Future work

In the presented studies, the concept of API-IL was focused on acidic APIs. The promising results suggest to expand the applicability of API-IL concept for converting crystalline basic APIs into IL form. Generally, basic APIs are ionized in in the acidic gastric fluids after oral administration and favored their dissolution and apparent solubility. ILs for basic APIs may be enhanced to stabilize these against precipitation in high pH environments.

The applicability of presented API-ILs were mainly in vitro topical/ transdermal applications.

These result suggest for in vivo studies for envisaging their commercial biomedical applications. In addition, API-ILs concept may be obviously usable for many drugs and extended their applications in other routes of delivery. The applicability of API-ILs in oral and/

or transdermal delivery may be enhanced by considering appropriate counterions, lipophilic counterions may be the promising candidate for designing the effective drug delivery of API-ILs. However, the newly synthesized NMP cation would be a potential counterion for delivering many acidic drugs, although their molecular IL formation mechanisms are yet explored. The applicability of this cation would be generalized for envisaging their biomedical applications.

The studies presented here may be regarded as a starting point for this novel formulation platform, leading to a better insight of ILs as a formulation strategy and promising new IL based drug delivery systems.

105 ABBREVIATIONS

API Active pharmaceutical ingredient

IL Ionic liquid

API-IL Active pharmaceutical ingredient ionic liquid FDA Food and drug administration

GRAS Generally regarded as safe GIT Gastrointestinal tract

PBS Dulbecco's phosphate buffered saline SIF Simulated intestinal fluid

SFG Simulated gastric fluid

MEM Minimum essential media

TDD Topical or transdermal drug delivery

SC Stratum corneum

MEs Microemulsions

AAE Amino acid ester

AspEt L-Aspartic acid diethyl ester ProEt L-Proline ethyl ester

Cho Cholinium

TMA Tetramethylammonium

TBP Tetrabutylphosphonium

EMI Trimethylimidazolium

IC50 Half maximal inhibitory concentration LD50 Half maximal lethal dose

Sal Salicylic acid

MTX Methotrexate

Ibu Ibuprofen

Sal-IL Salicylate ionic liquid MTX-IL Methotrexate ionic liquid TGA Thermogravimetric analysis DSC Differential scanning calorimetric

NMR Nuclear magnetic resonance spectroscopy FT-IR Fourier transform infrared

XRD X-ray diffractometer

106

LIST OF SCHEMES CHAPTER 2

Scheme 2.1. General synthetic procedure for amino acid alkyl ester. (a) Thionyl chloride-mediated esterification in alcohol on ice for 1 h, and then at room temperature for 24 h; (b) ammonia solution and diethyl ether at room temperature for 2 h; and (c) in the dark at 40 °C for 2 h.

CHAPTER 3

Scheme 3.1. General synthetic procedure for amino acid ethyl ester. (a) Thionyl chloride-interceded esterification in ethanol on ice for 1 h, and then at room temperature for 24 h; (b) ammonia solution and diethyl ether at room temperature for 2 h.

Scheme 3.2. General synthetic procedure for IL forming cation. Silver chloride was precipitated in water at room temperature for 2 h.

Scheme 3.3. Synthesize of MTX-ILs.

CHAPTER 4

Scheme 4.1. Synthetic scheme for the NMP-based IL. (a) Hydrochloric acid in water on ice for 1 h, and then at room temperature for 24 h; (b) silver oxide in water at room temperature for 2 h; (c) ibuprofen powder at 40 °C for 2 h.

LIST OF TABLES CHAPTER 1

Table 1.1. ILs as solubility enhancers in drug delivery.

CHAPTER 2

Table 2.1. Physical properties and water miscibility of Sal-ILs at room temperature.

Table 2.2. Cytotoxicities of the Sal-ILs towards L929 and HeLa cell lines with partitioning coefficient.

CHAPTER 3

Table 3.1. Physicothermal properties of the free hydrate MTX and MTX-IL moieties.

Table 3.2. Pharmacokinetic parameters of MTX-ILs and sodium salt of MTX in mice after oral administration at a dose of 30 mg/kg. Drug solutions was administrated at 100 µL per mice.

107

LIST OF FIGURES CHAPTER 1

Figure 1.1. Novel FDA approvals since 1993. Annual numbers of new molecular entities (NMEs) and biologics license applications (BLAs) approved by CDER.

Figure 1.2. A schematic representation of the main approaches used to improve drug solubility as well as drug delivery of poorly water-soluble drugs.

Figure 1.3. General aspect of prodrugs. A) FDA-approved prodrugs during 2010-2018. B) A schematic view of prodrugs concept.

Figure 1.4. Publication frequency of the term “Ionic Liquids” obtained from Scopus® database.

Figure 1.5. Examples of cations and anions commonly used in ILs.

Figure 1.6. Ionic liquids as component of drug formulations. A) Publication frequency of the term “Ionic Liquids in drug delivery system” and “Active pharmaceutical ingredients ionic liquids” obtained from Web of science® database. B) Active research directions for studies on biological activity of ionic liquids published in 2017–2018 (for illustrative purpose only).

Figure 1.7. Simulation snapshots. (A) IL ([C4MIM][N(CN)2]) in water; (B) Vanilla in water;

(C) IL and vanilla in water. (Light green): ionic liquid polar aggregates (strands); (blue): anion-water network; (light red) vanillin clusters.

Figure 1.8. (a) Schematic representation of ionic liquid-in-oil (IL/o) microemulsions containing drug molecules. Chemical structure of IL (b) and acyclovir (c).

Figure 1.9. Drug development platforms of API-ILs. A) API-ILs containing ionic API as anion, B) covalently linked API in the cation and C) API-ILs by combining both ways with dual activities.

Figure 1.10. A schematic view of prodrug strategy vs ionic liquid prodrug (API-IL prodrug) strategy.

CHAPTER 2

Figure 2.1. Structures, names, and abbreviations for (A) amino acid ester cations and (B) Sal-ILs used in this study.

Figure 2.2. Cytotoxicities of AAEs towards L929 and HeLa cell lines at pH 7.4. Effect of (A) alkyl chain length derived from alcohols, and (B) side chains derived from amino acids.

Figure 2.3. 1H NMR spectra of ProEt (A), free Sal (B), and [Sal][ProEt] (C).

Figure 2.4. FT-IR spectra of free Sal, AspEt, [Sal][AspEt], and [Sal][Na].

Figure 2.5. Thermogravimetric analysis of [Sal][AspEt], [Sal][ProEt], and [Sal][AlaEt].

108

Figure 2.6. Water miscibility of Sal-ILs. 1: Sal-IL viscous sample in transparent glass tube. 2:

Added water in Sal-IL sample, 3: Clear solution formed after shaking.

Figure 2.7. Skin permeation of Sal-ILs and [Sal][Na] after 60 h.

Figure 2.S1. Synthesized amino acid esters.

Figure 2.S2. Cytotoxicity of AAE cations on L929 at with and without control pH 7.4

Figure 2.S3. Cytotoxicity of proline-based AAE cations on mammalian cell lines (A) HeLa and (B) L929.

Figure 2.S4. Cytotoxicity of AAE cations on mammalian cell lines (A) HeLa and (B) L929.

CHAPTER 3

Figure 3.1. Structures, names, and abbreviations for the (A) IL-forming cations and (B) MTX-ILs used in this study.

Figure 3.2. ¹H NMR spectra of (A) the Cho cation, (B) free MTX and (C) [Cho][MTX].

Figure 3.3. FT-IR spectra of free MTX, ProEt, [ProEt][MTX] and [Na][MTX].

Figure 3.4. The XRD spectra of [AspEt][MTX], [Cho][MTX] and free MTX.

Figure 3.5. TGA and DTG thermograms of (A) free hydrate MTX and (B) [Cho][MTX]

measured in a nitrogen atmosphere with a heating rate of 10°C min^-1.

Figure 3.6. DSC thermograms of free MTX, [ProEt][MTX] and [Cho][MTX].

Figure 3.7. Solubility of MTX-IL moieties in buffers (SIF, SGF and PBS).

Figure 3.8. In vitro antitumor activity of MTX-IL moieties in HeLa cells. Effect of (A) cations derived from IL-forming cationic salts, and (B) MTX-IL moieties.

Figure 3.9. Time-dependent plasma drug concentration of MTX-ILs and sodium salt of MTX in C57BL/6 mice at single oral administration (at 30 mg kg⁻¹ of MTX drug).

Figure 3.S1. FT-IR spectra of (A) free MTX, AspEt cation and [AspEt][MTX], (B) Free MTX, choline cation and [Cho][MTX].

Figure 3.S2. FT-IR spectra of (A) free MTX, TMA cation and [TMA][MTX], (B) Free MTX, TBP cation and [TBP][MTX].

Figure 3.S3. FT-IR spectra of (A) free MTX, PheEt and [PheEt][MTX], (B) Free MTX, EMI cation and [EMI][MTX].

Figure 3.S4. XRD diffractograms of free MTX and MTX-ILs. A) Full spectrum and B) expanded regions of free MTX and MTX-ILs.

109

Figure 3.S5. TGA thermograms of free MTX (orange), [EMI][MTX] (tan), [TMA][MTX]

(green), [AspEt][MTX] (mustard), [PheEt][MTX] (black), [ProEt][MTX] (pal green) and [TBP][MTX] (blue).

Figure 3.S6. DSC thermograms of A) [TBP][MTX] and B) [PheEt][MTX]

Figure 3.S7. DSC thermograms of A) [TMA][MTX] and B) [EMI][MTX].

Figure 3.S8. DSC thermograms of [AspEt][MTX].

Figure 3.S9. Solubilities of MTX-IL moieties in water.

CHAPTER 4

Figure 4.1. Photographs of free ibuprofen powder (a), NMP cation (b), and IL [NMP][Ibu] (c).

Figure 4.2. ¹H NMR spectra of free ibuprofen in DMSO (A), NMP hydroxide in CD3OD (B), IL [NMP][Ibu] in DMSO (C), IL [NMP][Ibu] in CDCl3 (D), physical mixture of ibuprofen and NMP in DMSO (E), and in CDCl3 (F).

Figure 4.3. FT-IR spectra of free ibuprofen (Ibu), NMP cation and IL [NMP][Ibu].

Figure 4.4. TGA and DTG thermograms of A) free ibuprofen (Ibu) and B) IL [NMP][Ibu].

Figure 4.5. DSC thermograms of free ibuprofen (Ibu), [NMP][OH] cation and IL [NMP][IBU].

Figure 4.6. In vitro skin penetration profile of ibuprofenate ILs. A) FT-IR spectra of the lipid content for SC control sample (black line), IL [Cho][Ibu] (orange line), and IL [NMP][Ibu]

(green line) as measured by the peak heights of the CH2 asymmetric and symmetric stretching bands. B) Drug retention in the skin (epidermis and dermis) after 1 h.

Figure 4.7. Skin permeation of [NMP][Ibu] IL and [Cho][Ibu] IL

Figure 4.8. In vitro cytotoxicities of IL-forming cations with neutral NMP solvent in NIH3T3 and L929 cell lines.

110

ACKNOWLEDGEMENTS

I would like to express my heartfelt gratitude to Prof. Masahiro Goto for your kind support, guidance, and giving me an opportunity to work independently in your research groups. Your cordial cooperation, insightful ideas and constant enthusiasm toward research, perseverance for learning new things, and indispensable scientific intuition significantly influenced me to learn and become a skillful researcher during my PhD education. Without yours guidance, encouragement and constant feedback this PhD would not have been achievable. I am indebted to you for accepting me as a PhD student at a critical stage of research career. I would also like to sincerely thank to Prof. Noriho Kamiya for your thoughtful guidance, inspiration and continuous support throughout PhD course and for valuable advice and profound suggestion during the lab meetings and on my PhD thesis. I am really honored and fortunate to become a member of the Goto-Kamiya Lab. I would like to express my sincere gratitude to Prof. Hiroyuki Ijima for agreeing to be one of my PhD thesis committee. I really appreciate for his constructive comments and invaluable advice.

I would like to express my sincere thanks to Associate Prof. Muhammad Moniruzzaman at Universiti of Teknologi Petronans, Malaysia for your great cooperation, exchange of ideas and inspiring conversations, encouragement and constant support in my research and for the contributions to the manuscripts. I also would like to express my gratitude to Assistant Prof.

Rie Wakabayashi and Yoshiro Tahara for their contributions to the manuscripts and providing me the guidance from the initial stage of my research. I am very grateful to Assistant Profs.

Fukiko Kubota and Kosuke Minamihata for their support, conversations and valuable suggestions in many aspects of my research. I would also like to thank Dr. Momoko Kitaoka and staffs for their kind cooperation throughout my study in Kyushu University.