Elucidating the dehydration mechanism of ondansetron hydrochloride

Publication Date (Web): August 27, 2018 ... In drug development, evaluating the stability of the hydrate is particularly important because changes in ...
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Elucidating the dehydration mechanism of ondansetron hydrochloride dihydrate with a crystal structure Ryo Mizoguchi, and Hidehiro Uekusa Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b01014 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Elucidating the dehydration mechanism of ondansetron hydrochloride dihydrate with a crystal structure Ryo Mizoguchi†‡ and Hidehiro Uekusa*† †Department of Chemistry and Materials Science, Tokyo Institute of Technology, Ookayama 2-12-1, Meguro-ku, Tokyo 152-8551, Japan ‡Analytical Research Labs, Astellas Pharma Inc. 180, Ozumi, Yaizu-shi, Shizuoka 425-0072, Japan

In drug development, evaluating the stability of the hydrate is particularly important because changes in the hydration state could cause alterations of the physicochemical properties. In this study, it was found that ondansetron hydrochloride dihydrate, a competitive serotonin type 3 receptor antagonist, dehydrated to anhydrate in two steps with a hemihydrate form as an intermediate. The crystal structures of the unstable hemihydrate and anhydrate forms were successfully analyzed through the structure determination from powder diffraction data technique using high temperature synchrotron X-ray diffraction data. Comparison of the crystal structures of the dihydrate, hemihydrate revealed that in the first dehydration step the water molecule hydrogen-bonded to the imidazole ring was removed. In the second step, the remaining water molecule that was closely bound to the chloride anions was removed to form a void. Moreover, the molecular rearrangement caused by the dehydration was examined through analysis of the intermolecular interactions. Furthermore, the hygroscopic properties of the hydration and dehydration were investigated. These phase transitions triggered by dry and high temperature conditions are common in drug development. Therefore, the crystal structure and physicochemical properties of all phases of hydrates and anhydrates should be characterized.

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*Corresponding author Name: Hidehiro Uekusa Address: Ookayama 2-12-1, Meguro-ku, Tokyo 152-8551, Japan Phone: +81-3-5734-3529. Fax: +81-3-5734-3529. Email: [email protected].

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Elucidating the dehydration mechanism of ondansetron hydrochloride dihydrate with a crystal structure Ryo Mizoguchi†‡ and Hidehiro Uekusa*† †Department of Chemistry and Materials Science, Tokyo Institute of Technology, Ookayama 212-1, Meguro-ku, Tokyo 152-8551, Japan ‡Analytical Research Labs, Astellas Pharma Inc. 180, Ozumi, Yaizu-shi, Shizuoka 425-0072, Japan

In drug development, evaluating the stability of the hydrate is particularly important because changes in the hydration state could cause alterations of the physicochemical properties. In this study, it was found that ondansetron hydrochloride dihydrate, a competitive serotonin type 3 receptor antagonist, dehydrated to anhydrate in two steps with a hemihydrate form as an intermediate. The crystal structures of the unstable hemihydrate and anhydrate forms were successfully analyzed through the structure determination from powder diffraction data technique using high temperature synchrotron X-ray diffraction data. Comparison of the crystal structures of the dihydrate, hemihydrate revealed that in the first dehydration step the water molecule hydrogen-bonded to the imidazole ring was removed. In the second step, the remaining water molecule that was closely bound to the chloride anions was removed to form a void. Moreover, the molecular rearrangement caused by the dehydration was examined through

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analysis of the intermolecular interactions. Furthermore, the hygroscopic properties of the hydration and dehydration were investigated. These phase transitions triggered by dry and high temperature conditions are common in drug development. Therefore, the crystal structure and physicochemical properties of all phases of hydrates and anhydrates should be characterized.

Introduction Drug development is a long and slow process, which incurs considerable costs. In order for a drug to reach the market, it needs to go through several processes, including candidate selection, preclinical and clinical studies, and approval. Today, the success rate of finding a new drug candidate to enter the development stage is decreasing and the costs of this process continue to increase.1 Therefore, creating new strategies to overcome the decreasing success rate is paramount.2

Among many properties of the drug candidates, such as drug efficacy, toxicity, metabolic profile and marketability, the solid-state physicochemical properties are key in the drug development process.3 For example, the low solubility of a drug candidate can strongly impede the development process by making it difficult to attain good oral absorption and develop intravenous formulations.4

A well-known case, in which the solid-state properties of a molecule affected the drug discovery, is the development of ritonavir. Crystal form I of ritonavir was used as crystal form of drug substance. After the launch of the drug, crystal form II was discovered. As a result, crystal form I of ritonavir could not be obtained because of the presence of the more thermodynamically stable form II.5 This form, however, had lower solubility than the metastable form I. Thus, the

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exposure level in the blood at which the drug exerts its pharmacological effect could not be achieved. As a result, ritonavir had to be removed from the market, and additional clinical studies were needed.

Additionally, a thorough evaluation of the stability of the forms of a drug candidate is extremely important in the early drug discovery stages.6 The chemical stability of a potential drug such as the tendency of degradation by chemical reactions is predicted by accelerated stability studies in the early stages of the development process.7 However, the physical stability of the crystal forms such as phase transition among polymorphs or hydrates (dehydration and hydration) is not effectively investigated through these studies.8 In case the physical stability changes at room temperature, the drug form should be carefully evaluated.9

Evaluation of the stability of hydrate forms is essential because a change in the hydration state can have a serious impact on the solid-state properties. Such a change can easily occur when the humidity or temperature is changed.10-13 For example, FK041 forms a clathrate hydrate crystal which shows continuous variations in the amount of water with the changes in humidity.14 Moreover, the chemical stability of the FK041 hydrate exhibited strong dependence on the amount of water present in the crystal.14 This shows that dehydration and hydration processes can cause changes in the physicochemical properties of compounds, which can significantly affect drug discovery. Therefore, it is crucial to evaluate the physicochemical properties of both hydrates and anhydrates, and understand the mechanism of phase transition between them.15-19

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For these reasons, it is important to find the crystal structure of the unstable anhydrate form. However, observation of the dehydration and hydration phase transitions by single crystal X-ray diffraction analysis15 is often difficult. This is due to the fact that large crystal structure changes can lead to the degradation of the single crystalline form and the formation of a polycrystalline form. Also, the anhydrate form is often unstable at ambient temperature and humidity. In such cases, the structure determination from powder diffraction data (SDPD) method can be employed. It represents a powerful and useful technique for analyzing unstable polycrystalline forms such as dehydrated products.19-24 Recently, SDPD has become more widely used due to the development of new indexing methods, the direct space methods and Rietveld refinement.25, 26

Ondansetron (Figure 1), also known as Zofran, was developed by GSK and launched in the USA in 1991. Ondansetron is a competitive serotonin type 3 receptor antagonist and is effective in the treatment of nausea and vomiting caused by cytotoxic chemotherapeutic drugs.27 Patents for ondansetron have already expired, but Zofran was a blockbuster drug with peak annual sales of more than 1 billion US$. While the crystal structure of ondansetron hydrochloride dihydrate has been reported (CSD refcode: YILGAB),28 the dehydration behavior and crystal structure after dehydration have not yet been explored.28-30

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O

N1 C2

C3

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C4

N

N

C1

N

Figure 1. Chemical structure of ondansetron. Torsion angles: φ1 is formed by C1-C2-C3-N1; φ2 is formed by C2-C3-N1-C4.

The purpose of this study was to understand the mechanism of dehydration and hydration by comparing the crystal structures of active pharmaceutical hydrates. We used ondansetron hydrochloride dihydrate as a model compound (Figure 1) to try to clarify the mechanism underlying the transition of hydrates to anhydrates.

Experimental Materials Ondansetron hydrochloride dihydrate was purchased from Sigma Aldrich (St. Louis, MO, USA). All characterizations were performed using samples pulverized with an agate mortar. Samples were heated in a Fine Oven DF42 (Yamato Science, Tokyo, Japan).

Thermal analysis Differential scanning calorimetry (DSC) Thermal analysis was performed using TA Q20 DSC instrument that included a refrigerated cooling system (TA Instruments, New Castle, DE, USA). Temperature calibrations were carried

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out using the indium metal standard supplied with the instrument. Samples were weighed (about 3 mg) in aluminum pans and analyzed with a temperature range from 25 °C to 300 °C at a heating rate of 10 °C/min using a similar, but empty pan as a reference. An inert atmosphere was maintained in the calorimeter by purging with nitrogen gas at a flow rate of 50 mL/min.

Thermogravimetric analysis (TGA) TGA was performed using TA Q50 TGA (TA Instruments, New castle, DE, USA). Approximately 4 mg of the sample was loaded onto a platinum pan and heated to 300 °C at a rate of 10 °C/min. Measurements were carried out under nitrogen purge at a flow rate of 100 mL/min. Temperature calibrations were carried out using standard nickel. To identify the intermediate, a sample was heated in jump mode to 45, 50, and 55 °C, and kept at each temperature for 60, 40, and 20 min, respectively. Measurements were carried out under a nitrogen purge at a flow rate of 50 mL/min (sample purge gas only employed).

Water vapor sorption and desorption studies Dynamic vapor sorption experiments were performed on VTI SGA 100 (VTI corporation, Hialeah, FL, USA). Samples (about 10 mg) were studied over a selected humidity range [from 5−50% relative humidity (RH) to 5−95% relative humidity (RH)] at each temperature (30, 35, 40, 45, and 50 °C). For each humidity step, the equilibration was set to dm/dt 0.03%/min on a 5min time frame (maximum hold time 180 min).

Powder crystal X-ray diffraction measurement (PXRD)

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PXRD measurements were performed on TTR II (Rigaku, Tokyo, Japan) with Cu Kα radiation at 1.54184 Å at a voltage of 50 kV and current of 300 mA. Data were collected at a scan rate of 4º/min over a 2θ range from 2.5º to 40º. In variable temperature PXRD measurements, the simultaneous measurement of powder X-ray diffraction data and differential scanning calorimetry (XRD-DSC) was carried out on a SmartLab system (Rigaku, Tokyo, Japan) using Cu Kα radiation at 1.54184 Å at a voltage of 45 kV and current of 200 mA, with a DSC attachment and D/Tex Ultra as a detector. Samples were weighed (1.5–2.5 mg) in aluminum pans and analyzed at a heating rate of 2 °C/min using a similar, but empty pan as a reference. X-ray diffraction data were collected at a scan rate of 20°/min over a 2θ range of 10° to 25°. For the synchrotron X-ray measurements at SPring-8, the powder samples were enclosed in a 0.3 mm Lindemann glass capillaries. The X-ray powder diffraction data were collected at SPring-8 BL19B2,31 which is equipped with a high-resolution type Debye–Scherrer camera and a curved imaging-plate detector. The wavelength was set at 1.0000 Å. For the variable temperature measurements, it took one minute to set each temperature. This temperature was then maintained for 4 min to ensure equilibrium was reached before the measurements were taken. Data were collected for 5 min. During data collection, the sample was maintained at the set temperature and rotated at 1 r/min to reduce possible preferential orientation effects.

Structure determination from powder diffraction data (SDPD) The crystal structures of both the hemihydrate and anhydrate were determined from the PXRD data measured at SPring-8 BL19B2. Crystal structure analysis was carried out using the Powder Solve module of Materials Studio (BIOVIA, Tokyo, Japan).

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After selecting the peaks set, indexing was conducted in the XCELL module32 in order to introduce the unit cell and appropriate space group. The unit cell was refined by the Pawley refinement. The initial chemical structures of ondansetron and the water molecules were introduced using the Forcite module with COMPASS II33 as a force field. The initial crystal structure was introduced by the POWDER SOLVE module34 using the simulated-annealing approach and then optimized by the Rietveld refinement. Pareto optimization, a Rietveld refinement method that considers the energy of the structure calculated by a force field,35 was carried out at the final optimization step.

Results and Discussion The crystalline phase transitions of ondansetron hydrochloride dihydrate by heating were confirmed using thermal analysis (Figure 2). In Figure 2A, an endothermic peak with decreasing weight was observed starting from room temperature to ca. 120 ºC. The weight change in percentage was ca. 10% which is consistent with the calculated value for the dehydration of two water molecules from this dihydrate (calc. 9.85%). Therefore, the endothermic peak was assigned to the dehydration of the dihydrate to form the anhydrate. After this dehydration, the melting point of the anhydrate form was observed to be around 180 ºC. Moreover, the derivation curve of the TGA showed two peaks (ca. 80 and 100 ºC), indicating that the dehydration process was not simple, but occurred in two steps (Figure 2B). These have been looked at in detail and are described later in the text.

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Figure 2. (A) Thermal behavior of ondansetron hydrochloride dihydrate. Blue line indicates the differential scanning calorimetry curve, orange line indicates the thermogravimetric (TG) curve. (B) The TG curve (orange) was overwritten by the derivation of the TG (green).

In order to investigate the physicochemical properties, the stability of the anhydrate form was examined using the time course data of the PXRD (Figure 3). First, the dihydrate form was heated to 100 ºC for 30 min to form the anhydrate. The samples were then left at ambient conditions for varying amount of time, after which the PXRD patterns were measured. There was an apparent difference between the PXRD patterns of the anhydrate form (0 h; Figure 3, red

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pattern) and the dihydrate form before heating (Figure. 3 black pattern). However, the XRD patterns measured after 0.5 and 1 h (Figure 3; blue and green patterns, respectively) appeared the same as that of the dihydrate form. This result suggests that the anhydrate was highly unstable at ambient conditions and quickly transformed back into its initial dihydrate form. Therefore, further experimental investigation on the physicochemical properties of the isolated anhydrate form would be impossible.

Figure 3. Reversibility after heating confirmed with X-ray diffraction patterns: dihydrate as a reference (black), 0 h (red), 0.5 h (blue), 1 h (green) after heating at 100 ºC.

The two-step dehydration process of the dihydrate was further investigated by variabletemperature PXRD measurements with a range from room temperature to 95 ºC (Figure 4). There was a significant change of the PXRD patterns observed at around 45 ºC, while at around 70 ºC only a small change was present. These results were consistent with the TGA derivation curve (Figure 2B) and clearly indicate the existence of an intermediate form as a crystalline state

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in the dehydration of ondansetron hydrochloride dihydrate. Moreover, above 75 ºC, the PXRD patterns of the anhydrate form, which was also suggested by the result of the thermal analyses in Figure 2A, were observed.

Figure 4. Variable temperature X-ray diffraction by XRD-DSC measurement. Patterns of: dihydrate (red), intermediate (green), anhydrate (blue). Arrows and pale broken lines shows the change of characteristic peak positions.

In order to determine the stoichiometry of the hydration state of the intermediate form, timecourse TGA was carried out at fixed temperature (Figure 5) (45, 50, and 55 ºC, at which the intermediate form was stably observed in Figure 4). The consistent decrease in weight (%) for all three measurements corresponded to 1.5 water molecules, indicating that the intermediate was a hemihydrate form. At more than 60 ºC in TGA condition, an intermediate could not be obtained and a dihydrate directly transformed to an anhydrate (data not shown). Therefore, a two steps

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dehydration behavior of the ondansetron hydrochloride dihydrate was established and the intermediate form of the dihydrate to anhydrate transition was found to be a hemihydrate.

Figure 5. Identification of the intermediate using a thermogravimetric analysis. Changes in weight (%) at different temperatures: 45 ºC (green), 50 ºC (red), 55 ºC (blue).

The crystal structure analyses of both the intermediate (hemihydrate) and anhydrate were carried out using the SDPD technique. The measurements were carried out at high temperature because both phases were unstable at ambient conditions such as room temperature and humidity. The SDPD were conducted using the PXRD data of the hemihydrate (at 95 ºC) and anhydrate (at 140 ºC), which were measured at BL19B2 in SPring-8 (synchrotron X-ray experiments). The temperature for the measurements of the intermediate form was determined to be 95 ºC, which was higher than the temperature range indicated in the variable temperature PXRD (45 to 75 ºC, Figure 4) using the laboratory equipment. This could be due to a slight pressure difference in the two measurements. For high temperature measurements at BL19B2 in SPring-8, sample crystals were enclosed in a capillary with diameter of 0.3 mmφ. In high-

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temperature and dehydrating conditions, the pressure in the capillary is expected to be higher than that of the open pan, which was used for the thermal analysis (Figure 2) and the variable temperature PXRD (Figure 4). The increased pressure in the capillary would delay the dehydration transition because the dehydration behavior of crystalline phase strongly depends on pressure. Thus, the intermediate form would appear in the higher temperature condition. Other possible cause of the temperature variation may be the difference in the heating methods (SPring-8: N2 gas stream to capillary; laboratory system: DSC style heat conducting).

The crystal data from the SDPD measurements are presented in Table 1. The crystal structure analysis of the hemihydrate showed an occupancy of water molecules was fixed of 0.5 following the thermal analysis results (Figure 5), and the value was not optimized through Rietveld refinement. The final Rietveld refinement fittings are presented in Figure 6. The SDPD analysis showed that during the transition from dihydrate to hemihydrate the β angle of the unit cell became larger (from 101º to 115º) and the unit cell volume decreased by 74.7 Å3, which corresponds to a loss of 1.5 water molecules. In contrast, the transition from hemihydrate to anhydrate did not cause any large changes in the lattice parameters.

Table 1. Crystal data from the SDPD of the ondansetron hydrochloride hydrates Dihydrate28 Molecular formula

C18H20N3OCl.2H2O

Hemihydrate

Anhydrate

C18H20N3OCl.0.5H2O

C18H20N3OCl

Sample type Single crystal

Powder

Powder

Crystal system

Monoclinic

Monoclinic

Monoclinic

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Space group P21/c

P21/c

P21/c

a (Å)

15.082(3)

15.375(2)

15.084(4)

b (Å)

9.741(3)

9.907(1)

9.898(2)

c (Å)

12.734(3)

12.719(2)

12.913(3)

β (º)

100.83(1)

114.508(1)

114.717(2)

V (Å3)

1837.5(8)

1762.8(4)

1751.1(7)

R-Factor (%) 7

-

-

Rp (%)

-

7.16

7.39

Rwp (%)

-

10.30

10.16

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Figure 6. Final Rietveld refinement for ondansetron (A) hemihydrate (at 95 ºC) and (B) anhydrate (at 140 ºC). Red dots: measured data points; blue line: calculated pattern; black line: difference profile; green ticks: calculated peak positions; pink ticks: systematic absence markers.

Projection views along the b-axis are presented in Figure 7A. In the crystal packing, there are two layers of hydrophobic and hydrophilic nature, which are occupied by the ondansetron cation, and water and chloride anions, respectively. The total packing structures in all three phases appeared similar. In fact, the molecular conformation of the ondansetron cation and the π-π interaction between the tricyclic parts of the ondansetron cation were retained during the transition. Thus, the layer of the ondansetron cation (hydrophobic layer) did not change throughout the crystal phase transition, indicating that the structural transition mainly took place in the hydrophilic layer.

From the three structures in Figure 7A, further investigations were performed on the local structure consisting of two symmetry-related imidazole moieties, chloride anions, and water molecules that are connected by a network of hydrogen bonds (Figure 7B). Since hydrogen bonds were known to affect the formation of hydrate,36 the list of hydrogen bonds was shown in Table S1. In the first dehydration step, from dihydrate to hemihydrate, the water molecules that interact with the imidazole cation were removed. Also, the half occupancy of a water molecule, which interacts with two chloride anions, was removed. In the second dehydration step, from hemihydrate to anhydrate, the water molecule that interacts with two chloride anions was removed. As a result, a void space with a volume of 6.1 Å3 was left at the position that the water molecule occupied in the hemihydrate form. The presence of this small void may be the

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underlying reason for the instability of the anhydrate form and its rapid transformation back to the hydrate form.

(A)

hydrophilic hydrophobic

(B)

Figure 7. Comparison of the crystal forms observed throughout the crystal transition. (A) View along the b-axis. Hydrophobic and hydrophilic layers are indicated by the regions enclosed by the dotted lines. (B) Hydrogen bond networks of the dihydrate (red square), hemihydrate (green square) and anhydrate (blue square).

According to the results from the crystal structure analysis, the first dehydration step has also produced a void space. This, however, was filled by a translation of the imidazole rings to form

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a π-π stacking in order to stabilize the crystal structure. In contrast, in the second dehydration reaction, the structure around the imidazole rings was not changed (Figure 8).

Figure 8. Comparison of the crystal forms around the imidazole rings in the dihydrate (red square), hemihydrate (green square) and anhydrate (blue square) forms.

Due to the structural change induced by the dehydration transition, the distance between the chloride anions was reduced (dihydrate: 5.228 Å, hemihydrate: 4.917 Å, anhydrate: 4.755 Å in Figure 7B). In the first dehydration step, this distance was decreased by 0.31 Å, which is caused by the half-removal of the water molecule between the chloride anions to form the hemihydrate. In contrast, in the second dehydration reaction, the change in the distance was about 0.16 Å. This value was only a half of the decrease observed in the first dehydration step, although the removed amount of water between the chloride anions was same. This could be due to the repulsion forces between the two chloride anions, which resulted in the formation of the void space. The structural changes can also be explained from an intermolecular-interaction point of view (Figure 7B). In the first dehydration step, two hydrogen bonds were lost: the charge-assisted hydrogen bond between the imidazole cation and the water molecule (NH+…O), and the hydrogen bond between the water molecule and chloride anion (OH…Cl–). However, these losses were compensated by the formation of a new ionic interaction between the imidazole

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cation and chloride anion (NH+ and Cl–) and the π-π stacking between the two imidazole rings in the hemihydrate form. Additionally, the remaining water molecule had two charge-assisted hydrogen bonds to two symmetry-related chloride anions (OH… Cl–). These interactions could contribute to the formation of a diamond-shape like structure from one water molecule and two chloride anions in the hemihydrate form because the occupancy was 0.5. All these interactions could stabilize the water molecules in the diamond-shape like structure. However, as a negative factor to that, the ionic interaction between the imidazole cation and chloride anion (NH+ and Cl– ) in the hemihydrate form caused the electron-donating capacity of the chloride anions to be reduced. Thus, the OH…Cl– charge-assisted hydrogen bonds could be weaker than expected. As a result of these interactions in the intermediate hemihydrate form, the occupancy of the water molecule in the diamond-shape structure was not maintained as one, but became half.

As can be seen from the overlay of the ondansetron cation structure across the stages, no significant change of the molecular structure was observed in the two-step transition (Figure 9). An overlay of the ondansetron cation across the stages is shown in Figure 9. The torsion angles: φ1 (C1-C2-C3-N1) changed from 179º to -171º and φ2 (C2-C3-N1-C4) from 109º to 111º.

Figure 9. Overlay of the molecular structures of ondansetron in each crystal form: dihydrate (red), hemihydrate (green), anhydrate (blue).

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The dimer structure that was formed from the interaction of the tricyclic rings of the ondansetron cation was also kept across all crystalline phases. The distance between the rings was about 3.6 Å, allowing for the π−π stacking interaction to be maintained throughout the dehydration process (Figure 10). In addition, CH−π interaction was observed. The distance between H(C2) and the tricyclic ring was about 2.5 Å. These hydrophobic interactions contributed to the stabilization of the crystal structure. Also, the structural similarity of these three phases enabled the anhydrate to rapidly transform back to the dihydrate form when in the solid state.

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Figure 10. Stacking interaction between the tricyclic parts of ondansetron. (A) Top view, (B) side view. Dotted lines indicated the CH−π interaction.

The dehydration and hydration tendency (hygroscopicity) of the ondansetron hydrochloride dihydrate in various levels of relative humidity (5-50% RH) at 30 ºC were measured (Figure 11). Even at low humidity (5% RH), the dehydration was not complete and neither an anhydrate nor hemihydrate were formed. In the transition between dihydrate and hemihydrate, a significant structural change was observed, which included an alteration of the hydrogen bonds and translation of the imidazole rings. Due to this large modification, the phase transition took longer to complete, which led to a hysteresis in the water desorption and absorption chart in Figure 11.

Figure 11. Hygroscopicity of ondansetron hydrochloride dihydrate at 30 ºC.

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The temperature dependency of the hygroscopicity was measured using a similar process to the one in the hygroscopicity study in Figure 11, but the temperature was varied. The desorption and absorption curves that were obtained are shown in Figure 12A and 12B, respectively. Similar to the results from Figure 11, no anhydrate was observed even at higher temperature (50 ºC) and low humidity (5% RH). The results also indicated that the critical relative humidity (CRH) at which the phase transition occurs has the tendency to become higher at higher temperatures. Thus, the higher temperature leads to enhanced dehydration and hindered hydration. The phenomenon of hysteresis was also observed by comparing the desorption and absorption charts in Figure 12. The hysteresis did not depend on temperature, and the differences in CRH (RH at the beginning of dehydration minus the RH at the beginning of hydration) were about 5-10% RH.

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Figure 12. Temperature dependence of hygroscopicity in the (A) desorption process (from 50 to 5% RH) and (B) absorption process (from 5 to 50% RH). The arrows indicate the reaction direction.

In summary, ondansetron hydrochloride dihydrate transformed into a hemihydrate under drying conditions as shown in Figure 5 (drying under N2 gas), Figures 11 and 12 (RH-controlled measurement). From the various factors affecting the drug development, such as synthesis of drug substance, formulation process, and transportation conditions, moisture-free conditions were highly important in order for the transformation to a hemihydrate to occur. Therefore, in order to mitigate the risk in drug development, the chemical and physical stability, as well as other physicochemical properties of all three forms (dihydrate, hemihydrate, and anhydrate) should always be assessed.

Conclusions In this study, the mechanism of dehydration was successfully elucidated using crystal structures. Elucidation of the mechanism of dehydration can be useful in the drug discovery of

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hydrates. Additionally, we investigated the dehydration of ondansetron hydrochloride dihydrate and for the first time identified the presence of an intermediate hemihydrate form preceding the transition to the anhydrate. Both the hemihydrate and anhydrate were found to be unstable at room temperature and relative humidity and the transition back to the dihydrate was too fast for either form to be isolated. In spite of these difficulties, the crystal structures of the two forms were successfully analyzed through the SDPD technique using synchrotron radiation powder Xray diffraction data. It was found that the water molecules and chloride anions form hydrophilic layers between the hydrophobic layers comprising of ondansetron cations. In the dehydration process, the volume of the hydrophilic layer was reduced but did not disappear at high temperature. It was retained due to the formation of a void in the structure of the anhydrate. This void could be responsible for the instability of the anhydrate at room temperature and relative humidity. Moreover, the dehydration and hydration tendency (hygroscopicity) of ondansetron hydrochloride dihydrate was examined. The results indicated that at low relative humidity, the dehydration progressed to form the hemihydrate. Also, the increase in temperature enhanced the dehydration while hindering the hydration. A hysteresis was observed between the hydration and dehydration processes, but it showed no dependence on the temperature. Because the dry and high temperature conditions are rather common in drug development stages the dehydration and hydration phase transitions of the hydrate form of API should be regarded as common. The physicochemical properties of the hydrate and dehydrate phases would differ depending on the crystal structure. Similar to the study we performed in this work on ondansetron hydrochloride dihydrate, all crystalline phases including the polymorphs or pseudo-polymorphs should be considered and the stability and properties assessed in order to mitigate the risk in drug development.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supporting information (PDF) Accession Codes CCDC 1853609-1853610 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contracting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; FAX +44 1223 336033.

AUTHOR INFORMATION Corresponding Author *Corresponding author Name: Hidehiro Uekusa Address: Ookayama 2-12-1, Meguro-ku, Tokyo 152-8551, Japan Phone: +81-3-5734-3529. Fax: +81-3-5734-3529. Email: [email protected]. Author Contributions

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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The synchrotron radiation experiments were performed at the BL19B2 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2014B1867 and 2015A1744). ABBREVIATIONS API, active pharmaceutical ingredient; CRH, critical relative humidity; DSC, differential scanning calorimetry; PXRD, powder X-ray diffraction; RH, relative humidity; SDPD, structure determination from powder diffraction; TGA, thermogravimetric analysis

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For Table of Contents Use Only

Elucidating the dehydration mechanism of ondansetron hydrochloride dihydrate with a crystal structure Ryo Mizoguchi†‡ and Hidehiro Uekusa*†

SYNOPSIS The dehydration of ondansetron hydrochloride dihydrate was investigated and for the first time identified the presence of an intermediate hemihydrate form preceding the transition to the anhydrate. The crystal structures of the two forms were successfully analyzed through the SDPD technique using synchrotron radiation powder X-ray diffraction data. The mechanism of dehydration was successfully elucidated using crystal structures.

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