Enhancing the Solubility of Active Pharmaceutical Ingredients using Deep Eutectic Solvents to Develop Liquid Oral Formulations

doi:10.21203/rs.3.rs-1824852/v1

Few oral liquid formulations of poorly soluble drugs are available. In this study, we explore the use of several hydrophobic deep eutectic solvents (DESs) formed by the binary combination of thymol, menthol, and camphor (1:1 thymol:camphor, 2:1 menthol:camphor, and 1:1 thymol:menthol) to increase the solubility of furosemide, tetracycline, nitrofurantoin, carvedilol, and quercetin.

The maximum solubility of the studied drugs in different DESs was experimentally measured using a modification of the shake-flask method. The results indicate that the solubility of all five chemicals is more than 50 times higher than the aqueous solubility, and the solubility of quercetin in 2:1 menthol:camphor is especially remarkable (nearly 7000 times the aqueous solubility).

A calculation of the DES volume required for the maximum adult dose for each drug shows the oral liquid formulations can be prepared for tetracycline and carvedilol using the three studied DESs and for furosemide using 2:1 menthol:camphor. However, stability and formulation studies need to be performed to ensure that the oral liquid formulations prepared with these solvents comply with pharmacopeia requirements.

Hydrophobic DES

solubility

furosemide

tetracycline

nitrofurantoin

carvedilol

and quercetin

The difficulty of formulating poorly soluble active pharmaceutical ingredients (APIs) is a well-known problem that has been frequently studied in pharmaceutical science [1–3] (Class II and IV, Biopharmaceutical Classification System, BCS)[4]. Approximately 40% of APIs with market approval and almost 90% of medicines under development are poorly soluble in aqueous media[3]. The same problem with solubility arises with some important nutraceuticals: for example, natural antioxidants of phytochemicals can be biologically unstable and exhibit low solubility, stability, bioavailability and specificity[5]. The possibility of preparing oral liquid formulations are quite limited in these cases. However, liquid oral forms are the most recommended formulations for elderly and paediatric patients[6,7]. Other subpopulations of patients, such as those weakened by stroke, may also benefit from formulations specifically designed for children or elderly people.

To address these solubility and bioavailability issues, several approaches have been proposed within the pharmaceutical framework (science, industry, and regulatory agencies) [3,8–10]. The current scheme involves searching for the best formulation of poorly soluble and poorly bioavailable drugs from a range of options: salt forms and cocrystal formations, micronisation, use of cosolvents, complexation and lipidic formulation, emulsions, and suspensions[1–3,9]. In this study, we explored the use of novel solvents to improve the solubility profiles of selected APIs with poor bioavailabilty and solubility.

APIs.

Deep eutectic solvents (DESs) are used in this study. These solvents are mixtures of two or more compounds of natural origin that that exist as solids when pure but as liquids in mixtures (due to a decrease in the melting temperature of the compounds). The chemical structure of a DES is analogous to that of the well-known ionic liquid, because a DES is formed by the complexation of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD)[11]. DESs offer several advantages over other solvents[12,13]: DESs are not expected to react with water and have a high solvation capacity for a large variety of substances, including drugs and nutraceuticals[12,14]. DESs are economically competitive[15] and have significant green and safety potential: some DESs are easily biodegradable[16], and low (eco)toxicity profiles have been found for most cases[17–20].

The DESs used in this study are hydrophobic binary mixtures of thymol, menthol, and camphor. These DESs are composed only of molecular substances and have been previously categorised as Type V[21]. Among many applications, hydrophobic DESs can be useful for boosting the solubility of nonpolar substances because of the existence of π-interactions between a solute and a DES[22].

In this study, three hydrophobic DESs (binary mixtures of thymol, menthol, and camphor) were used to solubilise four APIs (furosemide, tetracycline, nitrofurantoin, and carvedilol) and one nutraceutical (quercetin). Table 1 provides details of the use and dosage of these APIs. Despite their importance, neither these APIs nor the nutraceutical are available in commercial liquid oral dosage forms. The working hypothesis of this study is that the selected DESs improve the aqueous solubility of the selected APIs up to the maximum dose needed for therapeutic purposes.

Table 1

Main uses, dosage forms, maximum dosing, class in the biopharmaceutical classification system and aqueous solubility (mg/l) of the studied APIs and nutraceutical.
Studied APIs and nutraceutical	Main use	Commercial oral dosage forms	Maximum adult dose	Maximum child dose	BCS Class	Aqueous solubility at 298.15 K (mg/l)
Furosemide	Diuretic Reduction of oedema Reduction of high blood pressure	Tablet	80 mg	40 mg	IV	18[23]
Tetracycline	Treatment of bacterial infections	Capsule	500 mg	500 mg	III	231^b
Nitrofurantoin	Treatment of urinary tract infections	Tablet^a	50 mg	10 mg	II	79,5^c
Carvedilol	Treatment of ischaemic heart disease Congestive heart failure Reduction of high blood pressure	Tablet	50 mg	-	II	88^d
Quercetin	Relief of allergy symptoms Adjuvant for treatment of gastrointestinal ulcers	Tablet Powder	1000 mg	-	IV	2,15[24]

^a Nitrofurantoin as an oral suspension has only been prescribed for use in the United States.

^b from Drugbank https://go.drugbank.com/drugs/DB00759 Accessed 21st April 2022.

^c at 297,17 K, from Drugbank https://go.drugbank.com/drugs/DB00698 Accessed 21st April 2022.

^d from Drugbank https://go.drugbank.com/drugs/DB01136

2.1 Reagents

Information about the pure chemicals and their chemical structures is provided in Table 2. All reagents were used without further purification.

2.2 DES preparation

Three different DESs were prepared as mixtures of thymol, menthol and camphor with the following compositions: thymol:camphor (1:1), menthol:camphor (2:1), and thymol:menthol (1:1). The DESs were prepared by measuring the components in the desired compositions using a PB210S Sartorius balance, followed by mixing under magnetic stirring and gentle heating (below 323 K) until a homogeneous liquid was formed. The uncertainty in the mass determination was ± 10^− 4 g. The mixtures were stored in the dark until use. Some information about the physicochemical properties of the studied DESs is provided in Table 3.

Table 3

Physicochemical properties of the studied DESs: molecular weight (Mw), melting point (Tm) and density (ρ) at 298.15 K.
DES	Composition	Mw (g/mol)	Tm (K)	ρ (g/ml)
Thymol:camphor	1:1	151.23	229[25]	0.96698
Menthol:camphor	2:1	154.92	280[26]	0.91241
Thymol:menthol	1:1	153.24	214.5[21]	0.93302

2.3 Solubility study

The solubility of the APIs in the hydrophobic DESs was measured using a modification of the shake-flask method[22]. The method is detailed below.

Calibration curves for each API in solution were constructed using ethanol as the solvent for furosemide, nitrofurantoin, carvedilol and quercetin and a mixture of isopropanol:water (3:1) for tetracycline. For each system, the wavelength corresponding to the maximum absorbance was used (as previously determined by a wavelength sweep). The maximum absorbance data, \({Abs}_{max}\), were plotted against the weight concentration, W (g solute/g solvent).

Supersaturated solutions of each API were prepared at a controlled temperature and protected from light using aluminium foil. At least 12 samples of each solution were placed in an ultrasound bath (Ultrasounds-H Selecta) for 15-minute cycles at 60 Hz. A second cooling bath was connected to the system to prevent heating by the ultrasonic treatment; thus, the temperature during the experiments was kept constant at 298.15 K. Every 15 minutes, 2 samples of each solution were centrifuged for 5 minutes at 5000 rpm (Nahita Model 2716). The supernatants were passed through a PES 0.22-µm syringe filter and analysed using UV-Vis spectroscopy with a double-beam spectrum VWR 6300 PC (u(λ) = ± 0.2 nm), where the absorbance was interpolated based on the previously obtained calibration curves. The results are presented as the median concentration of 2 samples. Samples with higher concentrations than those used to obtain the calibration curves were diluted. The process was repeated in 15-minute cycles until the final concentration did not increase over time. The final concentration was considered as the maximum solubility at the operating temperature.

2.4 Statistical analysis

The GraphPad Prism program was used to perform a statistical analysis of the solubility data using the one-way ANOVA method; the means of more than two groups were compared with each other, where the different groups consisted of the maximum solubilities of the different APIs in the same DES. Subsequently, a post hoc study adjusted to the Tukey–Kramer honest significant differences model was carried out to identify groups that differed significantly from each other.

The null hypothesis (H₀) is that there are no statistically significant differences between groups and therefore the groups are equivalent, and the alternative hypothesis (H₁) is that there are differences between groups. A 95% confidence interval is chosen, such that if p < 0.05, the null hypothesis is rejected, and the alternative is accepted.

3.1 Calibration curves

The calibration curves and validation parameters, such as coefficient of determination, R², limit of detection, LD, and limit of quantification, LQ, are shown in Table 4. None of the solvents used showed interference in the spectra.

Table 4

Calibration equation for each API/nutraceutical in the selected solvents, wavelength of maximum absorbance, λ *ABS*_max, and validation parameters: coefficient of determination, R², limit of detection, LD, and limit of quantification, LQ; ^a \(LD=\frac{x+3S}{m}\) ; ^b \(LQ=\frac{x+10S}{m}\), where \(m\) is the slope, and \(x\) and \(S\) are the average and standard deviation of the blank, respectively.
Studied APIs and nutraceutical	Solvent	Calibration equation	λ*(Abs_max)(nm)*	R²	*LD^a*	*LQ^b*
Furosemide	Ethanol	\({Abs}_{max}=12433\bullet W\)	342	0.9925	3.61· 10^− 7	9.78·10^− 7
Tetracycline	Isopropanol:water (3:1)	\({Abs}_{max}=32395\bullet W\)	364	0.9975	8.57·10^− 8	2.30·10^− 7
Nitrofurantoin	Ethanol	\({Abs}_{max}=61431\bullet W\)	365	0.9998	9.67·10^− 8	4.28·10^− 7
Carvedilol	Ethanol	\({Abs}_{max}=12507\bullet W\)	331	0.9963	4.08·10^− 7	1.14·10^− 6
Quercetin	Ethanol	\({Abs}_{max}=56245\bullet W\)	374	0.9998	1.48·10^− 8	7.70·10^− 8

3.2 Solubility

The mean experimentally determined maximum concentration (in mg/l) of each API in the studied DESs is shown in Table 5. The results are compared in Fig. 1. In general, the highest solubilities are found for tetracycline, whereas the lowest solubilities correspond to nitrofurantoin. Compared to the aqueous solubility, the highest increase in solubility for tetracycline and carvedilol is obtained using 1:1 thymol:menthol, whereas the highest increase in solubility for furosemide and quercetin is obtained using 2:1 menthol:camphor. The studied DESs do not produce any clear improvement in the nitrofurantoin solubility; however, 2:1 menthol:camphor produces the lowest solubilities for nitrofurantoin .

Table 5

Solubility results in mg/l for the studied APIs in different DESs (1:1 thymol:camphor, 2:1 menthol:camphor and 1:1 thymol:menthol), and a comparison of these results against the aqueous solubility (where ratio denotes the DES solubility/aqueous solubility).
Studied APIs and nutraceutical	Thymol:camphor 1:1		Menthol:camphor 2:1		Thymol:menthol 1:1
Studied APIs and nutraceutical	Solubility (mg/l)	Ratio	Solubility (mg/l)	Ratio	Solubility (mg/l)	Ratio
Furosemide	1014	56	6988	388	849	47
Tetracycline	24551	106	23166	100	37883	164
Nitrofurantoin	501	6	102	1	519	7
Carvedilol	12655	2876	24521	5573	28100	6388
Quercetin	1374	639	14902	6931	624	290

The results of the statistical study are shown in Table 6: all the experimental values are statistically different, except for the solubilities of furosemide and quercetin in 1:1 thymol:camphor.

Table 6

Results of the ANOVA test and the p values for a 95% confidence interval. The shaded cell indicates no statistical difference between the respective experimental values.
p values for the maximum solubilities of the studied APIs and nutraceutical in each DES
Thymol:camphor 1:1
	Furosemide	Tetracycline	Nitrofurantoin	Carvedilol	Quercetin
Furosemide	-	< 0.0001	0.017	< 0.0001	0.0677
Tetracycline		-	< 0.0001	< 0.0001	< 0.0001
Nitrofurantoin			-	< 0.0001	0.0016
Carvedilol				-	< 0.0001
Quercetin					-
Menthol:camphor 2:1
	Furosemide	Tetracycline	Nitrofurantoin	Carvedilol	Quercetin
Furosemide	-	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Tetracycline		-	< 0.0001	0.0002	< 0.0001
Nitrofurantoin			-	< 0.0001	< 0.0001
Carvedilol				-	< 0.0001
Quercetin					-
Thymol:menthol 1:1
	Furosemide	Tetracycline	Nitrofurantoin	Carvedilol	Quercetin
Furosemide	-	< 0.0001	< 0.0001	< 0.0001	< 0.0001
Tetracycline		-	< 0.0001	< 0.0001	< 0.0001
Nitrofurantoin			-	< 0.0001	< 0.0001
Carvedilol				-	< 0.0001
Quercetin					-

Bioavailability can be defined as the fraction of an administered dose that reaches the systemic circulation and reflects the rate at which the drug is made available. This parameter depends, among other factors, on the solubility of the API in the organism. Low solubility may imply low bioavailability[27]. For absorption (within the absorption, distribution, metabolism, and excretion scheme, ADME), a drug must be present as an aqueous solution at the site of absorption[28].

The APIs tetracycline, nitrofurantoin and carvedilol belong to class II of the BCS (corresponding to high permeability and low solubility), that is, the rate of absorption of these drugs is higher than the dissolution rate and controls the overall rate. Furosemide and quercetin belong to class IV of the BCS (corresponding to low permeability and low solubility). Class IV drugs cannot be effectively administered orally and are poorly absorbed by intestinal mucosa.

All the APIs and the nutraceutical that were studied were more soluble in the DESs than in water (Table 4). Although the increase in solubility for nitrofurantoin was small, the solubilities of all the other drugs were more than 50 times higher than the corresponding aqueous solubilities, where the solubility of quercetin in 2:1 menthol:camphor was especially remarkable (nearly 7000 times higher than in water). The polarities of the components of the three investigated DESs are low (all the components have a dipole moment µ < 3 D) and quite similar, and the polarity of all the APIs and the nutraceutical, except for carvedilol, is slightly higher than the polarity of the DESs.

Table 7 shows the calculated volume of each DES required for the adult maximum adult therapeutic dose. Considering only the required volume and the API solubility, the results indicate that oral liquid formulation is possible for both adults and children for furosemide using 2:1 menthol:camphor and for tetracycline and carvedilol using any of the studied DESs, among which the lowest volume is required for 1:1 thymol:menthol. Oral liquid formulation is also possible for nitrofurantoin in 1:1 thymol:camphor and 1:1 thymol:menthol for children. However, other pharmacopeia requirements should be analysed in terms of the maximum allowed dose of monoterpenes for oral liquid formulations.

Table 7: Volume required for therapeutic dose of each API in the different DESs.

Studied APIs and nutraceutical	Volume required for maximum therapeutic dose (ml)
	Thymol:camphor 1:1		Menthol:camphor 2:1		Thymol:menthol 1:1
	Adults	Children	Adults	Children	Adults	Children
Furosemide	79	39	11	6	94	47
Tetracycline	20	20	22	22	13	13
Nitrofurantoin	100	20	490	98	96	19
Carvedilol	4	-	2	-	2	-
Quercetin	728	-	67	-	1603	-

An attempt was previously made to formulate ethanol-free oral liquid furosemide for paediatric use[29]. That is, two formulas containing 2000 mg/l furosemide were developed for paediatric patients > 1 month old based on the strategy of increasing the pH to raise the aqueous solubility. Sodium hydroxide and disodium hydrogen phosphate were used to increase the pH. Other excipients were added to modify the taste and stability of the preparations. By comparison, 2:1 menthol:camphor can be used to therapeutically dose furosemide without the use of any other excipients or additives to modify the taste, which is a clear advantage.

To the best of our knowledge, no studies have previously been conducted on developing oral liquid formulations of tetracycline. There has been only one attempt to dissolve the salt form of this drug (tetracycline hydrochloride) using several Ora® syrups[30]. However, these preparations had limited stability, and the authors recommended the use of the nonionic form to increase stability.

Some studies have been performed on preparing oral liquid formulations of carvedilol using different strategies: self-emulsification[31,32], liquisolid techniques[31], coamorphisation of the drug with acidic amino acids[33] and nanomicelle/nanoemulsification formulations[34–38]. In all these studies, several vehicles were used to improve the bioavailability potential of carvedilol, and the API did not dissolve on its own but was incorporated into an emulsion/dispersion.

A preliminary study on the suitability of using DESs to prepare oral liquid formulations of poorly soluble APIs and a nutraceutical was carried out.

We proved our working hypothesis for the APIs carvedilol, tetracycline and furosemide (the selected DESs increase the solubilities of selected APIs relative to the aqueous solubility up to the maximum required dose needed for therapeutic purposes). For carvedilol and tetracycline, oral liquid syrups can be formulated using any of the three studied DESs, whereas 2:1 menthol:camphor can be used to prepare an oral liquid formulation of furosemide. It is remarkable that the solubility of all the studied chemicals in the DESs is more than 50 times higher than the aqueous solubility. However, stability and formulation studies must be performed to ensure these proposed oral liquid formulations meet pharmacopeia requirements.

Acknowledgement

All the authors gratefully acknowledge the financial assistance from Gobierno de Aragón and Fondo Social Europeo “Construyendo Europa desde Aragón” (E31_20R)

CRediT author statement

Leyre Pascual-Férnandez: Investigation, Formal analysis.

Fernando Bergua: Methodology, Formal analysis.

Manuela Artal: Conceptualization, Validation, Methodology.

Carlos Lafuente: Validation, Resources, Supervision.

Beatriz Giner: Data Curation, Validation, Writing - Original Draft, Visualization, Supervision.

D.J. Hauss, Advanced Drug Delivery Reviews 59 (2007) 667.
A.M. Kaukonen, Poorly water soluble substances: challenges, options and limitations for children. European Medicines Agency, 2010.
S. Kalepu, V. Nekkanti, Acta Pharmaceutica Sinica B 5 (2015) 442.
M.G. Papich, M.N. Martinez, Aaps Journal 17 (2015) 948.
S. Wang, R. Su, S.F. Nie, M. Sun, J. Zhang, D.Y. Wu, N. Moustaid-Moussa, Journal of Nutritional Biochemistry 25 (2014) 363.
A. Zajicek, M.J. Fossler, J.S. Barrett, J.H. Worthington, R. Ternik, G. Charkoftaki, S. Lum, J. Breitkreutz, M. Baltezor, P. Macheras, M. Khan, S. Agharkar, D.D. MacLaren, Aaps Journal 15 (2013) 1072.
Guideline on pharmaceutical development of medicines for paediatric use. European Medicines Agency, 2013.
J. Mitchel, D. Elder, American Pharmaceutical Reviews (2015).
T. Loftsson, International Journal of Pharmaceutics 531 (2017) 276.
P. Manikandan, N. Subramanian, International Journal of Pharmaceutical Sciences Review and Research 23 (2013) 220.
D. Lapena, L. Lomba, M. Artal, C. Lafuente, B. Giner, Journal of Chemical Thermodynamics 128 (2019) 164.
Y.T. Dai, J. van Spronsen, G.J. Witkamp, R. Verpoorte, Y.H. Choi, Analytica Chimica Acta 766 (2013) 61.
A. Paiva, R. Craveiro, I. Aroso, M. Martins, R.L. Reis, A.R.C. Duarte, Acs Sustainable Chemistry & Engineering 2 (2014) 1063.
H. Lores, V. Romero, I. Costas, C. Bendicho, I. Lavilla, Talanta 162 (2017) 453.
X. Tang, M. Zuo, Z. Li, H. Liu, C. Xiong, X. Zeng, Y. Sun, L. Hu, S. Liu, T. Lei, L. Lin, Chemsuschem 10 (2017) 2696.
K. Radosevic, M.C. Bubalo, V.G. Srcek, D. Grgas, T.L. Dragicevic, I.R. Redovnikovic, Ecotoxicology and Environmental Safety 112 (2015) 46.
B. Kudlak, K. Owczarek, J. Namiesnik, Environmental Science and Pollution Research 22 (2015) 11975.
I. Juneidi, M. Hayyan, O.M. Ali, Environmental Science and Pollution Research 23 (2016) 7648.
B. Giner, C. Lafuente, D. Lapena, D. Errazquin, L. Lomba, Ecotoxicology and Environmental Safety 191 (2020).
D. Lapena, D. Errazquin, L. Lomba, C. Lafuente, B. Giner, Environmental Science and Pollution Research 28 (2021) 8812.
D.O. Abranches, M.A.R. Martins, L.P. Silva, N. Schaeffer, S.P. Pinho, J.A.P. Coutinho, Chemical Communications 55 (2019) 10253.
F. Bergua, M. Castro, J. Munoz-Embid, C. Lafuente, M. Artal, Chemical Engineering Journal 411 (2021).
G.E. Granero, M.R. Longhi, M.J. Mora, H.E. Junginger, K.K. Midha, V.P. Shah, S. Stavchansky, J.B. Dressman, D.M. Barends, Journal of Pharmaceutical Sciences 99 (2010) 2544.
K. Srinivas, J.W. King, L.R. Howard, J.K. Monrad, Journal of Food Engineering 100 (2010) 208.
P. Makos, A. Przyjazny, G. Boczkaj, Journal of Chromatography A 1570 (2018) 28.
T. Phaechamud, S. Tuntarawongsa, P. Charoensuksai, Aaps Pharmscitech 17 (2016) 1213.
J.Y. Kim, S. Kim, M. Papp, K. Park, R. Pinal, Journal of Pharmaceutical Sciences 99 (2010) 3953.
E.M. Merisko-Liversidge, G.G. Liversidge, Toxicologic Pathology 36 (2008) 43.
L. Zahalka, S. Klovrzova, L. Matysova, Z. Sklubalova, P. Solich, European Journal of Hospital Pharmacy-Science and Practice 25 (2018) 144.
B.D. Glass, A. Haywood, Journal of Pharmacy and Pharmaceutical Sciences 9 (2006) 398.
T.M. Ibrahim, M.H. Abdallah, N.A. El-Megrab, H.M. El-Nahas, Drug Development and Industrial Pharmacy 44 (2018) 873.
L.A.D. Silva, S.L. Almeida, E.C.P. Alonso, P.B.R. Rocha, F.T. Martins, L.A.P. Freitas, S.F. Taveira, M.S.S. Cunha, R.N. Marreto, International Journal of Pharmaceutics 541 (2018) 1.
J. Mishra, K. Lobmann, H. Grohganz, T. Rades, International Journal of Pharmaceutics 552 (2018) 407.
F.S. Kuruvila, F. Mathew, S. Kuppuswamy, Indo American Journal of Pharmaceutical Sciences 4 (2017) 651.
M. Wegmann, L. Parola, F.M. Bertera, C.A. Taira, M. Cagel, F. Buontempo, E. Bernabeu, C. Hocht, D.A. Chiappetta, M.A. Moretton, Journal of Pharmacy and Pharmacology 69 (2017) 544.
S. Beg, S. Swain, H.P. Singh, C.N. Patra, M.E.B. Rao, Aaps Pharmscitech 13 (2012) 1416.
B. Singh, L. Khurana, S. Bandyopadhyay, R. Kapil, O.O.P. Katare, Drug Delivery 18 (2011) 599.
S. Chakraborty, D. Shukla, P.R. Vuddanda, B. Mishra, S. Singh, Colloids and Surfaces B-Biointerfaces 81 (2010) 563.

Table 2 is not available with this version

No competing interests reported.

Enhancing the Solubility of Active Pharmaceutical Ingredients using Deep Eutectic Solvents to Develop Liquid Oral Formulations

Status:

Version 1

Abstract

Figures

1. Introduction

2. Methodology

2.1 Reagents

2.2 DES preparation

2.3 Solubility study

2.4 Statistical analysis

3. Results

3.1 Calibration curves

3.2 Solubility

4. Discussion

5. Conclusions

Declarations

References

Tables

Additional Declarations

Status:

Version 1