INTRODUCTION:

Nowadays, the control of the rate and site of release of drugs in different dosage forms is of particular interest. The improvement of the release technologies aims above all to benefit the patient, taking into account the mechanisms related to the pH of the medium and the release time^1,2.

Polysaccharides, as polymers used in the controlled release of active ingredients, play an essential role. They are generally characterized by their swelling properties and are classified as hydrogels^3,4. Among the most interesting polysaccharides for biomedical applications are alginates, starches, cellulose, chitin, chitosan, cyclodextrins, pectin, guar gum, xylan, inulin and dextrans^3,4,5.

Starch is constituted of amylose and amylopectin. This report varies according to its biological sources⁶. It has been employed as core and coat materials in pharmaceutical oral dosage forms such as tablets, pellets, hydrogels, microparticles, and nanoparticles^3,4. Starch is among the most preferred biomaterials for biomedical applications due to their biocompatibility, biodegradability, and lack of toxicity^2,4. Starch has been utilized as a carrier material for anticancer therapeutics in the treatment of leukemia, breast, lung, liver, and colon cancers. With reference to oral colon-specific drug delivery, starch has been used as a matrix material for drug carriers for the treatment of colon cancer and related ailments³.

The use of native starch in some controlled drug release systems has limitations. It is characterized by low shear stress resistance, poor aqueous/organic solubility and gastrointestinal digestibility which limit its ease of processing and functionality display as an oral drug delivery vehicle⁶. Active ingredients tend to be released rapidly from native starch due to its swelling and rapid enzymatic degradation in biological media⁵.

To solve these problems, starch derivatives are often used in controlled release applications. These derivatives are generally more resistant to enzymatic degradation and offer a slower and more controlled release of drugs. When administered orally, starch derivatives are particularly suitable for achieving this goal.

Also worth mentioning is sodium alginate, another polysaccharide of interest in biomedical applications⁷. Sodium alginate is a biopolymer that is water-soluble, non-toxic, biocompatible, biodegradable, and non-immunogenic properties therefore having a high demand in the medical field^8,9,10.

Sodium alginate is derived from brown algae and is often used in the formulation of controlled release systems. It has swelling and gelling properties in the presence of calcium, which makes it possible to form matrices or gels for the controlled release of drugs. Sodium alginate is notably used for the encapsulation of drugs or the preparation of microspheres for prolonged release⁵.

In this work, we pursued the objective of developing pharmaceutical systems based on biodegradable polymers for a prolonged colonic release of the active ingredient, flubendazole, belonging to the anthelminthic family.

MATERIALS AND METHODS:

Reagents:

All chemicals and reagents used in this work were obtained from commercially available sources and used without additional purification of Ascorbic acid, Chloroacetic acid, and Flubendazole(Sigma), Sodium alginate, Starch (Aldrich).

Synthesis of carboxymethyl starch (CMS):

CMS was synthesized in aqueous medium from amylose-rich starch. Briefly, 40g of starch was dispersed in 100ml of distilled water with continuous stirring in a beaker at 55°C. For gelatinization, a volume of 135mL of 1.5M NaOH was incorporated with stirring (1 hour) followed by the addition of 30mL of 10M NaOH and 15g of sodium chloroacetate in a minimum volume of water. After 1h, a volume of 155ml of distilled water was added to the reaction, which was then neutralized with acetic acid. The CMS was precipitated with 80% methanol, then washed repeatedly with the 80% methanol solution until a conductivity of 50 µS/cm or less. Finally, it was washed with a methanol/acetone mixture (40:60, v/v), air-dried at 40°C for 24h¹¹. (Scheme1).

Scheme1. Synthesis of carboxymethyl starch (CMS)

Etherification Percentage:

The percentage of etherification (DE) were determined by titrimetry according to the following method¹²:

1.0g of CMS was placed in a 250ml flask and added to 50ml of ethanol (75% in distilled water). The mixture was stirred, heated at 50°C for 30 min. Afterwards, the mixture was cooled and then 40ml of 0.5 M KOH was added. The solution was titrated with 0.5 M HCl using phenolphthalein as an indicator.

The procedure is repeated for the etherified starch and the unmodified starch which will serve as a blank test.

Etherified % = [(V_Blank – V_sample)* Molarity of HCl* 0.043 * 100]/ sample weight

V _Blank, V _sample: are the titration volumes in ml, weight of the sample in grams.

Preparation of Flubendazole microparticules:

Two types of formulation have been proposed:

Formulation 1, based on: CMS/Sodium alginate/ Flubendazole/Ascorbic acid;

Formulation 2, based on: CMS/Sodium alginate/ Flubendazole/ Phloroglucinol.

The microparticules were prepared using sodium alginate and CMS in combination with ascorbic acid and phloroglucinol, as coating material by the ion gelation process.

The amount of sodium alginate, CMS, ascorbic acid and phloroglicinol are constant, introduced separately into purified water, and mixed properly to obtain a homogeneous polymer solution. Flubendazole was added to the polymer solution and then homogenized with an agitator to form a viscous dispersion. The dispersion thus obtained was then added dropwise in a solution of calcium chloride (10% w/v) via a syringe with a needle of size No. 24 (diameter).

The droplets are held in this calcium chloride solution for 15 minutes in order to complete the curing reaction and to produce rigid spherical microcapsules¹³. The microcapsules were collected by decantation, and the product thus separated was washed several times with distilled water and dried at a temperature not exceeding 45°C.

Analysis by optical microscopy:

We prepared suspensions of 0.1g of each sample (native starch, modified starch) in 10ml of distilled water, the suspension is observed under an optical microscope (Motic 200) with a magnification of x200.

Analysis by Fourier transform infrared spectroscopy (FTIR):

The IR spectra were recorded from the KBr pellets containing the different samples using a spectrophotometer (IRTF) (Bruker, Germany).

Molecular docking study:

Docking can be defined as the set of mechanisms and interactions involved in the formation of molecular complexes¹⁴.

In order to determine the antihelminthic activities of the compounds flubendazole, phloroglucinol and ascorbic acid, molecular docking studies were conducted. One enzyme was used in the docking study: Cruzain, the main papain-like cysteine protease from Trypanosoma cruzi.

Cruzaine is crucial for the survival and multiplication of the Trypanosoma cruzi parasite¹⁵. To understand the binding differences between the compounds and this protein, rigid docking of the receptors was performed.

For antihelminthic activity, docking was performed on the crystal structure of Trypanosoma cruzi (PDB: 3I06). The compounds (flubendazole, phloroglucinol, and ascorbic acid) were subjected to docking analysis, and the specifics of their interaction with these targets was identified.

Determination of drug in microparticules:

A UV-visible spectrophotometer with a spectral bandwidth of 2nm and an accuracy of ±0.5nm in wavelength was used. To measure the absorbance of the obtained solution, a pair of 1cm quartz cells was used¹⁶.

Preparation of the stock standard solution:

Flubendazole was accurately weighed and dissolved in 100ml of distilled water to give a stock solution (100 µg/ml). From the stock solution, the standard solutions were prepared to obtain solutions at 6.4/ 7.2/ 8.0/ 8.8/ 9.6 µg/ml which was analyzed for linearity.

Accuracy and precision:

To establish the accuracy of the proposed methods, studies of the recoveries were carried out by the standard addition method at five levels 80%, 90%, 100%, 110%, 120% from the standard standard solution at 8mg /ml.

The different proportions of the microparticules were weighed and the desired weight was calculated.

The micrparticules were ground into a fine powder. Powder equivalent to 100mg of drug was transferred separately into volumetric flasks of 100ml volume and in the ultrasonic bath for 10min. The resulting solution was then filtered through Whatmann filter paper (#41). The concentrations were determined by measuring the absorbance of the sample at 250.0nm in mode and the titer of the active ingredient is given by the following formula:

Encapsulation rate = T/T0

T0:the mass of encapsulated PA

T:the mass of drug

In vitro drug release study:

Preparation of buffer solution pH=1.2:

25ml of the 0.2M potassium chloride solution are mixed with 42.5ml of the 0.2M hydrochloric acid solution, then distilled water is added up to 200ml. The pH is adjusted using a pH meter up to the value of 1.2.

Preparation of buffer solution pH=6.8:

61.8ml of the 0.5M disodium sodium phosphate solution are mixedwith 9.1ml of the 0.5M citric acid solution, then distilled water is added up to 200ml. The pH is adjusted using a pH meter up to the value of 6.8.

Drug release study in simulated gastrointestinal conditions:

The in vitro drug release characteristics of microparticules were evaluated according to the method reported with little modification, 50mg of the prepared microparticules were placed in a 100ml beaker and kept constant with 50ml of elution medium with temperature 37°C and under shaking (200rpm).

RESULTS AND DISCUSSION:

Morphology:

Light microscopy is one of the most effective observations for native starch granules and CMS.

The micrograph of native starch granules and CMAs is presented in Figure 1(a, b,). Native starch and modified starch granules appear as distorted crystalline spheres and polygons, concentric layers around the hilum are visible after hydration (The hilum, the initial center of starch grain growth, is a region less organized which may contain some of the non-carbohydrate constituents). The particles of native starch are finer than those of modified starch.

The morphology of the starch grains etherified by the etherification reaction has a certain similarity. The particles are closer together in both cases but without agglomerating for that.

Figure 1: Microscopic analysis of native starch (a), Suspension of modified starch (b).

Particle size

The results determining the size of the particles of the different formulations obtained are of the order of 2 to 3 mm (Figure 2).

Figure 2:obtainedmicroparticules

IR spectra:

of the native starch, and the CMS are shown in Figures 3 and 4:

The FTIR spectra show an absorption peak was observed at 3357 cm^-1 and 2928 cm^-1which should be resulting from aliphatic -OH and -CH elongation vibration of the glucose unit.

Also the following bands were observed:

2818 cm^-1: sym (-CH-) (small shoulder), 1640 cm^-1: H₂O adsorbed (bound water), 1420 cm^-1: vibration CH₂, 1370 cm^-1: Asymmetric deformation vibration of CH₃, 1160 cm^-1(1080 cm^-1, 1010 cm^-1): Elongation vibration of the Acetal function, 1240 cm^-1: Stretching vibration of oxirane ethers, 700cm^-1: 620 cm^-1 for CH₂ swing.

Compared to native starch (a), a new absorption band at about 1170/1250 cm^-1, which is attributed to COC (Cyclic Ether) and which corresponds to the elongation vibration, and the strong bond at 1000cm^-1 indicate that the etherification reaction is well authenticated with the selected systems.

Figure 3:IR spectra of native starch

Figure 4:IR spectra of CMS

Percentage of etherification (DE)

The etherification of starch gives the CMS with a percentage of 16.125%. This result is in agreement with that of the literature, the percentage of etherification appears to be the factor responsible for the different properties of CMS. Depending on the chemical reaction chosen to modify the starch.

Encapsulation rate:

In view of the encapsulation rates obtained, we notice that the encapsulation rates of formulation1 are higher for the second formulation2.

This may be due to the ability of ascorbic acid to form a complex with sodium alginate. capable of tearing off the active principle, which increases the quantity of the active principle retained in the polymeric networks formed between the sodium alginate and ascorbic acid.

Table1: Encapsulation rate results.

Proportion(%)	Encapsulation rate(%)
Formulation 1	52.42
Formulation 2	46.92

Determination of antihelminthic activity using molecular docking:

The results in table 2 and 3, show significant amino acid residue interactions with the two ligands. In both cases (flubendazole and phloroglucinol), Lys(A:17) was found to be involved in hydrophobic interactions. Phloroglucinol interacts with cruzain, thereby acting as a hydrogen bond donor (HBD) at the receptor site interaction region involving the Glu (A:50) residue. The Lys(A:17) residue of Cruzain was also involved in the aromatic interaction with the benzelic moiety of the structure. The interaction of ascorbic acid with cruzain involves both hydrogen bond acceptor (HBA) interaction with Leu(A:118) and HBD with Glu(A:207), ASN(A:69), ALA(A:209).

Interaction of amino acid residues with flubendazole, Thr(A:186), ASN(A:47) have been shown to be involved in hydrophobic interactions.

Table 2: Binding affinity of the the tested compounds

Compounds	Affinity(kcal mol⁻¹)
Compounds	Cruzain from Trypanosoma cruzi (PDB: 3I06)
Flubendazole	-7.4
Phloroglucinol	-4.9
Ascorbic acid	-5.2

These interactions between the cruzain complex and the three compounds offer promising prospects for the development of new potential formulation for the treatments of this disease caused by the parasites.

Drug release study

The development of controlled or prolonged release systems is a tool to optimize the therapeutic effect, by maximizing the bioavailability of conventional drugs and reducing side effects. These systems include microparticiles, which have been the easiest strategy for controlled release systems¹⁷.

Figure 5: Flubendazole release from formulation 1.

Figure 6: Flubendazole release from formulation 2.

The in vitro release of drug from microcapsule is influenced by the test conditions, such as apparatus, shaking speed, volume, composition and temperature of dissolution fluid¹⁸.

CMS is a pH-sensitive excipient that also has interesting properties for colonic¹⁹ and prolonged²⁰release.

Hydration of the CMS leads to the formation of a gel layer which controls the rate of drug release²¹. When the penetration of water into the gel of the matrix is greater than a critical concentration, the polymer chains begin to separate, there is an extension of the spaces where the distribution of the active principle occurs, at this stage, there is to increases in erosion rate²².

In this study, from the dissolution profiles, we note for the formulations in the acid medium (pH=1.2) an absence of release of drug, thus an insufficient effectiveness.

In the colonic medium (pH=6.8):

It is noted that the kinetics of release of flubendazole in Formulations 1 and 2 has a certain proportionality in the variation of the concentration before the first 7hours. In fact, it is observed that F1 has a higher rate of release than that of F2. We record constant concentrations after 8 hours with values of 0.09 and 0.08mg/ml for F1 and F2 respectively, therefore it is confirmed that these formulations have sustained release characteristics.

The obtained results confirm the colonic release:

At pH=1.2 the CMS is in the protonated and compact state which prevents the diffusion of the buffer in the formulation and delays the release of the drug.

At pH=6.8 the carboxyl groups are in the ionized state (carboxylate) which promotes the diffusion of the buffer which allows the release of the drug, therefore the modified starch is used to slow down the release of drug. according to the following mechanism:

Figure 7: Proposed mechanism for Flubendazole release.

CONCLUSION:

The conventional forms on the market contain a large number of excipients to ensure the release of flubendazole, and the oral administration of these formulations requires daily doses, which raises the question of minimizing the excessive use of excipients, and to space out administration times. Moreover, this administration is associated with low bioavailability, high cost, more side effects, and an uncontrolled release profile. Therefore, the development of a new delivery system is of crucial importance. The process used in this study meets the requirements and problems posed.

CONFLICT OF INTEREST:

The authors have no conflict of interest. This article does not contain any studies with animal or human subjects.

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Received on 16.08.2023 Modified on 12.09.2023

Research J. Pharm. and Tech 2023; 16(11):5264-5270.

DOI: 10.52711/0974-360X.2023.00853

Compounds	3D molecular interaction	2D molecular interaction
Flubendazole
Phloroglucinol
Ascorbic acid