INTRODUCTION:

Achieving the necessary drug concentration in the systemic circulation for the intended pharmacological activity depends critically on solubility¹. Over 40% of nearly water-insoluble new chemical entities (NCEs) are produced by the pharmaceutical sector. Improving drug solubility and, therefore, one of the most difficult areas of pharmaceutical research is oral bioavailability, particularly for oral medication delivery².

Because it is the most practical and extensively used drug delivery method, oral administration has many advantages over other methods, including convenience of use,good patient compliance, economy of cost, lack of sterility issues, and compositional flexibility in dose form. Consequently, there is a greater likelihood that several generic drug manufacturers will create oral prescription formulations that are bioequivalent³. The main flaw in the design is that oral dosage forms have a limited bioavailability. Factors influencing oral bioavailability includedrug permeability, dissolving rate, first-pass metabolism, water solubility, and efflux mechanism susceptibility. Reduced oral bioavailability is most often caused by two factors: poor permeability and poor solubility. For formulation scientists, the solubility problem is a major concern⁴. Physical and chemical alterations of a drug, particle size reduction, crystal engineering, pH adjustment, cosolvency, hydrotropy, solid dispersion, surfactant applicationand complexation are all utilised to boost the medications' solubility that are poorly soluble^5,6. Physical modifications such as the addition of cyclodextrins, carbohydrates, hydrotropes, polyglycolized glycerides and dendrimers are used to improve formulation solubility. Due to unfavourable pharmacokinetics, approximately half of the drugs in clinical trials and one-third of those in development are water soluble. The inclusion complexation of drug (guest) with dendrimers (host) has been widely used among solubility improvement strategies^7,8.

Domperidone also known as “Motinorm” and 5-Chlor-1-{1-[3-(2-hydroxy-1H-benzimidazol-1-yl)propyl]-4-piperidinyl}-1H-benzimidazol-2-ol (Figure 1). With a molecular weight of 426 gm/mole, the medication is a derivative of benzimidazole and acts peripherally by inhibiting dopamine. Its actions on the chemoreceptor trigger zone and the motor function of the stomach and small intestine provide it antiemetic and prokinetic qualities. Because of its low blood-brain barrier penetration, unlike metoclopramide, it has no adverse neurological side effects. Because of this, it has a great safety record when taken orally over an extended period at the prescribed dosages^9,10. According to the BCS, DMP has a high permeability and a low solubility in water, making it a class II medication. Its pka value of 7.9 indicates that it is a weakly-basic medication with a very slow dissolving rate at relatively high pH values. One component in 50,000 parts of water makes it almost completely insoluble in water. This lowers the medication's bioavailability to 13–17% upon oral administration and restricts the pace of drug absorption. Moreover, DMP cannot be administered via buccal or rectal routes due to its weak solubility and rapid dissolution at high pH levels; strong solubilization methods would be required^11,9.

Figure 1. Structure of the Domperidone

Dendrimers have demonstrated their utility as solubilizers over the last few decades. Due to their special qualities, dendrimers are an effective drug solubilizing agent. These characteristics include outstanding host-guest chemistry, multivalent geometry, high water solubility, drug encapsulation efficiency, and adjustable surface design^12,13. A dendrimer's internal structure is often hydrophobic. Dendrimers are a great way to encapsulate hydrophobic medications or bioactive because of their hydrophobic interactions and hydrogen bond forms. Drugs that are weakly soluble in water can have their solubility increased by using dendrimers as excipients due to their high degree of control over dendritic shape. Many commercially available small-molecule medicines with antibacterial, anticancer, and antiemetic characteristics have been successfully created using poly(amidoamine) poly (propylene imine), and poly (etherhydroxylamine) dendrimers. Dendrimers are unimolecular micellar in nature, with hydrophilic exteriors. They create covalent and non-covalent compounds with medicinal molecules and hydrophobes, resulting in improved solubility. Higher generation dendrimers can encapsulate hydrophobic moieties more fully since they have higher surface area. Generally, a generation-dependent shift in a dendrimer's effectiveness and characteristics is seen when it is utilized for drug solubilization^14-16.

Triazine-based dendrimers are widely recognized. Triazine dendrimer production is easier than that of other types, such PAMAM dendrimer, since it does not need functional group modifications. Recently, triazine-based dendrimers have been used in many different fields, including molecular recognition, anti-tumor medication delivery, optics and catalysis^17,18. Earlier, the synthesis, characterisationand use of an s-triazine-based dendrimer for water remediation were described¹⁹.

This research looked at the possibility of using triazine-based dendrimers as domperidone drug carriers. The A-549 lung cancer cell lines were used in the published dendrimer hemolysis research and cytotoxicity tests, which showed that synthetic dendritic macromolecules outperformed PAMAM dendrimer, which is sold commercially. This work first investigates these studies. Using the Higuchi and Conner approach, reported nanostructured dendrimers generation 1.0, 2.0, and 3.0 are utilized to increase the solubility of domperidone (DMP). The impact of dendrimer concentration and production on domperidone's improved solubility was investigated. The experimental findings demonstrated that when domperidone's solubility grew, so did the dendrimer production and concentration. The infrared spectroscopy verified the complex development.

EXPERIMENTAL:

Materials:

Domperidone was generously provided by Aagya Biotech private limited, Roorkee.We bought Phosphate Buffer Saline (PBS) from Himedia Laboratories in India. Using the retro-orbital approach, blood was drawn from a healthy Sprague-Dawley rat for the hemolysis experiment. Dendritic macromolecules and their applications are synthesized using a variety of reagents and solvents, including methanol, acetone, dichloromethane, thiocarbamide, and triazine trichloride.

Dendritic macromolecules synthesis:

A hydroxy terminal nanostructured dendrimer was synthesized via the following process. At 0–5 ºC, thiocarbamide (0.01 mmol) and triazine trichloride (0.02 mmol) interacted to generate TG0.5, the starting point for the dendrimer production process. The TG0.5 dendrimer was purified following acetone and methanol washes. The synthesis of hydroxyl terminated generation 1 (TG1) dendrimer occurred by the reaction of diethanolamine (0.04 mmol) with TG0.5 (0.01 mmol). The TG1 dendrimer is dispersed and cleaned in dichloromethane. The same procedure as in the first stage was used to create chlorine terminated half generation TG1.5 dendrimer by reacting TG1.0 dendrimer (0.01 mmol) mixed with triazine trichloride (0.08 mmol) at 0–5ºC. Diethanolamine (0.16 mmol) and half generation dendrimer (TG1.5) interacted similarly to the second stage to create hydroxyl terminated dendrimers of the entire generation (TG2). To create full-generation TG3 and half-generation TG2.5 dendrimers, the two aforementioned processes were repeated.The synthesized dendrimer was extensively studied by ESI-Mass Spectrometry, 1H-NMR, 13C-NMR, and FT-IR²⁰.

Hemolysis assay:

The hemolytic impact of increasing concentrations of dendritic macromolecules on RBC suspension was investigated. To put it briefly, blood samples were centrifuged for five minutes at 1500 rpm. Following centrifugation, blood cells were resuspended in half a milliliters of PBS. and exposed to several dendrimer doses (0.01, 0.1, 1.0, 10, 100, and 1000 µg/ml) for a duration of one hour. After that, the plasma supernatant was collected. After the incubation time, the samples were centrifuged, the supernatant was discarded, and after diluting the supernatant with an equivalent volume of phosphate buffer saline (PBS), the absorbance at 540 nm was determined. As a positive control, distilled water was used to obtain the maximum inhibitory effect. The formula was utilized to determine how different dendrimer concentrations might affect hemolysis²¹.

Hemolysis percentage is equal to (test/control absorbance) * 100.

MTT Assay for Cell Viability Determination:

Human non-small cell lung cancer cells A-549 were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Antibiotic-Antimycotic solution at 37°C in a CO2 incubator with 5% CO2. To gauge the vitality of the cells, trypsin-treated cultured cells were gathered, 10,000 cells per well were sown in a 96-well plate, and the cells were incubated for 24 hours. After incubation, cells were exposed to different concentrations of dendritic macromolecules (10, 100, and 1000µg/ml) for a duration of 24 hours. In the control wells, DMEM was added. Following a 24-hour period, each well's media containing different dendrimer concentrations was withdrawn, and cells were exposed to 0.5 mg/ml MTT for four hrs at 37ºC. Following the incubation time, 100µl of DMSO was added to each well to dissolve the formazan crystals, and the MTT was then extracted. Every well's absorbance was calculated using an ELISA plate reader at 550 nm. The following formula was then used to calculate the percentage of viable cells²¹.

% Cell Viability = (Test Mean Absorbance / Vehicle Control Mean Absorbance)*100.

Estimation of Domperidone λmax:

The contents of a 100 ml volumetric flask were precisely weighed 10 mg of domperidone, which was then dissolved and the volume was adjusted with methanol. After 20 minutes of sonication, the combination yielded solutions containing 100µg/ml of domperidone. The domperidone stock solution was further diluted with solvent to get 3, 6, 9, 12, and 15µg/ml solution. To scan the solution, a Shimadzu UV-1800 spectrophotometer was utilized. across a 200–400 nm wavelength range²².

Study of solubility:

The solubility investigation was carried out in compliance with the recommendations made by Higuchi and Connors (1965). Excess domperidone was put into screw-capped vials with varying number of dendrimer concentrations (0.6to 3.0 mmole). Vials were retained in a bath of water bath with shaking for 48 hours at 37ºC. Once any remaining undissolved medication had been removed from the vials by centrifugation, a Shimadzu UV-1800 spectrophotometer was used to detect the absorbance of domperidone at its characteristic wavelength of 287 nm²³.

RESULT AND DISCUSSION:

Preparation mechanism:

TG1.0(OH8), TG2.0(OH32), and TG3.0(OH128) dendrimers have previously been synthesized and characterizedon the basis of thiocarbamide²⁰. The core component and half generation dendrimers were not soluble in water, whereas the full generation dendrimers, TG1.0(OH8), TG2.0(OH32) and TG3.0(OH128) were water soluble. Hemolysis and cytotoxicity of TG3.0 dendritic macromolecules were investigatedutilizing lung cancer cell lines A-549.The synthesized macromolecules functioned better than the commercially available PAMAM dendrimer, according to the results. For the purpose of medication solubilization, only complete generation dendrimers were used.

Hemolytic potential:

Figure 2 illustrates how hydroxyl-terminated dendritic macromolecules affect erythrocytes.The findings suggested that hemolysis could be caused by nanostructure dendrimer with hydroxyl ends in a concentration-dependent manner. But when compared to PAMAM dendrimer, TG3.0 dendrimer based on triazine showed much superior hemolysis results²⁴.Upon contact between the negatively charged surfaces of erythrocytes (RBCs) and the positively charged amine(NH₂) groups of PAMAM dendrimer²⁵. By contrast, the anionic OH-groups on the surface of TG3.0 dendrimers decrease contact with red blood cells (RBCs) and lead to much decreased toxicity.

Figure 2. Hemolysis of dendrimer

Cytotoxicity:

The cellular damage of nanostructured dendrimer (TG3.0(OH)128) on A-549 cell lines was assessed using the MTT test method. The yellow dye MTT dissolves readily in water. The reductive breakdown of the tetrazolium ring in live cells can convert MTT into water-insoluble, blue-colored formazan crystals. To assess the vitality of the cells, formazan crystals that were recovered using organic solvents and measured at a wavelength of 550 nm were connected to live cells. The cytotoxicity experiments showed that the TG3.0(OH)128 dendrimer exhibited over 90% cell remain viable at doses ranging from 10 to 1000 µg/ml. nanostructured dendrimer (TG3.0(OH)128) was hence far less cytotoxic. The A-549 cell line’s shape is shown under control and after treatment with different dendrimer concentrations. The density of cells decreases when nanostructured dendrimer concentration rises from 10 to 1000 µg/ml. Figure 3 and Figure 4 display the microscopic pictures of the control and 1000 µg/ml nanostructured dendrimer concentrations, respectively.

Figure 3. Cytotoxicity (Control)

Figure 4. Cytotoxicity of dendrimer(1000µg/ml)

λmax of Domperidone:

100µg/ml Domperidone were diluted with solvent to obtain 3-15µg/ml solution and scanned over wavelength range of 200-400nm using shimadzu UV-1800 spectrophotometer and λmax were found at 287nm. Absorption and overlay spectra of domperidone are shown in figure 5 and figure 6.

Figure 5. Absorption spectra of domperidone

Figure 6. Overlay Absorption spectra of domperidone

Drug solubility study:

One important obstacle to the successful creation of appropriate dosage forms for dopamine is its limited solubility in water. Measurements of solubility were conducted using the Higuchi and Connors technique²³. The water solubility of domperidone was determined to be 3.229µg/ml in the present experiment. Phase solubility diagrams were used to examine how dendrimer production affected domperidone's apparent solubility in water. The impact of nanostructured dendrimer concentration on the solubility of domperidone was investigated at 37°C (figure7, figure8 and figure9). Our results show that domperidone's solubility has been significantly enhanced by dendritic macromolecules. Domperidone's solubility rose linearly as dendrimer concentration increased across the concentration range of 0.6–3.0 mM. Domperidone's solubility was found to have increased by 3.0 mM. A dendrimer of TG3.0 is 53.273 µg/ml. figure 10 demonstrated how the several generations of dendritic macromolecules impacted domperidone's solubility. The solubility of domperidone increased by 3.0mM. At pH 1.2, the TG3.0 dendrimer is 60.318 µg/ml. The order of the several dendritic generations employed in this study's solubility improvement impact was TG3.0 > TG2.0 > TG1.0. For three weeks, the solubility and stability of the medication containing dendrimer were monitored, and there was no change in stability.Because of a cavity, hydrogen bonds, and electrostatic interaction between the molecules of drugs and dendrimer terminating functional groups, dendrimers promote drug solubility.This study found that dendrimer increased the solubility of domperidone more effectively than ꞵ-CD²⁶.

Figure 7. Solubility enhancement of domperidone by TG1.0

Figure 1. Solubility enhancement of domperidone by TG2.0

Figure 9. Solubility enhancement of domperidone by TG3.0

Figure 10. Effect of dendrimer generation

To determine which medicine included dendrimer, FT-IR was used to validate the drug's presence on TG3.0 dendrimer.The drug-containing dendrimers and pure generation 3.0 dendrimers were compared to each other's FT-IR spectra. From the FT-IR spectra of the pure thiogeneration(TG) 3.0 dendrimer, absorption bands were detected at 3314 cm-1 for hydroxyl group O-H stretching, C-O stretching appear at 1054 cm^-1, C=S stretching appear at 1456 cm^-1, and 1652 cm^-1 for C=N stretching, as indicated in figure 11.The pure domperidone FT-IR spectra revealed regions of absorption at 1694.03 cm^-1 for C=O stretching, N-H stretching appear at 2937.81 cm^-1, C-N stretching appear at 1148.84 cm^-1, and C-Cl stretching appear at 866.93 cm^-1(figure 12). The medicine containing dendrimers had almost all of the characteristic peaks of the treatment. The absorption bandsappears at 1669 cm^-1, 2935 cm^-1, 1122 cm^-1, and 865 cm^-1 for C=O stretching, N-H stretching, C-N stretching, and C-Cl stretching, respectively, are shown in figure 13. As a result, neither the TG3 dendrimer nor the domperidone-loaded dendrimer's overall characteristic bands changed as shown in the IR spectra. This results in hydroxyl groups on the outside of the dendrimer, which may promote hydrogen bonding, and a hydrophobic triazine ring inside, which may induce hydrophobic interactions. Thus, by hydrogen bonding, hydrophilic interactions, or both, dendrimers may improve domperidone solubility and encapsulation.

CONCLUSION:

Different generations of synthesized dendritic macromolecules were employed to increase domperidone's oral bioavailability. In the cytotoxicity and hemolytic tests, nanostructured dendrimerwere demonstrated to be significantly less hazardous and biocompatible, indicating that they may be employed as a possible drug delivery mechanism. How effectively a drug dissolves in an aqueous solution depends on its concentration and the number of dendritic macromolecules it produces. According to the current study, there is a great chance that the TG3.0 dendrimer will increase the solubility of domperidone.When it came to increasing the solubility of domperidone, dendrimer outperformed ꞵ-CD.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors wish to express their gratitude to the Principal, Institute of Science and Technology for Advanced Studies and Research (ISTAR), V.P and R.P.T.P Science College and Department of Pharmacy, Nirma University Ahmedabad. The spectroscopic analysis capability was provided by Sophisticated Instrumentation Centre for Advanced Research and Testing (SICART) at The CVM University, Vallabh Vidyanagar, and the API was provided by Aagya Biotech Private Limited, Roorkee. The authors would like to thank both organizations for their contributions.

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Received on 05.12.2023 Modified on 15.04.2024

Research J. Pharm. and Tech 2024; 17(11):5386-5392.

DOI: 10.52711/0974-360X.2024.00823