drugs are high energy forms, render ASDs advantageous in terms of solubility and bioavailability.12,13 Despite yielding relatively high supersaturation levels, recent studies have demonstrated that there is an upper concentration of “free” drug that can be achieved from the dissolution of ASDs.14,15 This concentration was found to correspond to the amorphous solubility of the API. Exceeding this concentration leads to the formation of a submicron drug-rich phase that is B



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pH 6.8 phosphate buffer solutions were prepared with a concentration of 50 mM and ionic strength (I) of 155 mM. pH 4.6 (I = 98 mM) citrate buffer solutions were prepared with a concentration of 50 mM. Preparation of Pure Amorphous and Amorphous Solid Dispersions of CRH1. To prepare amorphous CRH1, the crystalline free form was melted on aluminum foil and quench cooled using liquid nitrogen. A pestle and mortar were used to reduce the particle size of the glassy particles. Amorphous material was freshly prepared prior to use for all tests, and analysis by high performance liquid chromatography indicated that the material did not undergo degradation following melt quenching. Amorphous solid dispersions (ASDs) of CRH1 were prepared using HPMC as the polymer matrix. The drug/polymer mass ratio was 20:80. ASDs were prepared by rotary evaporating a 50/50 v/v methanol/dichloromethane solution of drug and polymer using a Buchi Rotavapor-R (New Castle, DE, USA) equipped with Yamato BM-200 water bath, under reduced pressure at 45 °C. The resulting dispersions were dried under a vacuum for 24 h to remove residual solvents. Freshly prepared ASDs were tested immediately after drying. UV/vis Spectrophotometry To Detect Phase Separation Phenomena. The following procedure was used to investigate phase separation phenomena occurring from supersaturated solutions of CRH1 prepared with a solvent-shift method. Supersaturation was generated by titrating solubilized drug (in methanol) to an aqueous buffered solution at a significantly faster rate compared to that of drug crystallization from solution. The concentration at which the intensity of light scattered from the solution sharply increases was taken as the point where the liquid−liquid phase separation (LLPS) occurs. The LLPS concentration of CRH1, a weak base with a measured pKa of 4.6, was determined in buffered solutions at pH 6.8 (where the drug is completely un-ionized) and pH 4.6 with 20 μg/mL of predissolved HPMC (conditions used in the dissolution experiments). A syringe pump was used to titrate 1 and 2 mg/mL solutions of drug in methanol into pH 6.8 and pH 4.6 + 20 μg/mL buffer solutions, respectively. The pump speed was 80 μL/min in the first case and 60 μL/min in the second. Solutions were kept inside a temperature controlled flask at 25 °C and stirred at a speed of 300 rpm. The intensity of light scattered from solution was measured with a SI Photonics UV/vis spectrometer (Tucson, AZ, USA) fiber optically coupled with a dip probe of path length 10 mm. Light scattering was detected by monitoring the extinction at a nonabsorbing wavelength (450 nm). Concentrations of un-ionized CRH1 at pH 4.6 were calculated using the Henderson−Hasselbalch equation. All UV extinction experiments were performed in triplicate. Fluorescence Spectroscopy To Investigate LLPS. This technique uses an environment-sensitive fluorescence probe, pyrene, to detect changes in local polarity as the solution undergoes liquid−

(4-(4-chloro-5-(2,6-dimethyl-8-(pentan-3-yl)imidazo[1,2-b]pyridazin-3-yl)thiazol-2-yl)morpholine; Figure 1), a poorly

Figure 1. Structure of CRH1.

soluble, weakly basic drug candidate, and to analyze the types of phase separation occurring during and after dissolution. This molecule was selected for study primarily because of its wide range of physical forms and their differing solubilities. The hypothesis to be tested is that the amorphous solubility is the upper limit of supersaturation achievable by dissolving any of the solid state forms (salts, ASDs, etc.). Phase separation phenomena (crystallization and LLPS) were analyzed using ultraviolet (UV) extinction and fluorescence methods. Maximum concentrations obtained from the dissolution of different solids were compared with the estimated amorphous solubility of the compound and the reference crystalline solubility of the free base form. The results supported the hypothesis, whereby the maximum free drug concentration observed never exceeded the estimated amorphous solubility. This was the upper limit, and some solid state forms did not reach this limit prior to crystallization occurring.

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MATERIALS AND METHODS

Materials. Acetonitrile, pyrene, and 1,5-naphthalenedisulfonic acid tetrahydrate (NAP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxypropylmethyl cellulose (HPMC) Pharmacoat grade 606 was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Methanol, N,N-dimethylformamide (DMF), and dichloromethane were obtained from Macron Chemicals (Phillipsbourg, NJ, USA). CRH1 was provided by Lilly Research Laboratories (Indianapolis, IN, USA) in the following solid forms: hydrochloride salt (CRH1 HCl), free base (CRH1 FB), heminapadisylate salt (CRH1 NAP), hydrobromide salt (CRH1 HBr), and adipic acid co-crystal (CRH1 CC). XRPD patterns of all solid forms are shown in Figure 2.

Figure 2. XRPD pattern of the various crystalline forms of CRH1: adipic acid co-crystal (top), hydrochloride salt (upper middle), hydrobromide salt (middle), heminapadisylate salt (lower middle), and free base (bottom). C



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liquid phase separation.32 Changes in the fluorescence spectrum of the probe will only occur if the probe molecules partition into a hydrophobic drug-rich phase. This is only possible if the new phase has liquid-like properties and provides a different environment from water. Therefore, if crystallization occurs from solution, no changes in the fluorescence characteristics are expected. Solutions of CRH1 were prepared at a range of concentrations by adding different volumes of solubilized drug, 1 and 2 mg/mL, to 10 mL of pH 6.8 phosphate buffer and 10 mL of pH 4.6 citrate buffer containing 20 μg/mL HPMC. Each buffered solution had predissolved pyrene with a final concentration of 2 μM. Solutions were stirred at a speed of 300 rpm. Fluorescence measurements were made with a Shimadzu Spectrofluorometer RF-5301 PC (Kyoto, Japan). The excitation wavelength was 332 nm. The emission spectra were collected and analyzed to determine the concentration where LLPS occurred. An increase in the ratio of intensities between the third (λ = 383−386 nm) and first (λ = 373−375 nm) emission peaks is expected upon liquid−liquid phase separation. Solubility Measurements. The equilibrium solubility of CRH1 free base crystals was determined at pH 6.8 where the drug is completely un-ionized and pH 4.6 to mimic the dissolution conditions. The saturated solutions of CRH1 prepared at pH 4.6 contained 20 μg/ mL HPMC. These solutions were shaken in a water bath at 25 °C for approximately 48 h. The supernatant was then separated from the excess solid by ultracentrifugation at 35 000 rpm in an Optima L-100 XP ultracentrifuge equipped with a Swinging-Bucket Rotor SW 41 Ti (Beckman Coulter Inc., Brea, CA) for 20 min. Triplicate samples were prepared for analysis. Drug concentration in the supernatant was determined using an Agilent high performance liquid chromatography (HPLC) 1260 Infinity system (Agilent Technologies, Santa Clara, CA). The separation column was an Agilent Poroshell 120 EC-C18, 2.7 μm, 4.6 × 50 mm (T = 25 °C). A 50/50 acetonitrile/acidified water (pH 2) mixture was used as the mobile phase. The injection volume was 10 μL, and the flow rate was 0.25 mL/min. The detection wavelength was 285 nm. Standard solutions of the drug were prepared in the mobile phase and analyzed with HPLC to prepare a calibration curve. All samples were analyzed in triplicate. The standard curve presented good linearity (R2 > 0.999) over the relevant concentration range. This method was also used to determine the concentration of drug in the aqueous phase in turbid supersaturated solutions (above the LLPS concentration). Ultracentrifugation was used to separate the two liquid-like phases, and the supernatant was analyzed with the HPLC method described above. A supersaturated solution of CRH1, with a total concentration of 36 μg/mL, was prepared in pH 4.6 buffer with 20 μg/mL HPMC using the solvent-shift method. The ionic strength of the solution was adjusted to 155 mM with NaCl. Moisture Sorption Analysis. The activity of the amorphous drug saturated with water (exp[−I(a2)] in eq 1) was determined using the method previously described by Murdande et al.33 The moisture sorption profile of the amorphous compound was determined using a gravimetric method that measured the water uptake as a function of relative humidity. From the measured activity of water as a function of composition, the activity of the drug saturated with water can be calculated by integrating the Gibbs−Duhem equation. A SGA-100 Symmetric vapor sorption analyzer (VTI Corp, Hialeah, FL) was used to measure the moisture sorption profiles of the amorphous drug as a function of relative humidity. Approximately 10 mg of amorphous CRH1 was placed in the SGA-100 sample holder. The analysis was performed at 25 °C from 0 to 95% RH (with 10% increases until 90% RH), and the equilibrium criterion was less than 0.01% weight change in 15 min with a maximum equilibration time of 500 min. DSC (Differential Scanning Calorimetry) Experiments. Differential scanning calorimetry was used to determine the melting temperature (Tm) and enthalpy of fusion (ΔfusH) of crystalline CRH1 free base, and the glass transition temperature (Tg) of the amorphous free base prepared by melt quenching. Heat capacity measurements were also performed to determine the fitting parameters (a, b, c, d, e, and f) that describe the change in heat capacity of samples with temperature.7 Thermal data were collected with a DSC Q2000 (TA

Instruments, New Castle, DE) equipped with a refrigerated cooling system RCS 90. Enthalpy and temperature calibrations were made with indium, while heat capacity calibrations were made with sapphire. Nitrogen was used as a purge gas at 50 mL/min. Each measurement was made in triplicate on approximately 5 mg of drug. A heating rate of 10 °C/min was used to determine the melting parameters of crystalline CRH1. Data were analyzed with the TA Universal Analysis 2000 software (TA Instruments, New Castle, DE). Heat capacity measurements were made using a modulated heating program (1 °C every 60 s) applied to an overall heating rate of 2 °C/min. The TA Universal Analysis software was used to separate the reversible signal from the irreversible heat capacity. Reversible Cp data were fitted to linear functions of temperature to obtain the fitting parameters a, b, c, d, e and f.7 Estimation of the Amorphous Solubility. The theoretical solubility of the amorphous drug (free base) was calculated using eq 1. Experimental values for the solubility of the crystal form (Ceq) and activity of the amorphous solute saturated with water (exp[−I(a2)]) were determined using the methods described above. The free energy of conversion of the crystalline drug to its amorphous form (ΔGc→a) was calculated using the Hoffman equation34 and the heat capacity (Cp) method described by Almeida e Sousa et al.7 The Hoffman equation (eq 2) is very convenient and easy to use as it only requires measurement of the melting temperature (Tm) and enthalpy of fusion (ΔfusH) of the compound.

ΔGc → a =

ΔfusH(Tm − T )T Tm 2

(2)

T is the experimental temperature. On the other hand, the Cp method uses information about the heat capacity change with temperature for the crystalline and amorphous forms, as well as melting data. This method is not only more accurate than the Hoffman approach, but it also allows the free energy difference between the crystal and either the supercooled liquid or glass to be determined. Dissolution Experiments. To evaluate the supersaturation potential of different solid forms of CRH1, dissolution experiments were performed in buffered solutions (50 mM) at pH 4.6, which corresponds to the pKa of CRH1. Under these conditions, the equilibrium concentrations of un-ionized and ionized drug are identical. In the case of CRH1 salts, the intent was to rapidly dissolve the highly soluble ionized species to achieve a high supersaturation before conversion of the salt to the free base. Other solid state forms (co-crystal, amorphous, and amorphous solid dispersions) were dissolved at the same conditions for comparison. The effect of a crystallization inhibitor, such as HPMC, on the dissolution profile of CRH1 was also investigated. HPMC was predissolved in pH 4.6 buffered solutions to generate a final concentration of 20 μg/mL. The following procedure was used to investigate the dissolution behavior of the different solid forms of CRH1. 50 mL of buffered solution was added to a temperature-controlled flask (25 °C) wrapped in aluminum foil and stirred at a speed of 200 rpm. With the exception of the co-crystal and hemi-NAP salt, approximately 10 mg of powder was added to the dissolution medium. Fifteen milligrams of co-crystal was added to compensate for the molecular weight of the coformer, adipic acid. Only 5 mg of the hemi-NAP salt was added because this form dissolves almost instantaneously and higher concentrations saturated the UV detector. For the same reason, 10 mg of the amorphous solid dispersion (ASD), containing 2 mg of CRH1, was added. To determine the concentration of drug during dissolution, a UV absorbance method was employed. A UV dip probe, with a 10 mm path length, was immersed in the dissolution medium, and the absorbance spectra were measured at regular time intervals with the UV spectrometer described above. Differences between the peak and baseline absorbance were calculated and converted to concentrations using calibration curves. Standard solutions were prepared in different dissolution media, and their UV spectra were utilized to prepare the calibration curves. Good linearity was obtained (R2 > 0.999). D



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Table 1. Physicochemical Properties of CRH1 Free Base crystalline

amorphous

MW (g/mol)

pKa

Tm(°C)

ΔfusH (kJ/mol)

Tg (°C)

e(−I(a2))

ΔGc‑sl (Hoffman) (kJ/mol)

ΔGc‑sl (Cp) (kJ/mol)

ΔGc‑g (Cp) (kJ/mol)

420.0

4.6 (B)

141.7

28.12

48.0

0.755

5.68

5.85

5.95

The maximum concentration of drug in solution was also measured using an ancillary HPLC method. Absorbance values, which are an indirect measure of solution concentration in turbid solutions where there may be spectral interference from particles or other scattering/ absorbing species, were monitored with the UV spectrometer. Immediately after the maximum absorbance was reached, a sample was taken from the dissolution vessel and centrifuged. The concentration of drug in the supernatant was determined using the HPLC method previously described. The type of phase transformations occurring during dissolution (crystallization or LLPS) was also investigated using the fluorescence probe technique described earlier. Pyrene was predissolved in the dissolution media to generate a final concentration of 2 μM. Following addition of the different solid state forms, samples were collected at regular intervals to measure the fluorescence properties of the predissolved probe.

from supersaturated solutions during preliminary studies and hence provides an opportunity to investigate the effect of polymers on the inhibition of crystallization from supersaturated solutions generated by dissolution of different forms. Key physicochemical properties of the model system are summarized in Table 1. Liquid−Liquid Phase Separation (LLPS) Studies. Supersaturated solutions of CRH1 were generated at both pH 6.8, where the drug is completely un-ionized, and pH = pKa = 4.6 (with 20 μg/mL HPMC), where the concentration of unionized and ionized species is similar, using the solvent-shift method. An increase in turbidity was observed at very high supersaturation, although no visible particles were observed with the naked eye. Subsequently, the media became clearer with the evolution of numerous visible particles. These observations are consistent with the occurrence of LLPS, followed by crystallization of the drug.6,7 The concentration at which LLPS occurred was determined using the UV extinction and fluorescence methods. Figure 3 summarizes the fluorescence and extinction data collected for CRH1 as a function of the un-ionized concentration. UV extinction and fluorescence data show very good agreement. An increase in extinction was detected around the same concentration where a change in the fluorescence emission properties was observed, suggesting that phase separation to a disordered drug-rich phase occurred at this concentration. Similar results were observed in a study where the phase separation behavior of six poorly soluble drugs was assessed using these two methods.7 The LLPS concentration of CRH1 was approximately 12 μg/mL (with respect to the concentration of un-ionized species), and good agreement was seen for the two pH conditions. Relative to the crystalline CRH1, this represents an approximately 10-fold solubility enhancement (LLPS concentration/crystalline solubility). To confirm that the LLPS concentration determined with the UV extinction and fluorescence methods corresponds to the maximum extent of supersaturation, the concentration of un-

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RESULTS AND DISCUSSION CRH1 was selected as the model API for a number of reasons. First, it is a poorly water-soluble compound (Table 2), Table 2. Measured Solubility of Crystalline CRH1 Free Base (Ceq) and Calculated Solubility of Amorphous CRH1 Free Base in Different Buffersa theoretical amorphous solubility using buffer solution pH 6.8 pH 4.6 + 20 μg/mL HPMC a

Ceq (μg/mL)

ΔGc-sl (Hoffman) (μg/mL)

ΔGc-sl (Cp method) (μg/mL)

ΔGc-g (Cp method) (μg/mL)

1.20 ± 0.05 1.50 ± 0.02

9.0 11.2

9.6 12.0

10.0 12.5

All concentrations refer to the unionized species.

representing the typical case of a molecule where solid form selection is considered during development to enhance drug solubility. Second, it is a weak base with a measured pKa of approximately 4.6 and can form salts or co-crystals. This drug was also chosen because it was observed to crystallize readily

Figure 3. UV extinction and fluorescence data for CRH1 in (a) pH 6.8 and (b) pH 4.6 + 20 μg/mL HPMC. E



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Figure 4. Summary of all experimental solubility measurements, predicted LLPS concentrations, and amorphous solubility estimates obtained for CRH1.

Figure 5. Dissolution of different solid forms of CRH1 in pH 4.6 buffer with no predissolved polymer (all concentrations refer to the un-ionized species). Error bars represent standard deviations obtained from triplicate measurements.

ionized drug in the bulk aqueous phase was assessed after removing the drug-rich phase using ultracentrifugation. The concentration of un-ionized drug in the bulk aqueous phase was 15 μg/mL. The small discrepancy between this value and the extinction/fluorescence values may be related to inefficient separation of the drug-rich phase from the aqueous drug-lean phase during ultracentrifugation. Theoretical Amorphous Solubility vs LLPS Concentration. Previous works by Ilevbare6 and Almeida e Sousa7 clearly showed that there is a strong correlation between the concentration at which LLPS occurs and the theoretical

solubility of the amorphous form. The estimated amorphous solubility of CRH1 was calculated using eq 1. The crystalline solubility (Ceq) and thermodynamic activity of the amorphous drug saturated with water (exp[−I(a2)]) were determined experimentally (Tables 1 and 2). The free energy difference between the crystalline and amorphous forms (ΔGc→a) was estimated using two different approaches: the Hoffman equation34 and the Cp method7 (Table 1). Table 2 shows data on the crystalline solubility of CRH1 free base in different media and the corresponding amorphous solubility estimated using the different prediction methods. F



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Figure 6. Dissolution of different solid forms of CRH1 in pH 4.6 buffer containing 20 μg/mL of HPMC (all concentrations refer to the un-ionized species). Error bars represent standard deviations obtained from triplicate measurements.

solution. Despite these four solid forms dissolving quickly to yield supersaturated solutions, solution concentrations never reached the estimated amorphous solubility of CRH1 (gray area in Figure 5). With respect to the hemi-NAP salt, interference from the counterion, which absorbed UV light and remained in solution when the free base crystallized, complicated interpretation of the data. This is discussed in more detail in the Supporting Information (Figure S1). The only form that was able to generate concentration levels that exceeded the estimated amorphous free base solubility was the ASD (orange squares in Figure 5). These results cannot be explained only by the fact that the drug was in its amorphous state because the glass form of CRH1 (blue squares in Figure 5) generated concentrations that never exceeded 4 μg/mL. Clearly the presence of the polymer (HPMC) in the ASD is important; this facilitated dissolution and delayed crystallization of drug from the supersaturated solution generated following dissolution and/or from the dissolving matrix. The polymers used to form ASDs are known to yield advantages in terms of the dissolution rate and crystallization inhibition.36 In this case, crystallization was delayed significantly resulting in higher maximum drug concentrations and larger areas under the concentration vs time curve, compared to other solid forms. The rapid crystallization of most solid state forms effectively prevents our hypothesis from being tested. Given the crystallization inhibitory properties of HPMC, the next logical step was to investigate the dissolution of the different solid forms of CRH1 in buffer containing predissolved polymer. Different polymers were tested (e.g., polyvinylpyrrolidone vinyl acetate copolymer, HPMC, and various grades of HPMC acetate succinate), but HPMC was selected based on its ability to delay crystallization significantly and solubility at pH 4.6. The concentration of HPMC in solution was also optimized. Concentrations higher than 20 μg/mL did not provide significant improvements in terms of delaying crystallization; therefore this concentration was used. Figure 6 shows the results obtained for the dissolution of all seven solid forms of CRH1 in the presence of the crystallization inhibitor (data on the dissolution of ASD in buffer without polymer−orange squares were added for comparison). With the exception of the CRH1 crystalline free base, the dissolution behavior of all solid forms was very different in the

Comparing the amorphous solubility estimates with the LLPS concentrations measured by fluorescence and UV extinction, a strong correlation is observed between the two sets of data. The amorphous solubility estimates shown in Table 2 ranged from 9 to 12.5 μg/mL, depending on the prediction method. These values were close to the LLPS concentrations measured by fluorescence and UV extinction (11−14 μg/mL). Small differences between the estimated and experimental values were not surprising because prediction methods always have a certain degree of uncertainty. Figure 4 compares all experimental and predicted concentrations obtained for CRH1. Dissolution Testing of Different Solid Forms of CRH1. Having established the maximum achievable supersaturation and the concentration at which LLPS occurs, the next step was to test the central hypothesis of this study: that the maximum achievable concentration upon dissolution of any solid state form of a given compound is dictated by the amorphous solubility. Therefore, the dissolution behavior of different solid forms of CRH1 was investigated to evaluate the dissolution rate, the extent of achievable supersaturation, and the longevity of any supersaturated solution. Dissolution results in pH 4.6 buffer containing no polymer are shown in Figure 5. The Y-axis is the apparent concentration, rather than the true concentration. Apparent concentrations are given because if LLPS occurs, very small species, in the nanometer size range are generated, and these species can absorb light in a manner similar to drug in solution, interfering with quantitative UV absorbance measurements.35 Thus, the concentrations observed during dissolution are representative of the true solution concentration only until the amorphous solubility is reached. Figure 5 shows that the dissolution behavior is quite different for the seven forms evaluated. The free base dissolved very slowly, reaching a plateau that corresponds to the equilibrium crystalline solubility. The UV absorbance determined concentration of the un-ionized species at this point is similar to the equilibrium solubility determined using the HPLC method (∼1.5 μg/mL). The co-crystal, glass, and the HCl and HBr salts dissolved much faster than the free base crystalline form and generated supersaturation almost instantly. The apparent solution concentration reached a maximum in less than 30 min, decreasing thereafter as a result of drug crystallizing from G



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Figure 7. Apparent un-ionized concentration measured by UV extinction and pyrene 3/1 fluorescence peak ratio during dissolution of different solid state forms of CRH1 (a) crystalline free base, (b) glass, (c) co-crystal, (d) HCl salt, (e) HBr salt, and (f) ASD in pH 4.6 buffer with 20 μg/mL HPMC. The red dashed line indicates the pyrene 3/1 ratio above which the probe is considered to be in a more hydrophobic environment. The black dotted lines in (d) and (e) show that the pyrene 3/1 ratio exceeds a value of 0.586 when the dissolved concentration exceeds ∼9 μg/mL.

reaching maximum concentration. We can divide the different solid forms into two groups according to their dissolution behavior: those that generated supersaturation at or above the estimated amorphous solubility and those that failed to do so. The ASD and all three salt forms of CRH1 are in the first group. The ASD and HBr salts generated supersaturated solutions exactly in the gray region representing the estimated amorphous solubility values, while the HCl salt dissolved to concentrations near the lower limit of this region. The hemi-

presence of HPMC. Significant increases were observed in terms of the maximum concentration achieved, crystallization induction time, and area under the curve. Figure 6 shows that the co-crystal, glass, and HBr and HCl salts dissolved initially at a similar rate. However, these forms resulted in different maximum concentrations because the onset of crystallization was observed at different times. Differences in crystallization induction time were observed between the triplicate measurements, and this explains why the error bars become larger after H



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Figure 8. Comparison of the maximum concentrations determined with the in situ UV and centrifuge/HPLC methods. CRH1 solid forms were dissolved in pH 4.6 buffer containing 20 μg/mL HPMC.

NAP salt generated “apparent” concentrations significantly above this region. If these were true solution concentrations, our hypothesis that the estimated amorphous solubility is the upper limit of supersaturation would not be true. However, it is highly probable that the submicron drug-rich phase that forms above this concentration also absorbs in the same manner as drug in solution.35 If this is the case, then the implication is that the solution concentration exceeded the amorphous solubility, resulting in LLPS and the generation of a new drug-rich phase. This will be further explored subsequently. Included in the group of solid forms that were not able to dissolve to concentrations near the estimated amorphous solubility are the crystalline free base (stable form), the glass, and co-crystal. Despite the low supersaturations obtained with the glass and co-crystal forms, we cannot conclude that these solids are not capable of dissolving to concentrations near the estimated amorphous solubility. The only conclusion that we can take from these results is that at these conditions, the kinetics of crystallization precluded the dissolution of these two forms to higher concentrations. In other words, the dissolution rate was slower than the crystallization kinetics. This is commonly observed when trying to dissolve amorphous solids to measure the amorphous solubility,37 although there are exceptions, and the amorphous solubilities of a limited number of slowly crystallizing compounds have been successfully measured using this approach.6,38 Often, a key issue with dissolving amorphous solids is that the solid itself crystallizes rapidly upon contact with water.39 The transformation of the amorphous material to a crystalline solid will then impact the extent of supersaturation generated. A further interesting observation is that the induction times for crystallization are different following dissolution of the various solid state forms and do not correlate with the level of supersaturation observed. It might be expected that the forms that dissolve rapidly and to high supersaturations would crystallize the most rapidly, as has been suggested in the

literature.40 However, within our ability to determine the rate of supersaturation generation, this is clearly not the case. For example, the HBr and HCl salts appear to dissolve at a similar rate, but the HCl salt system commences crystallization at a sooner time point than the HBr salt system. Given that the same crystallization inhibitor is present in the solution in both cases, this result suggests that crystallization may be initiated from undissolved solid phase, rather than from the solution phase. Crystallization from undissolved solid phase could also explain why the adipic acid co-crystal has the shortest induction time. Moreover, it is likely that for the crystalline systems, the undissolved material has converted to a different phase, since the salt or co-crystal is not a stable form at these solution conditions and will have a tendency to undergo disproportionation to the free base.21,41 The HCl salt has been reported to have a lower pHmax than the HBr salt,42 which would lead to an increased driving force for conversion back to the free base. This is because this salt is further away from the pH value below which it is the stable solid, and this may explain the shorter induction time for crystallization of the HCl salt observed relative to the HBr salt. The lower pHmax value in turn stems from the higher solubility of HCl salt (approximately twice as soluble as the HBr salt).42 If the undissolved solid converts to the crystalline free base rapidly, any supersaturation that has been generated will be rapidly depleted. Therefore, the dissolution rate of the solid form appears to be a key parameter that dictates the extent of supersaturation achieved when there is a risk of conversion to a more stable solid form. To further explore the hypothesis that LLPS occurred during the dissolution of certain solid state forms when the concentration exceeded the amorphous solubility, a fluorescence method that selectively detects the drug-rich phase produced by LLPS was used. Pyrene was predissolved in the dissolution media, and its fluorescence emission properties were measured during the dissolution experiment. If the ratio of intensities between peaks 3 and 1 of pyrene fluorescence I



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emission increases above 0.586 (the average I3/I1 ratio measured at LLPS; Figure 3b) during dissolution it strongly suggests that LLPS has occurred. The fluorescence results obtained from dissolution of all solid forms of CRH1 (except the hemi-NAP salt) are shown in Figure 7. Results for the hemi-NAP salt are shown in the Supporting Information (Figures S2−S4); fluorescence by the counterion required a different experimental procedure for this salt. Fluorescence data confirmed that LLPS did not occur during dissolution of CRH1’s crystalline free base, glass, and co-crystal forms. As indicated by the dashed red line in Figure 7, the I3/I1 of pyrene never reached 0.586 ± 0.009 for these solutions, the lowest ratio at which LLPS occurs in pH 4.6 buffer (Figure 3b). On the other hand, the pyrene ratios measured during dissolution of the ASD, HBr, and HCl salts were greater than 0.586, suggesting that LLPS occurred following dissolution of these solid state forms. According to Figure 7, the highest pyrene ratios were measured during dissolution of the amorphous solid dispersion (maximum I3/I1 = 0.71). These results suggest that more of the drug-rich phase was generated for this system. As shown in Figure S4, the hemi-NAP salt also dissolved rapidly to undergo LLPS. After demonstrating that LLPS occurs following dissolution of the ASD, HBr, HCl, and hemi-NAP salts, as long as a crystallization inhibitor (HPMC) is present, the concentration of drug coexisting in the bulk aqueous solution with the drugrich phase was determined by ultracentrifugation to separate the drug-rich phase, followed by analysis of the supernatant by HPLC. This method eliminates any UV absorbance effects that the drug-rich phase/counterion might have on the absorbance spectra measured with the in situ dip probe. Figure 8 compares the maximum concentrations obtained from dissolution of the different solids using the in situ UV probe and centrifuge/ HPLC methods. These results confirm once more that the crystalline free base, glass, and co-crystal did not dissolve to concentrations near the estimated amorphous solubility. On the other hand, all salt forms and the ASD were able to generate supersaturated solutions around the estimated amorphous solubility. In general, the concentrations determined with the centrifuge/ HPLC methods were very similar to those obtained with the in situ UV probe measurements. The only exception was the hemi-NAP salt. True solution concentrations were much lower than those measured with the UV dip probe and were in the region of the estimated amorphous solubility. These data provide strong support for the conjecture that, regardless of the solid form used, salt, ASD, co-crystal, amorphous, etc. it is clear that the upper limit of supersaturation that can be attained is governed by the solubility of the amorphous form. Dissolution of a solid state form to yield concentrations above this value leads to liquid−liquid phase separation, and the formation of a two phase solution consisting of a drug-rich phase and a bulk aqueous solution where the concentration of the drug is the same as the amorphous solubility. The addition of a crystallization inhibitor is clearly essential to enable salts or co-crystals of CRH1 to yield supersaturated solutions, and may provide a formulation strategy to utilize a salt or co-crystal form in combination with a polymer as an alternative to an amorphous solid dispersion.

water-soluble drugs for the purpose of improving bioavailability. Herein, it was observed that the rapid crystallization of the supersaturated solutions that evolved following the dissolution of different solid state forms of a model, weakly basic compound, CRH1, limited the observed supersaturation. By adding a polymeric crystallization inhibitor, higher supersaturations that persisted for a longer period of time were achieved in all cases. However, the upper limit of supersaturation that could be achieved upon dissolution of any solid state form was dictated by the amorphous solubility. Dissolution above this concentration led to liquid−liquid phase separation and the formation of a drug-rich phase. This was seen for some salts and an amorphous solid dispersion of CRH1. The pure amorphous, free base and co-crystal forms did not reach the amorphous solubility under the experimental conditions employed and yielded peak supersaturations less than the theoretical maximum, even in the presence of a crystallization inhibitor. These observations illustrate that the observed supersaturation profile depends on the competition between the dissolution rate of the given solid state form intended to generate a supersaturated solution, and the subsequent crystallization rate to the more stable form. Changing the solid state form, as well as employing crystallization inhibitors, can be used to manipulate both of these rate processes leading to optimally supersaturated solutions, important for improving the bioavailability of poorly water-soluble compounds.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01341. Information about methods related to evaluation of the hemi-Nap salt (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States. Tel: (765) 496-6614. Fax: (765) 494-6545. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge research funding from Eli Lilly and Company through the Lilly Research Awards Program (LRAP).

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DEDICATION With this paper, we wish to honor the life and legacy of Margaret (Peggy) C. Etter, whose pioneering work on host− guest chemistry and materials design through hydrogen-bond directed co-crystallization and formulation of hydrogen bonding rules have inspired our own work on amorphous solids and structure-based solid-state form design.

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REFERENCES

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CONCLUSIONS Solids such as salts, co-crystals, or amorphous solid dispersions are of interest to generate supersaturated solutions of poorly J



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Supersaturation Potential of Salt, Co-Crystal, and Amorphous Forms of

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