Highly efficient and low toxic skin penetrants composed of amino acid ionic liquids

Abstract

Herein, we introduce an amino acid ester (AAE) as a potent biocompatible counter ion to formulate ionic liquefied active pharmaceutical ingredients. By equimolar pairing of a non-steroidal anti-inflammatory drug (NSAID) as the anion and the AAE as the cation, the liquid formulation was feasibly obtained. In vitro drug permeation results using pig skin revealed a significant enhancement of the transdermal delivery of the ionic liquefied NSAIDs. The AAE showed much lower cytotoxicity to mouse fibroblast L929 cells when compared with that of conventional counter cations, indicating the significant potential of the AAE in the development of new ionic liquid drug formulations.

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Ionic liquids (ILs) have recently emerged as pharmaceutical ingredients in the development of effective drug formulation and delivery systems,¹ since the first proposal of the active pharmaceutical ingredients-ionic liquids (API-ILs) platform.² There are two ways to select the formulation of ionic liquefied drugs: (i) combining an ionisable drug with a proper counter ion or (ii) combining two pharmaceutically active ionisable drugs.^3,4 For the former case, lidocaine docusate, a hydrophobic room temperature IL, was successfully prepared from lidocaine hydrochloride and sodium dioctyl sulfosuccinate, and exhibited enhanced efficacy in topical analgesia as well as improved physical properties such as solubility and thermal stability.² Further design by replacing docusate with ibuprofenate yielded an API-IL with dual functionality.⁵ Because many types of drugs possess acid/base characters, API-IL platforms are highly promising approaches to upgrade conventional pharmaceuticals and those being investigated. ILs have also been used as additives and/or components of surfactant-based drug carriers to enhance the topical or transdermal delivery of small drugs^6,7 and transcutaneous vaccination of a model antigen.⁸

In comparison with the variety of anions available, such as carboxylate and sulphate, selection of biocompatible cations is rather limited. For example, tetrabutylphosphonium (TBP) salts were employed for the preparation of API-ILs, and their potential as a counter ion was demonstrated.^5,9 Although initial cell viability studies of TBP cations showed a rather benign safety profile, the requirement of extensive pre-clinical toxicological studies was also mentioned.¹⁰ When considering API-ILs with dual functionality, pharmaceutically active cations, such as lidocaine, could be selected for specific purposes; however, bifunctional characters may not be always required. From the viewpoint of a rather inert counter cation, cholinium has been widely accepted and recent reports clearly show its potential.^11,12 Cholinium geranate, developed for an antibacterial reagent and as a drug carrier, was found to exert excellent biocompatibility and multifunctionality in the dermal delivery of antibiotics.¹¹ Improved solubility and bioavailability of cholinium sulphasalazine compared with the free acid form was also demonstrated.¹² Therefore, new counter ion options, especially biocompatible cations, are desired and will further expand the potential utility of API-ILs.

Our goal in this study was to develop new API-ILs with biocompatible cations. We explored the new combination of acid–base complexes for API-ILs by focusing our search for new cations with minimum cellular cytotoxicity. Given the utility of natural constituents of ILs,¹³ we expected that amino acids would be potential candidates for our purpose. We reveal that an amino acid ester (AAE), proline ethylester (ProOEt), is a highly biocompatible and effective counter cation to formulate a non-steroidal anti-inflammatory drug (NSAID), as a model API, into the IL form.

Based on the design concept of amino acid ILs,¹³ ionic liquefied ibuprofen was prepared by the neutralisation of the free acid form of ibuprofen and the free base form of ProOEt (Scheme 1). In the synthesis of ProOEt, it was confirmed by HPLC analysis with an optical resolution column that racemization of ProOEt was not caused (ESI†). As shown in Scheme 1, the resulting product, ibuprofen–ProOEt, formed a transparent room temperature IL. The result suggests that a highly concentrated liquid formulation of ibuprofen with a low melting point can be feasibly obtained, even though ibuprofen is a solid, sparingly soluble pharmaceutical ingredient at room temperature. To confirm the generation of the IL form between the two constituents, ¹H NMR spectroscopic analysis was conducted with the ingredients and the product (Fig. S1 in ESI†). A new singlet peak was detected at 9.0 ppm, where no clear signal was observed for the ingredients, after formulation (Fig. 1), indicating ionisation by proton transfer between ibuprofen and ProOEt.

Scheme 1 Synthetic scheme of proline ethylester–ibuprofenate (ibuprofen–ProOEt), and photographs of (a) ibuprofen powder, (b) ProOEt, and (c) proline ethylester–ibuprofenate (ibuprofen–ProOEt).

Fig. 1 ¹H NMR spectra of ibuprofen–ProOEt (liquid form), ProOEt, and ibuprofen (free base and acid forms, respectively).

Next, for possible medical applications in transdermal drug delivery,¹⁴ we explored the functionality of ibuprofen–ProOEt as a skin penetrant. The ionic liquefied API was applied to the permeation study using purchased Yucatan micro pig skin (female, 5 months of age, Charles River Laboratories International, Inc., MA, USA). The skin permeation profile of the ionic liquefied API sample was almost the same as that of a control sample, a saturated PBS/EtOH solution, of the same drug, until 24 h (Fig. 2). Interestingly, when neat ibuprofen–ProOEt was applied to the skin, a marked amount of ibuprofen was detected in the receiver phase at a regular interval for 48–96 h, indicating the permeation of ibuprofen across the skin. Consequently, an approximately 10-fold enhancement in the cumulative amount of drug was observed at 96 h when compared with that of the control sample.

Fig. 2 Permeation profiles of ibuprofen–ProOEt and ibupurofen in a saturated PBS/EtOH solution across the skin.

In the form of an ionic liquefied API, a significant increase in the API concentration per unit volume in solution should be achieved, which could cause the remarkable concentration gradient between the outer and inner surface of the skin to promote the permeation rate.¹⁵ Furthermore, solubility of the API, which is sparingly soluble in aqueous milieu, could be improved by ionic liquefaction. A previous report suggested the presence of the neutral form of API-ILs, arising from the strong association of ion pairs, could be the key factor for the transport of the salt forms through a hydrophobic membrane.¹⁶ Given the marked skin permeation observed in Fig. 2, it is possible that ibuprofen–ProOEt is predominantly in the neutral form and this form promotes its delivery across the hydrophobic stratum corneum layer. In addition, it is also noted that an ester form of amino acids could be a suitable counter ion for generating the neutral form of API-ILs compared with that of natural amino acids with free carboxylic acid(s). It is likely that these features increase skin permeability.

Finally, to assess the cytotoxicity of ProOEt, we conducted cellular viability tests using mouse fibroblast L929 cells (RIKEN BRC CELL BANK, Ibaraki, Japan). We also evaluated the cytotoxicity of proline (Pro), proline tetrabutylphosphonium salt (Pro–TBP), triethanolamine and choline for comparison. First, Pro–TBP, an ionic liquefied Pro with the TBP cation, showed relatively high cytotoxicity at a sub-millimolar concentration (filled triangle, Fig. 3). This could be owing to the toxicity originating from the TBP cation because Pro showed little effect on the cell viability (filled circle, Fig. 3), implying that organic phosphonium may be unsuitable in drug formulation. Second, choline, which is a well-known biocompatible cation, was applied to the cell culture after neutralisation of choline hydroxide. The results showed a negative impact on cell viability at a somewhat high concentration range, i.e., over 20 mM. Triethanolamine also showed a trend similar to that of the cholinium cation. Conversely, ProOEt clearly showed a negligible effect on cell viability, similar to proline, and ∼80% of the cells are viable to 50 mM.

Fig. 3 Cytotoxicity of different types of cations, Pro(OEt), proline (Pro), Pro–TBP, triethanolamine and choline.

A recent histological study on the assessment of lidocaine ibuprofenate showed no significant damage to the epidermis when applied to hairless rats.¹⁴ The results suggest that lidocaine can be a potentially useful cation for the formulation of bifunctional API-ILs; however, the applicability would be limited to avoid undesired biological responses associated with the counter ion. Although cholinium showed a negative impact on cell viability at relatively high concentrations in this study, this cation has been widely used as a biocompatible counter ion in IL-based biotechnology. Our results demonstrated clearly the potential use of ProOEt as a cation with minimum cytotoxicity, which may not disturb the intrinsic pharmacological function of the API of interest, and thus provides a practical way for the design of new biocompatible IL formulations in biotechnological fields.

Conclusions

Herein, we introduced for the first time an AAE as a promising component of ionic pharmaceuticals with enhanced functionality in topical and transdermal drug delivery. When combined with ibuprofen as a model drug, ProOEt works as a counter cation and generated the IL form. Significant enhancement in skin penetration was observed with the IL formulation. It should be noted that ProOEt, a type of amino acid derivative, can be a highly biocompatible and safe constituent of API-ILs in comparison with conventional cations.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra16926k

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