Clinical translation of choline and geranic acid deep eutectic solvent

Abstract Choline geranate deep eutectic solvent/ionic liquid (CAGE) has shown several desirable therapeutic properties including antimicrobial activity and ability to deliver drugs transdermally in research laboratories. Here, we describe the first report of clinical translation of CAGE from the lab into the clinic for the treatment of rosacea, a common chronic inflammatory skin disorder that affects the face. We describe the seven steps of clinical translation including (a) scale‐up, (b) characterization, (c) stability analysis, (d) mechanism of action, (e) dose determination, (f) GLP toxicity study, and (g) human clinical study. We describe the challenges and outcomes in these steps, especially those that uniquely arise from the deep eutectic nature of CAGE. Our translational efforts led to a 12‐week open‐label phase 1b cosmetic study with CAGE1:2 gel (CGB400) in mild–moderate facial rosacea in 26 patients where CGB400 exhibited a marked reduction in the number of inflammatory lesions. These results demonstrate the therapeutic potential of CGB400 for treating rosacea as well as it provides insights into the translational journey of deep eutectic solvents, in particular CAGE, for dermatological applications.


| INTRODUCTION
Rosacea is a common chronic inflammatory skin disorder affecting 415 million people worldwide with highest prevalence in Caucasian adults (>10%) and clinical manifestations include facial erythema, papulopustular lesions, phymatous changes, facial telangiectasias, intermittent flares of acute vasodilation (flushing). 1,2 It is a phenotypically heterogenous disease with varying range of clinical manifestations and symptoms that makes the diagnosis and treatment challenging. 3,4 While the underlying causes of rosacea remain poorly defined, therapies that target multiple contributing factors may offer advantages over current therapies. 5,6 A few topical products have been approved by the FDA for the treatment of rosacea including Mirvaso (0.33% brimonidine) topical gel 7 and Rhofade (1% oxymetazoline hydrochloride) 8 for persistent (nontransient) facial erythema of rosacea in adults. Additional products include Soolantra (1% ivermectin cream), 9 Finacea (15% azelaic acid foam), 10 and Metrogel (1% metronidazole gel) 11 which have gained specific investigative interests for crucial role in modulating immune responses and selective antiparasitic activity in rosacea pathophysiology. 12,13 Mirvaso and Rhofade are vasoconstrictors and address only transient redness. They have limited efficacy against persistent redness, inflammatory papules or pustules. Soolantra, Finacea, and Metrogel have shown antimicrobial and anti-inflammatory effects in reducing the inflammatory papules and pustules associated with the disease; however, their effect on persistent facial erythema has been severely limited. 14 They are also limited by treatment cost, poor dermal permeation, and have certain regional restrictions of manufacturing. 15 Hence, the development of therapeutics that address these challenges can significantly improve the treatment and care of rosacea. 16,17 Recently, an ionic liquid/deep eutectic solvent comprised of choline and geranic acid (CAGE) has been shown to exhibit characteristics that make it a potential candidate for effective treatment of rosacea.
Specifically, prior in vitro studies have shown that CAGE exhibits excellent antimicrobial activity against a broad variety of pathogens 18,19 including Staphylococcus epidermidis which could be associated with pustules of rosacea. 20 Further, CAGE has been shown to exhibit deep penetration into the skin, thus suggesting its ability to treat pathogens residing deep into the skin. 21 Building on this scientific foundation, here we demonstrate successful clinical translation of this innovative technology into a formulation referred to as CGB400, comprising 40% w/w CAGE 1:2 (choline:geranic acid ratio of 1:2) gel formulated in common pharmaceutical excipients. Conversion of academic discoveries into clinical products is often limited by numerous hurdles of practical as well as fundamental origins. Specifically, academic innovations which are not usually constrained by issues of scalability, long-term stability and human safety may not be compatible with the requirements of a useful clinical product. Thus, many scientific discoveries, in spite of their significant potential, fail to reach the clinic. Here, we describe the journey of CAGE, which was first reported as an academic scientific discovery in 2014, 19 through the "seven steps of translation" including scale-up, characterization, stability, mechanism of action, dosing, preclinical toxicology in minipigs and finally, human volunteer studies on safety and efficacy in treating rosacea. Collectively, these studies report the first example of clinical translation of an ionic liquid-based approach for the treatment of dermatological conditions. We also share the learnings through these seven steps of translation that may be helpful for other translational activities in the field.
2 | RESULTS 2.1 | Translational step 1: CGB400 synthesis and scale-up Previous reports of synthesis of CAGE 1:2 was performed at a laboratory scale, with typical batch sizes of several grams. 19 While this is often adequate for research level use, this scale is not adequate for human testing. Scale-up of materials is often a complex process since some of the key functional aspects of the synthesis procedure may not scale proportionately. The active ingredient in CGB400, CAGE 1:2 , was synthesized in a commercial reactor at a kilogram scale using the same process as that used at a smaller scale. Specifically, CAGE 1:2 was synthesized by a one-step salt metathesis reaction between choline F I G U R E 1 Schematic illustration of mode of action of choline and geranic acid (CAGE) for rosacea. (a) Synthetic scheme of CAGE/CGB400 gel, deep eutectic mixture. (b) CGB400 upon topical application, extracts lipid from the lipid bilayer and penetrates deep into the skin layers. (c) CAGE neutralizes pathogens, modulates TLR2 signaling, and inhibits KLK5 and thus generation of pro-inflammatory peptides bicarbonate and geranic acid (Figure 1(a)). No specific issues were observed during scale-up. CAGE 1:2 is hygroscopic in nature and is miscible with water. During salt metathesis, evolution of CO 2 gas was monitored to assess the completion of the reaction. The resulting deep eutectic ionic liquid possessed both fluidity and transparency at room temperature with approximately 13% water content. The solution is a clear, colorless to yellow colored viscous liquid with a pH 8.5, conductivity~1.3 mS/cm, and a characteristic odor. Reaction duration and process parameters were optimized to improve manufacturing scale-up with the end product quality, defined by the water content, pH, conductivity and appearance. The initial scaled formulation (>3 kg) was designed to produce CAGE 1:2 with the desired homogeneity for nonclinical studies. CGB400 gel was manufactured by mixing the CAGE 1:2 solution with water, propylene glycol as co-solvent, d-limonene as a fragrance, and hydroxypropyl cellulose as a gelling agent. The inactive ingredients used in the CGB400 gel are inert in nature and were chosen to minimize dissociation of CAGE 1:2 or have minimal effect on the CAGE 1:2 structure.
Then, 5 kg batches of CGB400 gel were manufactured at an experienced contract manufacturing organization in California for the human studies. Preservatives are often needed to protect against the growth of microbes in the formulation. However, since CGB400 gel itself possessed antimicrobial properties due to the inherent antimicrobial activity of CAGE 1:2 , the formulation did not require additional preservation; keeping it paraben-free. All excipients used were USP/NF grade, except the fragrance agent which is a food-grade material.
2.2 | Translational step 2: Characterization of CAGE 1:2 Adequate characterization steps form the foundation of product development. For small molecules drugs, methods such as chromatography and NMR, among others, have been well-established over the years. As newer drugs such as biologics emerged on the landscape, the field of drug development has collectively rushed to develop the necessary methods for such products. This is one of the key challenges for deep eutectic solvents/ionic liquids. As an emerging category of actives, methods for characterizing these materials as drug products are in infancy. A key challenge is that the standard chemical methods are not adequate since no new covalent bond is formed during the salt metathesis between choline and geranic acid, thus making over reliance on conventional methods characterization challenging. CAGE 1:2 was chemically characterized using NMR and HPLC analyses ( Figure S1). 1H NMR spectra of CAGE 1:2 indicated notable differences in the chemical shifts compared to the individual components. These chemical shifts are pronounced for the protons localized adjacent to the quaternary nitrogen atoms indicating the strong anion effect of the anion, geranic acid. HPLC did not exhibit a clear signature of CAGE compared to its individual components. Specifically, the retention times (RT) of major peaks in the chromatogram of CAGE 1:2 corresponded to that obtained in choline and geranic acid standard solutions. Both methods confirmed the presence of choline and geranic acid in CAGE 1:2 . However, in the absence of a covalent bond between them, these methods were deemed necessary but not sufficient.
The physical and chemical properties of CAGE 1:2 were additionally evaluated using modulated differential scanning calorimetry (MDSC) and thermal gravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR). MDSC indicated a glass transition temperature (Tg) around −68 C ( Figure S2). This is illustrated in both the reversible heat flow and reversible heat capacity curves in both heating and cooling. It is likely that the liquid CAGE 1:2 formulation solidifies into an amorphous solid. The small exotherm during cooldown at −80 C (only apparent at 1 C/min) might be associated with an ordering event to an extent, like partial crystallization ( Figure S3). From the TGA data, it is evident that CAGE 1:2 lost approximately 4% weight at approx. 140 C, likely water. The sample completely evaporated or decomposed starting around 165 C. No hysteresis was observed in the sorption/desorption isotherm of the gravimetric vapor sorption which indicated that CAGE 1:2 is hygroscopic ( Figure S2). The salt metathesis reaction was also monitored via FTIR spectroscopy ( Figure S4). The broad peak at In case of CAGE 1:2 , it has been observed that partial dissociation of the anion and cation occurs in the presence of water. We anticipate that a dynamic equilibrium exists between CAGE 1:2 , individual components and water. At concentrations below 20%, CAGE 1:2 transitions from a bulk deep eutectic/ionic liquid structure into vesicles and transitions to microemulsion at much lower concentrations. 22

| Translational step 3: Stability under forced degradation conditions
Long-term stability of the drug product is a key requirement. It should be noted that the components of CAGE 1:2 , that is, choline and geranic acid are GRAS materials with well-established safety profiles. No specific toxic impurities in either of these materials have been reported. Impurities in CAGE 1:2 and CGB400 were quantified using validated Solution stability of CGB400 gel was also tested. The stored samples and standards met the 95.0-105.0% recovery of initial concentration of choline and 97.0-103.0% recovery of initial concentration of geranic acid per the validation protocol criteria. The chemical and physical stability of CGB400 gel was evaluated over a 12-month period. The CGB400 gel was found to be stable in accelerated (40C/75% RH), intermediate conditions (30C/65% RH), and long-term storage conditions (25C/60%RH) with relatively low increase in impurities level (total impurities level was increased from 0.42 to 2.5%). Moreover, microbial limit that was tested at every time point of the stability study was found to be in the acceptance range for topical gel products suggesting that CGB400 gel is well preserved for a period of at least 12 months.
There was a slight increase in the pH (~0.4) and change in viscosity  While not an essential element of the translational path, an understanding of the mechanism of action can provide the knowledge that enables making rational choices when multiple dosing or formulation options are feasible. The biological origin of rosacea is relatively less understood. However, studies have clearly demonstrated the role of resident bacteria and the activity of kallikrein 5 enzyme (KLK5). 23,24 Previous studies have demonstrated excellent antiparasitic efficacy of CAGE 1:2 against a breadth of bacterial, fungal and viral species. 18 The ability of CAGE 1:2 to inhibit P. acnes as a model gram positive bacterium was tested using the CLSI standard broth microdilution methodology (BMD) (described in SI). CAGE 1:2 demonstrated high antibacterial activity against P. acnes isolates (Figure 2 respectively, indicating that CAGE 1:2 is effective in inhibiting hKLK5 activity, although its potency is lower than that of aprotinin ( Figure 2 (b) and (c), Figure S5, and Table S1).

| Translational step 6: GLP dermal toxicity in minipigs
Minipigs are considered an excellent model for studying toxicity of topical skin formulations. 26,27 GLP toxicity studies were performed in Göttingen minipigs by applying the CGB400 gel once a day for 91 consecutive days. Total of 1 ml CGB400 was applied over 10% of the total body surface area of the animal. All animals survived to scheduled study termination and there were no macroscopic findings. No clinical signs, changes in body weight, food consumption, ocular, electrocardiography, hematology, coagulation, clinical chemistry, urine analysis, and organ weights parameters were observed. Dermal changes including edema, erythema, fissuring, and focal red spots were observed in CGB400-treated animals and completely disappeared by the end of 14-day recovery period. Overall, CGB400 was well tolerated in minipigs.
Toxicokinetic (TK) parameters were determined from plasma con- TK studies in pigs also included two additional doses, one at 1.5× of CGB400 (i.e., 60% CAGE 1:2 ) and one at about twice that of CGB400 (i.e., 87% CAGE 1:2 , which is pure CAGE 1:2 which naturally absorbs water to form a 87% mixture). Generally, similar results were obtained at these doses and the plasma concentrations of geranic acid generally scaled with the dose ( Figure S6).

| Translational step 7: Clinical evaluation in human volunteers
In an IRB-approved 12-week open label cosmetic study for rosacea, subjects with a mean baseline lesion count (papules and pustules) of 13.4 ± 6.7 lesions were presented. Three sites were used for the study and the median age of subjects was 57 years (Figure 4(a)). After 2 weeks of therapy with CGB400 gel, the mean papules and pustules count was reduced by 34.6% (p = 0.0018) with further reductions in lesion counts being observed at each subsequent study visit. The greatest reduction in lesion counts was seen at Week 12 (Visit 5) where a mean drop from baseline of 71.9% (p < 0.0001) was observed ( Figure 4(b)).
It is worth noting that the CGB-400 gel dose applied daily on the minipig's skin was 1 g, where each gram of gel contained 0.4 g of CAGE. At the highest concentration (87%), the applied dose of CAGE 1:2 was 0.87 g per day, which is equivalent to 69.3 mg/kg/day, assuming 12.55 kg as an average weight of a minipig. For the studies in human volunteers,~0.2 g of a drug product was applied twice a day, that is, 0.08 g of CAGE 1:2 per application or 2.3 mg/kg/day for a 70 kg adult. As previously established, considering minipig as the most predictive animal model for human percutaneous absorption, the target human dose is~×30 lower than the highest dose evaluated in the repeat dose toxicity study in minipigs and thus justifies the translational potential of CGB400 gel. [28][29][30] In addition, investigator global assessment (IGA) responder rates (i.e., ≥1-point decrease from baseline) as well as the proportion of subjects with IGA scores of "clear" or "almost clear" were assessed. Both IGA measures demonstrated accumulated effects throughout the study. Therapeutic response was noted as early as    (Table S2).

| DISCUSSION
Here, we report clinical translation of a novel ionic liquid/deep eutectic solvent CAGE for the treatment of rosacea. CAGE has been previously reported as an engineered ionic liquid/deep eutectic solvent that possesses excellent skin permeation, antimicrobial activity and the ability to enhance drug delivery into skin. 31    after drying on the TGA which helped in removing most of the moisture and water associated thermal events (there may be a very low fraction of water left, that is, <0.5%). There were two parts to the MDSC analysis including run at 3 C/min followed by run at 1 C/min. The run at 1 C/min was better controlled over a wider range. It should be noted that the modulation signal gets a little distorted <-80C during the cooling cycle, so does not read the fine details below −80C. The TGA was ran in Hi-Res mode, that is, a dynamic (variable) heating rate where the heating rate is inversely proportional to the weight loss rate. On the Y-2 axis of the TGA plot, the derivative of the temperature vs time was plotted. This was simply to illustrate the instantaneous heating rate (degrees/min) at any particular temperature during the run. This shows that the dynamic heating rate varied from nearly 0 (around 160 C-when the weight dropped off the cliff) to 50 C/min near the end of the run. DVS, using gravimetric moisture sorption (GVS) analysis was also conducted. Both TGA and GVS analyses have been considered since, TGA measures volatiles in % wet weight, whereas GVS measures water uptake with respect to dry weight.
Additionally, FTIR analysis of choline, geranic acid and CAGE 1:2 were carried out and compared using FTIR Spectrometer, PerkinElmer Spectrum One. Report builder, Rev 2.01 (software) was used for spectra analyses. Considerable precipitate was observed when each of the test components was added to the media at a 1:2 dilution. Therefore, preliminary concentration checks were performed to determine the highest concentrations of the liquid to be utilized.

| hKLK5 enzymatic assay
Test compound CAGE 1:2 (87.07% w/v) was diluted to 0.99 mM and control compound aprotinin was prepared at 6 μM in di-water. A twofold serial dilution was then performed in di-water to generate 10 concentrations for CAGE 1:2 (87.07% w/v) and 8

| Forced degradation and stability validation studies
The content of choline and geranic acid in CGB400 gel following stability and forced degradation studies were analyzed via matrix-

| Determination of dermal toxicity and TK profile in minipigs
For GLP studies, the test and control/vehicle items were administered to groups of minipigs daily by nonoccluded dermal application for 13 weeks or 91 consecutive days. The volume of test or control/vehicle items (1 ml) was applied to the dermal test site and spread uniformly over the surface of the skin, using a gloved finger but ensuring that excessive residue did not remain on the glove. Gloves were changed between each group of animals. The same dermal test site was used for all doses. Assessment of toxicity was based on mortality, clinical observations, body weight, food consumption, dermal changes, ophthalmology, electrocardiography, and clinical and anatomic pathology.
During the GLP dermal toxicity study in Gottingen minipigs, a

| Phase Ib cosmetic study design
The goal of this study was to evaluate the ability of CGB400 in reducing facial redness, bumps/blemishes (inflammatory lesions such as papules and pustules) and determine its safety and tolerability. A multicenter, open-label, single-group POC study using CGB400 Gel to reduce facial redness, bumps, and blemishes was conducted. Twenty-five subjects with redness, but without bumps/ blemishes, received study treatment for 4 weeks and attended a total of five study visits (i.e., BL, W1, W2, W4, and posttreatment follow-up at W5). Twenty-seven subjects with redness and bumps/ blemishes received study treatment for 12 weeks and attended a total of six study visits (i.e., BL, W2, W4, W8, W12, and posttreatment follow-up at W13. CGB400 gel was applied topically to the face twice per day, once in the morning after a shower or washing the face, and once in the evening after washing the face. It was a POC study and a formal sample size justification was not provided.
It was the opinion of the sponsor that 40-50 subjects would be sufficient to achieve study objectives.

Diagnosis for inclusion included:
• Subjects with facial redness and bumps/blemishes: Facial redness associated with rosacea.
• Subjects with facial redness and without bumps/blemishes: Facial redness associated with rosacea.
Facial redness (IGAR) score of 2 or 3 (i.e., mild or moderate). and data analysis. Samir Mitragotri was involved in characterization, design of phase Ib study, and data analysis. Abhirup Mandal was involved in data summary and writing of the manuscript.

DATA AVAILABILITY STATEMENT
Any data associated with this study are available from the authors upon reasonable request.