Salt Disproportionation in the Solid State: Role of Solubility and

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Salt Disproportionation in the Solid-State: role of solubility and counter-ion volatility Naveen Kumar Thakral, Robert J Behme, Aktham Aburub, Jeffrey A Peterson, Timothy A. Woods, Benjamin A Diseroad, Raj Suryanarayanan, and Gregory A Stephenson Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00745 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 21, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Salt Disproportionation in the Solid-State: role of solubility and counter-ion volatility Naveen K. Thakral1,2†, Robert J. Behme1††, Aktham Aburub1, Jeffrey A. Peterson1, Timothy A. Woods1, Benjamin A Diseroad1, Raj Suryanarayanan2, Gregory A. Stephenson1* 1. Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States 2. Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States † Presently at Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States †† Presently at Eurofins Lancaster Laboratories PSS, 2425 New Holland Pike, Lancaster, PA 17601, United States *Corresponding Author ABSTRACT Disproportionation propensity of salts (HCl, HBr, hemi-napadisylate) and adipic acid co-crystal of corticotropin releasing hormone receptor-1 antagonist, was studied using model free kinetics. Using thermogravimetic weight loss profile or heat flow curves from differential scanning calorimetry, an activation energy plot for salts and co-crystal was generated based on model free kinetics. This activation energy of disproportionation provided qualitative information about the solid state salt stability. To ensure the stability throughout the shelf life, “prototype” formulations of salts and co-crystal in tablet form, were stored at 40 °C and several water vapor pressures. Disproportionation kinetics were studied in these prototype tablet formulations using two-dimensional X-ray diffractometry. Formulations containing the adipic acid co-crystal or hemi-napadisylate salt did not show disproportionation of API when stored at 40 °C/ 75% RH, for 300 days. On the other hand, formulations containing HCl or HBr salt disproportionated. Though isostructural, the disproportionation propensity of HBr and HCl salts were quite different. The HCl salt highlighted the important role that volatility of the counter-ion plays in the physical stability of the formulations. Solution state stability (i.e. in dissolution medium) of salts and co-crystal was also assessed and compared with solid state stability, by determining their solubility at different pH’s, and intrinsic dissolution rate.

KEYWORDS: salt, co-crystal, disproportionation, model free kinetics, volatilization, solubility, intrinsic dissolution, X-ray diffractometry.

1 ACS Paragon Plus Environment

counter-ion,


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 2 of 28

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION With the advent of high throughput screening (HTS) and combinatorial chemistry in sourcing the drug leads, empirical screening methods have become less important1. HTS methods are efficient in selection of new drug candidates, but often result in high molecular weight lipophilic molecules with low aqueous solubility2. An analysis of approved drugs by the FDA in the last thirty years indicated a trend of continuous rise in lipophilicity (log P), and decrease in aqueous solubility3. Following oral administration, absorption of these compounds is expected to be dissolution rate and/or solubility limited. These active pharmaceutical ingredients are usually weak electrolytes and their corresponding acidic or basic salts exhibit improved aqueous solubility, and hence potentially improved oral absorption4,5. Also, during the “lead optimization stage” of drug discovery, the drug candidate must have adequate aqueous solubility to achieve sufficiently high exposure to observe a toxic effect in animals to establish its margin of safety. This can be achieved by making salts of the weak electrolytes. However, due to processing conditions or reaction with formulation ingredients, salts disproportionate, that is dissociate and revert back to the less soluble, unionized form6. This reaction can be brought about when the pH of the aqueous microenvironment is more basic than the pHmax of the salt (of the weakly basic drug). Trace levels of basic impurities in the excipients, for example in magnesium stearate, can cause such a pH shift. Salt disproportionation may adversely affect both bioavailability and chemical stability of an active pharmaceutical ingredient (API)7,8. Early formulations for “first-in-human” use are quite simple - commonly drug in capsule. While selecting the form of the API (this term encompasses both the physical and chemical form) for commercial development, physical and chemical stability typically over a shelf life of two years must be ensured9. Disproportionation issues detected in late stages of development can dramatically increase the cost of development and may result in significant delays. During lead optimization, when only a limited amount of material is available, simple slurry of the candidate salt form in water, measurement of the solution pH and identification of the form in equilibrium with the water can be useful. Similarly, pH solubility profile of a candidate salt form, where the residual crystalline form in equilibrium with the water is identified, provides an early indication of the potential for salt disproportionation. We further advocate the use of “prototype” formulations to provide more meaningful information, as disproportionation in our experience is observed within the first two weeks and almost certainly within one month of equilibration9. Thermal analysis using model free kinetics10-15 may provide qualitative information about the solid state salt stability, and can be used during formulation development to ensure the stability of salt throughout the shelf life of the formulation. Similarly, our current simplistic model based primarily upon solution state equilibria within the water contained in the formulation and the solubility of the constituents of the formulation has been useful in predicting disproportionation in the solid formulations16. There are limitations in our understanding of the chemistry of the system that need to be incorporated into the model if it is to be relied upon. The appropriate modifications to the model will be the subject of a future publication. In this 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

investigation, we will specifically address the role of counter-ion volatility on salt disproportionation reaction. In the present study, a poorly water soluble, weakly basic compound (corticotropin releasing hormone receptor-1 antagonist; CRH-1 (Fig. 1), three of its salt forms (HCl, HBr, heminapadisylate) and a co-crystal (adipic acid co-crystal) were used to study disproportionation/dissociation occurring in the solid state.

Figure 1. Corticotropin releasing hormone receptor-1 antagonist, 4-(4-chloro-5-(2,6-dimethyl-8(pentan-3-yl)imidazo[1,2-b]pyridazin-3-yl)thiazol-2-yl)morpholine; (C20H26ClN5OS), molecular weight 419.98. One particularly interesting aspect of the study is that two of the salts, the hydrobromide and the hydrochloride, are isostructural. This affords the rather unique opportunity to compare the disproportionation tendency of two salts from structures of nearly identical packing and highlight the important role that volatility of the counter substance plays in the physical stability in formulations. The objectives of the present research were: i) to assess/predict the disproportionation in dry neat salts by model free kinetics using thermogravimetric/differential scanning calorimetry data, ii) study the disproportionation kinetics in the solid state in “prototype” formulations containing different salts and the co-crystal of CRH-1, at elevated temperature and humidity conditions, iii) generate single crystal data of HCl and HBr salts of CRH-1 to verify their isostructural nature, and iv) study the effect of counter-ion volatility on disproportionation kinetics. EXPERIMENTAL Materials CRH-1 free base was synthesized by Eli Lilly and Company’s Research Laboratories and provided for the study. The HBr salt was prepared by suspending 545 mg of free base in 3 mL of isopropyl alcohol and stirred at 60 °C. To this mixture, 150 µL of 48% HBr acid (pKa -9) was added and it clarified quickly before solid precipitation. The precipitation continued with the addition of excess diethyl ether. The mixture was slurried and evaporated at ambient temperature. 4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The hemi-napadisylate salt was prepared by suspending 528.8 mg of freebase in 2 mL of acetone to which 340 µL of 3.8 M naphthalene-1,5-disulfonic acid (pKa -3) in methanol was added. Within 30 seconds, there was precipitation from solution and 2 mL of methanol was added with further precipitation noted. The liquid was decanted and solids were dried under nitrogen. The solids were suspended in 10 mL of ethanol and slurried overnight at ambient temperature before vacuum filtration and drying. The HCl salt was prepared by addition of 47 µL of HCl acid (pKa -7) to an acetone solution of the free base (420 mg in 2 mL) at a low temperature (0-10 ⁰C). The solution was then warmed to 20 ⁰C and filtered. The adipic acid co-crystal was prepared by suspending 5.8 g of free base in 11.5 mL of ethyl acetate. To this slurry, 2.02 g of adipic acid in 1:4 acetone:methanol (v/v) solution was added. The clear solution was then filtered into a vial containing seeds of the adipic acid co-crystal. While some seeds persisted, the bulk dissolved so the liquor was concentrated at 55 °C by ~50%. The clarified solution was filtered again and seeded again and cooled to RT overnight. Approximately 1.5 g of adipic acid co-crystal formed along the bottom of the vial and was isolated and dried under vacuum at 65 °C. Microcrystalline cellulose (MCC; Avicel PH-102; FMC BioPolymer), hydroxypropyl methyl cellulose (HPMC; Methocel K4M; Dow Chemical Company), polyvinylpyrollidone (PVP; Kollidon 30; BASF), and magnesium stearate (Fisher Scientific Company; lot # 740042), were used as received. Thermogravimetric analysis with (TGA/sDSC) or stand-alone DSC

simultaneous

differential

scanning

calorimetry

Samples were heated usually in open 40 µL aluminum pans without lids (Mettler Toledo TGA/SDTA851e) except in one case where a lid was hermetically sealed. TGA results were acquired at a variety of heating rates from as low as 2 ˚C/minute to as high as 20 ˚C/minute. For each TGA weight loss profile at a desired heating rate, an analysis of an empty pan under the same conditions was subtracted from the sample TGA thermal curve. Performance qualification of the TGA balance was conducted with standard weights. The experimental weight loss observed using sodium tartrate dihydrate and barium chloride dihydrate as standards were in excellent agreement with the theoretical weight loss. The TGA temperature axis was calibrated using the melting points of indium, tin and zinc melting point standards accessed by simultaneous DSC analysis. Samples were heated at 2, 5, 10, 20 and 100 ˚C/minute. Performance qualification of the heat flow axis and the temperature axis utilized indium, tin and zinc at heating rates of 2, 5, 10, 20, 50, 100 and 200 ˚C/minute. The onset melting point and apparent heat of fusion data from multiple melting point standards at all heating rates were entered into the FlexCal software. The elaborate calibration procedure ensured that regardless of heating rate over wide temperature 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ranges any instrumental thermal lag was compensated, and differences detected for materials could be attributed to kinetics. Model free kinetics (MFK) and software Model free kinetics, sometimes called iso-conversion kinetics, was applied to the conversion curves of the TGA weight loss profiles (or DSC profiles) obtained at different rates. The MFK module calculates apparent activation energy for a family of curves obtained at different heating rates at each iso-conversion point. The MFK application is a module within the Mettler Toledo software suite (STARe software; version 13.00a) that assumes the kinetic process is the same across the family of curves at each iso-conversion point. However, it does not assume a single process across the entire conversion profile. Weight loss profiles at different heating rates, were plotted together. In each thermal curve, the weight loss profiles were truncated to focus on the specific weight loss region of interest. The software enabled normalization of the data by converting the weight loss to percent conversion profiles. Model free kinetics software calculated activation energies at each iso-conversion point and plotted the iso-conversion activation energy values versus percent converted. Model free kinetics in the advanced MFK software option requires at least three curves based on different temperature programs. The curves can be isothermal or dynamic or include a combination of isothermal and dynamic segments. The curves are evaluated by numerically calculating the minimum of the following integral.

1, () 2 , ()

I (Eα) = Where,

1, () =

#

#$∆

2, () =

#

#$∆

."(#) &

."(#) &


Page 6 of 28

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


where, ‘T’ is temperature [K], ‘t’ is time [sec], ‘α’ is the fraction converted, ‘J1’ and ‘J2’ are sub-integrals, ‘Eα’ is activation energy as a function of conversion, and ‘R’ is the universal gas constant. Crystal structure determination- HBr salt A clear colorless prismatic-like specimen of C20H27BrClN5OS, approximate dimensions 0.060 mm x 0.070 mm x 0.080 mm, was used for the X-ray crystallographic analysis. The dataset was collected at 296K using a copper source and wavelength of 1.5418 Å. The total exposure time was 2.82 hours. A total of 3383 frames were collected and integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 14134 reflections to a maximum θ value of 66.71° (0.84 Å resolution), of which 4023 were independent (average redundancy 3.513, completeness = 95.8%, Rint = 6.28%, Rsig = 5.98%) and 3238 (80.49%) were greater than 2σ(F2). The final cell constants of a = 13.3301(5) Å, b = 16.8060(6) Å, c = 11.6385(4) Å, β = 114.292(2)°, volume = 2376.47(15) Å3, are based upon the refinement of the XYZ-centroids of 6111 reflections above 20 σ(I) with 7.275° < 2θ < 130.3°. Data were corrected for absorption effects using the multi-scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.696. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.7210 and 0.7792. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P2(1)c, with Z = 4 for the formula unit, C20H27BrClN5OS. The final anisotropic fullmatrix least-squares refinement on F2 with 271 variables converged at R1 = 7.08%, for the observed data and wR2 = 22.16% for all data. The goodness-of-fit was 1.025. The largest peak in the final difference electron density synthesis was 0.687 e-/Å3 and the largest hole was -0.495 e/Å3 with an RMS deviation of 0.088 e-/Å3. On the basis of the final model, the calculated density was 1.400 g/cm3 and F(000), 1032 e-. The pentyl group is disordered within the crystal structure, giving rise to high peak in the final difference map and relatively high wR2. The remainder of the molecule and the counter ion refined well. The structure was sufficient for reasonably calculation of its theoretical X-ray powder diffraction pattern, calculation of its true density, and to confirm the isostructural nature of the HCl and HBr salts. The structure parameters at room temperature are reported. Crystal structure determination - HCl salt A clear colorless prismatic-like specimen of C20H27Cl2N5OS, approximate dimensions 0.020 mm x 0.070 mm x 0.080 mm, was used for the X-ray crystallographic analysis. The dataset was collected at 296 K using a copper source and wavelength of 1.5418 Å. The X-ray intensity data were measured. A total of 2832 frames were collected. The total exposure time was 2.36 hours. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 10341 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

reflections to a maximum θ angle of 64.22° (0.86 Å resolution), of which 3704 were independent (average redundancy 2.792, completeness = 95.9%, Rint = 6.56%, Rsig = 8.39%) and 2525 (68.17%) were greater than 2σ(F2). The final cell constants of a = 13.1687(4) Å, b = 16.6379(5) Å, c = 11.5914(3) Å, β = 114.243(2)°, volume = 2315.70(12) Å3, are based upon the refinement of the XYZ-centroids of 2321 reflections above 20 σ(I) with 9.914° < 2θ < 120.4°. Data were corrected for absorption effects using the multi-scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.653. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.7777 and 0.9490. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P21/c, with Z = 4 for the formula unit, C20H27Cl2N5OS. The final anisotropic fullmatrix least-squares refinement on F2 with 271 variables converged at R1 = 9.18%, for the observed data and wR2 = 29.72% for all data. The goodness-of-fit was 1.087. The largest peak in the final difference electron density synthesis was 0.959 e-/Å3 and the largest hole was -0.482 e-/Å3 with an RMS deviation of 0.159 e-/Å3. On the basis of the final model, the calculated density was 1.309 g/cm3 and F(000), 960 e-. The pentyl group is disordered within the crystal structure, giving rise to high peak in the final difference map and relatively high wR2. There was no improvement by collecting data for a longer time or at a lower temperature. The rest of the molecule and counter ion refined well and the site of protonation was found in the difference map. The structure was not deemed of sufficient quality for deposition in the Cambridge Structural Database. The structure was sufficient for reasonably calculation of its theoretical Xray powder diffraction pattern, calculation of its true density, and to confirm the isostructural nature of the HCl and HBr salts. The room temperature structure’s parameters were reported. Synchrotron X-ray diffractometry (SXRD)

The powder samples, ∼20 mg, were filled in aluminum pans (used for differential scanning calorimetry, TA Instruments), crimped hermetically at room temperature and placed in a specially fabricated sample holder. Experiments were performed in the transmission mode in the 17-BM-B beamline at Argonne National Laboratory (Argonne, IL, USA). A monochromatic Xray beam [wavelength 0.72808 Å; beam size 250 µm (horizontal) × 160 µm (vertical)] and a twodimensional area detector (XRD-1621, PerkinElmer) were used. A triple-bounce channel-cut Si single crystal monochromator with [111] faces polished was used which limited the line broadening to its theoretical low limit, i.e., the Darwin width. The flux of the incident X-ray was 8 × 1011 photons/s at 17 keV. Calibration was performed using an Al2O3 standard (SRM 674a, NIST). Using a stepper motor, the sample was oscillated (±1 mm from the center along the horizontal axis) during data collection. Each sample was scanned 30 times, with an exposure time of 1 s for each scan, and the results were averaged. The raw images were integrated to yield one dimensional d-spacing (Å) or 2θ (degree) scans using the FIT2D software developed by A. P. Hammersley of the European Synchrotron Radiation Facility17,18. Commercially available software (JADE 2010, Material Data, Inc.) was used for determining the integrated peak intensities. 8 ACS Paragon Plus Environment

Page 8 of 28

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation of tablets APIs’ and excipients were passed through sieve # 170 (D90 = 90 µm). API (40% w/w) and placebo (60% w/w) were mixed manually. The placebo composition was: MCC (70.0% w/w), HPMC (23.5% w/w), PVP (5.0% w/w), and magnesium stearate (1.5% w/w). The blend (100 mg) was filled in circular stainless steel die (8 mm). Tablets were prepared using a universal material testing machine (Zwick/Roell, Zwick GmbH & Co., KG, Ulm, Germany), equipped with 8 mm diameter flat-faced punches. During compression, the lower punch was stationary while the upper punch moved at a constant speed of 10 mm/min. Tablets were compressed under ambient conditions (25 °C; 45% RH) to 100 MPa. The upper punch displacement was measured using a linear variable differential transformer (LVDT) position gauge, attached to the compression platens. The upper punch was allowed to retract, as soon as the desired compression force was achieved. For ejection of the compressed tablets from the die, the lower punch was removed and a force was applied to the upper punch using a hydraulic press (Carver Model C Laboratory press, Menomonee Falls, WI, USA). Three different tablets of each formulation composition were prepared. Solid state stressed stability testing Three tablets of each formulation were kept in open pans in glass chambers maintained at different relative humidity (RH) conditions, using saturated salt solutions (lithium chloride, 11% RH; potassium acetate, 22% RH; magnesium chloride hexahydrate, 32% RH; sodium chloride, 75% RH) at 40 °C. Tablets containing the HCl salt were stored at 11, 32, and 75% RH conditions, tablets containing HBr salt were stored at 22, and 75% RH conditions, while tablets containing hemi-napadisylate salts and adipic acid co-crystal were stored only at 75% RH. Tablets were withdrawn at specific time intervals and were analyzed using two dimensional Xray diffractometry. After analyses, the tablets were placed back in the chamber. The top surface of each tablet was marked so as to analyze the exposed surface at each time point. Two dimensional X-ray diffractometry (2D-XRD) Intact tablets were exposed, at room temperature, to CoKα radiation (1.78899 Å; 35 kV X 40 mA) in a two dimensional X-ray diffractometer (D8 Discover 2D, Bruker with a 140 mm diameter window VÅNTEC-500 detector). XRD patterns were collected for 100 seconds, using a 0.8 mm collimator set at 4° angle of incidence and an area detector (angular range 36°) set at an angle of diffraction at 8° 2θ. The irradiated area can be described by an ellipse with a major axis of 13.91 mm and minor axis of 0.97 mm. To keep the X-ray irradiated area of tablet constant, five random points on centerline of the upper surface of each tablet were analyzed at each time point (supporting information; Fig. S1). Since no internal standard was used, it was necessary to detect and correct for any short and long term instrumental drift. On each day before and at the end of the experiment, the instrument was calibrated using Corundum (SRM 1976a, NIST). By setting the Ω at 20º, and 2θ at 40º (scan time 50 seconds), the integrated 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

intensity of the 41.02º 2θ (2.55 Å) peak was determined. The coefficient of variation of such measurements was found to be 0.92%. There was no measurable short term instrumental drift during the time of analysis of each sample. Quantification of free base in intact tablets: generation of standard curves Tablets containing known compositions of free base and salt (0-40% w/w; total concentration 40% w/w), and placebo (60% w/w) were prepared (11 compositions; 3 tablets of each composition). Two dimensional XRD was used to collect patterns from 5 locations (along the center line of top surface) in each of the 3 tablets. The intensity (integrated) of the 7.6° 2θ peak of the free base was determined and averaged for the 15 determinations and plotted as a function of free base concentration. This experiment was performed separately for HCl and HBr salts. The relationship between integrated peak intensity (y) and free base fraction (x; % w/w) was expressed as y = 2.1024x + 0.9086, in case of HBr salt and y = 2.0834x + 5.1613, in case of HCl salt. These standard curves were used to determine the free base concentrations, formed due to disproportionation of HBr or HCl salt in tablets. Determination of microenvironmental pH by slurry method To determine the microenvironmental pH, 500 mg of HBr or HCl salt was suspended in 5 ml distilled water. On similar lines, slurry of formulations of both the salts were prepared by suspending 500 mg of each formulation in 5 ml of distilled water. The pH of the slurry was measured (pH meter, Mettler Toledo), in an instrument calibrated with buffers of pH 2, 4, 7 and 10. After measuring the pH at time ‘0’, each slurry vial was stored in an oven maintained at 40 °C. The vials were withdrawn at specific time intervals and the pH was measured. pKa determination Neat compound was weighed into sample vial and titrated in triplicate from pH 2 to 10 using Sirius Automated Titrator Instrument (GLpKa with DPAS) in the presence of co-solvent (methanol). Approximate wt% of methanol for the three titrations was 27, 36, and 47. YasudaShedlovsky extrapolation was used to extrapolate the pKa values obtained using different concentrations of co-solvent to 0% co-solvent19. Solubility determination Equilibrium solubility of CRH-1 salts and co-crystals was determined at different pH values. For solubility determination at lower pH values (≤ 3), different molar concentrations of corresponding acids forming counter-ions were used. One exception to this was in case of adipic acid co-crystal, where the solubility study below the pHmax was determined in HCl solution as it was not possible to achieve low pH using adipic acid. Solubility was also determined in phosphate buffer (pH 4, 6, and 8), and deionized water. Excess solids were weighed in HPLC vials, and solvent was added. Samples were rotated using laboratory shaker (Barnstead 10 ACS Paragon Plus Environment

Page 11 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thermolyne Labquake Rotisserie Shaker) for 24 hours at RT. After 24 hours, samples were filtered using Ultrafree-MC centrifugal filter devices (0.22 µm) using centrifuge (5415C Eppendorf centrifuge) for 5 min. The pH of the filtered samples was determined using a calibrated pH meter (Orion 7110 BH), dilutions were made using 50/50 (acetonitrile/water) + 0.1% trifluoroacetic acid, and concentrations were determined using HPLC.

Intrinsic dissolution rate (IDR) To assess the effect of salt and co-crystal formation on dissolution rate, CRH-1, its different salts and co-crystal were subjected to IDR analysis. Accurately weighed quantity of free base, salts or co-crystal (n=3, each) was transferred into the die cavity of a Wood’s apparatus. The powder was compressed in a hydraulic press (Carver Press) at 2,000 psi for 1 min. The pellets thus formed were mounted on the shafts of a dissolution apparatus (Varian, VK7000). The dissolution medium used was 500 mL of 0.01 N HCl (37 °C; vacuum degassed before using), and the experiment was conducted for 4 h at 100 rpm. Data was collected every minute up to 4 hours using inline UV probe (head size 5 mm; wavelength 285 nm). RESULTS AND DISCUSSION Solution stability of the solid state forms of CRH-1 The solubility of a weak base is influenced by its solubility product (Ksp), pKa(s), intrinsic solubility (free base solubility) and pH. To calculate the pH-solubility profile of a weak base, only the solubility product, ionization constant(s), and intrinsic solubility are needed to be known. Region 1 of the pH-solubility profile of a diprotic weak base is where solubility of the free base (intrinsic solubility) is limiting, that is when pH > pHmax and can be described mathematically as shown below (details in supporting information). Region 1: Solubility description when pH > pHmax for a diprotic weak base:

( = )*+,-./. 0 + )*+,- / 0 + )*+,0 (1)

( = )*+,0 . 2 6

5

34

78 675

+ 6 + 19 34

75

(2)

As [Base] = S0 ( = (: . ;10(=678 /=675 $.=3) + 10(=675 $=3) + 1> 11 ACS Paragon Plus Environment

(3)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 28

where S is solubility, S0 is intrinsic solubility, Ka1 and Ka2 are the acid dissociation constants of the diprotic weak base. The pH solubility profile is identical, irrespective of the salt (HCl, HBr, hemi-napadisylate) or co-crystal (adipic acid), when pH > pHmax (Fig. 2). The data was fitted (Excel Solver function, MS Office 2003) using equation 3 and pKa1, pKa2 and intrinsic solubility were estimated to be 0.6, 4.8 and 0.0016 mg/mL; respectively. The pKa values are consistent with those determined using potentiometric titration and in silico (0.4 and 4.8 for pKa1 and pKa2; respectively). Region 2 of the pH-solubility profile of a diprotic weak base is where solubility of the protonated form is limiting, that is when pH < pHmax and can be described mathematically for the HCl and HBr salts as shown below.

Region 2: Solubility description when pH < pHmax for a diprotic weak base: ?@= =

)*+,-/ 0. )ABCDEBD$ 0

?@= = ( . F

5

678 3 4

3 4 /678 3 4 /678 675

G . )ABCDEBD$ 0

5 3 4 /678 3 4 /678 675 678 3 4

( = ?@= . 2

(4)

(5)

9 . )HIJ #KLI M0

(6)

)ABCDEBD$ 0 = ( + )-A0 (7)

( = ?@= . F

:M5OP / :M(OQ78 4OP) / :M(OQ78 4OQ75 )

:M(OQ78 4OP)

G . )HIJ #KLI M0

(8)

Here [HC] is the concentration of acid (i.e. HCl, HBr) in which the solubility experiment was conducted. Since S was determined experimentally, and the concentration of acid in which the solubility experiment was conducted is known, the concentration of counter-ion as a function of pH was plotted (supporting information, Fig. S2) and an equation that describes the concentration of counter-ion as a function of pH was developed. This equation (concentration of counter-ion as a function of pH) was then used to replace [Counterion-] in equation (8).


Page 13 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Since the napadisylate salt is a hemi-salt, two drug molecules are associated with one napadisylate molecule. This is mathematically described below: ?@= = )*+,-/ 0. . )ABCDEBD$. 0

?@= = ( . . F

3

45

678 3 4

/678 3 4 /678 675

( = F)HIJ #KLI M5 0G 6RO

:.S

.

(9)

G . )ABCDEBD$. 0

5 3 4 /678 3 4 /678 675 678 3 4

. 2

(10)

9

(11)

)ABCDEBD$ 0 = (0.5). ( + )-2A0 (12) ( = F)HIJ #KLI M5 0G 6RO

:.S

. F

:M5OP / :M(OQ78 4OP) / :M(OQ78 4OQ75 )

:M(OQ78 4OP)

G

(13)

Below pHmax, the pH solubility profiles of the HCl and HBr salts were generated (Excel Solver function, MS Office 2003) using Equation 8. The pH solubility profile of the hemi-napadisylate salt was generated using Equation 13. A summary of the solubility product, pHmax and acid dissociation constants can be seen in Table 1. The pH solubility profile of the adipic acid co-crystal overlaps with that of the HCl (Fig. 2). This is expected above pHmax since the observed solubility is intrinsic solubility limited. Below pHmax, the adipic acid co-crystal solubility study was conducted in HCl solutions since it was not possible to achieve low pH using adipic acid (adipic acid has low solubility). Hence, the cocrystal was converted to the HCl salt. Table 1. Solubility Product, pHmax and pKa(s) for the HCl, HBr and hemi-napadisylate Salts. Ksp and pKa(s) values were estimated from fitting pH-solubility data using equations 8 (HCl and HBr salts) and 13 (hemi-napadisylate salt). Salt HCl HBr Hemi-napadisylate

pHmax 1.0 1.3 2.5

Ksp 2.8 X 10

-3

7.7 X 10

-4

-10

8.0 X 10

pKa1

pKa2

2

0.4

4.6

2

0.6

4.8

3

2.1

4.8

[M] [M]

[M]



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

Figure 2. pH solubility profiles (HCl, HBr, hemi-napadisylate salts, and adipic acid co-crystal). The curves were generated from the experimental data.

The intrinsic dissolution rates (IDR) of CRH-1 free base, its salts and co-crystal were determined. This provided a measure of their propensity for disproportionation during dissolution. The IDR (mcg/min.cm2; 1-10 min.) of CRH-1 was found to be 40.8 (±1.63); for HCl salt 202.4 (±15.31); for HBr salt 232.8 (±13.46); for hemi-napadisyalte salt 63.9 (±0.48), and for adipic acid co-crystal 164.3 (±7.71). Though the solubility of the HCl salt was more than that of the HBr salt, the lower IDR of the HCl salt is likely due to common ion effect (0.01 N HCl dissolution medium).


Page 15 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The IDR of the HCl salt could be described by two linear profiles (Fig. 3a). From 0 to 10 min, the drug dissolution rate was ~101 mcg/min., but during the time period of 20-240 minutes, its dissolution rate dropped to ~ 20 mcg/min. The dissolution rate during 20-240 min. was similar to that of the free base which was ~16 mcg/min. This strongly suggested that HCl salt disproportionated to the free base after ~ 10-20 min. in the dissolution medium.

a b

Figure 3. a) IDR of CRH-1-HCl and CRH-1 free base. Disproportionation was observed in HCl salt after ~ 10-20 minutes during IDR analysis. b) comparison of IDR of CRH-1 salts, and adipic acid co-crystal up to 90 minutes showing disproportionation. A similar trend was observed with the HBr salt (Fig. 3b). In Fig. 3b, IDR data is provided only up to 90 minutes to enable the visualization of the disproportionation/dissociation. Complete IDR profiles are presented in the supplementary information (supporting information; Fig. S3). The IDR profile of hemi-napadisyalte salt does not indicate disproportionation in dissolution medium, as seen in solid state stability studies, whereas the HCl and HBr salt forms and the adipic acid co-crystal forms do disproportionate/dissociate shortly after exposure to the dissolution medium. In case of HCl and HBr salts, there is a substantial difference in their solubilities, as compared to free base. The pHmax of the HCl and HBr salts (1.0 and 1.3 respectively) is lower than the pH of the dissolution medium (2.0), hence the free base form is the thermodynamically favored form. On the other hand, pHmax of hemi-napadisylate salt is 2.5, which is higher than that of the dissolution medium. Consequently, the hemi-napadisylate salt form is thermodynamically favored to exist in its ionized salt form in the dissolution medium. Interestingly, the adipic acid co-crystal has the coformer present in an unionized form, as such it does not have a true pHmax per se. Under these conditions, adipic acid is more soluble than the free base and results in its transformation to the less soluble free base form.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

Studies of CRH-1 Physical Stability in the Solid State X-ray Diffraction The two dimensional SXRD patterns and corresponding one dimensional patterns of CRH-1 free base, HCl, HBr, hemi-napadisylate salts, and adipic acid co-crystal are depicted in Fig. S4(A) and S4(B), respectively (supporting information). The calculated XRD patterns of HCl and HBr salts are presented in Fig. S4(C). The HCl and the HBr salts of CRH-1 are isostructural, wherein the unit cell dimensions and the molecular arrangement within the unit cell are virtually identical, with the exception that in one structure the counter-ion is a chloride ion and in the other it is a bromide ion (Fig. 4). Due to the lack of hydrogen bond donating groups in the molecule itself, the only formal hydrogen bond found is between the counter-ion and the protonated imidazo[1,2-b]pyridazin-3-yl nitrogen atom. In the HCl salt, the Cl to N(19) distance was 2.970 Å, having a hydrogen bond motif designation of D1,1(2)a, whereas for the larger bromide ion distance to the adjacent nitrogen atom was 3.148 Å [Br(1)….N(19)]. This is a discrete hydrogen-bonding pattern, where there is a single acceptor and a single donor atom, with each of the two atoms participating in the hydrogen-bonding motif20. Also this is considered to be the simplest form of hydrogen bonding that consists merely of a protonated drug molecule associated with anion, due to the scarcity of hydrogen bond donating groups. Isostructural pharmaceutical salts are known to exhibit substantial differences in physicochemical properties21-23. It is therefore of interest to see if isostructural salts exhibit similar disproportionation behavior.

Figure 4. Microscopic image (20x; Olympus BX51), and unit cell diagram of the HBr salt (a & c) and HCl salt (b & d) of CRH-1. The unit cells of the two salt forms viewed along their lattice c-axis demonstrates the similarity (isostructural). 16 ACS Paragon Plus Environment

Page 17 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thermal Analysis of Neat API In the early stages of development, thermal analysis is particularly useful in the characterization of crystalline salts. Thermally stable crystalline salts melt congruently with no change in composition across the melting temperature range and its molten liquid retains the counter-ion. On cooling below the melting point, the salt recrystallizes. In other words, the salt is thermally stable both in its crystalline phase and as a molten liquid. This process is reversible and repeatable assuming crystallization kinetics are favorable and crystallization occurs at a reasonable rate. The fragility of the super-cooled melt and its proclivity to crystallize has been the subject of much interest in the literature. Congruently melting crystalline salts melt consistently at a single temperature with a constant heat of fusion. This type of behavior is well documented in the pharmaceutical literature, which is often assumed to be what occurs during conventional thermal analysis. We observed the congruent melting of CRH-1 free base at different heating rates (supporting information; Fig. S5). On the other hand, some salts may be stable in solid state, but quite unstable when melted. If the counter-ion is volatile, it will begin to vaporize with the onset of melting and continue until the crystalline solid has melted completely. While this unstable liquid phase may not negatively impact the measurement of the melting point, it seriously affects the heat of fusion measurement because it is the sum of the heat of fusion and the heat of disproportionation. In addition, there will be contribution from the heat of vaporization of the counter-ion. So in this case, the crystalline phase is incongruently melting at, during, and above its melting point. If the liquid phase cools and spontaneously crystallizes, the crystals are the free form of the drug if the counter-ion volatilized or may be a solution of molten drug and counter-ion if the counter-ion did not completely volatilize. Another possible scenario is disproportionation as was observed in the HCl salt. Spontaneous disproportionation begins with complete loss of the volatile HCl gas, resulting in collapse of the crystalline phase, forming a molten liquid of the free base. For such a salt, incongruent melting (disproportionation) and liquefaction occurs below the theoretical congruent melting point of the crystalline material. This can be practically determined as explained in Fig. 5. The upper panel of the figure shows TGA weight loss profiles while the lower panel contains the simultaneously collected DSC heat flow signals. In an open pan, spontaneous disproportionation results with a weight loss equivalent to the mono-hydrochloride (8%) beginning at about 150˚ and continuing up to 240 ˚C. The simultaneously collected DSC curve shows an endothermic event in this same temperature range. When the HCl salt, in a hermetically sealed pan was analyzed, volatile loss (endothermic event) was inhibited, that is, these thermal events did not occur until above 250˚C. The initial volatile loss of HCl fills the head space of the hermetic pan and prevents further reaction. As the temperature increases, eventually the pressure limit is exceeded with sudden loss of about 8% weight, attributed to complete loss of HCl, over a narrow temperature range. This experiment suggests the back pressure in the hermetic pan inhibits disproportionation as has occurred in the open pan and perhaps allows the crystalline HCl salt to approach its “true” 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

congruent melting point. When the HCl salt is subjected to unencumbered heating in an open pan, spontaneous disproportionation ensues and incongruent melting occurs at about 100˚C or well below the inhibited melting point in the hermetic pan. Volatile loss attributed to the disproportionation of HCl gas in Fig. 5 was confirmed by simultaneous TGA/Mass Spectrometry (supporting information, Fig. S6).

Figure 5. TGA/DSC demonstrating spontaneous volatile loss of the HCl counterion. Upper half of the figure are the TGA curves while the lower half of the figure shows simultaneously collected DSC curves. The red curves are results for material heated in an open pan while black curves are for material in a hermetically sealed pan.

DSC heat flow curves of the HBr salt, heated at different rates show differences in onset temperature or melting point depression at slower heating rates (Fig. 6A). This is due to incongruent melting (disproportionation) of the HBr salt. The HBr and HCl salts (Fig. 6B) share this common feature.


Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

Figure 6. DSC heat flow curves of (A) CRH-1 HBr salt, and (B) CRH-1 HCl salt, heated in open pans at five different heating rates. Model free kinetics (MFK) was applied to the conversion curves of the TGA weight loss profiles of HCl and HBr salts. An initial evaluation using MFK is a rather small investment in time to gain some important empirical information. The MFK module calculates apparent activation energy for a family of curves at each iso-conversion point. MFK is especially useful in the evaluation of solid state reactions in materials that may undergo more than one type of process or when the mechanism of the reaction changes throughout the heating range. Materials can be analyzed more generally without an in depth knowledge of the underlying processes. For example, it would be plausible to hypothesize that disproportionation and volatile loss of either HCl or HBr from the parent molecule would be more facile from the surfaces of the drug particles and that the rate of loss might change as diffusion of the gases from inside particles lowers the rate. It might also be expected that the rate of volatile loss would be different initially from crystalline particles than from a collapsed liquid that could form throughout the complete process. In the bottom panel of Fig. 7, the activation energy is plotted as a function of the extent of conversion. There was a gradual increase in the apparent activation energy through the first 25% disproportionation of the HCl salt and then it leveled out. Between 10 and 90% conversion, the activation energy was 122 ± 23 kJ/mol. The weight loss profiles for HBr salt occurred at higher temperatures indicating that it was more thermally stable than HCl salt. The activation energy for HBr was 210 ± 10 kJ/mol roughly twice that for the HCl salt.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

Figure 7. TGA weight loss profile (top panel; acquired at several heating rates), was used to create conversion curve (mid panel), showing % converted as a function of time. Activation energy plots (bottom panel), created using conversion curve, reveal that the activation energy for the HBr salt disproportionation is about twice that of the HCl salt. MFK of the HCl salt by TGA weight loss profiles was corroborated using the simultaneous DSC heat flow curves. The DSC profiles produced an activation energy plot similar to that produced by the TGA data (supporting information; Fig. S7). It is both interesting and important that similar kinetic information was obtained for a volatile counter-ion to provide some confidence that MFK disproportionation kinetics could be obtained by DSC even for a non-volatile counterion. TGA/sDSC of hemi-napadisylate salt showed that the melting endotherm and the weight loss occurred approximately over the same temperature range. This was independent of the heating rate (supporting information; Fig. S8). More importantly, the temperature at which the weight loss was initiated was lower than the temperature of the melting endotherm onset. These results strongly suggest that decomposition is initiated before the onset of melting. TGA weight loss profiles for the hemi-napadisylate material occurred at progressively lower temperatures as heating rates decreased. MFK applied to these time and temperature dependent weight loss profiles for hemi-napadisylate salt revealed the conversion rate was not constant across the entire range. It was higher at the outset and through about 50% of the weight loss and then decreased. The activation energy for hemi-napadisylate was 360 ± 44 kJ/mol, much higher than for HCl or the HBr salt (supporting information; Fig. S9, S10). TGA/sDSC of the adipic acid co-crystal (supporting information; Fig. S11) indicates that endothermic melting is complete before the sample decomposes (weight loss in TGA). 20 ACS Paragon Plus Environment

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Therefore, there is no effect of heating rate on its melting transition. Based on thermal analyses, the adipic acid co-crystal, though with the lowest melting point, was the most thermally stable physical form.

Counter-ion volatility The Litmus Paper Test To confirm the volatility of HCl counter-ion, as observed during DSC, we conducted a simple litmus paper experiment. A saturated solution of sodium chloride was filled in a small glass vial and this open vial was placed in a bigger glass vial, containing 200 mg of powdered tablet of HCl or HBr salt formulation (at RT). The open small glass vial containing saturated salt solution was used to create an environment of ~ 75% RH inside the capped large glass vial. A blue litmus paper was suspended in the glass vial, capped and kept at 80 °C. After 24 hours of storage, the blue litmus paper had turned red in the vial containing the HCl salt, due to volatilization of HCl gas. On the other hand, in the vial containing the HBr salt, no color change of the litmus paper was observed.

Figure 8: (a) no change in litmus paper color in vial containing HBr salt, and (b) blue litmus paper changed to red in the vial containing the HCl salt.

Physical Stability in “Prototype” Formulations by Quantitative XRPD The tablets containing prototype formulations were subjected to accelarated stability testing, in an open pan. As predicted by thermal analysis, formulation containing the adipic acid co-crystal did not show any evidence of free base formation, even after storage for 300 days at 40 °C/75% RH. This was a bit surprising, given the solution state data, but consistent with thermal data obtained under a dry environment without the presence of excipients. Hence, the co-crystal appears to be suitable as a crystalline API. However there would be concern for its dissociation in the solution state and potential in vivo influence. In case of the hemi-napadisylate salt, thermal analysis showed degradation even before completion of its melting, and predicted dispropotionation propensity. But with no free base 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

formation, the formulation containing hemi-napadisylate salt, was also found to be stable after storage for 300 days at 40 °C/75% RH. This may be due to the fact that the hemi-napadisylate salt may be unstable at very high temperatures - conditions not relevant to storage of pharmaceutical dosage forms. This physical stability data is also consistent with the solution stability data, which indicates that the hemi-napadisylate salt form is stable even when in equilibrium with water at a pH of 2. The formulation containing the HBr salt, when subjected to accelerated stability testing at 40 °C and 22% or 75% RH conditions, was unstable and disproportionated to the free base. The disproportionation kinetics are depicted in Fig. 9. In tablets stored at 75% RH, the disproportionation rate was the highest during the first 2 days of storage. On the other hand, in tablets stored at 22% RH, disproportionation could be discerned after 2 days. This is likely due to the much lower water content in the sample stored at the lower RH. Following storage at 75 and 22% RH for 2 days, the water content in the tablets containing HBr salt were ~10% and ~0.5%% w/w respectively (determined by the water sorption experiment). Disproportionation is reported to be a solution mediated transformation16,24,25.

Figure 9. Free base (%, w/w) formed in tablets containing HBr salt (n=3) stored at 40 °C and 22 or 75% RH. Inset shows early time-points to for onset of disproportionation It is interesting to note that irrespective of the RH conditions, the disproportionation reached a plateau after few days of storage. Slurry pH measurements were done as a surrogate to microenvironmental pH measurement. At time ‘0’, the slurry pH of the formulation containing 22 ACS Paragon Plus Environment

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the HBr salt was found to be 2.28, which is higher than the slurry pH of the HBr salt alone, which was found to be 1.63. A drop in the slurry pH of the formulation started and continued until it reached a constant pH of ~ 1.65 within 26 hours (supporting information; Fig. S12). Dissolution of the dissociated HBr counter-ion from the salt, within the mobile water may have brought down the microenvironmental pH and stopped further disproportionation reaction, resulting in the observed plateau in the disproportionation kinetics of HBr salt. The formulation containing the HCl salt, when subjected to accelerated stability testing at 40 °C and 11%, 32% or 75% RH conditions, disproportionated to the free base. The disproportionation kinetics are depicted in Fig. 10. In case of samples stored at 32 and 75% RH, disproportionation was observed within 4 hours of storage. But in case of samples stored at 11% RH, no disproportionation was observed within 24 hours of storage. Table 2 contains the slope values of the trend-lines of initial and later time points in disproportionation kinetics data. Table 2. Slope of disproportionation kinetics plots of formulation, containing HCl salt. RH (%) 11 32 75

Slope (initial time points) Slope (later time points) 0.31 0.02 1.98 0.05 3.39 0.05

Figure 10. Free base (%, w/w) formed in tablets containing HCl salt (n=3) stored at 40 °C and 11, 32 or 75% RH. Inset shows early time-points for onset of disproportionation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

In contrast to the HBr salt, where a plateau was observed in the disproportionation, the HCl salt disproportionation reaction does not plateau. We shall discuss this in detail later. The initial time points in the curve (Fig. 10) indicate rapid disproportionation. A similar trend was also observed in the slurry pH measurement that was done as a surrogate to microenvironmental pH measurement (supporting information; Fig. S13). In later time points during stability study, HCl vaporization may be the driving force for the disproportionation (Fig. 10), and may be the rate limiting step. Volatilization of HCl, following disproportionation of HCl salt, has been reported in the literature26-30. Hydrochloride (pKa = - 6.1), and methanesulfonate (pKa = - 1.2) salts of triazole antifungal agent of α-styrylcarbinol class (pKa = 1.7 – 2.2) were prepared, and stored at 60 °C. A loss of 12.7% of chloride ion was observed after 6 hours of storage, in case of its HCl salt. On the other hand, the methanesulfonate salt with 5% w/w water was stable after 3 weeks of storage26. Other examples also indicate that the primary driving force for disproportionation reactions in HCl salts is liberation of HCl gas. The liberated hydrogen chloride gas either escapes from the system or reacts with other components of the formulation27,28. A proprietary hydrochloride salt, REV-6000-A(SS), was found to liberate hydrogen chloride gas and thereby cause the rusting of tablet punches and dies. The thermogravimetric analysis of the salt indicated the loss of a mole of hydrogen chloride between 147° and 175° C29. Volatilization of HCl may also influence the chemical stability of an API. Lyophilization of quinapril HCl, in the absence of pH control, produced an amorphous mixture of salt and free base due to volatilization of small amount of HCl. As amorphous free base is chemically less stable, the overall degradation rate increased relative to quinapril HCl salt alone30. HCl and HBr are salts of mineral acids, and moisture associated with these salts can be very acidic. When compressed as a solid dosage form, these acidic salts share the available moisture with other basic formulation excipients. As compared to sulfonate or carboxylate salts, salts of these mineral acids can produce highly unfavorable stability environment in dosage forms31. Though HCl and HBr salts of CRH-1 are isostructural, their disproportionation kinetics were quite different. The released HBr remained in the system caused a pronounced lowering of the microenvironmental pH below pHmax, and stopped further disproportionation. But in the case of the HCl salt, the released HCl volatilized and escaped the system, keeping the microenvironmental pH well above its pHmax. HCl vaporization became the driving force for continuous (or larger degree) of disproportionation as HCl was quantitatively removed from the system. SIGNIFICANCE Disproportionation of salts of weakly basic drugs can be modulated by microenvironmental pH. While the HCl and HBr salts of CRH-1 are isostructural, they exhibit a profound difference in their disproportionation behavior. When stored at 40 °C/75% RH, in case of the HBr salt, disproportionation was self-limiting and occurred only up to ~ 9%. We hypothesize that the HBr 24 ACS Paragon Plus Environment

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


formed following disproportionation, dissolved in the sorbed water, and its ionization caused a dramatic lowering of the microenvironmental pH and stopped further disproportionation reaction. Thus the reaction byproduct makes disproportionation self-limiting. But in case of the HCl salt, the disproportionation reaction did not exhibit a plateau. In this case, HCl left the “system” by volatilization. As a result, the disproportionation reaction continued. Interestingly, no disproportionation was observed in case of the hemi-napadisylate salt and adipic acid co-crystal in solid state. However, the co-crystal dissociated readily in the dissolution medium during IDR analysis. If the salt to free base solubility ratio is very high, the pHmax is reduced to well below the pKa of the compound. When the salt form is taken to a pH where the free base is the thermodynamically stable form, disproportionation occurs spontaneously. This was observed in case of the HCl and HBr salts. Therefore, the objective of salt selection should be to identify counter-ions which yield the desired rather than very high aqueous solubility. Very high salt solubility (with respect to the free base) can translate to an increase in physical instability and lead to salt free base conversion. CONCLUSION 1) Disproportionation propensity of salts (HCl, HBr, hemi-napadisylate) and adipic acid cocrystal of corticotropin releasing hormone receptor-1 antagonist, was studied using MFK. The DSC heat flow curves of HCl, HBr and hemi-napadisylate salts showed incongruent melting (disproportionation). Based on thermal analysis, though the adipic acid co-crystal had the lowest melting point, it was the most stable crystal form. 2) To ensure stability in solid state, tablets of “prototype” formulations containing salts and co-crystals were subjected to accelerated stability studies. As predicted by MFK, formulations containing the adipic acid co-crystal did not show disproportionation of API even after 300 days of storage. Similarly formulations containing the HCl or HBr salts, disproportionate as predicted by MFK. Though isostructural, the disproportionation kinetics of HBr and HCl salts were quite different, as the HCl counter-ion is volatile. 3) Formulation containing the napadisyalte salt, also did not disproportionate after 300 days of storage. But this contradicted the findings of model free kinetics. Finally solution state stability of salts and co-crystal was also assessed by determining their solubility at different pH values, and intrinsic dissolution rate using 0.01 N HCl. In the present study, all the salts and co-crystal, except the hemi-napadisylate salt, disproportionated in dissolution medium.

Acknowledgements N.K.T. was sponsored by the Lilly Innovation Fellowship Award. The work was partially supported by the William and Mildred Peters Endowment Fund. The authors thank Jeremy 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

Merritt for the consultations and enlightening discussion. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Science, under contract No. DE-AC02-06CH11357. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program.

Supporting Information Explanation of solubility description when pH > pHmax for a diprotic weak base. Fig. S1: Tablet top surface exposed to X-rays during stability study, and major and minor axis of the ellipse formed by incident X-ray beam on tablet surface. Fig. S2: Counter-ion concentration vs pH. Fig. S3: Comparison of IDR of CRH-1 free base, its salts, and adipic acid co-crystal upto 90 minutes showing disproportionation. Fig. S4: SXRD of CRH-1 free base and salts. Fig. S5: DSC heat flow curves of CRH-1 free base heated in open pans at three different heating rates. Fig. S6: Simultaneous TGA/Mass Spectrometry of HCl salt. Fig. S7: Model Free Kinetics of the HCl salt by DSC profile. Fig. S8: TGA/sDSC of hemi-napadisylate salt. Fig. S9: TGA weight loss profiles for the HCl, HBr, and hemi-napadisylate salts. Fig. S10: Effect of heating rate on the TGA of the hemi-napadisylate salt and determination of activation energy for disproportionation, using model free kinetics. Fig. S11: TGA/sDSC of Adipic acid co-crystal. Fig. S12: Slurry pH of CRH-1 HBr salt (neat) and its formulation, as a function of time. Fig. S13: Slurry pH of CRH-1 HCl salt (neat) and its formulation, as a function of time.

REFERENCES 1. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev. 2012, 64, 4-17. 2. Thayer, A.M. Finding solutions. Chemical & Engineering News 2010, 88(22), 13-18. 3. Crew, M. Bioavailability enhancement - A new year for solubility enhancement. Drug Development and Delivery 2014. 4. Serajuddin, A.T. Salt formation to improve drug solubility. Adv. Drug Delivery Rev. 2007, 59(7), 603-616. 5. Engel, G.L.; Farid, N.A.; Faul, M.M.; Richardson, L.A.; Winneroski, L.L. Salt form selection and characterization of LY333531 mesylate monohydrate. Int. J. Pharm. 2000, 198(2), 239-247.


Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6. Stephenson, G.A.; Aburub, A.; Woods, T.A. Physical stability of salts of weak bases in the solid‐state. J. Pharm. Sci. 2011, 100(5), 1607-1617. 7. Rohrs, B.R.; Thamann, T.J.; Gao, P.; Stelzer, D.J.; Bergren, M.S.; Chao, R.S. Tablet dissolution affected by a moisture mediated solid-state interaction between drug and disintegrant. Pharm. Res. 1999, 16(12), 1850-1856. 8. Hsieh, Y.L.; Yu, W.; Xiang, Y.; Pan, W.; Waterman, K.C.; Shalaev, E.Y.; Shamblin, S.L.; Taylor, L.S. Impact of sertraline salt form on the oxidative stability in powder blends. Int. J. Pharm. 2014, 461(1), 322-330. 9. Baertschi, S.W.; Alsante, K.M.; Reed, R.A. Pharmaceutical Stress Testing: predicting drug degradation. CRC Press 2011. 10. Vyazovkin, S.; Wight, C.A. Kinetics in solids. Annu. Rev. Phys. Chem. 1997, 48(1), 125149. 11. Vyazovkin, S.; Wight, C.A. Model-free and model-fitting approaches to kinetic analysis of isothermal and nonisothermal data. Thermochimica Acta 1999, 340, 53-68. 12. Vyazovkin, S. Reply to “What is meant by the term ‘variable activation energy’when applied in the kinetics analyses of solid state decompositions (crystolysis reactions)?”. Thermochimica Acta 2003, 397(1), 269-271. 13. Vyazovkin, S. Thermal analysis. Analytical chemistry 2006, 78(12), 3875-3886. 14. Zhou, D.; Schmitt, E.A.; Zhang, G.G.; Law, D.; Wight, C.A.; Vyazovkin, S.; Grant, D.J. Model‐free treatment of the dehydration kinetics of nedocromil sodium trihydrate. J. Pharm. Sci. 2003, 92(7), 1367-1376. 15. Zhou, D.; Schmitt, E.A.; Zhang, G.G.; Law, D.;Vyazovkin, S.; Wight, C.A.; Grant, D.J. Crystallization kinetics of amorphous nifedipine studied by model‐fitting and model‐free approaches. J. Pharm. Sci. 2003, 92(9), 1779-1792. 16. Merritt, J.M.; Viswanath, S.K.; Stephenson, G.A. Implementing quality by design in pharmaceutical salt selection: a modeling approach to understanding disproportionation. Pharm. Res. 2013, 30(1), 203-217. 17. Hammersley, A. P. Fit2D: an introduction and overview; ESRF internal report, ESRF97HA02T; 1997. 18. Hammersley, A. P.; Svensson, S. O.; Hanfland, M.; Fitch, A. N.; Hausermann, D. Twodimensional detector software: from real detector to idealised image or two-theta scan. High Pressure Res.1996, 14 (4−6), 235−248. 19. Avdeef, A.; Comer, J.E.; Thomson, S.J. pH-Metric log P. 3. Glass electrode calibration in methanol-water, applied to pKa determination of water-insoluble substances. Analytical Chemistry 1993, 65(1), 42-49. 20. Motherwell, W.S.; Shields, G.P.; Allen, F.H. Graph-set and packing analysis of hydrogen-bonded networks in polyamide structures in the Cambridge Structural Database. Acta Crystallographica Section B: Structural Science 2000, 56(5), 857-871. 21. Wood, P.A.; Oliveira, M.A.; Zink, A.; Hickey, M.B. Isostructurality in pharmaceutical salts: How often and how similar?. CrystEngComm 2012, 14(7), 2413-2421. 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

22. Ong, W.; Cheung, E.Y.; Schultz, K.A.; Smith, C.; Bourassa, J.; Hickey, M.B. Sodium and potassium salts of bumetanide trihydrate: Impact of counterion on structure, aqueous solubility and dehydration kinetics. CrystEngComm 2012, 14(7), 2428-2434. 23. Galcera, J.; Molins, E. Effect of the counterion on the solubility of isostructural pharmaceutical lamotrigine salts. Crystal Growth and Design 2008, 9(1), 327-334. 24. Guerrieri, P.; Jarring, K.; Taylor, L.S. Impact of counterion on the chemical stability of crystalline salts of procaine. J. Pharm. Sci. 2008, 99(9), 3719-3730. 25. Hsieh, Y.L.; Taylor, L.S. Salt stability-effect of particle size, relative humidity, temperature and composition on salt to free base conversion. Pharm.Res. 2015, 32(2) (2), 549-561. 26. Maurin, M.B.; Addicks, W.J.; Rowe, S.M.; Hogan, R. Physical chemical properties of alpha styryl carbinol antifungal agents. Pharm. Res. 1993, 10(2), 309-312. 27. Elder, D.P.; Delaney, E.D.; Teasdale, A.; Eyley, S.; Reif, V.D.; Jacq, K.; Facchine, K.L.; Oestrich, R.S.; Sandra, P.; David, F. The utility of sulfonate salts in drug development. J. Pharm. Sci. 2010, 99(7), 2948-2961. 28. Lin, S.L.; Lachman, L.; Swartz, C.J.; Huebner, C.F. Preformulation investigation I: relation of salt forms and biological activity of an experimental antihypertensive. J. Pharm. Sci. 1972, 61(9), 1418-1422. 29. Narurkar, A.N.; Purkaystha, A.R.; Sheen, P.C. Effect of various factors on the corrosion and rusting of tooling material used for tablet manufacturing. Drug Dev. Ind. Pharm. 1985, 11(8), 1487-1495. 30. Guo, Y.; Byrn, S. R.; & Zografi, G. Effects of lyophilization on the physical characteristics and chemical stability of amorphous quinapril hydrochloride. Pharm. Res. 2000, 17(8), 930-936. 31. Gould, P. L. Salt selection for basic drugs. Int. J. Pharm.1986, 33(1), 201-217.


Salt Disproportionation in the Solid State: Role of Solubility and

Recommend Documents