Solubility improvement of atorvastatin using deep eutectic solvents

Abstract

Deep eutectic solvents (DESs) in pharmaceutical investigations are still largely unknown in terms of safety, environmental acceptability, and practical use. The investigation focus is primarily of attempts to improve solubility is examined using the co-crystallization approach, which follows similar principles to the production of eutectic mixtures. We will look into the eutectic mixture interaction between selected coformers (nicotinamide [NA], isonicotinamide, and citric acid as acceptors of hydrogen bond and atorvastatin calcium trihydrate [ATCH] as donors of hydrogen bond in the DES system, assisted by propylene glycol [PG]). The study found that the optimum interaction in the DES system for ATCH is using NA in PG as a carrier, with evaluation by polarization microscopy, Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC), verifying that the drug and the DES components have formed H-bonds. H-bond interactions had been recognized through FT-IR and DSC, revealing that the DESs with NA coformer can increase ATCH solubility, as shown in the phase diagram. The increase in solubility was fairly considerable, allowing ATCH to dissolve at high concentrations in DESs, with a solubility of 0.158 ± 0.098 mg/mL compared to 0.000597 ± 0.003 mg/mL in water. Thus, it is obtainable to conclude that among the selected coformers, the DES system with the potential for improving the solubility of ATCH is DESs with NA coformer assisted by PG.

INTRODUCTION

Enhancing the bioavailability and dissolution of hydrophobic medications is a primary goal in pharmaceutical research and production. Drugs with poor solubility in water significantly limit bioavailability and can cause serious adverse effects, necessitating formulations to address this. To address the issue of poor active pharmaceutical ingredients (API) solubility, a variety of delivery techniques were established, which may be classified into two groups. The first kind involves physically and chemically changing the API molecules, whereas the second employs a formulation method. One example that will be investigated is atorvastatin (ATV), which has undergone numerous procedures in order to improve its solubility.[1]

ATV, the most commonly prescribed statin, was created and originally sold by Pfizer under the trade name Lipitor^® and was the best-selling medicine in the United States until its patent expired in 2011. This medicine stood out as the most profitable drug worldwide from 2002 to 2009, achieving amazing amounts total sales of about 9.3 billion dollars.[2] It functioned as an initial lipid-lowering medication by competetively inhibiting the enzyme hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonic acid. This conversion was a critical metabolic event that creates multiple chemicals involved in lipid metabolism and transport. It is the rate-limiting step in hepatic cholesterol processing.[3]

However, due to a number of biopharmaceutical issues, ATV belongs to the category of a biopharmaceutics classification system class II (low solubility/high permeability), as its solubility is affected by pH and it dissolves quickly. The combination of extensive intestine cleansing and first-pass metabolism in the liver results in low bioavailability both absolutely and systemically of around 12% and 30%, respectively. ATV has minimal solubility in aqueous solutions below pH 4 and limited solubility in water and phosphate buffers at pH 6.8. High intestinal permeability is linked to physiologically relevant pH levels. Due to its poor solubility, the absorption is primarily controlled by meal consumption, with decreased rates of 25% and 90%, respectively. This leads to uneven clinical usage. As additional information regarding toxicity, the use of high doses of ATV for chronic treatment can cause liver and muscle damage, including rhabdomyolysis, reducing its therapeutic usefulness.[2] As the consequence, a breakthrough is required to achieve the greatest level of solubility, allowing it to take effect immediately and minimizing the potential negative effects.

After the patent expired, plenty of companies began producing ATV with a selection as atorvastatin calcium trihydrate (ATCH), which is currently available in polymorph I as stable crystalline or amorphous form.[4] Presently, as for trying of addressing the aforementioned challenge, some physically and chemically modified, then at least 60 polymorphic forms/solvates/hydrates have been patented,[5,6,7] also including changing particle size,[8] co-grinding,[9,10,11,12] crystal modification,[13,14] and numerous pharmaceutical institutions are still working on generic medication formulations based on many different atorvastatin calcium polymorphs.[15,16,17]

Another technique that had not been attempted on ATCH to improve its solubility is to combine it with safe chemicals, which can lower the melting point at a certain molar ratio while causing eutectic behavior. In certain situations, the mixture’s melting point is dropped below room temperature, resulting in a liquid at room temperature, known as a deep eutectic solvent (DESs). The intermolecular interactions of the API and the coformer in the DES system are based on hydrogen bonds, alkyl–alkyl interactions, and Van der Waals forces with hydrogen interactions, which are the fundamental causes of the lowering of the melting point of eutectic mixtures to room temperature.[18,19] DESs are presently widely employed in a range of industrial applications, including the extraction of biomolecules in natural goods[20] extraction of nonhydrocarbon species from petroleum products, and metal extraction.[21] Moreover, DESs are commonly used in electropolishing,[22] desulfurization,[23] polymer synthesis, metal recycling, and metal electrodeposition.[24] However, the ideas of DESs may also be employed in the pharmaceutical sector to overcome the solubility and permeability difficulties of API different studies have reported on different APIs, which are thus named Therapeutics DESs (TheDESs), or Pharmaceuticals DES (PDESs).[25,26] Therefore, the aim of this investigation was carried out concerning the development of DES from ATCH with several chosen excipients as coformers and cosolvents.

DESs have been described as an appealing solution for improving pharmaceuticals solu-bility and/or permeability while also influencing bioavailability. Among the several potential varieties of DESs, THEDESs, a novel type of DES with at least one active ingredient, has been developed throughout the years. As due to of their natural and therapeutic attributes as well as their high biodegradability and biocompatibility, the DESs created by these combinations may be considered safe, nontoxic, and beneficial for any applications, such as pharmaceutical delivery systems. THEDESs or DESs are distinct from the co-amorphous system; THEDESs is more thermodynamically stable since DESs does not crystallize.[25,27] The distinction between DESs and co-amorphous systems is that DES is liquid at room temperature, whereas co-amorphous systems are solid at ambient temperature.[25,26]

MATERIALS AND METHODS

Materials

ATCH (DSM Anti-Infectives India Ltd., India), Nicotinamide (NA) (ex Westerndrug, India), Isonicotinamide (INA) (ex Yisheng Pharma, China), citric acid anhydrous (Pyridam Farma), Propylene glycol (PG) (ex Dow Chemical Pacific, USA), Methanol (ex Merck), distilled water.

Preparation of eutectic mixture

Eutectic mixing took place when the solid form of ATCH is heated and stirred with specified coformers on a heating plate and magnetic stirrer. A heating plate is used to melt varying weight ratios of ATCH solids and coformer. The temperature was gradually changed until it melts the combination, then recorded and utilized as data to create a phase diagram.

Phase diagram of eutectic mixture

Melting point data for each recorded combination of weight ratio composition are used to generate the phase diagram. The phase diagram developed allowed for the determination of the optimal mixture of compounds between ATCH and coformers, with the composition that results in the lowest melting point. The lowest melting point of this mixture is the eutectic point of the ATCH combination.

Preparation of deep eutectic solvents

DESs was prepared by combining PG as a cosolvent with coformers INA, NA, or citric acid. Before mixing, the mole fraction of the mixture is determined to establish the optimal ratio for forming the eutectic point between the two compounds. After calculating the mole fraction, the two compounds were heated and stirred using a magnetic stirrer. Following that, the mole ratio composition was measured, resulting in a clear solution after 1 day of cooling. The DES solvent was then used to dissolve ATCH and analyzed using different equipment.[31]

Characterization of deep eutectic solvents

Polarisation microscopy

Small droplets of the produced DES (PG: Coformers) were placed on a microscope slide and observed at 200x and 400x magnification. A polarizing microscope (Olympus BX53 model U-LH100-3 microscope) is used to capture pictures of polarized light. The lack of a solid crystalline structure is demonstrated by entirely clear polarized light photographs. This demonstrates that ATV and DES do not react, resulting in the formation of a cocrystal molecule.

Fourier transform infrared spectroscopy

The eutectic mixture was characterized using Fourier transform infrared spectroscopy (FT-IR) spectroscopy (IR Prestige-21 Shimadzu, Japan) to detect interaction among functional groups. The resulting DES is analyzed using combination of 1 mg sample at 60 mg potassium bromide and compacted into pellets. Infrared spectroscopy was utilized to measure wavelengths ranging from 4,000 to 400 cm⁻¹ at a speed of 0.5 cm/s.

Differential scanning calorimetry

A total of 5–9 mg of sample was placed in a tightly closed sample container before thermal analysis using the differenital scanning calorimetry (DSC) instrument (DSC-60A PLUS, Shimadzu). The thermogram is set at a temperature range of 20°C–250ºC at a rate of 10°C/min to detect all changes in the melting point of the substance.

Solubility test

A specific quantity of ATCH was weighed and added into 1 mL of the DES mixture until saturated, then agitated for 24 h to achieve equilibrium at room temperature. After mixing, the sample was filtered using a PTFE filter needle with a 0.45 μm membrane to remove insoluble ATCH solids. The liquid phase was then diluted with methanol, and the quantity of dissolved ATCH was measured using a ultraviolet-Vis spectrophotometer (Analytical Jenna Specord 200). The absorbance of the solution was measured at ATCH’s maximal absorbance (246 nm). To determine the concentration of ATCH in the solvent, a calibration curve was developed. The solubility of ATCH in this eutectic solvent was then compared to that of ATCH in PG and water.

RESULTS AND DISCUSSION

The word “eutectic” itself is an ancient Greek word “eútēktos” which means easy to melt.[32] Eutectics can include both inorganic and organic substances. Mixtures of organic and inorganic, or inorganic and inorganic, can be used to develop eutectics. This makes it possible for a wide variety of compound combinations to be eutectic and appropriate for certain applications.

In the pharmaceutical industry, eutectic mixtures are not as commonly employed as cocrystallization development. This is because eutectic mixes are difficult to create, it is impossible to forecast if eutectics are present, and characterizing these combinations takes time and resources.[33]

Even though eutectic and cocrystallization processes are closely connected. It’s simply that the melting point reduction of the two-component chemicals varies. The distinction between eutectic and cocrystallization is the synthesis of molecular compounds and a corresponding reduction in melting points.

Cocrystallization produces a molecular product with an unique crystal structure and a lower melting point than the parent molecule. Meanwhile, the eutectic system does not alter in terms of crystal structure; rather, some combination compositions can drastically reduce the melting point. A possible technique is to generate a phase diagram of the two combined chemical compositions, as shown in Figure 1.[34]

Figure 1.

Difference between (a) co-crystal and (b) eutectic mixture in phase diagrams

The melting points of the two components, compound A and compound B, are significantly dependent on their interaction. The more deformation between both, the greater the drop in melting point. The eutectic point, also known as the eutectic temperature, is the lowest melting point that may be obtained by combining chemicals A and B. The phase diagram depicts three phases: liquid, mixed, and solid. The liquid phase is formed when chemicals A and B are melted. When solid A starts to solidify while B remains liquid as the temperature drops, the mixed phase, liquid + solid A, is created at temperatures below the liquidus curve. The solid phase is the temperature at which both compounds become solid.[21] To convert the eutectic mixture into THEDES or DES, a natural solvent is required to maintain the composition liquid. Solvents often employed include choline chloride, many high-density alcohols (mannitol, sorbitol, PG, etc.), fatty acids, and other corresponding molecules that can operate as hydrogen bond acceptors as like in Table 1.[35]

Table 1.

The following are some cases of the use of deep eutectic solvents in pharmaceuticals

API	DESs (mol ratio)	Solubility in DES (mg/mL)	Solubility in water (mg/mL)	References
Acetylsalicylic acid	PG – ChCl (2:1)	202	7.03	[28]
Itraconazole	Malonic acid – ChCl (1:1)	22	<0.001	[28]
Benzoic acid	Urea – ChCl (2:1)	229	3	[29]
Piroxicam	Glycolic acid – ChCl (2:1)	9.9	0.023	[30]

API: Active pharmaceutical ingredients, DES: Deep eutectic solvents, PG: Propylene glycol, ChCl: Choline chloride

Phase diagram of eutectic mixture

The ATCH eutectic mixture with the specified coformer is prepared by heating and stirring with a heating plate and magnetic stirrer. Heating is carried out gently when the ratio of each coformer to ATCH reaches 300 mg at temperatures ranging from 90°C to 161°C. The melting temperature is then measured, and a phase diagram appears, as seen in Figure 2.

Figure 2.

Phase diagram represent-tation of atorvastatin calcium trihydrate and selected coformers ((a) isonicotinamide, (b) nicotina-mide, and (c) citric acid)

The phase diagram revealed a significant reduction in melting point for the three coformers with a eutectic system profile. The lowest melting point drop is achieved with a 1:1 eutectic combination of ATCH and NA up to 105°C, as opposed to the same ratio in INA, which gives a temperature range of 130°C. This is due to changes in intermolecular forces, which influence how molecules are organised in the solid state. Stronger hydrogen bonding interactions in the INA construct a more ordered and denser crystal structure, resulting in a higher melting point.[36]

Polarisation microscopy

A polarisation microscopy was used to study the melt of the two coformers to determine whether there is any just emerging crystallisation or if it has entirely melted, as shown in Figure 3. As seen in Figure 3, the picture appears clean and purple, indicating that the melting occurred successfully and left no crystals.

Figure 3.

Observation results of polarisation microscopy (a) Atorvastatin calcium trihydrate (ATCH) – Deep eutectic solvent (DES) (×200), (b) ATCH – DES (×400)

The eutectic mixture is composed of molecules that serve as hydrogen acceptors and donors. Hydrogen acceptors are electronegative atoms such as N, O, or F. Examples of hydrogen donors include NH₃, H₂O, ROH, NH (R₂), and NH₂R.[37] The fundamental difference between INA and NA is the location of the amide group (CO-NH₂) in their structures. In NA, the amide group is in position 4 of the pyridine ring, whereas in NA, it is in position 3. The amide group in INA is at position 4, which allows the development of stronger and more effective hydrogen bonding between neighbouring molecules than the amide group at position 3 in NA.[38]

Based on this, we exclusively investigate the eutectic mixtures with the best melting point decrease, namely the ATCH and NA eutectic mixtures, in addition to the DES results. Figure 4a depicts the findings of studying functional groups using FT-IR, which reveal that there is no observable interaction between functional groups that produces new functional groups. It’s merely that highly observable hydrogen bond interactions are seen in the 2800–3600 cm⁻¹ range, which corresponds to the elongation vibration of the DESs component’s hydroxyl group (-OH). It was observed that in that wave number, ATCH did not have a significant peak, but appears in ATCH-DES when interacting with DES (NA and PG), which resembles the DES pattern. In the infrared spectrum, the elongation vibration peak of the hydrogen-bonded OH group is typically larger and lower on the frequency scale.

Figure 4.

Characterization analysis results from (a) Fourier transform infrared spectrum, (b) differenital scanning calorimetry thermogram. ATCH: Atorvastatin calcium trihydrate, DES: Deep eutectic solvent

Differenital scanning calorimetry

The DSC thermogram results in Figure 4b show significant differences from ATCH, where the THEDES thermograms from ATCH and NA no longer show the typical endothermic peaks of ATCH at temperatures of 117°C and 151°C, but instead resemble the endothermic profile of DES with a lower endothermic temperature at 186°C and 220°C, while DES itself is in the temperature range of 191°C and 224°C. This demonstrates that the interaction between DES and ATCH is beneficial and the melting point reducing is lower than DES.[39]

Solubility test

The solubility test findings after 24 h of mixing in DES and water revealed that the solubility of ATCH in DES was 0.158 ± 0.098 mg/mL higher than in water, at 0.000597 ± 0.003 mg/mL. Meanwhile, attempts to dissolve ATCH in PG alone were unsuccessful, resulting in a hazy gel that was difficult to handle.

Thus, it has been proved that ATCH is more soluble in DESs than in water. This dissolving ability can serve as a foundation for the future development of medicinal formulations for both oral and topical administration.

CONCLUSION

In this work, we showed that NA: PG as DES could offer a promising system for dissolving ATCH at high concentrations, with a solubility of 0.158 ± 0.098 mg/mL compared to its solubility in water (0.000597 ± 0.003 mg/mL). This important increase in solubility was due to the capacity of the DES components to establish hydrogen bonds with the medication, as confirmed by FT-IR. The analysis of DSC data validated the eutectic mixture’s performance in increasing drug solubility, in spite of the lack of the molecule’s fusion endotherm. Finally, we discovered that ATCH may significantly enhance solubility using a simple eutectic mixture consisting of NA and PG as THEDESs or PDESs.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil