Enhanced Solubility of Famotidine through Salification

Abstract

Famotidine (FMT) is an active pharmaceutical ingredient that exhibits poor aqueous solubility and poor permeability. Molecular electrostatic potential and pK_a calculations were used to guide a salification strategy for FMT and afforded five salts featuring carboxylic acid-containing coformers. All solids feature charge-assisted hydrogen bonds between the guanidinium group of FMT and carboxylate of the coformer. All salts exhibited good thermal and benchtop stability, and a six- to 20-fold increase in aqueous solubility was achieved, depending on the coformer used. The best performing salts include coformers on the FDA’s generally recognized as safe list, making the solids promising for pharmaceutical applications.

Graphical Abstract

1. INTRODUCTION

Famotidine (FMT, Figure 1), the active pharmaceutical ingredient (API) present in the commercial drug, Pepcid, is a histamine H₂-receptor blocking agent utilized to treat heartburn,¹ gastric ulcers,² and other gastrointestinal disorders related to hypersecretion of stomach acid. It works to reduce the volume and production of stomach acid by selectively binding to H₂ receptors present in the cells lining the stomach walls.^3,4 Compared to other H₂ antagonists, FMT shows higher potency and a longer duration of action⁵ while having fewer drug interactions, making it a competitive choice over drugs such as cimetidine and ranitidine.⁶ According to the biopharmaceutical classification system (BCS), FMT is a class IV drug, meaning that it has poor aqueous solubility and poor permeability, effectively limiting its therapeutic efficacy.^7–9 Additionally, FMT exists primarily as two polymorphic forms, A and B, with form B being metastable, but more bioavailable.¹⁰ Form B is the commercially marketed form; however, there are concerns with form B converting to form A during storage and handling.¹¹ A third form of FMT, form C, has been discovered, but has not been well explored or characterized structurally.¹²

Figure 1.

Chemical structures of FMT (highlighting key functional groups) and the chosen coformers. The asterisk (*) denotes coformers not on the GRAS list.

Approximately 40% of APIs on the market, and an estimated 90% of APIs in development stages, exhibit poor water solubility, either falling into BCS class II or IV.^13,14 Thus, strategies that improve drug properties and enhance pharmaceutical efficacy are needed. Several methods to improve drug performance have been investigated and are often based on modification to the formulation (e.g., nanosuspensions, solid dispersions, particle-size reduction)^15,16 or the phase (e.g., excipients, salts, cocrystals).^17–20 Cocrystallization, combination of two or more components into a unique solid phase, has been previously applied to FMT and afforded solids with improved water solubility.^9,21,22 Several of the known multicomponent phases were prepared through salification of FMT, affording salts, wherein charge transfer occurs between the components. For example, Zeleke and Sarma prepared six FMT salts with hydroxybenzoic and aminobenzoic acids that exhibited improved solubilities ranging from approximately four- to 23-fold.²³ Solubility enhancement of FMT has also been seen in salts with m-nitrobenzoic acid,²⁴ maleic acid,²⁵ and several more solids, with carboxylic acids predominantly serving as the second component. In these FMT salts, proton transfer occurs at the guanidine moiety, and improved solubility has been attributed to higher water solubility of the second component in the solid (i.e., coformer), as well as formation of hydrogen bonds between the components.

Here, we aimed to improve the aqueous solubility of FMT through crystallization with coformers containing carboxylic acids, which exhibit higher aqueous solubility and are expected to form strong hydrogen bonds with FMT. With this goal in mind, we chose both aromatic and aliphatic acids including nicotinic acid (NA), adipic acid (Adi), and acetic acid (AA), which are all on the FDA’s generally recognized as safe (GRAS) list²⁶ (Figure 1). To further investigate structure–property relationships, cocrystallization of FMT with two isomers of NA, namely, picolinic acid (PA) and isonicotinic acid (INA), was also conducted. Each new solid was characterized structurally, spectroscopically, and thermally. By controlling the noncovalent interactions between the API and the coformer, we demonstrate enhanced aqueous solubility across the board and either increased or decreased solubility under strongly acidic conditions, depending on the coformer identity. We also discuss correlations between thermal stability, solubility, and lattice energy. Overall, the work describes the improvements in drug properties that can be achieved through a salification approach using small molecule coformers.

2. EXPERIMENTAL SECTION

2.1. Salt Preparation

Salts of FMT and NA or Adi were prepared by liquid-assisted grinding (LAG) of FMT and the appropriate coformer in a 1:1 molar ratio at 1500 rpm for 10 or 6 min, respectively. Ethanol (30 μL) was used for NA and methanol (30 μL) was used for Adi. FMT-AA was prepared by LAG using excess AA at 900 rpm over 1 h.²⁷

The resulting solids obtained from LAG experiments were dissolved in acetonitrile/H₂O (volume ratio 1:1, FMT-NA·H₂O) or methanol (FMT-Adi·MeOH and FMT-AA) and allowed to slowly evaporate for single crystal growth. Salts of FMT and INA or PA were synthesized by dissolving a 1:1 molar ratio of FMT and the coformer in acetonitrile/H₂O (volume ratio 1:1). Both solutions were allowed to evaporate at room temperature, yielding suitable single crystals within a week. Each new phase was characterized by nuclear magnetic resonance (NMR) spectroscopy, powder X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, and single-crystal X-ray diffraction (SCXRD). Each phase was further characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).

2.2. pK_a Calculations

To assess if FMT and a given coformer would be expected to produce a salt or a cocrystal, ΔpK_a studies were carried out using the Chemaxon protonation calculator.²⁸ According to the ΔpK_a rule,^29,30 if the ΔpK_a is less than −1 then a cocrystal will form, and if the ΔpK_a is greater than 4 then a salt will form. If the ΔpK_a falls somewhere in the middle, then either type of solid could form and the probabilities of each can be calculated. The equation for determining ΔpK_a and the probabilities are below.³⁰ Equation 1 affords the ΔpK_a value. (Equations 2 and 3) calculate the probability of a cocrystal (2) and a salt (3) if the ΔpK_a value falls between −1 and 4.

$$\text{Δp}{K}_{\text{a}}=\text{p}{K}_{\text{a}}(\text{protonated base})-\text{p}{K}_{\text{a}}(\text{acid})$$ (1)

$$P_{\text{obs}}(\text{AB})=-17\text{Δp}{K}_{\text{a}}+72$$ (2)

$$P_{\text{obs}}({\text{A}}^{+}{\text{B}}^{-})=17\text{Δp}{K}_a+28$$ (3)

2.3. Lattice Energy Calculations

Lattice energy calculations were conducted using the Mercury program from the Cambridge structural database portfolio. A Gavezzotti force field³¹ was used with a limiting radius of 30 Å and electrostatic correction was done using the Evjen method.³²

2.4. Electrostatic Potential Calculations

Gas-phase energy calculations in the ground state were performed using the Spartan′24 graphical user interface.³³ The geometry of the structure was optimized using a semiempirical method with the AM1 basis set.^34,35 Once optimized, a DFT calculation was performed using the B3LYP functional^36–38 and the 6–311++G** basis set.³⁹ The electrostatic potential map was computed and mapped using the isodensity surface 0.0020 e/au³.

2.5. Stability Studies

Benchtop stability studies of FMT-NA·H₂O, FMT-PA, FMT-INA· H₂O, and FMT-AA were carried out by subjecting each solid to three different environments, and stability was evaluated over 14 days. The environments involved: (I) placing samples into an open vial at ambient temperature, (II) placing samples in an open vial while heating at 40 °C (accelerated thermal), and (III) placing samples in a small vial that was put inside a larger, closed vial containing water while heating to 40 °C (accelerated thermal and humidity). The stability was assessed via PXRD on day 0, 7, and 14. For salt-hydrates, TGA studies were carried out on day 14 to evaluate water loss.

2.6. Solubility Studies

Solubility studies were conducted at 25 °C using the shake-flask method^40,25 with DI water or pH 1.2 buffer. An excess amount of each solid and 2 mL of medium (water or buffer) were added to 20 mL glass scintillation vials. The vials were capped, and the mixtures were stirred and maintained at 25 °C for 24 h (water) or 1 h (buffer). FMT gradually decomposes in solution at pH 1.2,⁴¹ necessitating a shorter experiment time compared to water. After stirring, each solution was allowed to rest for 30 min (water) or 15 min (buffer). Then, the suspended solid was removed by filtration through a Fisherbrand 13 mm, PTFE, nonsterile (0.2 μm pore size) syringe filter. The filtrate was appropriately diluted and analyzed by UV–vis spectroscopy based on the Beer–Lambert law. The measurements were conducted on an Agilent 8453 UV–vis Spectrophotometer, and the maximum absorption wavelength for each salt in each medium was used. The absorption coefficient of each salt was measured using calibration curves in DI water and pH 1.2 buffer.

3. RESULTS AND DISCUSSION

3.1. Calculations

Although multicomponent solids of FMT are known,^9,22–24,41 the drug contains several hydrogen-bond donors and acceptors, which makes choosing suitable coformers that will form hydrogen bonds at specific sites more challenging. Thus, molecular electrostatic potential (MEP) calculations were employed to understand which hydrogen-bonding sites should be targeted on FMT. The electrostatic potential map showed a weaker positive potential on the amine hydrogen that is closest to the nitrogen in the thiazole group (117 kJ/mol). Based on previously reported FMT crystal structures, this hydrogen often forms an intramolecular hydrogen bond with the thiazole nitrogen to yield a six-membered ring;^23,24 thus, this amine was excluded as a strong hydrogen-bond donor. Most notably, the MEP map showed an area of high negative potential on the imine nitrogen within the guanidine group (−142 kJ/mol) and an area of high positive potential on the neighboring NH₂ group (201 kJ/mol), indicating a strong potential for a two-point hydrogen bond to occur at this location (Figure 2, top). The oxygens located on the sulfonamide group also demonstrated high negative potentials; however, previously reported salts and cocrystals with FMT rarely showed interactions between the sulfonamide group and the coformer. Instead, the sulfonamide group tends to form intramolecular interactions or interact with a sulfonamide group on a neighboring FMT molecule in the solid state. This led us to target the guanidine group on FMT as a site of hydrogen bonding using carboxylic acid coformers, which often form two-point hydrogen bonds.

Figure 2.

MEP map of FMT with potential values labeled (top) and FMT showing calculated pK_a values for protonation at the indicated site (bottom).

Aromatic carboxylic acids have been used by Sarma and coworkers to form several salts with FMT that exhibit improved solubility, and many coformers contained an additional hydrogen-bond-donor moiety (e.g., phenol, amine).^22,23 Here, we selected carboxylic acids from the FDA’s GRAS list that had not previously been crystallized with FMT as coformers and included Adi, AA, and NA. Two isomers of NA, PA and INA, were also chosen to examine the effect of slight structural modifications and the presence of hydrogen-bond acceptor moieties on solid-state properties. To determine the likelihood of obtaining a salt or cocrystal with FMT and each coformer, the ΔpK_a values²⁸ were calculated (eq 1, Table 1). If the ΔpK_a is less than 0 or −1, a cocrystal is expected to form, while a ΔpK_a greater than 4 predicts salt formation.^29,30 The guanidine present in FMT has a pK_a value of 8.44 (protonated imine nitrogen, Figure 2, bottom), and, as shown in Table 1, crystallization of FMT with the aromatic acids NA, PA, and INA is predicted to form salts. For solids formed with the aliphatic acids, Adi and AA, the ΔpK_a lies between −1 and 4; thus, the probability was calculated using eqs 2 and 3. For Adi and AA, formation of a salt was 93% and 94% likely, respectively.

Table 1.

Calculated pK_a Values for the Coformers and Corresponding ΔpK_a Values Using FMT (Guanidine Site) as the Base^b

Coformer	Coformer pKa	ΔpKa
NA	2.31	6.13
PA	1.48	6.96
INA	2.01	6.43
Adia	4.62	3.82
AA	4.54	3.90

^aFor Adi, only a single deprotonation was used for the calculation, and the value shown is pK_a1.

^bThe pK_a value for FMT is shown in Figure 2.

3.2. Preparation and Characterization of FMT Salts

Crystallization of FMT-NA·H₂O, FMT-AA, and FMT-Adi·MeOH were accomplished using LAG experiments followed by slow solvent evaporation from acetonitrile/H₂O (for NA) or methanol (for AA and Adi) at room temperature to yield single crystals. PXRD revealed new peaks for each solid when compared to the starting materials following LAG experiments, confirming the presence of a new crystalline phase. The single crystals of FMT-PA and FMT-INA·H₂O were obtained by slow evaporation from solutions of acetonitrile/H₂O and methanol, respectively, at room temperature. SCXRD demonstrated that each solid was a salt and showed inclusion of water/solvent for some solids (Tables S1–S2, Figures S1–S5). FMT-NA·H₂O and FMT-INA·H₂O crystallized with water included in the lattice, and FMT-Adi·MeOH crystallized with methanol in the solid. Proton transfer between the carboxylic acid of the coformer and the imine nitrogen of the guanidine moiety occurred, as predicted by the ΔpK_a calculations and the electrostatic potential map of FMT. Furthermore, the PXRD pattern of the bulk crystalline solids matched with the simulated PXRD pattern for each single crystal (Figures S6–S11). The stoichiometric ratio between FMT and the coformer was confirmed via ¹H NMR spectroscopy for each solid and showed good correlation with the ratio determined by SCXRD (Figures S12–S17).

FTIR experiments for each solid demonstrated a shift in the C═O stretch when compared to the coformer, indicative of the formation of an intermolecular interaction (Figures S18–S22). Additionally, each of the salts showed a carboxylate stretch in the 1610–1540 cm⁻¹ region, confirming the occurrence of a proton transfer. FMT exhibits signals at 3504 cm⁻¹, 3399 cm⁻¹, and 3355 cm⁻¹, characteristic of the N–H vibrations of the guanidine group. All salts showed shifts within this region, indicating intermolecular interactions involving the guanidine moiety.

3.3. FMT Salts with Isomers of Pyridyl Carboxylic Acids

SCXRD experiments were employed to confirm the solid-state structure of each of the obtained salts with NA, INA, and PA (Figure 3). Two solids, FMT-NA·H₂O and FMT-PA, crystallize in the space groups, P2₁/n and P2₁/c, respectively. FMT-INA·H₂O crystallizes in the P-1 space group. The asymmetric unit of FMT-NA·H₂O contains one molecule each of FMT, NA, and water, while FMT-INA·H₂O contains two crystallographically unique molecules of FMT, INA, and water. The asymmetric unit of FMT-PA includes one molecule of each type. In all three solids, the acid group of the coformer forms a two-point hydrogen bond with the guanidine group of FMT. Proton transfer is first evident in each solid by the carbon–oxygen bond lengths within the coformer, which are nearly equal for each unique molecule (lengths differ by 0.006–0.018 Å) and indicate delocalization of electron density and deprotonation. Second, after protonation, the guanidinium is stabilized by resonance, and in all structures, the bond between the carbon and an NH₂ group is the shortest of the three C–N bonds, indicating double bond character (Table S3). The other hydrogen bonding interactions and structural assembly differs when the coformer is changed.

Figure 3.

X-ray crystal structures showing hydrogen-bonded chains within (a) FMT-NA·H₂O, (b) FMT-INA·H₂O, and (c) FMT-PA. Hydrogen bonds are shown with green dashed lines.

Within FMT-NA·H₂O, the pyridine nitrogen engages in a hydrogen bond with the NH₂ group of the amidine of FMT. The interactions involving the pyridine and carboxylate of NA facilitate formation of hydrogen-bonded chains between the two components that extend approximately along the a axis (Figure 3a). The water molecule in FMT-NA·H₂O plays a key role in bridging the chains along the c axis and facilitating assembly into a 2D hydrogen-bonded network. Each water molecule forms four hydrogen bonds with four different molecules. The water serves as a hydrogen-bond donor to the carboxylate group of NA and to a sulfonamide oxygen atom in a neighboring FMT. The sulfonamide NH₂ group of FMT donates two hydrogen bonds to adjacent water molecules, which also forms 1D hydrogen-bonded chains. Hydrogen bonds between neighboring FMT molecules at the sulfonamide oxygen atoms and NH₂ groups of the guanidine moiety further assist in formation of the sheet. Weak C–H contacts dominate interactions between the 2D networks.

The water molecule containing oxygen O10 in FMT-INA·H₂O was determined to be partially occupied due to spontaneous dehydration when handling the single crystals. While this water molecule donates strong hydrogen bonds, it only interacts with species that are stabilized by other strong hydrogen bonds and could be removed without disrupting the packing, allowing it to dehydrate and become partially occupied. The site occupancy for the molecule was allowed to freely refine to a stable occupancy (0.695). The two crystallographically unique INA molecules engage in the carboxylate-guanidinium hydrogen bonds with two different FMT molecules, and one water molecule acts as a bridge between the two sets of INA and FMT pairs (Figure 3b). The water molecule donates a hydrogen bond to the pyridine nitrogen in one INA molecule and accepts a hydrogen bond from the amidine NH₂ of an FMT. The pyridine nitrogen on the second INA molecule in the chain accepts a hydrogen bond from an amidine NH₂ of an FMT to propagate along the a axis. The second water molecule donates hydrogen bonds to the carboxylate of an INA molecule, as well as to the water molecule lying in the 1D chain. This water in the chain further donates to the carboxylate of an adjacent INA to connect neighboring chains along the c axis. Lastly, the sulfonamide and amidines of FMT engage in hydrogen-bonded dimers to further facilitate self-assembly.

The location of the pyridyl nitrogen in PA (at the 2-position) alters the extended assembly within FMT-PA compared to the other two solids. The pyridine and carboxylate of PA form a second, two-point hydrogen bond with the NH₂ groups on the guanidine to facilitate formation of infinite hydrogen-bonded chains that extended along the b axis (Figure 3c). The sulfonamide groups on neighboring FMT molecules form dimers and neighboring FMT molecules are further connected along the a axis through interactions between the sulfonamide and amidine groups.

3.4. FMT Salts with Aliphatic Carboxylic Acids

The salts FMT-AA and FMT-Adi·MeOH crystallized in the space groups P2₁/c and P2₁/n, respectively, as evidenced by SCXRD. The asymmetric unit of FMT-AA contains one molecule of each type, while FMT and Adi crystallize as a methanol salt-solvate with one FMT molecule, one-half of an Adi molecule, and 1.33 methanol molecules present in the asymmetric unit. The methanol molar ratio was confirmed by PLATON SQUEEZE,⁴² which estimated 91 e⁻ per unit cell of disordered matter (see refinement details in SI). Both salts exhibit a two-point hydrogen bond between the carboxylate and guanidinium moieties, similar to the pyridine-based coformers (Figure 4). The guanidium groups are also stabilized through resonance in both salts (Table S3).

Figure 4.

X-ray crystal structures of (a) FMT-AA and (b) FMT-Adi·MeOH with disorder omitted for clarity. Hydrogen bonds are shown with green dashed lines.

The coformer AA lacks additional hydrogen-bond donors/acceptors. In FMT-AA, the carboxylates of AA accept additional hydrogen bonds from the NH₂ groups of the guanidine and amidine functional groups of FMT. FMT molecules further engage in hydrogen bonding via the sulfonamide and amidine groups to form a hydrogen-bonded network (Figure 4a).

In FMT-Adi·MeOH, the methanol molecules were disordered, partially occupied, and trapped in an isolated cavity within the unit cell. The SQUEEZE⁴² command was implemented to remove the solvent. The Adi molecule was found to be disordered over three positions, and since only half of the molecule is crystallographically unique, Adi engages in the guanidium-carboxylate interactions on both sides of the molecule (Figure 4b). Each carboxylate of Adi further accepts a hydrogen bond from the NH₂ group within a neighboring FMT sulfonamide group. Similar to FMT-AA, the FMT molecules in FMT-Adi·MeOH engage in substantial hydrogen bonding interactions involving the sulfonamide, amidine, and guanidine NH₂ groups, facilitating self-assembly in 3D. Notably, solvent exchange studies were attempted with FMT-Adi·MeOH to change the MeOH to a more pharmaceutically suitable solvent; however, the solid loses crystallinity when attempting to introduce a different solvent into the system. Desolvation studies were also carried out, but when methanol was fully removed from FMT-Adi·MeOH via heating, a physical mixture of the two starting materials was obtained.

3.5. Thermal Analysis

FMT has two well-characterized polymorphs, A and B, with distinct melting points (171 and 162 °C).^43,44,41 The commercially obtained FMT is a mixture of form A and B, and we observed both melting signals using DSC. Further analysis using DSC revealed a unique melting point for each salt, confirming a new phase (Figure 5, top). All of the salts exhibited a lower melting point than FMT and the respective coformer, except for FMT-PA, which has a melting point that is higher than both polymorphs of FMT and PA (Table 2, Figures S23–S28). Surprisingly, FMT-AA exhibited a melting point of 143 °C, representing a dramatic increase in thermal stability compared to AA alone. AA has a melting point of 17 °C and is a liquid at room temperature.

Figure 5.

DSC (top) and TGA (bottom) thermograms for FMT from the bottle (mixture of form A and B) and the salts. The inset in part b highlights the region from 25 to 145 °C.

Table 2.

Melting Point and Onset Temperature for the FMT Salts^a

FMT Salt	Melting Point (°C)	Onset Temperature (°C)
FMT-NA·H2O	124	162
FMT-INA·H2O	137	174
FMT-PA	180	152
FMT-AA	143	106
FMT-Adi·MeOH	138	155

^aFor hydrates/solvates, the onset temperature corresponds to the first mass loss following solvent loss. For solvent loss onset temperatures, see TGA discussion or SI.

TGA was utilized to investigate the thermal stability of the salts, as well as the loss of water/solvent upon heating (Figure 5, bottom). FMT-INA·H₂O showed higher thermal stability in comparison to FMT by itself (onset 174 °C) whereas the other salts demonstrated a lower onset temperature (Table 2, Figures S29–S34). FMT-NA·H₂O showed a loss of water at 86 °C with a weight loss of 3.5%, which is comparable to the theoretical loss of 3.8%. The onset temperature for the remaining solid is 162 °C. FMT-INA·H₂O showed a loss of water at 46 °C, corresponding to a weight loss of 1.9%. Although the observed loss is less than the theoretical loss of 3.15%, the result is consistent with the observation of spontaneous dehydration of the salt when handling. The onset temperature for the remaining solid was 174 °C. FMT-Adi·MeOH showed a loss of methanol at 82 °C with a percent weight loss of 8.4%. This is comparable to the calculated loss of 9.4% when considering 1.33 methanol molecules per asymmetric unit. The onset temperature for the remaining solid is 155 °C. The salts FMT-PA and FMT-AA each exhibit a single onset temperature due to no solvent inclusion in these solids.

3.6. Stability and Solubility of the FMT Salts

To further investigate the potential pharmaceutical relevance of the FMT salts, we investigated stability and solubility. The benchtop stabilities of FMT-NA·H₂O, FMT-INA·H₂O, FMT-PA, and FMT-AA were determined under ambient, accelerated thermal (40 °C), and accelerated thermal with humid conditions over a period of 14 days (SI section 10, Figures S35–S51). The aqueous solubilities of these four solids were determined using DI water or pH 1.2 buffer at 25 °C via the shake-flask method. FMT-Adi·MeOH was excluded from stability and solubility measurements due to the presence of methanol in the crystal lattice, which is not useful for pharmaceutical applications. Notably, several crystallizations were attempted for FMT and Adi using other solvents/conditions, in addition to solvent exchange and desolvation experiments, but none were successful.

PXRD data demonstrated that FMT-NA·H₂O and FMT-INA·H₂O are stable at ambient temperatures over 2 weeks. The ambient stability of the salt-hydrates was further confirmed via TGA following the two-week period. FMT-NA·H₂O showed a 3.4% mass loss at an onset temperature of 71 °C and for FMT-INA·H₂O, a 1.8% mass loss occurred at an onset temperature of 42 °C. Both transitions correspond to the loss of water and are comparable to the initial experiments (Figures S47 and S49). Under accelerated thermal conditions (40 °C), FMT-NA·H₂O appears to convert to a new crystalline phase as evident by PXRD on day seven. This new phase may be an anhydrous phase, but we have been unable to isolate suitable single crystals thus far. However, at 40 °C under humid conditions, FMT-NA·H₂O maintained crystallinity over 14 days and showed a loss of water (3.5%, onset temperature of 74 °C) that was comparable to initial solid as evidenced by TGA on day 14. The PXRD pattern of FMT-INA·H₂O following accelerated thermal treatment revealed moderate stability with the solid beginning to show signs of slight amorphization by day seven, which was supported by decreased thermal stability in the TGA. Under humid conditions at 40 °C, FMT-INA·H₂O intakes a small amount of water (2.3% mass loss vs 1.9% initial mass loss), which supports the observation of partially occupied water molecules in the solid-state structure. FMT-PA showed variation in peak intensity over the 14 days, suggesting reduced stability over time in all environments. PXRD characterization of FMT-AA showed appearance of a peak at 5.96° 2θ on day seven, corresponding to FMT. This suggests FMT-AA began to exhibit partial disassociation into the starting materials; however, PXRD characterization on day 14 did not show further disassociation of the salt under ambient conditions. In accelerated thermal and humid conditions, FMT-AA exhibited a slight reduction in peak intensity accompanied by broadening, suggesting a slight lack of stability under stressed conditions.

The solubility of FMT (from the bottle) was determined to be 1.07 ± 0.13 mg/mL in water, which is comparable to previously reported solubilities.^9,21,25 All four salts exhibited an improvement in solubility in DI water compared FMT (Figure 6, top, Table S4). FMT-AA showed the largest improvement with over a 20-fold increase in aqueous solubility. The salts containing the aromatic acid coformers showed a six- to 8-fold increase in aqueous solubility compared to FMT, and the hydrate with NA (FMT-NA·H₂O) exhibited the best performance of the set. For studies conducted in pH 1.2 buffer, chosen to be representative of the lower portion of the stomach,⁴⁵ FMT was determined to have a solubility of 35.9 ± 0.2 mg/mL. The significant increase in solubility for FMT under acidic conditions is due to protonation of the guanidine group, which enhances its solubility. All four salts also exhibited a higher solubility in the acidic medium when compared to their solubility in DI water. FMT-NA·H₂O showed enhanced solubility in acidic media by 1.4 times compared to FMT. The other three salts showed lower solubility in acidic medium compared to FMT (Figure 6, bottom, Table S5).

Figure 6.

Solubility of FMT and salts in DI water (top) or pH 1.2 buffer (bottom). Note that the y-axis scales differ between the plots.

3.7. Structure–Property Relationship

The origin of increased solubility in multicomponent solids has been attributed to a variety of reasons: higher water solubility of coformers, the introduction of charged species (salts), and changes involving intermolecular interactions, among others.^46–48 Salts have been recognized as frequently having higher solubility in comparison to their neutral counterparts. The introduction of charged species has been seen to increase dissolution rates, credited to the favorable intermolecular interactions that form in solution between water and the charged species. Furthermore, both forms of FMT contain extensive, multipoint hydrogen bonding interactions. Disruption of the strong FMT-FMT interactions in the solid state via introduction of a coformer into the phase can lead to increased solubility, as exhibited by these salts. For example, in FMT-NA·H₂O, the addition of NA and water minimizes the strong hydrogen bonding interactions between neighboring FMT molecules that are seen in both solid forms of FMT,^49,50 affording an increased solubility of the salt overall.

In all the FMT salts described here, the primary recognition site between the drug and coformer is unaffected when the coformer structure is changed because all coformers contain a carboxylic acid group. However, within the three salts containing pyridine-based coformers, modification to the isomer structure does influence the extended supramolecular assembly in the solid state due to the differences in location of the pyridyl nitrogen atom and its propensity to form hydrogen bonds. All three of the pyridine-containing FMT salts exhibit similar aqueous solubilities, even though two of the salts are hydrates and one is not, likely due to the presence of ionic hydrogen bonds in all three solids. A more significant difference in solubility under acidic conditions is observed, with the hydrated solids exhibiting higher solubility. The thermal stabilities are relatively similar in these three solids, but the solid with PA does not lose water upon heating and exhibits a melting point higher than the FMT. Overall, in this case, the small structural changes can afford differences in assembly and resulting properties.

To further understand the increased solubility in the salts, we turned to lattice energy calculations. Higher lattice energy is correlated with a decreased solubility for salts.⁵¹ The polymorphs of FMT had a calculated lattice energy of −233.3 kJ/mol (form A) and −221.6 kJ/mol (form B), which are both higher than the lattice energies for the salts obtained here (Table 3). Furthermore, a plot of lattice energy versus solubility demonstrated that FMT (form A and B) and the salts follow the trend of decreasing lattice energy affording increased aqueous solubility, with FMT-AA being an outlier (Figure S52). FMT-AA exhibited the highest aqueous solubility of the four FMT salts, which is attributed to the coformer, AA, having a considerably higher aqueous solubility⁵² compared to the other coformers used here. Within the four FMT salts, an increase in lattice energy value also correlates with an increase in melting point (Tables 2 and 3).

Table 3.

Aqueous Solubility and Lattice Energy Values for FMT Polymorphs and the FMT Salts in This Work

Solid	Aqueous solubility (mg/mL)	Lattice energy (kJ/mol)
FMT form Aa	0.768	−233.3
FMT form Ba	0.984	−221.6
FMT-NA·H2O	8.92	−129.3
FMT-INA·H2O	7.51	−130.6
FMT-PA	6.61	−183.3
FMT-AA	22.2	−153.9

^aSolubilities of FMT form A and B.²⁵

4. CONCLUSION

This work sought to improve the solubility of FMT through the modification of intermolecular interactions in the solid state via a cocrystallization strategy. We produced five new salts with FMT, including two hydrates and one methanol solvate through LAG and slow evaporation methods. The new phases were characterized through spectroscopic methods, X-ray diffraction, and thermal analysis. The FMT salts exhibited good thermal stability, benchtop stability under ambient conditions, and the hydrates exhibited reasonable stability under hot and humid conditions. All the salts demonstrated an increased aqueous solubility in DI water compared to both forms of FMT, with FMT-NA·H₂O exhibiting an 8-fold improvement and FMT-AA showing over a 20-fold increase in solubility. The improvement in solubility was further substantiated by lattice energy calculations. All solids also exhibited enhanced solubility under acidic conditions when compared to DI water. The most promising solids are FMT-NA·H₂O and FMT-AA given the inclusion of these coformers on the GRAS list. Increased solubility in these solids suggests an improvement in the bioavailability of FMT; however, further pharmacokinetic studies would be needed to confirm this. This study demonstrates the utilization of cocrystallization or salification methods to overcome poor drug solubility.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.cgd.6c00704.

Experimental details; single-crystal and powder X-ray data; thermal data; and calculation details (PDF)