Co-amorphous formation of high-dose zwitterionic compounds with

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Co-amorphous formation of high-dose zwitterionic compounds with amino acids to improve solubility and enable parenteral delivery Saijie Zhu, Huisheng Gao, Sreehari Babu, and Sudhakar Garad Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00738 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Molecular Pharmaceutics

Co-amorphous

formation

of

high-dose

zwitterionic compounds with amino acids to improve

solubility

and

enable

parenteral

delivery Saijie Zhua, Huisheng Gaoa, Sreehari Babua and Sudhakar Garadb* a

Chemical and Pharmaceutical Profiling, Technical Research and Development, China

Novartis Institutes for Biomedical Research Co., Ltd, Shanghai, China b

Chemical and Pharmaceutical Profiling, Technical Research and Development, Novartis

Pharmaceuticals, Cambridge, MA 02139, United States *

Corresponding author

KEYWORDS Co-amorphous; ofloxacin; amino acid; parenteral; solubilization; molecular interaction

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

ABSTRACT Solubilization of parenteral drugs is a high unmet need in both pre-clinical and clinical drug development. Recently, co-amorphous drug formulation has emerged as a new strategy to solubilize orally dosed drugs. The aim of the present study is to explore the feasibility of using the co-amorphous strategy to enable the dosing of parenteral zwitterionic drugs at a high concentration. A new screening procedure was established with solubility as the indicator for co-amorphous co-former selection, and lyophilization was established as the method for co-amorphous formulation preparation. Various amino acids were screened, and tryptophan was found to be the most powerful in improving the solubility of ofloxacin when lyophilized with ofloxacin at a 1:1 weight ratio, with more than 10 times solubility increase. X-ray powder diffraction showed complete amorphization of both components, and an elevated Tg compared with the theoretical value was observed in differential scanning calorimetry. Fourier-transform infrared spectroscopy revealed that hydrogen bonding and π−π stacking were possibly involved in the formation of co-amorphous system in the solid state. Further solution state characterization revealed the involvement of ionic interactions and π−π stacking in maintaining a high concentration of ofloxacin in solution. Furthermore, co-amorphous ofloxacin/tryptophan at 1:1 weight ratio was both physically and chemically stable for at least 2 months at 40°C/75%RH. Lastly, the same screening procedure was validated with two more zwitterionic compounds, showing its promise as a routine screening methodology to solubilize and enable the parenteral delivery of zwitterionic compounds.


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1. INTRODUCTION

The number of new chemical entities (NCEs) is increasing every day in drug discovery after the introduction of combinatorial chemistry and high throughput screening, which has also resulted in the increasing number of poorly soluble ( 98.0%) was purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). All the amino acids including lysine (LYS), arginine (ARG), histidine (HIS), serine (SER), isoleucine (ILE), methionine (MET), phenylalanine (PHE), tryptophan (TRP) and tyrosine (TYR), as well as urea, HPLCgrade trifluoroacetic acid (TFA), deuterium oxide (D2O, 99.9 atom % D, 0.05% sodium trimethylsilyl propionate-d4 (STSP, as internal reference) and HPLC-grade acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Theophylline (purity > 99%) was purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) pH 7.4 (1×) was purchased from Life Technologies (Grand Island, NY, USA). Water was purified using a Millipore filtration system



(Millipore Corporation, Billerica, MA, USA). All other reagents used were of analytical grade.

2.2. Determination of the equilibrium solubility of OFX. Approximately 10 mg of OFX was weighed into a glass vial containing a stirring bar and 0.5 mL of aqueous solution containing various concentrations of amino acids were added. The slurry was stirred overnight at room temperature and filtered through a 0.45 µm membrane and the filtrates were diluted appropriately before UPLC analysis.

2.3. Preparation of lyophilized mixture of OFX and amino acids. OFX and amino acids were first weighed into a glass bottle at different ratios listed below. Water was added to dissolve both components with sonication. The resulting solution was further filtered to remove any undissolved particles to give a clear solution, which was then lyophilized with a Labconco lyophilizer (Labconco Corporation, MO, USA). Off-white to white solid was obtained after lyophilization.

To evaluate the effect of different amino acids on the solubility of OFX, OFX was lyophilized with HIS (1:1 and 1:3.33 by weight), SER (1:3.33 by weight) and TRP (1:1 by weight). To further evaluate the effect of the weight ratio of TRP on the solubility of OFX, OFX was lyophilized with TRP at the weight ratios of 1:0.25, 1:0.5, 1:1, and 1:1.5. To confirm the solubilization effect after lyophilization, the physical mixture was prepared by blending crystalline OFX and TRP at the weight ratios of 1:0.25, 1:0.5, 1:1, and 1:1.5, and the solubility after 24 h of stirring was determined. To investigate the molecular level interaction of the lyophilized mixture, OFX was lyophilized with TRP at


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the weight ratios of 95:5, 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9 and 5:95. In addition, OFX and TRP were lyophilized at the molar ratio of 1:1, 1:2 and 1:3 to evaluate the stoichiometric effect on solubilization. Samples prepared by weight ratio are designated hereafter as OFX/TRP_x/y, where x and y are the relative weights of OFX and TRP in the lyophilized mixtures, respectively. Likewise, OFX/TRP_mole_x/y designates samples prepared by molar ratio, where x and y represent the relative moles of OFX and TRP in the lyophilized mixtures, respectively.

2.4. Determination of the kinetic solubility of lyophilized OFX. The lyophilized mixture of OFX and amino acids was reconstituted with water to give a target concentration of 15 mg/mL or 30 mg/mL of OFX. Samples were taken at predetermined time points, filtered, diluted and analyzed by UPLC.

To investigate the molecular interaction of OFX and TRP in the solution state, agents known to disrupt different non-covalent interactions in the aqueous solution were used to reconstitute the lyophilized solid of OFX/TRP_1/1, and the kinetic solubility was determined.

2.5. Ultra-performance liquid chromatography (UPLC). A UPLC method was established to simultaneously determine the concentration of OFX and TRP using a reverse-phase Waters Acquity UPLC system (Waters Corporation, MA, USA). OFX and TRP were separated by an ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 µm) and detected at 280 nm. Mobile phase A was 0.05% TFA in 95% water/5% ACN, and mobile phase B was 0.05% TFA in 95% ACN/5% water. A linear gradient of 95%



mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B over 2.5 min was applied at the flow rate of 0.5 mL/min. The column temperature was set at 30°C. The injection volume was 0.5 µL.

2.6. X-ray powder diffraction (XRPD). XRPD measurements were performed using a Bruker D8 GADDS Discover X-ray diffractometer (Bruker AXS Inc., Madison, WI, USA) with CuKα radiation of 1.54184 Å, acceleration voltage and current of 45 kV and 40 mA, respectively. The samples were scanned in reflectance mode between 5° and 45° 2θ with a scan rate of 10° 2θ/min and a step size of 0.02°. The data was collected and analyzed using Bruker EVA v.13.0.0.3 software (Bruker AXS Inc., Madison, WI, USA).

2.7. Differential scanning calorimetry (DSC). DSC thermograms (DSC Q2000, TA Instruments, USA) were obtained under a nitrogen gas flow of 50 mL/min. Sample powders (1 to 2 mg) were crimped in a standard aluminum pan and heated at a rate of 10°C/min from 0 to 300°C with a modulation amplitude of 1.0°C and a period of 60 s. The glass transition temperature (Tg) and crystallization temperature (Tc) were determined using TA Universal Analysis software, version 4.5A. The Tg was determined as the midpoint of the change in heat capacity of the sample, while the Tc was determined as the onset temperature.

The theoretical Tg values of the lyophilized mixtures of OFX and TRP were calculated based on the FOX equation (see below) assuming there were no intermolecular interactions between the components.12

1 / Tg = w1 / Tg 1 + w2 / Tg 2


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Where w1 and w2 are weight fractions and Tg 1 and Tg 2 are glass transition temperatures (°C) of each component.

2.8. Fourier Transform Infrared Spectroscopy (FTIR). Infrared spectra were recorded on a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and evaluated using OMNIC 9.2.41 software (Thermo Fisher Scientific Inc., Waltham, MA, USA). Each spectrum was scanned in the range of 650−4000 cm−1 with a resolution of 0.09 cm−1 and a minimum of 16 scans were collected and averaged. The spectra were normalized and the background was corrected.

2.9. Solution state nuclear magnetic resonance (NMR). The NMR experiments were performed on a Bruker Avance III 400 MHz spectrometer (Bruker GmbH, Germany). The 1H NMR spectra of OFX, TRP, OFL/TRP_1/1 at various concentrations in D2O were recorded at 298 K and all chemical shifts were measured relative to STSP. NMR data were processed using ACD/Spectrus Processor 2014 software (Advanced Chemistry Development, Inc. Toronto, Canada).

2.10. Stability. The lyophilized samples including OFX/TRP_9/1, 1/1 and 1/9 were placed in a screw-capped vial and stored at 4°C, 25°C/60%RH and 40°C/75%RH. The samples were analyzed after 1 week, 2 weeks, 1 month and 2 months by XRPD and UPLC to check the physical and chemical stability, respectively. The onset of recrystallization was reflected by the appearance of recrystallization peaks arising from the amorphous halo in the XRPD diffractograms. The chemical stability was evaluated by comparing the UPLC chromatograms before and after stability tests.



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2.11. Data analysis. All data were presented as mean ± standard deviation (SD). Statistical analyses were completed by performing analysis of variance followed by Fisher’s protected least significant difference procedure. A P value of ≤ 0.05 (two-tail) was considered significant.

3. RESULTS AND DISCUSSION

3.1. The effect of amino acids on the equilibrium of solubility of OFX. Commercially available OFX was obtained as a white crystalline powder, with a melting point of 275.1°C and enthalpy of 155.2 J/g. The solubility in water at room temperature was determined to be 2.86 ± 0.07 mg/mL with a final pH of 7.37 ± 0.03. A total of 9 amino acids, including LYS,13,14 ARG,13,15 HIS,13

SER,14

PHE,15 ILE,16 MET,16

TRP,15,17 and TYR,15 were selected based on literature reports to form co-amorphous systems with drugs.

As outlined in Figure 1, LYS and ARG significantly increased the solubility of OFX in a concentration-dependent manner, with a maximal solubility of 17.00 ± 0.45 mg/mL achieved at 50 mg/mL of ARG (P < 0.01). The improved solubility could be explained by the elevated pH (close to 10) which was caused by the basic nature of the two amino acids. The pH value was in agreement with the zwitterionic nature of OFX and the determined pH-solubility profile (data not shown). Amino acids including SER, PHE, ILE, MET and TYR didn’t change the solubility of OFX, whereas a significant improvement of solubility was observed with HIS and TRP without a pH change. A slight increase in solubility was observed with HIS up to 15 mg/mL, and a 2.67-fold increase was observed when HIS concentration was further increased to 50 mg/mL (P
0.05). Thus, ionic interactions might be responsible for the solubilization of OFX and TRP in water.

Methylxanthines, including caffeine and theophylline have been reported to form ππ stacking

complexes with aromatic mutagen/carcinogen molecules,31 and caffeine

decreases the complexation between daunomycin and flavin-mononucleotide, a Vitamin B2 derivative in solution.32 As outlined in Figure 8, the solubility of OFX showed dependency on concentration and time when theophylline solution was used to reconstitute the co-amorphous OFX/TRP_1/1. The target concentration of 30 mg/mL of OFX was achieved for at least for 4 h in the presence of theophylline, while precipitation was observed with longer incubation time (16 h), with OFX concentration reduced to


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21.40 ± 1.10 mg/mL and 10.68 ± 0.95 mg/mL in the presence of 1 and 6 mg/mL theophylline, respectively (P < 0.01 vs. the control at 16h). We hypothesized that OFX and TRP, both containing aromatic rings in their structures, might form heteroassociation complexes via π-π stacking in the aqueous solution, and the higher solubility of the complex was responsible for the solubility improvement. Theophylline might disrupt the complex between OFX and TRP either by forming a less soluble complex with OFX or competing and displacing OFX from the OFX/TRP complex, and consequently reducing the solubility of OFX.

Urea is a powerful protein denaturant as it disrupts the non-covalent bonds, mainly hydrogen bonds in proteins due to the existence of both hydrogen bond donor and acceptors in its structure.33,

34

It had also been used to disrupt the hydrogen bonds

between small molecules, such as β-cyclodextrins.35 As depicted in Figure 8, urea didn’t significantly reduce the solubility of OFX in the co-amorphous system at either high or low concentrations (P > 0.05), indicating that the contribution of hydrogen bonding to solubility improvement was limited. Yet the contribution of hydrogen bonding to the solubilization of the co-amorphous system could not be completely excluded since hydrogen bonding is weaker in polar media, such as water, than non-polar media. The disruption of an interaction of less contribution may not significantly reduce the solubility.



Figure 8. The effect of noncovalent interactions on the solubility of co-amorphous OFX/TRP_1/1 (n = 3). a, P < 0.01 vs. the control at the same time point.

3.5.3. Solution state NMR. Proton NMR has been previously used to investigate the self and hetero-association of aromatic molecules in the solution state. The extent of change in the chemical shift represents the strength of association and the extent of intermolecular proximity.36 Self-association in the aqueous solution was unlikely for OFX or TRP (Figure 9A), as there was a negligible chemical shift change for OFX and TRP within the concentration range of 0.5-2 mg/mL and 0.5-15 mg/mL in D2O, respectively (data not shown). As demonstrated in Figure 9B, a general decrease in


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proton chemical shift values was observed for both OFX and TRP when they were dissolved in D2O as co-amorphous OFX/TRP_1/1 at 2 mg/mL by OFX, compared with their respective solutions at 2 mg/mL in D2O, indicating the possible association of OFX and TRP in the aqueous solution. Similar upfield shifts (decrease in chemical shift) were observed for the self and hetero-association of a couple of aromatic drug molecules,37, 38 which could be explained by greater shielding of the protons in the complex than their respective monomers. The chemical shift changes were more significant when the concentration of OFX/TRP_1/1 increased to 15 mg/mL by OFX (Figure 9B), which was characteristic of the molecular association due to the π-π stacking interaction.37 Furthermore, it was also observed that the chemical shift of protons distal from the aromatic rings, e.g. OFX-7’ and TRP-2 (Figure 9A), showed almost no change at low concentration (2 mg/mL) (Figure 9B), while there was observable chemical shift changes for protons on and adjacent to the aromatic rings. Overall, the findings further supportthe π-π stacking interaction and hetero-association between OFX and TRP in the aqueous solution.

Addition of theophylline (TEO) into the D2O solution of the co-amorphous OFX/TRP_1/1 (15 mg/mL by OFX) significantly changed the chemical shift of the protons from all three components including OFX, TRP and TEO. As shown in Figure 9C, all the protons of TEO and TRP showed decreased chemical shifts, while the chemical shifts of the protons of OFX generally increased. A possible explanation is that TEO formed even stronger complexes with TRP and displaced OFX from the OFX/TRP complex, which is in agreement with its solubility-reducing effect as previously observed (Figure 8).



Figure 9. (A) Chemical structure and assignment of protons of OFX, TRP and theophylline (TEO). (B) The concentration-dependent chemical shift change of the protons of co-amorphous OFX/TRP_1/1 in D2O (n = 3). (C) The effect of TEO on the chemical shift of co-amorphous OFX/TRP_1/1 in D2O (n = 3).

3.6. Stability. Preliminary physical and chemical stability tests of OFX/TRP_1/1 were carried out to explore the developability of the mixture as a drug product. As shown in Figure 10A, co-amorphous OFX/TRP_1/1 was stable for up to two months at 4°C and two other conditions (25°C/60%RH and 40°C/70%RH) required by the FDA for drug products, as evidenced by the amorphous halo in the XRPD diffractograms. The chemical stability was confirmed by UPLC analysis, with no obvious degradation after two months of storage at all the above conditions. The preliminary stability data, in addition to a superior solubility profile, demonstrated the developability of the co-amorphous


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OFX/TRP_1/1 as a drug product. On the other hand, OFX/TRP_1/9 and 9/1 were much less stable than OFX/TRP_1/1.

As depicted in Figure 10B, the initial sample of

OFX/TRP_9/1 showed a small reflection corresponding to OFX in the XRPD diffractogram and larger reflections were observed at 25°C/60%RH and 40°C/70%RH after one week of storage. Similarly, reflections were observed in the XRPD diffractogram of OFX/TRP_1/9 after one week of storage at 40°C/70%RH (Figure 10C). The best stability profile corresponding to OFX/TRP_1/1 was in agreement with the highest positive deviation in Tg and highest Tc. Once again, these results emphasize the importance of selecting an appropriate ratio of co-amorphous components when designing a co-amorphous system in order to achieve best performance.



Figure 10. XRPD diffractograms of (A) OFX/TRP_1/1 after two months of storage at different conditions (see below), (B) OFX/TRP_9/1 after one week of storage at different conditions, and (C) OFX/TRP_1/9 after one week of storage at different conditions. Blue line: initial sample; green line: 4°C; black line: 25°C/60%RH; red line: 40°C/70%RH.


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3.7. Assay limitation and applicable domain. The same screening procedure was applied to another two commercially available fluoroquinolones (Enrofloxacin and Ciprofloxacin), and the solubilization effect was confirmed (supporting information Table S1). Therefore, we hypothesized that zwitterionic compounds could be solubilized by certain amino acids to enable its parenteral delivery at high dose. The solid and solution state characterization of OFX/TRP co-amorphous system provided preliminary insights into the molecular mechanism. First of all, it appeared the compounds needed to be ionizable in the physiological pH condition, thus could interact with zwitterionic amino acids by electrostatic interaction. Secondly, aromatic rings seemed to be necessary to provide molecular interaction both in the solid and solution state by π−π stacking interaction. However, the current hypothesis is only based on the limited number of compounds with similar chemical structure, more structurally diverse compounds need to be tested to further validate this hypothesis.

4. CONCLUSIONS

In our work, a new solubility-based co-amorphous screening and optimization method was proposed for the solubilization of parenteral drugs using amino acid as the coamorphous co-former. The optimized formulation OFX/TRP_1/1 (1:1 by weight) showed a >10 times increase in solubility for OFX. Solid state characterization indicated the involvement of hydrogen bond and π−π stacking interaction in the co-amorphous formation. Further solution state characterization revealed the possible hetero-association between OFX and TRP via ionic and π−π stacking interactions, which might be responsible for maintaining a high concentration of OFX in solution. The co-amorphous



OFX/TRP_1/1 mixture was both physically and chemically stable for at least two months under standard stability conditions, showing the potential to be developed into a drug product. More importantly, the co-amorphous screening methodology could be a routine procedure to solubilize and enable the parenteral delivery of poorly soluble zwitterionic compounds.

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Figure 1. The effect of amino acids on the equilibrium solubility of OFX (n = 3). a, P < 0.01, b, P < 0.05 vs. the solubility in water. 1076x456mm (120 x 120 DPI)



Figure 2. The effect of amino acids on the kinetic solubility of lyophilized OFX (n = 3). a, P < 0.01 vs. the control at the same time point. 545x379mm (150 x 150 DPI)


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Figure 3. (A) Kinetic solubility profile of OFX in the lyophilized mixture of OFX and TRP at different ratios (n = 3). (B) Kinetic solubility profile of TRP in the lyophilized mixture of OFX and TRP at different ratios (n = 3). (C) Kinetic solubility of OFX at 24 h in the lyophilized mixture and physical mixture of OFX and TRP at different weight ratios (n = 3). All ratios are expressed as OFX:TRP. a, P < 0.01 vs. the control, b, P < 0.01 vs. the solubility in physical mixture at the same ratio. 624x1214mm (120 x 120 DPI)



Figure 4. (A) XRPD diffractograms of lyophilized OFX and TRP. (B) XRPD diffractograms of lyophilized mixture of OFX and TRP at different ratios. 314x477mm (120 x 120 DPI)


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Figure 5. (A) DSC thermogram of OFX/TRP_1/1. Blue and green arrows indicate the glass transition and recrystallization events upon heating, respectively. (B) Glass transition temperature (red line) and recrystallization temperature (blue line) as a function of the proportion of TRP in the co-amorphous samples ( n = 3). Theoretical Tg was calculated by the FOX equation (green line). 266x356mm (150 x 150 DPI)



Figure 6. FTIR spectra of co-amorphous OFX/TRP_1/1 (blue line), physical mixture of amorphous OFX and TRP (1:1 by weight) (green line), amorphous TRP (OFX/TRP_5/95) (purple line) and amorphous OFX (OFX/TRP_95/5) (red line). 655x424mm (120 x 120 DPI)


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Figure 7. (A) Kinetic solubility of the stoichiometric co-amorphous mixture of OFX and TRP (OFX/TRP_mole_1/1, 1/2 and 1/3) (n = 3). Solid and dashed lines represent the concentrations of OFX and TRP, respectively. (B) XRPD diffractograms of the precipitates from OFX/TRP_mole_1/1, 1/2 and 1/3 4 h after reconstitution. 291x421mm (120 x 120 DPI)



Figure 8. The effect of noncovalent interactions on the solubility of co-amorphous OFX/TRP_1/1 (n = 3). a, P < 0.01 vs. the control at the same time point. 190x187mm (150 x 150 DPI)


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Figure 9. (A) Chemical structure and assignment of protons of OFX, TRP and theophylline (TEO). (B) The concentration-dependent chemical shift change of the protons of co-amorphous OFX/TRP_1/1 in D2O (n = 3). (C) The effect of TEO on the chemical shift of co-amorphous OFX/TRP_1/1 in D2O (n = 3). 612x1076mm (120 x 120 DPI)



Figure 10. XRPD diffractograms of (A) OFX/TRP_1/1 after two months of storage at different conditions (see below), (B) OFX/TRP_9/1 after one week of storage at different conditions, and (C) OFX/TRP_1/9 after one week of storage at different conditions. Blue line: initial sample; green line: 4°C; black line: 25°C/60%RH; red line: 40°C/70%RH. 303x693mm (120 x 120 DPI)


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Table of content 364x149mm (120 x 120 DPI)


Co-amorphous formation of high-dose zwitterionic compounds with

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