ENHANCE THE AQUEOUS SOLUBILITY OF DICLOFENAC THROUGH THE SYNTHESIS OF DICLOFENAC-INOSITOL PRODRUG

Hemn Latif Qader^1*, Omer Abdulrahman Alsheikhly², Hewa Abdulla Hamadameen Hama¹, Adnan Burhan Qader¹

¹Department of Pharmaceutics, College of Pharmacy, Hawler Medical University, Erbil, Kurdistan Region, Iraq

²Department of Pharmaceutical Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Kurdistan Region, Iraq.

Corresponding Author Email: hemn.latif@hmu.edu.krd

Received: 28 Jul. 2024 / Accepted:26 Sep., 2024 / Published: 10 Nov., 2024. https://doi.org/10.25271/sjuoz.2024.12.3.1360

ABSTRACT:

Due to limited aqueous solubility, poor bioavailability makes oral dosage formulations difficult to formulate. Chemical alteration of medicinal compounds improves solubility. Prodrug design is a popular molecular modification method that improves solubility and oral bioavailability. This study aims to synthesize a diclofenac prodrug to enhance aqueous solubility. In this study, diclofenac was esterified with inositol to make a prodrug (DIP), which was identified by ¹H-NMR and FT-IR. A computational pharmacokinetic software was used to study DIP's pharmacokinetic profile, and saturation solubility was measured in phosphate buffer (pH 6.8) and 0.1 HCl (pH 1.2) solutions. The ester bands of (C=O) stretch at 1739 cm^-1 and the elimination of H signals of carboxylic acid at 10-12 ppm in the ¹H-NMR spectrum proved the synthesis of (DIP). Diclofenac solubility increased 827-fold in phosphate buffer solution (p < 0.05) from 0.059± 0.0164 mg/ml to 48.8± 0.034 mg/ml, primarily due to polarity change. The solubility of diclofenac and (DIP) in 0.1 N HCl (pH 1.2) was 0.016± 0.0031 and 0.018± 0.002, respectively. The improvement in solubility in the acidic medium was non-significant (p > 0.05) due to acid hydrolysis of the ester bond between inositol and the drug. The synthesis of diclofenac prodrug can greatly enhance its water solubility.

KEYWORDS: Aqueous solubility, Diclofenac, esterification reaction, inositol, prodrug,

1. INTRODUCTION

Due to its ease of use, high patient acceptability, affordability, minimal sterility requirements, and flexibility in dosage form design, oral administration is the most practical and widely used method of drug delivery (Sopan et al., 2021). Consequently, a lot of pharmaceutical companies are inclined to create bioequivalent oral medicine products. Essential factors that govern the bioavailability of therapeutic compounds in systemic circulation for optimal pharmacological activity are their soluble and dissolvable state at the site of absorption in the gastrointestinal tract (Yadav et al., 2021). However, their low bioavailability is the main barrier to the development of oral dosage forms. The hepatic first-pass effect, permeability through the gastrointestinal wall, drug release in the biological fluid, and water solubility are some factors that affect the bioavailability of medications taken orally (Khan, 2021).

The biopharmaceutical classification system (BCS) divides medicinal compounds into four classes; class II medicines being distinguished by strong membrane permeability and poor aqueous solubility (Daravath et al., 2017). Class II medicines have low oral bioavailability and lower pharmacological effects due to rate-limiting phases in the absorption process, namely solubility and dissolving rate (Rubim et al., 2014). Drug substance alterations on a physical, chemical, and molecular level are the many categories of solubility improvement techniques. Drug dispersion in carriers, particle size reduction, and drug crystal habit modification are examples of physical modification techniques. Chemical modification strategies, on the other hand, include complexation, pH changes, and drug salt forms (Khatri et al., 2022). Prodrug design is a well-known molecular modification technique that aims to optimize the physical, chemical, and biological properties of drugs to decrease toxicity and boost solubility and pharmacokinetic characteristics (Jornada et al., 2016).

"Pharmacologically inactive moiety which is converted to an active form within the body" is how Albert initially defined the term "prodrug" (Liu et al., 2019). The primary goal of creating a prodrug is to alter the chemical structure of a drug molecule to impart desirable physicochemical and/or pharmacokinetic properties through the use of precursor-based or carrier-linked techniques (Shah et al., 2017). One of the first medications to be synthesized as a prodrug to increase its water solubility and bioavailability was phenytoin, a medication which is poor water soluble. A research team under the direction of Valentino J. Stella developed prophenytoin, a phenytoin prodrug that is soluble in water, in 1975. In one of the first and comparatively complete works on prodrugs, the synthesis method, physicochemical characteristics, activity, and pharmacokinetic profile of prophenytoin after oral and parenteral administration were described (Stella, 2020).

Diclofenac is a nonsteroidal anti-inflammatory medication (NSAID) with analgesic, anti-inflammatory, and antipyretic properties that is a member of the phenylacetic acid class (Figure 1) (Kaynak et al., 2015). It was selected as the model drug for this study since BCS(Pund et al., 2021) indicates that it is classified as a class II drug. As previously indicated, for this category to achieve meaningful bioavailability following oral administration, the solubility must be improved.

A naturally occurring substance, inositol is involved in lipid production, cell development, morphogenesis, and cytogenesis(Bizzarri et al., 2016). Long regarded as a B vitamin (Vitamin B8), inositol is a cyclic carbohydrate with six hydroxyl groups (Figure 1) that controls numerous vital cellular functions, such as neuron activation, secretion, contraction, gametogenesis, and cell proliferation and development (Suliman et al., 2022). In numerous trials, including patients with insulin resistance and polycystic ovarian syndrome, inositol has been used as a dietary supplement safely for decades (DiNicolantonio & O’Keefe, 2022). In this study, inositol was added to a novel diclofenac prodrug formulation to improve its oral bioavailability and water solubility, in addition to its clinical applications.

This study aims to enhance water solubility class II drug diclofenac acid through the synthesis of prodrugs with inositol.

2. Materials and Methods

Materials

Diclofenac was kindly gifted from Awamedica Pharmaceutical Company, Erbil. Iraq. The chemical solvents and inositol were received from the commercial suppliers Sigma-Aldrich, Merck, and Alpha. Distilled water was prepared in the laboratory. The chemical structures and the chemical schemes were drawn by the Chemoffice 12 program, the identification of the new products was performed using the FT-IR JASCOFT/IR-4600 spectrometer (Japan) and ¹H-NMR Bruker spectrometer (300 MHz) (Germany).

Chemical Preparation of diclofenac from diclofenac sodium

The chemical conversion of diclofenac sodium shown in scheme 1, which is one of the methods for chemical preparation of diclofenac according to literature, is accomplished by dissolving precisely 10g of diclofenac sodium in a mixture of absolute ethanol and Tetrahydrofuran (THF) at a volume ratio of 3:1. The mixture was lowered to 18°C. After that, 15 milliliters of 2N HCl were added. After agitating the mixture for ten minutes, 150 milliliters of cold water were added (Hassan & Elias, 2014). The precipitate was filtered and dried without additional purification to produce diclofenac acid, which was used in the next step.

Synthesis of diclofenac prodrug

The Diclofenac-inositol prodrug (DIP) molecule was synthesized by dissolving 8g of Diclofenac in 120 ml of dry chloroform scheme 2. The resulting solution was then placed in a round bottom flask and cooled to a temperature of below 0°C, which was maintained throughout the procedure. Exactly 4 milliliters of thionyl chloride were gradually added over 20 minutes while continuously being stirred. Once all of the thionyl chloride had been added, the mixture was subjected to reflux for 17 hours at a temperature of 65°C, with continuous stirring. The solvent was permitted to undergo evaporation, and the

powder was dissolved again in anhydrous chloroform many times to ensure the complete elimination of any remnants of thionyl chloride.

The diclofenac acid chloride was dissolved in 25 milliliters of dry chloroform. The resultant solution was then added to a combination containing 3.5 milliliters of triethylamine, 25 milliliters of dry dichloromethane, and 4.87 g of inositol that had been previously chilled to temperatures below 0°C. The reaction mixture was agitated for an extended period of time, followed by three washes with 20 ml of hydrochloric acid solution with a concentration of 5% volume/volume. Subsequently, it was washed with a 20 ml sodium hydroxide solution with a concentration of 5% weight/volume. Finally, it was rinsed with 5 ml of cooled distilled water and the result (DIP) was dehydrated using anhydrous sodium sulphate (Hassan & Elias, 2014).

The pure compound was obtained using column chromatography with a mobile phase consisting of n-hexane and ethyl acetate at a ratio of 7:1(Alsheikhly et al., 2024).

Identification of the prepared prodrug compound.

Fourier transforms infrared spectrophotometry (FT-IR)

The identification of the prepared prodrug was performed by obtaining FT-IR spectrum in a range of 4000–400 cm^-1 using JASCOFT/IR-4600 spectrometer (Japan). The main objective of FT-IR study was to detect the formation of ester bond between diclofenac and inositol.

Proton-Nuclear magnetic resonance (¹H-NMR)

Further identification of the prepared prodrug was performed by obtaining ¹H-NMR in a range of 1-19 ppm using Bruker spectrometer (Germany).

Computational pharmacokinetic studies

The evaluation of pharmacokinetic properties is crucial in the process of drug development, as it facilitates the assessment of the biological components of potential drug candidates. Lipinski's rule of five was employed to assess the suitability of a substance for oral bioavailability. The study focused on examining the ADME characteristics by using the Swissadme program to gain a better understanding of the drug-likeness and pharmacokinetics profiles. The colored portion in the obtained radar image represents the ideal physicochemical conditions for maximizing bioavailability(Unnisa et al., 2022).

Evaluation of Diclofenac acid and Diclofenac prodrug

Determination of maximum absorbance (λ max) and calibration curve

The Maximum absorbance and calibration curve of diclofenac and DIP were determined in two different aqueous media, namely phosphate buffer (pH 6.8) and 0.1 N HCl (pH 1.2). A standard stock solution of diclofenac and DIP was prepared by dissolving a 20 mg of each one in 10% v/v of aqueous methanol solution, which further diluted with phosphate buffer and 0.1N HCl respectively to obtain a total of four standard stock solutions of the drug and the prodrug at a concentration of 200 mcg/ml(Rashid et al., 2023). Solutions were prepared by suitable dilution of corresponding stock solution with phosphate buffer and 0.1N HCl: the prepared solutions were scanned using (Lambda 25 double beam UV/Vis spectrophotometer) in the range of 200-400 nm to determine the wavelength of maximum absorbance (ƛ max) of the drug and the prodrug in both mediums.

For the construction of calibration curves a series of solutions at concentration of 5, 10, 15, 20, 25 and 30 µg/ml were prepared for both drugs in the corresponding mediums by diluting the standard solutions and then the obtained solutions were scanned at the ƛ max of the drugs (Yilmaz & Ciltas , 2014).

Saturated solubility studies of Diclofenac acid and Diclofenac prodrug

Saturated solubility studies were performed to obtain the maximum amount of diclofenac and diclofenac prodrug which could be dissolved per unit volume under specific conditions. Solubility of the drug and its prodrug were obtained in phosphate buffer (pH 6.8) and 0.1N HCl by adding access amount of them to 50 ml graduated conical flasks containing 20 ml of phosphate buffer and 0.1N HCl (pH 1.2) respectively. The solutions were shaken and left for 24 hours with continuous stirring at room temperature, after that the obtained suspension was filtered using Whatman filter paper No.1 (Lobo et al., 2013).

Samples were taken from the filtrate for the estimation of drug quantity per unit volume by obtaining absorbance of the samples using a UV-VIS spectrophotometer at the at ƛ max of diclofenac and diclofenac prodrug. The results are illustrated in Table

Statistical analysis

The effect of the new prodrug formulation on the solubility of diclofenac was tested for significance by using paired samples t-test with the aid of the statistical package for the social sciences (SPSS 21) program. The difference was considered statistically significant when (P = 0.005). The calibration curves of diclofenac and DIP in different aqueous media were constructed using Microsoft Excel 2019 program. The correlation coefficient (r²) and standard curve equations for diclofenac and DIP were determined to measure the degree to which the values of x and y are linearly correlated. A correlation coefficient close to 1 shows the strength of the correlation between x and y values. The correlation coefficient was determined using Microsoft Excel 2019 program.

Figure 2: the FT-IR spectrums of Diclofenac (A), Inositol (B) and the prodrug compound DIP (C)

Compound	molecular Formula	Chemical name	FT-IR bands (cm^-1)	¹H NMR signals (DMSO) (ppm)
DIP	C₂₀H₂₁Cl₂NO₇	2,3,4,5,6-pentahydroxycyclohexyl 2-(2-(2,6-dichlorophenylamino) phenyl) acetate	3382 (N-H) stretching vibration of amine group, 3251 (O-H) stretching vibration of alcohol ,1743 (C=O) stretching vibration of ester group, 1565 (C=C) aromatic stretching	2.89-2.92 H of methylene group, 3.09-3.7 H of cyclohexane group, 4.62-4.75 H of alcohol group (OH), 6.2-7.44 H of aromatic group

Table 1: The molecular Formula, Chemical name, FT-IR bands and 1H-NMR signals of the compound DIP

Figure3: ¹H-NMR spectrum of the synthesized compound DIP

Figure 4: The chemical structures and bioavailability radar images of Diclofenac acid (A) and the synthesized compound DIP (B).

Saturated solubility studies were carried out for diclofenac and DIP in phosphate buffer and 0.1 N HCl (pH 1.2) solutions at room temperature as demonstrated in Table 2. The solubility data is expressed as mean±SDE (n=3). The solubility enhancement ratio which shows the number of folds increment in the solubility of diclofenac and DIP in different dissolving medium are also shown in Table 3. Comparison and statistical differences in the mean solubility between DIP and diclofenac in the two were shown in Table 3.

According to the results of solubility studies, Diclofenac is considered as practically an insoluble drug with a solubility of 0.059 mg/ml in phosphate buffer and 0.021mg/ml in 0.1 N HCl (pH 1.2) solutions(Mirza, 2021). Enhancement of diclofenac solubility in phosphate buffer was achieved after its preparation as a prodrug with inositol with a solubility of 48.8 mg/ml which is equivalent to (31.5 mg) of pure diclofenac. This enhancement in the solubility is due to modification of drug lipophilicity and the increase in the polarity of the parent drug(Shah et al., 2017), while the result of Table 2 indicates that the formulation of prodrug does not affect diclofenac solubility in an acidic medium which is mainly due to the hydrolysis of the basic ester bond between the drug and the career(Wang et al., 2015) Diclofenac solubility also decreased form 0.059 mg/ml in phosphate buffer to 0.016 mg/ml in an acidic medium Table 2, this is because

the drug is a weak organic acid that undergoes a partial ionization in an alkaline medium and it will be more soluble in alkaline than in acidic medium(Taylor & Aulton, 2021). The results in Table 3 illustrate the solubility enhancement ratio between the pure drug and the prepared prodrug in different mediums as well as the statistical difference between them. The result indicates a significant improvement (p < 0.05) and 825 folds increase in solubility when the compound DIP is compared with the pure drug in phosphate buffer and 533 folds increase in solubility when the equivalent weight of diclofenac in compound DIP is compared with diclofenac acid. Also, a decrease in the solubility of compound DIP in 0.1N HCl solution was observed when it compared to the solubility in phosphate buffer and this significant change (P< 0.05) is due to the breakdown of the ester bond between the drug and inositol in the acidic medium, as mentioned before.

A non-significant improvement (p> 0.05) in the solubility between compound DIP and diclofenac in an acidic medium was obtained, as demonstrated in Table 3. This result indicates that this approach of solubility enhancement is preferred in an alkaline medium where the basic ester bond remains stable and efficient to bring the lipid-soluble drug into a solution which finally results an increasing the bioavailability and pharmacological action which is completely consistent with the findings of the earlier study (De Souza et al., 2022)

4.CONCLUSION

The formulation of diclofenac-inositol ester prodrug (DIP) induces a great enhancement in aqueous solubility which may give a higher potency in the inhibition of inflammatory process than free diclofenac or diclofenac salt forms. This approach enhanced the solubility of the drug because the drug is conjugated to the freely water-soluble carrier that modulates the physicochemical properties of the drug and makes it more soluble, effective, and less irritant at the site of absorption.

5. RECOMMENDATION

Based on the results of this study, the efficiency and stability of this technique are preferred in an alkaline medium; therefore, a successful oral delivery system containing this prodrug should be coated with an enteric coating solution which improves the stability and allows the drug to be released in the alkaline pH of the intestine.

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Formula	Dissolving medium	Solubility mg/ml^*
Diclofenac	Phosphate buffer (pH 6.8)	0.059±0.0164
DIP	Phosphate buffer (pH 6.8)	48.8± 0.034
Diclofenac	0.1N HCl (pH 1.2)	0.016±0.0031
DIP	0.1N HCl (pH 1.2)	0.018±0.002

Formula	Solubility (mg/ml)	Solubility enhancement ratio	P Value
DIP / Diclofenac in (phosphate buffer)	48.8 mg / 0.059 mg	827 folds	0.002
DIP / Diclofenac in (phosphate buffer/ 0.1 N HCl)	48.8mg/0.012	4066 folds	0.002
DIP / DIP in (phosphate buffer/ 0.1 N HCl)	48.8 mg/ 0.016 mg	3050 folds	0.002
DIP / Diclofenac in (0.1 N HCl)	0.018mg/0.016mg	1.12 folds	0.52