Predicting the Physical Stability of Amorphous Tenapanor

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Predicting the Physical Stability of Amorphous Tenapanor Hydrochloride using Local Molecular Structure Analysis, Relaxation Time Constants and Molecular Modelling Sanjeev Kothari and Radha R. Vippagunta* Pharmaceutical Chemistry and Formulations, Ardelyx, 34175 Ardenwood Blvd, Fremont, CA 94555, USA. *Corresponding Author: [email protected]; Phone :5104567717 Sanjeev Kothari, and Radha Vippagunta Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00853 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 1, 2019

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Molecular Pharmaceutics

Predicting the Physical Stability of Amorphous Tenapanor Hydrochloride using Local Molecular Structure Analysis, Relaxation Time Constants and Molecular Modelling Sanjeev Kothari and Radha R. Vippagunta* Pharmaceutical Chemistry and Formulations, Ardelyx, 34175 Ardenwood Blvd, Fremont, CA 94555, USA.

KEYWORDS. Conformational flexibility, tenapanor, amorphous, local molecular structuring, stability, relaxation time and density functional theory ABSTRACT: The conformational flexibility of organic molecules introduces more structural options for crystallization to occur, but has potential complications, such as, reduced crystallization tendency and conformational polymorphism. Although, a variety of energetically similar conformers could be anticipated, it is extremely difficult to predict the crystal conformation for conformationally flexible molecules. The present study investigates differences in thermodynamic parameters for the free base, c-FB and an amorphous dihydrochloride salt, a-Di-HCl, of a conformationally flexible drug substance, tenapanor (RDX5791). A variety of complementary techniques such as, thermal analysis, powder X-ray diffraction, and molecular modeling were used to assess the thermodynamic properties and the propensity of crystallization for a-FB and a-Di-HCl, tenapanor. Molecular modeling and total scattering measurements suggested that the a-Di-HCl salt exists in an open elongated state with local 1D stacking, which extends only to the first nearest neighbor, while the aFB shows local stacking extending to the third nearest neighbor. The overall relaxation behavior which typically, is an indicator for physical stability, as measured by modulated temperature differential scanning calorimetry and powder X-ray diffraction (PXRD) suggested a non-typical dual relaxation process for the dihydrochloride salt form. The first relaxation was fast and occurred on warming from the quench conditions without any thermal annealing, while, the second relaxation step followed a more traditional glass relaxation model, exhibiting an infinite relaxation time. Similar analysis for the a-FB suggested a comparatively shorter relaxation time (about nineteen days) that results in its rapid crystallization. This observation is further validated with the extensive amount of physical stability data collected for the a-Di-HCl salt form of tenapanor under accelerated and stress stability conditions, as well as, long term storage for more than 3 years that show no change in its amorphous state.

INTRODUCTION Various solid forms of an active pharmaceutical ingredient (API) often display different mechanical, thermal, physical and chemical properties that can remarkably influence its bioavailability, hygroscopicity, stability and other performance characteristics. Hence, a thorough understanding of the relationship between the particular solid form of an API and its functional properties is important in selecting the most suitable form of the API for development into a drug product. Although, a crystalline form of active compound is generally preferred, assessing the viability of an amorphous formulation strategy is of great importance in an era of drug discovery where a large percentage of new molecules have solubility limited dissolution rates 1-3. However, physicochemical processes leading to instability of amorphous drug products such as recrystallization and chemical degradation, offsets the potential benefit of greater aqueous solubility4.

*Corresponding Author: [email protected]; Phone :5104567717

Despite progress in recent years, the fundamental understanding and therefore the predictability of physical and chemical stability of amorphous phases, is one of the main challenges in developing an amorphous drug product. Molecular mobility is generally thought to be a key factor governing the stability of amorphous phases and has been the subject of many studies 5-11. Although, a detailed understanding of the relationship between mobility and stability of amorphous materials is still on-going, especially below the glass transition temperature, Tg, there is enough evidence to support a strong connection between the molecular mobility and the stability of amorphous materials 12-18. However, structural complexity and the flexibility of the molecule, which manifests itself as an entropic barrier to crystallization 19-26, has rarely been discussed. We describe a study wherein a simple hydrochloride

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salt of a small molecule, tenapanor (RDX5791, a- DiHCl) with a relatively large molecular weight (1197 daltons), resists crystallization, although its free base form (c-FB) exists in a crystalline state. RDX5791 (tenapanor), CAS name: (12,15-Dioxa2,7,9-triazaheptadecanamide,17-[[[3-[(4S)-6,8dichloro-1,2,3,4-tetrahydro-2-methyl-4isoquinolinyl]phenyl]sulphonyl]amino]-N-[2-[2-[2[[[3-[(4S)-6,8-dichloro-1,2,3,4-tetrahydro-2-methyl-4isoquinolinyl]phenyl]sulphonyl]amino]ethoxy]ethoxy ]ethyl]-8-oxo-,hydrochloride (1:2), as shown in Figure 1, is currently in late-stage clinical trials for multiple indications. As the amorphous dihydrochloride salt was chosen for development due to better in vivo performance linked to better solubility and dissolution properties compared to the free base form, understanding its physical stability was of utmost importance. The current study attempts to assess the propensity of a-Di-HCl to crystallize.

Figure 1 The chemical structure of tenapanor dihydrochloride

Many analytical techniques have been developed to monitor and investigate the re-crystallization of amorphous drugs. These techniques include: thermal analysis such as differential scanning calorimetry (DSC), solution calorimetry, powder x-ray diffraction (PXRD), and spectroscopic techniques, such as, NMR (nuclear magnetic resonance), FT-IR (Fouriertransform infrared spectroscopy), NIR (near- infrared), and Raman spectroscopy 27-33. In general, thermal analysis techniques have proven to be very useful in assessing the bulk thermal properties of the solid sample associated with recrystallization processes. The average rate of molecular motions at any given temperature is probably the most important parameter to know for amorphous pharmaceutical materials, and it can be used to explain and even predict the stability of amorphous systems 25,27. It can be used to monitor the extent of relaxation at temperatures below glass

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transition (Tg), as well as, heat capacity changes as a function of temperature 12-18. However, these thermal techniques are not able to give specific information related to molecular and crystal structures. PXRD measurements combined with computational approaches can provide additional insight to assess the physical state of an amorphous solid. Materials that exhibit no local molecular order in the amorphous state have been observed to exhibit the minimum propensity for crystallization24. In order for crystallization to occur, local molecular order must first develop. PXRD has been used on amorphous materials to probe the degree of local order and provide information on the propensity to crystallize 24. Especially, when crystal structure models are not available, individual molecular units can be derived using molecular structure minimization of a given molecule and can be tested against the measured total scattering signal to identify the most self-consistent molecular unit. Recent advances in solid-state modelling via density functional theory (DFT) have led to an increase in accuracy and efficiency in producing and predicting the most stable conformers31.Once, the most selfconsistent molecular model is identified, lattice function can be derived. The lattice function defines the local, inter-molecular packing in the noncrystalline state. The form of the lattice function is thus a sensitive probe of the inherent propensity for crystallization and is a precursor indicator of physical instability of the sample. The present study combines results from thermal analysis, PXRD and molecular modelling along with predictions on conformational preferences to assess, the propensity of the a-Di-HCl to crystallize as compared to the a-FB. EXPERIMENTAL Materials Crystalline tenapanor free base (c-FB) and amorphous tenapanor dihydrochloride (a-Di-HCl) samples were used in this study. The crystalline tenapanor free base (c-FB) was converted into non-crystalline tenapanor free base (a-FB) by melt-quenching. Differential Scanning Calorimetry (DSC) DSC analyses were carried out for both, crystalline tenapanor free base (c-FB) and amorphous tenapanor dihydrochloride (a-Di-HCl) using a TA Instruments Q2000. The instrument temperature calibration was

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performed using indium. The sample was heated from ambient temperature to 350 °C at a rate of 10 °C per minute. The DSC cell was kept under nitrogen purge of about 50 mL per minute during each analysis. Modulated Differential Scanning Calorimetry (MDSC) The presence of significant volatile content contribution in the DSC heat flow data between 40 and 110 °C, resulted in effectively hiding the glass transition event for the dihydrochloride (a-Di-HCl) and for the amorphous tenapanor free base (a-FB) obtained after a heat-cool-heat run of crystalline tenapanor free base (c-FB). To enhance the visibility of the glass transition, the free base (a-FB) and amorphous dihydrochloride (a-Di-HCl) was equilibrated at 170°C which is above the observed melting temperature of the crystalline free base form (150 °C). After equilibration within the DSC unit, the samples were rapidly cooled to -40 °C (to quench the non-crystalline state) and held isothermally for 15 minutes. The modulated DSC scans ran from -40 °C to 170 °C at a rate of 4 °C/min with a modulated amplitude of 0.8 °C and modulation period of 40 seconds. Differential Scanning Calorimetry (DSC) – Absolute Heat Capacity To better determine the change in heat capacity between the non-crystalline forms of free base (a-FB) and the HCl salt (a-Di-HCl), a series of absolute heat capacity measurements were performed on TA Instruments Q2000. The absolute heat capacity determination follows the ASTM E1269-11 procedure and makes use of sapphire and empty pan heat flow measurements to correct the baseline and scale individual heat flow results for each sample. Due to the dominant volatiles contribution close to the expected glass transition event, absolute CP measurement for a-Di-HCl was performed by first equilibrating the a-Di-HCl at 100 °C for 15 minutes before quenching to -90 °C. The 100 °C equilibration step was designed to remove the volatiles without thermally stressing the salt. The c-FB was first equilibrated at 170 °C for 15 minutes before performing the absolute CP measurements. The equilibration temperature is above the free base melt (150 °C) and lower than the thermal degradation events. After equilibration, the liquid free base was

quenched to -90 °C and held for 15 minutes before heating to the starting temperature of 5 °C. The quenched free base was held at 5 °C for 15 minutes before beginning the DSC scan from 5 °C to 200 °C at 10 °C per minute. Powder X-ray Diffraction The Rigaku Smart-Lab X-ray diffraction system, used for this study was configured as a Bragg-Brentano system using a line source X-ray beam. The X-ray source is a Cu Long Fine Focus tube that was operated at 40 kV and 44 mA. The data collection range was extended to 80 °2θ in order to support the normalization procedures used during molecular modeling. A reflection X-ray geometry with low background silicon sample holders was used to better control the variable background observed. The BraggBrentano configuration utilized is a clean optical system with the only active optical component being a beta filter. The D’teX detector used in this configuration was a position sensitive detector (PSD) that captures diffraction events over a relatively large diffraction range. Data Analysis: Methodology The measured PXRD data files collected on the noncrystalline forms represent the total scattering signal from the sample. This data forms the basis of the total scattering modeling to characterize the local molecular structure within the non-crystalline state. Total scattering data analysis is an iterative procedure, wherein data pre-processing, and Debye calculations are performed together within each iterative step in the following sequence25 along with molecular modeling: a. Data correction/pre-processing. b. In parallel, DFT/molecular modeling is carried out by: i) Generation of molecular models by DFT calculations. ii) Use of the Debye-Menke total scattering approach to simulate the separate Debye and Menke contributions for selected molecular models. iii) Combination of both single curves to simulate the Debye-Menke total scattering curve. c. By combining the experimentally corrected XRPD pattern recovered at point “a”, with the



Debye-Menke contribution generated at point “b”, and by using of the formula given in next sections, the lattice function can be recovered. For this an additional normalization step is necessary. d. Extraction of information about local order from the lattice function. e. When a non-crystalline system becomes physically unstable and begins to undergo crystallization, the lattice function describing the arrangement of molecular units must begin to develop local inter-molecular order.

Equation 1: The Debye equation 𝐼𝐷𝑒𝑏𝑦𝑒 =

∑

𝑓2𝑖 𝑖=1

𝑛

+2

∑

𝑀𝑒𝑛𝑘𝑒 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑖𝑜𝑛 =

𝑓𝑖𝑓𝑗𝑠𝑖𝑛(𝑄𝑑𝑖𝑗) (𝑄𝑑𝑖𝑗)

𝑖,𝑗 𝑖 ≠ 𝑗 1

This assumes an atomic level molecular model for the complete sample of interest, where: n is the number of atoms in the model (with indices i and j), fi is the individual atomic form factor for atom i, and dij is the distance between atoms i and j. A more tractable approach to the calculation of the total scattering signal for a non-crystalline system is to use the lattice function formulism which can be used with a single representative molecular unit, shown in Equation 2. The lattice function approach assumes that the non-crystalline state can be described by a single average molecular unit that is arranged throughout the solid sample according to a random lattice function.

∑ ∑𝑓 𝑓

𝑖 𝑗

𝑖 = 1𝑗 = 1

sin (𝑄𝑙𝑐𝑖)sin (𝑄𝑙𝑐𝑗) 𝑄𝑙𝑐𝑖

𝑄𝑙𝑐𝑗

Divisions of the measured data by the Debye models leave the lattice functions, which are parts of the diffraction responses resulting from molecular packing as per Equation 3.

Equation 3 𝑄 (𝑖𝑛Å) =

Total scattering measurements are utilized in order to probe the local structure of non-crystalline systems. To extract the local structure from the observed data, computational modeling of the observed total scattering signal is required. Total scattering is usually modeled utilizing the Debye diffraction equation (Equation 1)37 either in the form of a Pair-wise Distribution Function (PDF) or through direct molecular modeling. 𝑛

𝑛

1

Total scattering Modeling

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(4𝜋𝜆)sin (2𝜃2 )

The data pre-processing is performed iteratively with the total scattering calculations to determine the most self-consistent molecular model. Experimental Data pre-processing In order to compare experimentally determined PXRD data for non-crystalline materials with computationally calculated data from the total scattering method, the measured data has to be rendered into an instrumental free form called a reduced structure factor. The preprocessing steps applied are: 1) Instrumental background removal and digital filtering 2) Correction for instrument intensity function (Lorentz-Polarization + optics) 3) The normalization step is carried out to transform the Y-axis from measured intensity to electron units 4) Removal of Compton scattering 5) Debye Normalization

To ensure self-consistency between the derived total scattering responses for these data files, an additional high angle normalization was performed. The normalization step transforms the Y-axis from measured intensity to electron units. With the data transformed into electron units, the Compton scattering correction can be performed. As such, this step of the Equation 2: The Lattice Function Formalism 𝐼𝑇𝑜𝑡𝑎𝑙 𝐷𝑖𝑓𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 = (𝐼𝐷𝑒𝑏𝑦𝑒 ― 𝐼𝑀𝑒𝑛𝑘𝑒) × 𝐿𝑎𝑡𝑡𝑖𝑐𝑒_𝐹𝑢𝑛𝑐𝑡𝑖𝑜𝑛 data pre-processing is performed iteratively with the molecular modeling and total scattering calculations. where IMenke is the small angle scattering correction for With the removal of Compton scattering, the prethe chosen molecular unit. Both the Debye equation processed data can be directly compared to the total and Menke correction can be calculated from a given 38 scattering Debye-Menke response for the selected molecular model . Menke correction is used to molecular model. This allows a final normalization remove coherent SAXS response due to shape and step where the pre-processed data is scaled to give a mean number density.


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best match to the Debye-Menke total scattering response for the selected molecular unit. This preprocessing step is again an iterative step performed in combination with molecular modeling and total scattering calculations. After Debye normalization, the lattice function can be derived based upon the total scattering Debye-Menke curve for the selected molecular unit model. This methodology was employed for all PXRD data files presented in this study.

Figure 2 Modulated DSC analysis of the noncrystalline dihydrochloride (a-Di HCl) after equilibration at 100°C.

Molecular Modeling To enable interpretation of the total scattering data for a non-crystalline system, development of molecular models that best describe the essential molecular unit underlying the non-crystalline state are required. Individual molecular units were derived using molecular structure minimization of a given molecule. Given the molecular structure of tenapanor dihydrochloride as depicted in Figure 1, a molecular model was constructed for both, the free base and the dihydrochloride salt using the Spartan molecular modeling package. RESULTS AND DISCUSSION In order, to broadly define a range of conditions under which the non-crystalline forms of a-Di HCl and a-FB were well behaved, the initial study was carried out at non-ambient conditions. For a-Di HCl, the loss of volatiles under 150 °C interfered with observation of a glass transition (Tg) temperature in that region. Therefore, before analyses by MDSC, samples of a-Di HCl were equilibrated at 170 °C, as shown in Figure 2. a-FB samples analyzed by MDSC were prepared by equilibrating c-FB at 170 °C, which is above the melting point of free base (150 °C), and quenching at −40 °C, as shown in Figure 3.

Figure 3 Modulated DSC analysis of the noncrystalline free base (a-FB) after equilibration at 170°C.

The non-crystalline dihydrochloride (a-Di-HCl) has glass transition around 115 °C (Figure 2) which is significantly higher than that for the amorphous free base (a-FB) which is 60°C (Figure 3). The absolute change in heat capacity (δCp) during each Tg event was obtained from the reversing heat flow curves, as shown in Table 1. These changes provide a relative measure of the molecular mobility in the glassy phase close to ambient conditions. a-FB exhibits a substantially-larger δCp than does a-Di HCl, suggesting that a-FB is a more fragile glass that would be expected to rapidly develop local order upon cooling.



Table 1

MDSC results at glass transition event

Sample Free Base (a-FB) Dihydrochlor ide Salt

Tg (°C)

δCp (J/

at center

g•°C)

60.03

0.3640

2.174

115.48

0.2835

2.453

H (J/g)

(a-Di HCl)

Relaxation Time Constants Relaxation time constants below Tg were determined using the method of Hancock 14, which utilizes aging experiments. In MDSC, the non-crystalline forms (aFB and a-DiHCl) were freshly prepared in-situ by heating each sample to 170 °C and holding them isothermally for 15 minutes. The samples were then rapidly cooled at -20 °C / minute to -90 °C where they were again held isothermally for 15 minutes. After cooling, each sample was slowly warmed at 4 °C / minute to the target annealing temperature. The target annealing temperatures were selected to be 12 °C, 24 °C and 48 °C below the glass transition event for each non-crystalline sample. A series of different annealing times were selected for each target temperature. For the initial study, all samples were annealed for 0, 2, 4 and 8 hours. However, for the dihydrochloride salt, no relaxation was observed over these annealing times for the Tg-48 °C target temperature. For this specific Tg48 °C temperature point, the samples were annealed for 0, 8, 16 and 32 hours. The MDSC data obtained from the above samples were utilized to determine two experimental parameters: 1) absolute change in heat capacity (δCp) on passing the glass transition event and 2) relaxation enthalpy (δH) released. The individual δCP values are averaged to give a single number for the dihydrochloride salt a-Di HCl and the free base a-FB. The calculated mean δCP values are presented in Table 2.

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Assuming ideal linear relaxation behavior, the δCp values (from Table 2) can be used to determine the maximum amount of relaxation enthalpy generated for infinite annealing times (δH∞) depending on the temperature below Tg at which the annealing was performed (Tanneal); using Equation 4. Equation 4: δH∞ = (Tg – Tanneal). δCp Calculated values for δH∞ as per Equation 4 are shown in Table 3 for the dihydrochloride salt a-Di HCl and the free base a-FB for all annealing temperatures used.

Table 3 Calculated δH∞ values for dihydrochloride and free base Samples Dihydrochloride Salt (a-Di HCl)

Free Base (a-FB)

Temperature

δH∞(J/g)

Tg-48

12.24

Tg-24

6.12

Tg-12

3.06

Tg-48

16.03

Tg-24

8.02

Tg-12

4.01

The individual δH values as determined from the nonreversing heat flow curves, are presented in Table 4 and Table 5. It was noted that even for the 0-hour hold time experiments, a significant relaxation enthalpy was determined for both, a-Di HCl and a-FB. For the free base, this starting enthalpy was essentially constant for all runs (~ 2.1 (J/g)). The starting enthalpy for the dihydrochloride salt varied for each of the annealing cycles. This type of behavior is not representative of ‘ideal’ relaxation phenomena. The enthalpy values for the extended hold time experiments on the a-Di-HCl are shown in Table 5 and continued to show variability.

Table 2 Calculated mean δCp values for free base and dihydrochloride salt 39 Samples

δ Cp (J/(0C.g)

Free Base (a-FB)

0.334

Dihydrochloride Salt (a-Di HCl)

0.246


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Table 4 δH values as determined from nonreversing heat flow curves Samples Dihydrochlo ride Salt (a-Di HCl) Free Base (a-FB)

Temper ature

0 Hours

2 Hours

4 Hours

8 Hours

Tg-48

2.65

2.53

2.52

2.37

Tg-24

2.49

3.87

4.55

5.11

Tg-12

1.56

3.67

3.87

4.23

Samples

Temperat ure

0 Hours

8 Hours

16 Hours

32 Hours

Tg-48

2.04

2.17

2.22

2.07

Tg-24

2.07

3.23

3.59

4.27

Dihydrochlor ide Salt (aDi HCl)

Tg-48

0.00

0.02

0.74

0.76

Tg-12

2.14

4.71

5.25

5.92

Samples

Temper ature

0 Hours

8 Hours

16 Hours

32 Hours

Dihydro chloride Salt (aDi HCl)

Tg-48

3.05

3.07

3.79

3.80

The extent of relaxation at each annealing point (ΦT) is determined using Equation 5. Equation 5: ΦT = 1 – (δH /δH∞)

Table 6 Extent of relaxation (ΦT) determined for each annealing experiment

Free Base (a-FB)

Equation 6: ΦT = exp(-(t / η)β) For small molecule systems in general, a typical value of the distribution parameter is around 0.5, with a practical range of between 0.3-0.5 near Tg and approaches 1.0 at high temperatures14. The values for the distribution parameter and mean relaxation time constants for each annealing run are presented in Table 8.

Table 8 Distribution parameters and time

The extent of relaxation numbers presented in Table 6 gave numbers less than zero for the Tg-48 °C dihydrochloride annealing experiment.

Dihydroc hloride Salt (a-Di HCl)

Table 7 Extent of relaxation (ΦT) determined for longer hold time experiments

The extent of relaxation as a function of annealing time (t), was modeled using Equation 6 to determine optimum values of the mean relaxation time constant (η) and relaxation time distribution parameter (β).

Table 5 Extended hold time δH values for dihydrochloride salt.

Samples

relaxation. For this reason, the more extended hold times were employed in a follow up measurement; as shown in Table 7.

Temp eratur e

0 Hours

2 Hours

4 Hours

8 Hours

Tg-48

0.00

-0.12

-0.13

-0.28

Tg-24

0.00

1.38

2.06

2.62

Tg-12

0.00

2.11

2.31

2.68

Tg-48

0.00

0.13

0.18

0.04

Tg-24

0.00

1.16

1.51

2.20

Tg-12

0.00

2.57

3.10

3.78

This indicates no relaxation was occurring within the experiment time scale beyond the initially observed

constants derived from annealing studies Samples Dihydrochl oride Salt (a-Di HCl)

Free Base (a-FB)

Distributio nβ

0.47

0.65

Temperatu re

Time Constant (hrs)

Tg-48

11718

Tg-24

26.4

Tg-12

1.37

Tg-48

13462

Tg-24

44.3

Tg-12

1.96

The relaxation time constants as a function of the annealing temperature can be described by the empirical Vogel-Fulcher-Tammann (VFT) stretched exponential equation (Equation 7), where, the preexponential factor η0 has been related to the single molecule relaxation times. In this equation, D*, is the kinetic fragility parameter and T0 is a fictive temperature where the energetic barriers to relaxation effectively become infinite. The kinetic fragility



parameter D* will typically take values between 2.0 and 100.0. For organic molecular glasses in general, D*, is about 10.034. The fictive temperature T0 is generally approximated as Tg – 50 °K34.

Table 9 VFT fitting parameters

Equation 7: η = η0 exp(D* T0 / (T – T0)) Excel® solver was used to model the observed relaxation times with respect to Equation 7 giving the output curve fits presented in Figure 4 and Figure 5 for the a-Di-HCl and a-FB, respectively.

log10 relaxation time (hours)

Figure 4 VFT modeling of relaxation time for a-Di-HCl dihydrochloride_relaxation

4

Calc_VFT

2 0

0

20

40

60

T-Tg (°C)

Figure 5 VFT modeling of relaxation time for a-FB 6 free-base_relaxation

4

Calc_VFT

2 0

0

10

20

30

40

50

T-Tg (°C)

The derived VFT parameters for each material are shown in Table 9.

Samples

η0(10E-9 hours)

T0(°K)

D*

Dihydrochlo ride Salt (aDi HCl)

0.86

251.0

10.1

Free Base (a-FB)

0.83

192.0

13.9

The VFT curve fitting resulted in fictive temperatures (T0) for both the free base and dihydrochloride at about Tg – 135 °K. These are considerably lower than the more traditional Tg – 50 °K values. This suggests that some relaxation processes are still active at relatively low temperatures. The kinetic fragility parameters (D*) were within the expected ranges34,35. For dry ambient conditions, from the VFT model, the mean relaxation time for the free base at Tg – 37 °K is estimated to be about 460 hours (19 days). Under the same dry conditions, the dihydrochloride salt at Tg – 90°K is predicted to have an infinite mean relaxation time. Thus, based on the MDSC data, the dihydrochloride salt form seems to show a non-typical dual relaxation process. The first relaxation was fast and occurred on warming from the quench conditions without the need for any thermal annealing. The second relaxation step followed a more traditional glass relaxation model, exhibiting the long relaxation times, as predicted. Traditionally, relaxation times are critical indicators for physical stability. This, however, is based upon the existence of a crystalline form that exists with lower free energy than the relaxed glass. In several crystallization attempts, a stable dihydrochloride salt could not be successfully isolated as a crystalline solid 33. This is consistent with the long relaxation time for a-Di-HCl calculated from the VFT model.

6

log10 relaxation time (hours)

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

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60

X-ray Powder Diffraction The measured PXRD for the non-crystalline dihydrochloride (a-Di HCl), crystalline free base (cFB) and amorphous free base (a-FB) are shown in Figure 6.


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Figure 6 Measured PXRD data for noncrystalline dihydrochloride (a-Di HCl), crystalline free base (c-FB) and amorphous free base (a-FB).

The PXRD data obtained from a-FB and a-Di HCl were processed to provide lattice functions, which were used to assess the extent of local order in these materials. In order to compare the experimentallydetermined, PXRD data for non-crystalline materials with data calculated by the total scattering method, measured data were processed to remove instrumental contributions and normalized according to the procedure outlined in the experimental section. This data forms the basis for what is called total scattering modeling, which is used to characterize the local molecular structure within each non-crystalline material. Total scattering modeling is an iterative procedure with data pre-processing, molecular modeling and Debye calculations being performed together within each iterative step.

experimentally observed data. Different potential conformers were first isolated using a simple molecular dynamics model with the phenomenological MMFF (Merck Molecular Force Field). In an inert local environment (neutral like nitrogen atmosphere or vacuum), both tenapanor and tenapanor dihydrochloride were observed to fold up into a compact (closed) conformer to minimize interaction energies. In an interacting local environment, such as a polar solvent (water), tenapanor exists in an elongated (open) conformer. Representative open and closed conformers were selected for c-FB and a-Di HCl, for full quantum mechanical structure minimization. The minimization was performed using density functional theory (DFT) with the basis set EDF2 6-31G*. The resulting minimized molecular structures are presented in Figure 7, Figure 8, Figure 9 and Figure 10.

Figure 7 Minimized structure for open tenapanor dihydrochloride (DFT EDF2 6-31G*).

Figure 8 Minimized structure for closed tenapanor dihydrochloride (DFT EDF2 6-31G*).

Normalization depended on the molecular model used. The first step was to generate a Debye model, which is simply the PXRD pattern expected from a single molecule in a single conformation (molecular unit), without consideration of packing. In order to describe the molecular unit underlying the non-crystalline state, various molecular units of tenapanor were derived using molecular structure minimization. In the absence of known crystal structures, energy-minimized, singlemolecule conformers represent the best choice for a molecular unit. Molecular models were defined for both ‘open’ and ‘closed’ conformers of tenapanor free base and tenapanor dihydrochloride and the calculated total scattering curves were compared with the



Figure 9 Minimized structure for open tenapanor free base (DFT EDF2 631G*).

Figure 10 Minimized structure for closed tenapanor free base (DFT EDF2 631G*).

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Figure 11 Final processed total scattering signal for non-crystalline tenapanor free base and dihydrochloride salt.

The lattice functions derived from the PXRD data of aFB and a-Di HCl are shown in Figure 12. The positions of the large peaks at about 1.5 Q indicate there is more local order in a-FB than in a-Di HCl.

Figure 12 The lattice functions derived from the PXRD data of a-FB and a-Di HCl

Modeling was carried out using the Debye-Menke method. The Debye equation (Equation1) assumes an atomic-level molecular model of the entire sample, whereas, the lattice function formalism (Equation 2) utilizes a single average molecular unit arranged throughout the sample according to a random lattice function. The Debye–Menke total scattering curves calculated using the minimized molecular conformers of a-FB and a-Di HCl (in both open and closed forms) showed that the open conformer of tenapanor is the best molecular unit for amorphous tenapanor, as can be seen in Figure 11.

( 𝟏)

∗ 𝒕𝒉𝒆 𝒂𝒙𝒊𝒔 𝒊𝒔 𝒊𝒏 𝑸 𝒊𝒏Å , 𝒘𝒉𝒊𝒄𝒉 𝒊𝒔 𝒓𝒆𝒍𝒂𝒕𝒆𝒅 𝒕𝒐 𝟐𝜽, 𝑸 =

(𝟒𝝅𝝀)𝒔𝒊𝒏 (𝟐𝜽𝟐)

(Equation 3)

Different solid structural models were used to calculate lattice functions and compared to the lattice functions derived from the measured data. The best fit was attained using a para-crystalline lattice function. Paracrystalline materials have short and medium range ordering in their lattices (similar to mesophases), putting them between frozen liquids and crystalline solids 36. In Figure 13, the calculated lattice functions are displayed with respect to a para-crystalline, one-


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dimensional, random lattice function for a-FB and aDi-HCl.

Figure 13 Derived lattice function for meltquenched, non-crystalline tenapanor free base and dihydrochloride plotted with respect to para-crystalline model

potential for three-dimensional stacking due to ordering achieved up to the third nearest neighbor that could result in achieving the closed energy minimized structure (Figure 10).

Figure 14 Lattice function PDF derived from non-crystalline free base and dihydrochloride lattice functions.

1NN 2NN

The para-crystalline lattice function allowed two variables (stacking distance and damping factor) to be changed to alter the lattice function curve to better fit the measurement-derived curves. Each best-fitting model has a one-dimensional stacking distance of about 3.5Å. Agreements between the calculated lattice functions and the para-crystalline models are reasonable, suggesting that some local nearestneighbor alignment of the open tenapanor molecules is taking place in both a-FB and a-Di HCl. The paracrystalline stacking distance suggests that the molecules are lined up with respect to each other, effectively stacking normal to their long axes. The degree of local order is minimal and can be considered only a nearest-neighbor phenomena. PDF lattice functions were generated from the paracrystalline lattice functions, which provide a visual indication of the distances over which the molecules are ordering in the glassy state; (Figure 14). For the dihydrochloride, the local order barely extends beyond nearest neighbor. The free base exhibits a more extensive range of local order out to at least the 3rd nearest neighbor. The PDF lattice function calculations (Figure 14) can be used to conclude that the amorphous dihydrochloride salt only shows the potential for one-dimensional stacking that accounts for its open elongated energy minimized structure (Figure 7) while, the amorphous free base develops the

3NN

Storage Stability of a-Di-HCl The physical stability of the a-Di-HCl has been evaluated under ICH long term storage stability conditions and data collected for at least three years has shown no change in the PXRD pattern. Additionally, aged lots when tested with solid-state 13C NMR spectroscopy showed no spectral changes or appearance of crystallinity. CONCLUSIONS A variety of complementary techniques such as thermal analysis, powder X-ray diffraction and molecular modeling were used to assess the crystallization propensity of two non-crystalline systems containing a relatively large MW drug substance, tenapanor (RDX5791). Tenapanor, is known to exist as crystalline anhydrous free base form (c-FB) and as an amorphous powder of dihydrochloride salt (a- DiHCl). Molecular modeling and total scattering measurements suggested that the non-crystalline tenapanor dihydrochloride exists in an open elongated state with local 1D stacking in agreement with the paracrystalline model. The extent of stacking repeat is limited to its nearest-neighbor. Further, modulated DSC measurements were used to determine the relaxation dynamics of the non-crystalline forms below the glass transition event. The overall relaxation behavior was well described by the VFT stretched exponential model. The predicted mean relaxation



time for the dihydrochloride salt under dry ambient conditions was infinite due to the observed high Tg. For tenapanor dihydrochloride, there is no evidence that a stable, crystalline form of lower energy than the relaxed glass can be formed. This is consistent with the observed physical stability data on several lots of tenapanor dihydrochloride under ICH accelerated and long-term storage conditions. ASSOCIATED CONTENT Supporting Information. Additional experimental details on X-ray diffraction, solid state NMR data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Radha R. Vippagunta: [email protected] Phone :5104567717 Author Contributions The manuscript was written through equal contributions of both authors. Both authors have given approval to the final version of the manuscript. ACKNOWLEDGEMENTS The authors would like to thank Triclinic labs for generating the data and their contributions. ABBREVIATIONS DFT, density functional theory; PXRD, powder X-ray diffraction; PXRD; PDF, Pair-wise Distribution Function. REFERENCES 1. Babu, N.J. and A Nangia. "Solubility

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Predicting the Physical Stability of Amorphous Tenapanor

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