A study of monophasic supramolecular formulation of Sulpiride with P-glycoprotein efflux inhibitor to enhance solubility and intestinal permeability with molecular modeling insights

doi:10.21203/rs.3.rs-2964902/v1

Download PDF

Research Article

A study of monophasic supramolecular formulation of Sulpiride with P-glycoprotein efflux inhibitor to enhance solubility and intestinal permeability with molecular modeling insights

https://doi.org/10.21203/rs.3.rs-2964902/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The aim of the study was to design and develop a supramolecular formulation of sulpiride (SUL) to enhance its solubility, dissolution, and permeability. This was accomplished by co-amorphizing SUL with Naringin (NARI) to improve its physicochemical properties. In addition, NARI was incorporated as a co-former to enhance the drug's intestinal permeability by targeting P-glycoprotein (P-gp) inhibition. To confirm the intermolecular interaction and formation of the co-amorphous system, extensive characterization was performed. This included quantum mechanics-based molecular dynamics simulation studies investigating intermolecular interactions and phase transformation. The findings of the miscibility study, Radial Distribution Function (RDF) analysis, and quantitative simulations of hydrogen/π-π bond interactions helped in comprehending the co-amorphization aspects of SUL-NARI systems. Molecular docking studies were conducted to predict the in-silico biological activity. The solubility, dissolution, and ex-vivo permeability studies were performed to determine the extent of improvement in solubility, dissolution and flux, demonstrating 31.88-fold, 9.13-fold, and 1.91-fold increments, respectively. In conclusion, this study demonstrates the benefits of synthesizing a drug-nutraceutical supramolecular formulation to enhance the solubility and permeability of poorly soluble and permeable drug.

Sulpiride

Naringin

Supramolecular

Solubility

Permeability

P-gp efflux

Sulpiride (SUL), marketed as Dogmatil®, is a benzamide derivative that antagonists D2/D3 dopamine receptors in the gastrointestinal tract (GIT) and the central nervous system (CNS). It is commonly used to treat gastric and duodenal ulcers, migraine, antiemetic, anti-depressive, and anti-dyspeptic conditions. However, the clinical use of SUL is limited due to its low oral bioavailability, typically around 20–30% [1]. The poor oral bioavailability of SUL can be attributed to its poor aqueous solubility and limited permeability in the GIT. Additionally, SUL acts as a substrate for the P-glycoprotein (P-gp) efflux mechanism, further restricting its oral absorption and thus its efficacy. The poor solubility and permeability of SUL in the GIT often necessitate high doses and frequent dosing schedules, which can negatively impact patient compliance and result in unwanted side effects. In particular, high doses of SUL are known to cause extrapyramidal symptoms (EPS) and movement disorders such as Parkinsonism[2]. According to the Biopharmaceutics Classification System (BCS), SUL is classified as a Class IV drug [1, 3]. Furthermore, it has been reported that poor intestinal bioavailability of drugs can be improved by administering P-gp efflux inhibitors or substrates, such as verapamil. The co-administration of P-gp substrates similar to verapamil, such as cyclosporine A and quinidine, suggests that enteral P-gp efflux significantly contributes to poor oral bioavailability. Thus, improving the solubility and intestinal permeability of SUL are key requirements to enhance its bioavailability. Recently, numerous SUL formulations, which include liposomes [4], gastric retained form [5], self-emulsifying drug delivery systems [6], solid lipid nanoparticles [7], and solid dispersions [8] have been developed to improve oral exposure of SUL.

Poor solubility and poor permeability of BCS class II and class IV drugs could be enhanced by the amorphous solid dispersion (ASD) approach. The scale-up of ASD could be possible by using the hot melt extrusion (HME) and spray drying methods [9]. However, ASD has some limitations, such as low drug loading capacity, phase separation, poor miscibility of the drug with the polymer, lower glass transition temperature (Tg) during stability due to its hygroscopic nature of polymer, which may induce crystallization during processing, transport, utilization, and storage. Additionally, ASD formulations often contain major large molecular weight polymers that can make the formulations bulky [10–12].

In recent years, there has been an increased interest in supramolecular formulations such as co-amorphous (CAM) systems containing two small molecules. This approach has become promising due to the challenges associated with ASD. CAM systems use small molecules such as amino acids, organic acids, nutraceuticals (natural compounds), and drugs as stabilizing agents instead of polymers [13]. Therefore, small molecules with higher glass forming ability (GFA) and P-gp efflux inhibitor activity can be utilized to develop a co-amorphous system for poor solubility and permeability drugs such as SUL [14, 15]. CAM systems have been prepared using the same techniques as ASDs, such as the HME and spray drying methods. However, drug degradation can occur during manufacturing of CAM system by HME process due to the high-temperature melting required for CAM synthesis. On the other hand, CAMs prepared using the solvent evaporation method can be easily scaled up to a commercial level by spray drying and freeze-drying and can be more readily formulated into a solid dosage form, such as tablets and capsule [9, 16].

The stabilizing agent in the CAM system was selected to meet the two main criteria: first, it must produce enough disorder in SUL to create a stable amorphous combination, and second, it must have the ability to prevent SUL's P-gp efflux. Naringin (NARI), a flavonoid primarily found in fruit, was chosen as it has numerous biological activities, including anti-apoptotic, anti-oxidant, and anti-inflammatory activities [17]. In addition, it exhibits anti-parkinsonian and neurotrophic factor enhancer activity, which makes it suitable for use in CAM systems for its potential to stabilize the CAM systems and inhibit P-gp efflux [15, 18]. Naringin is a bioflavonoid that belongs to the Generally Regarded as Safe category. It has been shown to increase oral exposure to poorly permeable drugs when given concomitantly. Naringin also exhibits P-gp efflux inhibition as well as anti-parkinsonian and neuroprotective activities [18, 19]. The CAM of talinolol-naringin developed by the solvent evaporation method exhibited a significant improvement in talinolol solubility and permeability compared to crystalline talinolol. This improvement is attributed to the co-amorphization of talinolol with the P-gp efflux inhibitor NARI [15]. The CAM system of SUL with NARI is a viable combination for the improvement of both solubility and permeability, and has not been reported previously. Therefore, this study represents the first report of a CAM system of SUL and NARI for the purpose of enhancing aqueous solubility and permeability.

In the present investigation, a CAM system of SUL incorporated with NARI as a stabilizer and P-gp inhibitor was developed with the aim of enhancing the aqueous solubility and intestinal permeability of SUL. The CAM system was prepared using the solvent evaporation method with various SUL and NARI molar ratios. The solid-state characteristics were characterized using Powder X-ray Diffraction (PXRD), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), Solution ¹H Nuclear Magnetic Resonance (NMR), Polarized Light Microscopy (PLM), Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR), and Scanning Electron Microscopy (SEM). The supportive and insightful investigation involved molecular dynamics simulations with miscibility analysis, Radial Distribution Function (RDF) analysis, and quantitative H-bond interactions and π-π interactions, which built a strong interactions-based theory for the co-amorphization of SUL and NARI. Furthermore, the CAM systems were evaluated for solubility and dissolution profile and compared with the crystalline SUL and physical mixture. Additionally, the effect of NARI on SUL intestinal permeability was assessed using the non-everted sac method and compared with the crystalline SUL and physical mixture. In-silico studies were conducted to assess the biological activities via D2/D3 dopamine interactions and Structure-Activity Relationship (SAR). The maximum level of residual solvents for finished products is documented by the ICH Q3C(R8) guidelines. GC-FID is one of the universal techniques employed for the quantitative analysis of methanol and dichloromethane (DCM) as residual impurities [20]. The molecular structures of SUL and NARI with proposed intermolecular interactions are shown in Fig. 1 for molecular interaction.

Sulpiride (SUL) and Naringin (NARI) were obtained from Arrow Chemicals, India, and Sigma Aldrich, Germany, respectively. Solvents for HPLC and other analysis were procured from Merck, Mumbai, India, and SRL India, respectively. Throughout the study, Mili Q® system prepared water was used.

2.1 Analytical method development

Samples were analyzed using an HPLC instrument (e2695 Waters) equipped with an XBridge BEH C18 Column (4.6 mm X 150 mm) and a UV detector (2998 PDA). The drug signal was calculated as the peak area using EMPOWER 3 software. Primary stock solutions (1000 ppm) and working stock solutions were prepared in methanol. The detailed HPLC method is provided in the supplementary materials (Table S1). NARI was well separated from the SUL peak, and the resolution was acceptable, as shown in (Fig. S1)

2.2 Preparation of co-amorphous system (CAM)

The solvent evaporation method was used to prepare different molar ratios of CAM of SUL and NARI. Briefly, a mixture of SUL and NARI at varying molar ratios (SUL to NARI ratio; 1:1, 1:2, 1:3, and 2:1 M) was dissolved in a 50 mL solvent mixture containing a 50:50 proportion of Methanol and DCM in a round bottom flask (RBF) to form a transparent solution. The solvent was evaporated using a rotary evaporator under vacuum at 45°C and 150 rpm for 30 min. The obtained powder was placed in a desiccator until further use. Amorphization of solvent evaporated samples was confirmed by PXRD, PLM, and DSC.

3.1 Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was performed using a Discovery 5500 instrument (TA Instruments ®, New Castle, USA) with a N₂ gas flow rate of 25 mL/min and a ramp rate of 10°C over a temperature range of 30 to 250°C. The sample, with an equivalent weight of 10 mg, was placed in a platinum pan and data were analyzed using the TRIOS software.

3.2 Differential scanning calorimetry (DSC)

The Mettler-Toledo DSC system, equipped with STARe software, was used to evaluate the thermal properties of the samples. In standards of indium (In) and zinc (Zn) were employed to calibrate heat flow and enthalpy. Approximately 3–5 mg of each sample was placed in an aluminum pan and sealed with an aluminum lid. The samples were then subjected to a heating rate of 10°C/min, while nitrogen was flowed through the DSC cell during the experiment.

3.3 Powdered X-ray diffraction (PXRD)

The diffraction pattern or crystallinity of the samples was determined using a Bruker D2 Advance diffractometer (Bruker AXS, Karlsruhe, Germany). The instrument was operated at room temperature and utilized Cu Kα monochromatic radiation (1.54 A; 40 kV × 30 mA). Fixed time scanning was used to measure the Bragg diffractions within an angle range of 5–40° 2θ with a dwell time of 0.2 s.

3.4 Polarized light microscopy (PLM)

The Nikon Eclipse-LV100 N POL microscope (Nikon, USA) operated with Linkam processing software was used to capture morphological images of the samples. The crystallinity was analyzed in the initial and stability samples. Approximately 5 mg of the sample was placed on a glass slide, and images were captured using 5x magnification.

3.5 total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy

The ATR-FTIR spectra of the samples were recorded using an FTIR instrument (PerkinElmer) equipped with an ATR accessory. The spectra were recorded over a range of 4000–500 cm^− 1 with a resolution of 4 cm^− 1 and 16 scans per sample. Each sample was placed on the diamond crystal of the ATR accessory and a pressure of about 1000 psig was applied. Please note that "diamond shape" is not accurate as the ATR accessory typically has a diamond crystal.

3.6 Nuclear magnetic resonance (NMR) Spectroscopy

The ¹H NMR spectra of SUL, NARI, and their CAM systems of SUL and NARI were recorded on Bruker ADVANCE III 500 MHz spectrometer operating at 500 MHz and temperature at 298k. A powdered sample equivalent to 10 mg was dissolved in 0.8 mL of Dimethyl sulphoxide (DMSO) and transferred to an NMR tube. The ¹H NMR spectra of four ratios SUL-NARI CAM systems were investigated by taking the ¹H NMR spectra of SUL and NARI, which are referred to as standard spectra.

3.7 Scanning Electron Microscope (SEM)

Characteristic features like surface morphology and shape attributes of plain SUL, physical mixture (PM), and CAM systems were explored and characterized by SEM (Quanta 250, FEI, USA) operating under a high vacuum (HV) and a 5 kV accelerating voltage applied. The samples were anchored on the copper grid using double-sided carbon adhesive tape. Prior to analysis, the sample was made electrically non-conductive by coating it with gold while being maintained for 10 min under a vacuum (8 × 10^− 3 mbar).

3.8 Quantum mechanics contingent molecular dynamics study

For the molecular dynamics (MD) study, the Material Science Suite version 2022 (©2022 Schrödinger, Inc.) was used. Amorphous cells of SUL and NARI CAM systems consisting of 64 molecules were created using the Disordered System Builder module with the OPLS4 force field. The systems were simulated using Boltzmann statistics at a temperature of 300 K, an initial density of 0.5 g/cm³, and Orthorhombic periodic boundary conditions (PBC). The Disordered System Builder was used to obtain a tangled chain system of solvents comprising 500 and 400 molecules in the S-OPLS forcefield for analyzing the solubility of methanol and DCM. CAM systems of SUL-NARI were generated using MD simulation through the Classical Mechanics module for the miscibility study. The molecular dynamics simulations were performed using the Desmond software integrated module. The simulation was initiated with the Compressive Relaxation protocol, followed by the Molecular dynamics stage with a simulation time of 1.2 ns and a recording interval of 4.8 ps. Approximately 100 frames were obtained under NPT (isothermal) conditions, with a temperature of 318 K and a pressure of 1.01325 bar. The time steps were set at 2 fs. The Classical mechanics panel was used to analyze the Bulk properties, including density (g/cm³), cohesive energy (kcal/mol), and solubility parameter (MPa^1/2). The Radial Distribution Function (RDF) analysis was performed on the Radial Distribution Function panel by selecting specific atoms from the MD simulated trajectory. The Interactions function was used to calculate H-bond interactions and π- π interactions.

3.9 Residual solvents analysis by GC-FID

Residual solvent analysis by gas chromatography (GC) was carried out on an Agilent 8890 GC system integrated with a flame ionization detector (FID) and Agilent 7697A Headspace sampler. The GC cycle was set for 20 minutes, including twelve minutes of run time and five minutes of equilibration time. After each run, the cooling cycle was run at 40°C, and the detector and inlet flow were turned off. An Agilent DB-624 (30 m x 320 µm x 1.8 µm) column was used for chromatographic runs with a set temperature of 40°C and a ramp rate of 30°C /min to a maximum temperature of 240°C. Standards containing 100 ppm DCM and 500 ppm MeOH were prepared using DMSO as a diluent.

3.10 Saturation solubility

The shake flask method was used to determine the saturated solubility of powder samples (Plain SUL, PM, and CAM systems). An excess amount of the powder sample was added to a glass vial and mixed with 5 mL of Mili Q water by vortexing. The vials were placed on an orbital shaker (IKA, Germany) and rotated at 100 rpm while maintaining the temperature at 37°C for 48 hours. The samples were then centrifuged at 12,000 rpm for 15 minutes using an advanced centrifuge (SIGMA 3 K30, Shropshire, UK). The resulting clear supernatant was filtered through a 0.45-micron membrane syringe filter (Millipore, Bedford, MA) and subjected to HPLC analysis in triplicate (n = 3), as described in the chromatography section.

3.11 In vitro dissolution

The dissolution of CAM systems was conducted in a USP dissolution apparatus II (LAB INDIA DS–8000, India) and compared with plain SUL and physical mixture (PM) of SUL and NARI. An equivalent quantity of 25 mg of SUL was kept in 900 mL of Mili Q water (dissolution medium) for each CAM system, SUL, or PM. Paddles rotated at 50 rpm, and the temperature of the dissolution medium was maintained at 37 ± 0.5°C. At predetermined time points (5, 10, 15, 30, 45, 60, 90, and 120 mins), 5 mL samples were collected during dissolution testing and filtered via 0.45-micron membrane syringe filters (Millipore, Bedford, MA). The dissolution media was replenished to maintain the sink condition by adding an equal amount of fresh dissolution media. The concentration of SUL in filtrate after suitable dilution was quantified by the validate HPLC method. The dissolution profiles of all the samples were determined for various dissolution parameters, particularly f2 (similarity factor) and DE₅ (% dissolving efficiency at 5 min), using DDsolver [21].

3.12 Ex-vivo permeability

The non-everted gut sac model was used to evaluate the ex-vivo permeability of the CAM system of SUL. Ex-vivo permeability studies were conducted on adult male Wistar rats in accordance with CPCSEA criteria and with approval from the Institutional Animal Ethics Committee (Protocol number: NIP/11/2022/PE/522) at NIPER Hyderabad. The rats used for the ex-vivo permeability investigation were 7–8 weeks old, weighed around 200–220 gm, and were starved for 12–16 hours before being euthanized by cervical dislocation. The small intestine was removed by a midline incision in the abdomen, flushed carefully with cold saline solution, and cut into a 7 ± 0.2 cm length sac and 1 ± 0.1 cm diameter. Each sac's one end was hermetically tied with a thread at another end. A suspension of SUL, PM, and SUL-NARI 1:1 CAM equivalent to 2 mg/mL of SUL were loaded into a small intestine sac with a volume of 1 mL. The effective length of the sac was fixed at 5 cm for permeation after the other end was tied. The sac was treated with 50 mL of Tissue Culture Buffer in a beaker (TCB). The assembly was maintained at 37 ± 2°C on a hot plate operating at a speed of 100 rpm, and aeration set a 10 to 15 bubble/min using an aerator. The assembly is aerated at 10 to 15 bubbles/min using a laboratory aerator and operated at 100 rpm while maintaining a temperature of 37 ± 2°C on a hot plate. The TCB solution is used to ensure tissue viability and metabolic activity. Approximately 3 mL sample is withdrawn from outside the sacs at predetermined time points (15, 30, 45, 60, 90, and 120 mins), and an equal volume of fresh TCB buffer is added. Subsequently, the samples are filtered through a syringe membrane filter with a 0.45-micron pore size. The HPLC method is used to quantify the amount of drug permeated through the intestine. The permeation flux (F, µg/min) is calculated as the slope, which is determined from linear regression analysis of the cumulative amount of SUL permeated versus time (min). All the experiments are performed in triplicates and reported as mean ± SD; n = 3.

3.13 In-silico biological activity studies

The online web application PASS (Prediction of Activity Spectra for Substances) is used for predicting biological activity based on Structural Activity Relationship (SAR), with a mean accuracy of 95% [22].

Molecular docking studies were carried out using the Maestro Software with the Glide module. The dopamine D2 (PDB ID:6CM4) and D3 (PDB ID:3PBL) receptor structures were downloaded in .pdb format with their previously docked state from the RCSB Protein Data Bank (https://www.rcsb.org). The 3D structures of SUL and NARI were downloaded in SDF format from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). The Protein Preparation module was used to preprocess the receptor structure and fix any structural defects, while keeping all parameters in their default state. The LigPrep module was used to obtain the ligand structure, with the forcefield set to OPLS4 at pH 7.0 ± 2.0. The Receptor Grid Generation module was used to specify the receptor and set the boundaries for docking, using the centroid of the ligand to define the receptor site, with grid box dimensions kept in their default state (10Å x 10Å x 10Å). The Ligand Docking module was used to run the docking experiment with a tolerance value of 0.1Å [23]. The docking score, Glide energy (kcal/mol), and interaction matrix were obtained after post-docking analysis. The Discovery Informatics and QSAR module was used to obtain clustering statistics and distance matrix plots. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile was predicted using pkCSM, a machine-learning platform[24].

3.14 Physical stability

The physical stability assessment of the prepared CAM systems was conducted by storing them at room temperature (25 ± 3ºC) for three months. The samples were then analyzed using PLM, PXRD, and DSC to determine the degree of crystallinity at the end of the stability period.

3.15 Statistical analysis

To conduct a non-parametric t-test, we used the trial version of GraphPad Prism 9.4.1 software (GraphPad Software Inc., USA). The significant differences between the plain drug, PM, and CAM systems (1:1, 1:2, 1:3, and 2:1 M) were determined. Differences were considered statistically significant when P < 0.05.

4.1 Solid state characterization of CAM

The CAM system was mainly prepared by combining two methods: (i) solvent-based and (ii) heating-based, including solvent evaporation, freeze-drying, and quench cool methods. Compared to solvent evaporation, quench-cool and freeze-drying techniques have excellent scalability and efficiency. However, quench cooling cannot always be employed because the drug(s) may undergo higher temperature treatment, which increases the probability of drug degradation during processing [9, 15, 25–27]. Here, the solvent-based technique was employed to synthesize CAMs of SUL and NARI in molar ratios of 1:1, 1:2, 1:3, and 2:1 because naringin is a heat-labile substance.

4.2 TGA

TGA analysis was performed to determine a suitable temperature range for the DSC experiment. This was achieved by investigating the decomposition behavior of SUL, NARI, and the CAM systems of SUL and NARI. A thermogram was recorded from 30 to 250°C. The absence of weight loss (%) observed in the thermograms indicates that the samples do not decompose between 30–250°C, as shown (Fig. S2)

4.3 DSC

DSC thermochemical analysis was used to describe the thermal characteristics of plain SUL, plain NARI, and CAM systems. The absence of melting endotherm and the presence of glass transition temperature (Tg) manifesting the amorphous nature of CAM [28]. The thermogram of the CAM system is displayed in Fig. 2.

SUL and NARI exhibited their corresponding melting endotherms at 187.65°C and 155.38°C, respectively, indicating the crystalline nature of SUL and NARI [8, 15]. The PM had a melting endotherm at 78.71°C and 183.25°C (Fig. S3). DSC profile of the prepared CAM systems of SUL-NARI showed the disappearance of the melting endotherm of the parent compound due to the formation of a binary amorphous phase.

The Tg values of SUL-NARI 1:1 CAM, SUL-NARI 1:2 CAM, and SUL-NARI 1:3 CAM were 86.14°C, 116.27°C, and 119.22°C, respectively. However, the system with an excess of SUL in SUL-NARI 2:1 CAM exhibited an exothermic recrystallization peak at 133.93°C, followed by an endotherm at 169.20°C. The excess amount of SUL induced recrystallization in the amorphous form due to high molecular mobility at a temperature higher than the Tg of the system, which was attributed to a lack of molecular interactions [29, 30]. The highest Tg value recorded in this study was 119.22°C for the CAM system with a 1:3 ratio, in which NARI was present in a higher proportion. This result demonstrates that the Tg values of the CAM systems increased as the proportion of NARI increased, indicating an anti-plasticization effect of NARI on the stabilized CAM systems [18, 31]. The ability of SUL to form intermolecular interactions in both solution and solid states provides an additional stability-enhancing effect, in addition to the anti-plasticization effect of NARI [32]. The intermolecular interactions of SUL, NARI, and CAM systems must be understood for supramolecular chemistry. To achieve this, ATR-FTIR analysis and solution state ¹H NMR analysis were performed.

4.4 PXRD

The PXRD patterns of the plain SUL, NARI, and CAM systems are depicted in Fig. 3. The diffractogram of plain SUL and plain NARI exhibited sharp diffraction patterns confirming their crystalline structure, as shown in (Fig. 3A) [8, 33]. The conversion of SUL into an amorphous state resulted in CAM systems with the ratio of 1:1, 1:2, and 1:3 exhibiting halo patterns and

The conversion of SUL into an amorphous state resulted in CAM systems with a ratio of 1:1, 1:2, and 1:3 exhibiting halo patterns and no diffraction peaks, indicating an amorphous phase. On the other hand, SUL-NARI 2:1 CAM (Fig. 3B) had characteristic peaks of the drug with lower intensity, suggesting that the drug presented in a semi-crystalline state and was not completely converted into the amorphous form. Thus, the drug in SUL-NARI CAM at a 2:1 ratio was inadequate for the complete conversion of SUL to the amorphous form. The sample's PXRD and the DSC analysis findings are found to be in excellent agreement.

4.5 PLM

PLM measurements were conducted on the samples to evaluate the completion of the conversion of components to a CAM state based on birefringence phenomena. As shown in Fig. 4, plain SUL and plain NARI appeared in the crystalline phase under a polarized microscope by showing a birefringence pattern. Amorphous SUL and amorphous NARI were prepared by the same rotary evaporation method as described in the CAM systems preparation section, and the prepared amorphous individual compounds were subjected to PLM. Amorphous SUL under PLM showed signs of birefringence, indicating it is semi-crystalline. However, amorphous NARI showed no signs of birefringence, indicating it is amorphous (Fig. 4). SUL-NARI CAM systems (1:1, 1:2, 1:3 M) showed no birefringence pattern under PLM, confirming the complete amorphization of CAMs due to molecular interactions (Fig. 4E-G). The PLM measurement revealed that the SUL-NARI 2:1 CAM system (Fig. 4H) exhibited a slight birefringence pattern, indicating that the system had not completely transformed into an amorphous state. The PLM finding was in line with the thermal and PXRD analysis results,

4.6 ATR-FTIR

ATR-FTIR experiments were performed to investigate the plausible molecular interactions between SUL and NARI in the CAM systems. Analyzing the structure of SUL and NARI (Fig. 1), it can be observed that SUL has both hydrogen acceptors and hydrogen bond donors. In contrast, NARI has hydrogen bond acceptors, suggesting the potential for hydrogen bond formation between SUL and NARI. This was confirmed by studying the structure of SUL and NARI (Fig. 1). The three peaks at 3369.05 cm^− 1, 3250.70 cm^− 1, and 3108.51 cm^− 1 in the plain SUL spectrum (Fig. 5) are attributed to the N-H stretching of primary sulfonamide, secondary amine group, and an aromatic group, respectively. The spectrum also displays two major sharp peaks at 1616.78 cm^− 1 for the C = O group and at 1551.77 cm^− 1 for the N-H group of amide (-CONH). The symmetric SO₂ stretching was observed at 1087.07 cm^− 1. Additionally, other absorption peaks of plain SUL were present at 2967.48 cm^− 1 to 2814.95 cm^− 1 (C-H stretching of methylene and methyl groups) and 1166.02 cm^− 1 (C-O stretching of the methoxy group) [7, 32]. The ATR-FTIR spectra of plain NARI (Fig. 5) showed a characteristic sharp peak at 1644.62 cm^− 1 for the stretching of the C = O group and a broad peak at 3343 cm^− 1 for the phenolic hydroxyl (O-H) groups. The alkyl(-R) stretching (sp3 hybridized) peak is observed at 2932.50 cm^− 1 and 2887.80 cm^− 1 [15]. The assigned data and relevant peak shifts are shown in Supplementary Table S2.

The spectra of the CAM systems for SUL and NARI exhibited certain changes with respect to the peaks (Fig. 5). In the ATR-FTIR spectrum of the CAM systems, the peaks corresponding to the secondary amine (N-H) stretching of primary sulfonamide (3369.05 cm^− 1), secondary amine (3250.70 cm^− 1), and aromatic amine (3108.51 cm^− 1) of SUL disappeared compared to the plain SUL spectra. Interestingly, a red shift was observed for the carbonyl group stretching of NARI (1644.62 cm^− 1→1634 cm^− 1) associated with decreased peak intensity upon amorphization into the CAM form. These results reveal the formation of intermolecular interactions between SUL and NARI in the CAM systems. Hydrogen bond between the sulphonamide group of SUL and the carbonyl group of NARI, which contributes to the co-amorphization of CAM systems [25]. The participation of the C = O stretching of the amide in SUL in the CAM system was ascertained by the disappearance of the peak at 1616.78 cm^− 1 in the spectra of the CAM systems [34]. The peaks attributed to the O-H stretching of NARI at 3343.62 cm^− 1 shifted to higher frequencies at 3360.37 cm^− 1, 3367.90 cm^− 1, 3368.53 cm^− 1, and 3367.38 cm^− 1 in the CAM systems of SUL-NARI 1:1, 1:2, 1:3, and 2:1, respectively. Additionally, the secondary amine (N-H) stretching peak of SUL at 1551.77 cm^− 1 was reduced in intensity and shifted to a higher wavenumber (1573.2 cm^− 1) in the CAM systems. The peaks of alkyl/aromatic C-H stretching of NARI at 2932.50 cm^− 1 and 2887.80 cm^− 1 were merged, reduced in intensity, and shifted to a lower frequency (2879.8 cm^− 1) in the 1:1, 1:2, and 1:3 systems, respectively, while in the 2:1 system, they were slightly shifted to 2883.7 cm^− 1 in the spectra of the CAM systems, suggesting the existence of π-π intermolecular interactions between the components of the CAM systems. Furthermore, the addition of NARI resulted in a red shift in the SO₂ stretching of SUL in the CAM systems, as the wavelength decreased with

increasing amounts of NARI (Fig. 5).

The peaks at 1087.07 cm^− 1 (symmetric SO2 stretching) in SUL shifted to 1058.95 cm^− 1, 1058.62 cm^− 1, and 1055.76 cm^− 1 in the CAM SUL-NARI 1:1 CAM, 1:2 CAM, and 1:3 CAM systems, respectively, compared to plain SUL. In contrast, the peak in SUL-NARI 2:1 CAM did not undergo any significant shift (1087.07 cm^− 1 → 1086.28 cm^− 1), remaining at the same position as plain SUL. These shifts suggest the involvement of the SO2 group in the formation of intermolecular interactions in SUL-NARI CAM systems [25, 32].

Similarly, increasing the percentage of NARI in the CAM systems resulted in a bathochromic shift in the C-O stretching of SUL. The characteristic peak of C-O stretching at 1166.02 cm^− 1 and 1125.93 cm^− 1 shifted to 1169.30 cm^− 1 and 1128.05 cm^− 1 (1:1); 1170.10 cm^− 1 and 1128.78 cm^− 1 (1:2); and 1171.00 cm^− 1 and 1129.43 cm^− 1 (1:3) in CAM, whereas no significant shift was observed in the case of 2:1 (1166.02 → 1168.61 cm^− 1 and 1125.93 cm^− 1 → 1127.25 cm^− 1) for the C-O stretching of the methoxy group (Table S2).

These results suggest that the interaction between SO₂ and C-O groups is responsible for the complete amorphization of SUL in 1:1, 1:2, and 1:3 mixtures as strong interactions are present, leading to stabilized co-amorphous systems, while weak interaction of these groups in 2:1 leads to a semi-crystalline nature. The FTIR analysis results are in line with DSC, PXRD, and PLM results, confirming the formation of a CAM system of SUL with NARI. In addition to NARI's anti-plasticization effects, ATR-FTIR studies showed that intermolecular interaction between SUL and NARI is essential for developing and stabilizing the CAM system. The formation of CAM systems could also be observed visually through apparent color changes. The CAM systems, after rotary evaporation, were characterized by a shiny yellow color, while the crystalline individuals were white (SUL) or milky white (NARI) in powder form (Fig. 6). The coloration phenomenon could be due to the absorption of visible light or a bathochromic shift [35, 36]. The color change observed in the CAM system provides an interesting research topic for future studies aimed at discovering the mechanism underlying this phenomenon.

4.7 NMR

The potential intermolecular interactions between SUL and NARI in CAM systems were investigated by using ¹H NMR analysis. The ¹H NMR spectra of the SUL, NARI, and CAM systems are presented in Fig. 7 (see also Fig. S4 and S5). The same interactions are anticipated to persist during CAM generation and influence the physical stability of CAM systems. ¹H NMR analysis was conducted on the samples in DMSO-d6 to solubilize the two components and obtain clear chemical shifts. Both SUL and NARI gave well-resolved 1H NMR spectra at 500 MHz. The peaks in the SUL and NARI 1H NMR spectra were assigned based on previously reported assignments [37, 38] and predictions based on ChemDraw® Software [39] to their corresponding proton, as shown in Fig. S5. By utilizing information from existing studies and computational models, we made predictions regarding the ¹H positions in SUL and NARI. The co-amorphization of SUL and NARI resulted in several chemical changes, leading to the shifting of characteristic signals and changes in electron density around various functional groups. Due to the variability in chemical shift positions, the assignment of -OH and amine functional groups in NMR spectroscopy can be complex. As a result, we have chosen to focus solely on certain characteristic peaks and functional groups in our analysis. Additional detailed descriptions of assignments can be found in Fig S6.

This approach is commonly used in NMR spectroscopy, where the chemical shifts of -OH and amine functional groups can be influenced by the specific molecular environment. Focusing on the characteristic peaks and functional groups made it easier to assign these signals and interpret the NMR data accurately [40, 41].

The C5-OH (δ 9.61 ppm), C5’-OH (δ 4.73 ppm), and C5"-OH (δ 4.67 ppm) peaks show an upfield chemical shift due to intermolecular interaction and hydrogen bonding. The peak at δ 8.35 ppm was assigned to the proton of the secondary amine (-NH) in SUL, and this peak showed a relatively downfield shift in CAM systems, suggesting the involvement of the SUL secondary amine in intermolecular interaction with the NARI molecule [34].

The proton signals of SUL at δa 7.61 ppm, δb 7.71 ppm, and δc 7.73 ppm showed changes in chemical shift, which may be attributed to the π-π interaction and rearrangement of the π-cloud in the CAM system of SUL and NARI [32, 34]. The peaks at 4.12 ppm attributed to -OCH₃ of SUL and at 6.88 ppm attributed to C-H of NARI shifted to a lower energy region in the NMR spectra of CAM systems due to a decrease in electron density caused by intermolecular interactions between the two hetero-synthons. The peak intensity at 4.12 ppm remained the same in the 2:1 molar ratio, while in the other CAM ratios (1:1, 1:2, and 1:3), the intensity decreased when compared to plain SUL. These results are consistent with ATR-FTIR results, which also showed incomplete conversion of SUL into the amorphous form in the 2:1 ratio. The peak assigned to the phenol group (-OH) proton in NARI shows a chemical shift drift in the CAM systems, which is ascribed to hydrogen bonding of the phenol group of NARI with SUL in the CAM system [42]. The results obtained from ATR-FTIR and solution NMR spectroscopy clearly indicate the formation of intermolecular interactions between SUL and NARI, leading to the successful development of CAM systems.

4.8 SEM

SEM analysis was carried out for crystalline SUL, amorphous SUL, crystalline NARI, amorphous NARI along with the different ratios of CAM system, and SEM image is presented in Fig. 8. Crystalline SUL exhibited irregular plate-shaped crystals with rough surfaces, while

crystalline NARI displayed ball-shaped structures covered with thin needle-like structures. After evaporation, the morphology of amorphous SUL and amorphous NARI was modified, resulting in irregular flakes with smooth surfaces (Fig. 8B and 8D). A distinctive morphology of evaporated amorphous samples could be easily identified. The prepared CAM systems exhibited similar morphologies as the ones recorded for amorphous SUL and amorphous NARI, which were dissimilar from the original crystalline SUL and crystalline NARI materials (Fig. 8E-H). Another indication of the formation of CAM systems between SUL and NARI can be observed in the morphologies of the evaporated samples.

4.9 Quantum Mechanics contingent Molecular Dynamics

4.9.1 Miscibility analysis

Miscibility analysis of the co-amorphous system (Fig. S6) was performed based on the Hildebrand solubility parameter principle, which depends on density (g/cm³). The cohesive energy of the system is 43 kcal/mol. To predict the miscibility of SUL-NARI CAM systems, a comparison of the solubility parameter (MPa^1/2) between the co-amorphous systems and solvents (MeOH and DCM) was performed to calculate the difference between solubility parameters with MeOH (∆Sm) and DCM (∆Sd) in kcal/mol. The calculated differences were found to be 2.4 kcal/mol (SUL-NARI 1:1), 2.31 kcal/mol (SUL-NARI 1:2), 2.85 kcal/mol (SUL-NARI 1:3), and 2.06 kcal/mol (SUL-NARI 2:1) for methanol and − 5.40 kcal/mol (SUL-NARI 1:1), 5.49 kcal/mol (SUL-NARI 1:2), -4.95 kcal/mol (SUL-NARI 1:3), and − 5.74 kcal/mol (SUL-NARI 2:1) for dichloromethane (see Table S4). The difference between ∆Sm and ∆Sd is < 7 MPa^1/2, indicating that the formed CAM systems are miscible with methanol and dichloromethane [43].

4.9.2 Molecular interaction investigation by RDF analysis

The RDF analysis investigates the probability of molecular interactions by examining the probability of an atom, represented by g(r), being found at a certain radius (r) from another interacting atom. This analysis is primarily based on atomic density [44, 45]. The peaks appearing before 5 Å indicate positive intermolecular interactions [46]. The results of the RDF analysis are presented in Fig. 9, which demonstrate the presence of hydrogen bond interactions between SUL and NARI in different CAM systems, with varying intensities. In Fig. 9A, an intense peak is observed at ≤ 1.8 Å, which is due to the presence of a strong hydrogen bond interaction between the oxygen atom of the carbonyl (= O) group of SUL and the hydrogen atom of the phenyl hydroxyl group (-OH) of NARI. In (Fig. 9B), two molecular interactions are assessed for the sulphonamide group of SUL and the hydroxy chromanone moiety of NARI. Intense peaks around 4.5 Å and broader, small observable peaks near 2 Å are due to weaker intermolecular interactions between the -SO2NH2 of SUL and the -C = O and -OH of NARI. In (Fig. 9C) shows the formation of H-bonds between the secondary amine (-NH) of SUL and the hydroxy (-OH) group of the pyranol moiety. The intense peak observed at 2.0 Å indicates a strong intermolecular H-bond interaction between the hydrogen atom of the secondary amine (-NH) of SUL and the oxygen atom located at the seventh position (-OH) from the pyranol moiety of NARI [47–50]. This intense peak is absent in the SUL-NARI (2:1) co-amorphous system, possibly due to the presence of a condensed phase in the system and the overlay of other intermolecular and intramolecular interactions [51]. The pattern observed in the RDF analysis for various ratios indicates short-range order, which corresponds to the amorphous nature of the systems [52].

4.9.3 Hydrogen-bond interactions and π-π interactions quantitative simulations

The hydrogen-bond interactions and π-π interaction counts are plotted for quantitative analysis of interactions. As demonstrated in Fig. 10A, H-bond interactions have appeared in order as SUL-NARI co-amorphous system (1:3 > 1:2 > 1:1 > 2:1). The number of H-bond interactions was observed and demonstrated in Fig. 10, which shows that as the percentage of NARI increases in the co-amorphous system, the number of H-bond interactions also increases. However, in the SUL-NARI (2:1) system, the lowest number of H-bond interactions was found, which is probably due to the high degree of condensation. The plot of π-π interaction counts (Fig. 10B) shows the order of the SUL-NARI co-amorphous systems (1:3 > 1:2 ≈ 1:1 > 2:1). The SUL-NARI co-amorphous systems of 1:1 and 1:2 exhibit almost an equal number of π-π interactions.

4.10 Residual solvents analysis by GC-FID

A residual solvent analysis was carried out as per ICH Q3C(R8)[20]. The results of GC-FID analysis chromatography are shown in Fig.S8. Based on the chromatograms of DCM and MeOH standard solutions, the retention time (min) of DCM was found to be 5.50, and that of MeOH was 4.36. When SUL, NARI, and different ratios of SUL-NARI co-amorphous systems (1:1, 1:2, 1:3, and 2:1) were injected, no peaks were observed at 5.50 min and 4.36 min. Therefore, it was concluded that the prepared co-amorphous systems do not contain any residues of DCM or MeOH [53].

4.11 Equilibrium solubility

The equilibrium solubility of plain SUL, physical mixture (PM), and CAM systems was determined in water, and the comparative solubility profiles are presented in Fig. 11. The solubility of plain SUL and physical mixtures was determined to be 0.89 ± 0.14 mg/ml and 0.90 ± 0.10 mg/ml, respectively. The solubility of SUL from the PM was not found to be statistically significant (P > 0.05) compared to plain SUL, suggesting no improvement in solubility in the presence of NARI. On the other hand, the CAM systems showed a significant improvement (P < 0.0001) in SUL solubility compared to plain SUL. Specifically, the SUL-NARI 1:1 CAM exhibited the highest drug solubility, approximately 31.88-fold, followed by SUL-NARI 1:3 CAM (28.13-fold) > SUL-NARI 1:2 CAM (27.41-fold) > SUL-NARI 2:1 CAM (16.04-fold) compared to its crystalline counterpart. These results support the hypothesis that CAM formation can contribute to improved solubility of poorly aqueous-soluble drugs, as the solubility of SUL was not found to be significantly different in PM compared to plain SUL despite containing a similar amount of NARI. Thus, the observed solubility advantage is not due to the exhibition of the co-solvency phenomenon, but rather originates from the co-amorphization effect [54].

4.12 In vitro dissolution

The dissolution profiles of SUL in the CAM systems were performed under sink conditions and compared with plain SUL and physical mixture (PM). The dissolution profiles of the CAM systems are presented in Fig. 12. The plain SUL showed complete release of the drug after 120 minutes. The physical mixture (PM) of SUL and NARI displayed a dissolution profile similar to plain SUL with a significant difference observed at the end of 120 minutes (P < 0.05) with a f2 value greater than 50, as shown in (Table S5). The determined DE5% values of plain SUL and PM (Table S5) also reflected the same. Within 5 min, approximately 100% of the drug was released from SUL-NARI 1:1 CAM and SUL-NARI 1:3 CAM, whereas 100% of the drug was released from SUL-NARI 1:2 CAM within 10 min, showing a statistically significant difference (P < 0.05). However, a different release pattern of SUL-NARI 2:1 CAM was observed compared to other CAM systems and plain SUL. The release of SUL from the SUL-NARI 1:1 CAM, SUL-NARI 1:2 CAM, and SUL-NARI 1:3 CAM systems was found to be 9.13, 8.35, and 9.04 times higher than that of plain SUL, respectively, as determined by the DE5% values (Table S5). The rapid release of SUL from these CAM systems may result in immediate action after administration, which could be advantageous for enhancing therapeutic efficacy and safety. The significant enhancement is attributed to intermolecular interactions between SUL and NARI in the amorphous form present in CAM, which leads to faster SUL drug release [30]. The dissolution profiles of CAM systems were in consistent with their respective solubility profile.

4.13 Permeability study

The ex vivo model is a widely used approach to predict in vivo drug absorption and assess various human drug transport mechanisms. Both everted and non-everted gut sac techniques are employed in this model. In this study, the non-everted gut sac technique was used due to its several advantages over the everted gut sac method, such as its simple procedure, lower test sample requirement, ease of serosal sample collection, and reduced risk of morphological damage to the intestinal tissue [55, 56]. SUL-NARI 1:1 CAM was chosen for further investigation due to its superiority in drug loading, solubility, dissolution, and stability compared to the other ratios, as demonstrated in Fig. 13. The results of the ex-vivo permeability study of SUL from the plain drug, PM, and SUL-NARI 1:1 CAM. Compared to the plain drug and PM suspension, the SUL-NARI 1:1 CAM system showed a statistically significant improvement in flux (P < 0.05). There is not a significant difference in flux between the SUL and a physical mixture. In comparison to the plain drug and physical mixture, the CAM system demonstrated an increase in SUL flux by 1.9-fold and 1.83-fold, respectively.

Baluom et al. (2001) and Kim et al. (2016) have demonstrated that the permeability of SUL could be improved by the presence of P-gp inhibitors such as verapamil, quinidine, and TPGS. This improvement is attributed to the inhibition of P-gp-mediated efflux [1, 57]. Furthermore, NARI has also been reported to inhibit the P-gp efflux pump with different molecules, which can eventually lead to increased drug absorption [58, 59]. The literature suggests that the permeability of poorly water-soluble drugs can increase linearly with NARI concentration [60]. Teza et al. (2015) and Uppala et al. (2022) reported significant increases in the permeability of fexofenadine and talinolol, respectively, in the presence of NARI as a P-gp efflux inhibitor, which is consistent with our findings [15, 18].

The first-generation P-gp inhibitors include verapamil, quinidine, and cyclosporine, which are most widely used to improve the effectiveness of treatment or therapy [61–63]. However, the use of these inhibitors is limited due to their high doses, pharmacological activity, and potential side effects, which can pose a challenge for long-term treatment [14, 64]. In this study, CAMs of SUL with NARI exhibited significantly improved permeability of SUL, which suggests that CAM systems containing similar flavonoids have the potential to serve as substitutes for first-generation P-gp inhibitors. The observed improvement in SUL flux could be attributed to enhanced solubility, resulting in a higher driving force for permeation due to the supersaturated solution, as well as the inhibition of P-gp efflux pump transport of SUL by NARI.

4.14 In-silico biological activity studies

The biological activities of SUL and NARI were predicted using PASS Online, and evaluated based on the values of Pa (to be active) and Pi (to be inactive). Table S6 summarizes the significant predicted biological activities of SUL and NARI based on the Pa > Pi values, along with the potential adverse effects of SUL. The molecular docking of the SUL-NARI system was performed on the D2 and D3 dopamine receptors. The results for different poses are described in Table S7, which includes the docking score (kcal/mol) and glide energy (kcal/mol). Based on the docking score and glide energy, the two best stable poses from D2 and D3 receptors were selected for post-docking analysis. For the D2 receptor, the SUL-NARI system exhibited two significant poses in molecular docking. In one pose, SUL interacts with ASP114, SER193, and TRP386 residues of the D2 receptor via H-bonding, pi-pi stacking, and salt bridge intermolecular interactions. The docking score and glide energy for this pose are − 6.547 kcal/mol and − 41.673 kcal/mol, respectively. In another pose, naringin interacts with ASP114, ILE184, and ASN396 residues of the D2 receptor via H-bonding intermolecular interactions. The docking score and glide energy for this pose are − 6.731 kcal/mol and − 48.384 kcal/mol, respectively (Fig. 14A).

The interaction matrix plot indicates a high number of positive interactions. The clustering analysis revealed that the optimal number of clusters is 5 based on the Kelley penalty plot, and the corresponding distance matrix is illustrated in Fig. 14B [65]. For D3 Receptor, the SUL-NARI system shows two significant poses in molecular docking. In which one pose, sulpiride shows interactions with ASP110, PHE345 and TYR373 residues of D3 Receptor by H-bonding, pi-pi stacking and salt bridge intermolecular interactions; it has a docking score of -6.647 kcal/mol and glide energy of -45.289 kcal/mol. In the other pose, naringin interacts with GLU90 and GLY94 residues of the D2 Receptor through H-bonding intermolecular interactions, resulting in a docking score of -6.400 kcal/mol and a glide energy of -47.604 kcal/mol, as shown in Fig. 15A. The interaction matrix plot indicates a positive number of interactions. The clustering analysis reveals that the optimal number of clusters is 7.5 according to the Kelley penalty plot, and the associated distance matrix is displayed in Fig. 15B [65]. The results of additional ADMET predictions related to our approach are as follows: -2.92 log mol/L solubility, P-glycoprotein II inhibitor activity, 0.496 log L/kg volume of distribution, 9 -2.821 logBB, and no observed hepatotoxicity (Table S8).

4.15 Physical stability

The physical stability of the amorphous SUL and SUL-NARI CAM systems was assessed at room temperature for a duration of three months using PLM and PXRD. The results showed that the amorphous SUL completely recrystallized after three months of storage, whereas amorphous NARI exhibited partial recrystallization, as observed by the birefringence patterns in PLM. On the other hand, the CAM systems were found to remain in the amorphous form even after three months of storage at room temperature, as confirmed by the results of PLM and PXRD (refer to Figs. 16 and 17). The PLM and PXRD data indicated that the CAM systems remained amorphous after three months of storage at room temperature, as shown in Figs. 16 and 17. No birefringence or diffraction patterns were observed after three months under PLM and PXRD for the 1:1, 1:2, and 1:3 M ratios. The DSC thermogram also demonstrated that all three ratios (1:1, 1:2, and 1:3 M) did not exhibit the endotherm of the parent compounds (see Fig S9). As expected, the SUL-NARI 2:1 CAM exhibited birefringence patterns and intensified diffraction peaks, indicating that the residual amount of SUL was not sufficient to stabilize the CAM system during the stability period and resulting in the crystalline nature of this system.

To conclude briefly, we successfully synthesized the CAM system of (SUL-NARI) using solvent evaporation technique. The CAM systems were amorphous at molar ratios of 1:1, 1:2, and 1:3, and semi-crystalline at a molar ratio of 2:1, as confirmed by DSC, PXRD, and PLM. In addition to the anti-plasticization effect, ATR-FTIR revealed that intermolecular interactions between SUL and NARI played a vital role in the formation and stabilization of the amorphous system. The NMR results demonstrate characteristic changes in the chemical shifts of various functional groups, indicating intermolecular H-bond interactions and π-π interactions. Furthermore, molecular dynamics studies support the interactions responsible for the co-amorphization of various SUL and NARI CAM systems. The solubility and dissolution of SUL from the CAM systems significantly improved compared to plain SUL and the physical mixture (PM). The CAM systems maintained physical stability for up to 3 months when stored at room temperature. A nearly 1.91-fold increase in flux were observed for SUL from the CAM system. The present investigation reveals that the synergistic interaction between amorphized SUL and NARI has P-gp efflux potential and contributes to the increased permeability of SUL in the CAM system. Molecular docking and biological activity predictions explain the benefits of the SUL-NARI system by targeting adverse effects of SUL (i.e., EPS) using NARI. These results highlight the advantages of developing a drug-nutraceutical CAM system for BCS class IV drugs to increase drug aqueous solubility, permeability and stability of co-amorphous system.

GIT Gastrointestinal tract

CNS Central nervous system

SUL Sulpiride

P-gp P-glycoprotein

EPS Extrapyramidal symptoms

BCS Biopharmaceutical classification system

ASD Amorphous solid dispersion

HME Hot melt extrusion

Tg Glass transition temperature

CAM Co-amorphous

GFA Glass forming ability

NARI Naringin

PXRD Powder X-ray diffraction

TGA Thermogravimetric analysis

DSC Differential scanning calorimetry

NMR ¹H Nuclear magnetic resonance

PLM Polarize light microscopy

ATR- FTIR Attenuated total reflectance-fourier transform infrared

SEM Scanning electron microscope

RDF Radial distribution function

SAR Structure-activity relationship

DCM Dichloromethane

MD Molecular dynamics

GC Gas chromatography

FID Flame ionization detector

f2 Similarity factor

DE Dissolution efficiency

TCB Tissue culture buffer

PASS Prediction of activity spectra for substances

D2, D3 Dopamine receptor

DMSO Dimethyl sulphoxide

Ethics approval and consent to participate: Studies in accordance with CPCSEA criteria and with approval from the Institutional Animal Ethics Committee.

Consent for publication: Not applicable

Availability of data and materials: Not applicable

Competing interests: The authors have no conflicts of interest to declare that are relevant to

the content of this article.

Funding: Not Applicable

Authors’ contributions: The idea for the research studies by Ekta Pardhi and Neelesh Kumar Mehra. The literature search and data analysis were done by Ekta Pardhi, Devendra Singh Tomar, Rahul Khemchandani. The article was drafted by Ekta Pardhi. The article is critically reviewed and revised by Gananadhamu Samanthula^b, Shashi Bala Singh and Neelesh Kumar Mehra.

Acknowledgements:

The authors would like to acknowledge the financial support from the Department of Pharmaceuticals (DoP), Ministry of Chemicals and Fertilizers, Govt. of India.

Kim DS, Kim DW, Kim KS, Choi JS, Seo YG, Youn YS, Oh KT, Yong CS, Kim JO, Jin SG. Development of a novel l-sulpiride-loaded quaternary microcapsule: Effect of TPGS as an absorption enhancer on physicochemical characterization and oral bioavailability. Colloids Surf B: biointerfaces. 2016;147:250–7. http://doi.org/10.1016/j.colsurfb.2016.08.010.
Lai EC-C, Hsieh C-Y, Kao Yang Y-H, Lin S-J. Detecting Potential Adverse Reactions of Sulpiride in Schizophrenic Patients by Prescription Sequence Symmetry Analysis. PLoS ONE 2014, 9 (2), e89795 DOI: https://doi.org/10.1371/journal.pone.0089795.
Ayoub AM, Ibrahim MM, Abdallah MH, Mahdy MA. Sulpiride microemulsions as antipsychotic nasal drug delivery systems: In-vitro and pharmacodynamic study. J drug delivery Sci Technol. 2016;36:10–22. https://doi.org/10.1016/j.jddst.2016.09.002.
Shukr MH, Ahmed Farid OA. Amisulpride–CD-loaded liposomes: optimization and in vivo evaluation. AAPS PharmSciTech. 2018;19(6):2658–71. https://doi.org/10.1208/s12249-018-1079-z.
Kohri N, Naasani I, Iseki K, Miyazaki K. Improving the oral bioavailability of sulpiride by a gastric-retained form in rabbits. J Pharm Pharmacol. 1996;48(4):371–4. https://doi.org/10.1111/j.2042-7158.1996.tb05935.x.
Chitneni M, Peh KK, Darwis D, Abdulkarim M, Abdullah GZ, Qureshi MJ. Intestinal permeability studies of sulpiride incorporated into self-microemulsifying drug delivery system (SMEDDS). J Pharm Sci. 2011;24(2):113–21. PMID: 21454158.
Ibrahim WM, AlOmrani AH, Yassin AEB. J. I. j. o. n., Novel sulpiride-loaded solid lipid nanoparticles with enhanced intestinal permeability. 2014, 9, 129 DOI: https://doi.org/10.2147/ijn.s54413.
Kim DS, Choi JS, Kim DW, Kim KS, Seo YG, Cho KH, Kim JO, Yong CS, Youn YS, Lim S-J. J. I. j. o. p., Comparison of solvent- wetted and kneaded l-sulpiride-loaded solid dispersions: Powder characterization and in vivo evaluation. 2016, 511 (1), 351–8 DOI: https://doi.org/10.1016/j.ijpharm.2016.07.006.
Tomar D, Singh PK, Hoque S, Modani S, Sriram A, Kumar R, Madan J, Khatri D, Dua K. Amorphous systems for delivery of nutraceuticals: challenges opportunities. Crit reviews food Sci Nutr 2020, 62 (5), 1204–21 DOI: https://doi.org/10.1080/10408398.2020.1836607.
Dengale SJ, Grohganz H, Rades T, Löbmann K. Recent advances in co-amorphous drug formulations. Adv Drug Deliv Rev. 2016;100:116–25. https://doi.org/10.1016/j.addr.2015.12.009.
Mizoguchi R, Waraya H, Hirakura Y. Application of co-amorphous technology for improving the physicochemical properties of amorphous formulations. Mol Pharm. 2019;16(5):2142–52. https://doi.org/10.1021/acs.molpharmaceut.9b00105.
Kasten G, Löbmann K, Grohganz H, Rades T. Co-former selection for co-amorphous drug-amino acid formulations. Int J Pharm. 2019;557:366–73. https://doi.org/10.1016/j.ijpharm.2018.12.036.
Yarlagadda DL, Anand VSK, Nair AR, Sree KN, Dengale SJ, Bhat K. Considerations for the selection of co-formers in the preparation of co-amorphous formulations. Int J Pharm. 2021;602:120649. https://doi.org/10.1016/j.ijpharm.2021.120649.
Wei Y, Zhou S, Hao T, Zhang J, Gao Y, Qian S. Further enhanced dissolution and oral bioavailability of docetaxel by coamorphization with a natural P-gp inhibitor myricetin. Eur J Pharm Sci. 2019;129:21–30. https://doi.org/10.1016/j.ejps.2018.12.016.
Teja A, Musmade PB, Khade AB, Dengale SJ. Simultaneous improvement of solubility and permeability by fabricating binary glassy materials of Talinolol with Naringin: Solid state characterization, in-vivo in-situ evaluation. Eur J Pharm Sci. 2015;78:234–44. http://dx.doi.org/10.1016%2Fj.ejps.2015.08.002.
Pandi P, Bulusu R, Kommineni N, Khan W, Singh M. Amorphous solid dispersions: An update for preparation, characterization, mechanism on bioavailability, stability, regulatory considerations and marketed products. Int J Pharm. 2020;586:119560. https://doi.org/10.1016/j.ijpharm.2020.119560.
Bharti S, Rani N, Krishnamurthy B, Arya DS. Preclinical evidence for the pharmacological actions of naringin: a review. Planta Med. 2014;80(06):437–51. https://doi.org/10.1055/s-0034-1368351.
Uppala S, Vullendula SKA, Yarlagadda DL, Dengale SJ, Technology. Exploring the utility of co-amorphous materials to concurrently improve the solubility and permeability of Fexofenadine. J drug delivery Sci Technol. 2022;72:103431. https://doi.org/10.1016/j.jddst.2022.103431.
Kumar N, James R, Sinha S, Kinra M, Anuranjana P, Nandakumar K. Naringin exhibited Anti-Parkinsonian like effect against haloperidol-induced catalepsy in mice. Res J Pharm Technol. 2021;14(2):662–6.
IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R8). ICH HARMONISED GUIDELINE. 2021, https://www.ema.europa.eu/documents/regulatory-procedural-guideline/ich-guideline-q3c-r8-impurities-guideline-residual-solvents-step-5_en.pdf.
Zhang Y, Huo M, Zhou J, Zou A, Li W, Yao C, Xie S. DDSolver: an add-in program for modeling and comparison of drug dissolution profiles. AAPS J. 2010;12(3):263–71. https://doi.org/10.1208/s12248-010-9185-1.
Filimonov DA, Lagunin AA, Gloriozova TA, Rudik AV, Druzhilovskii DS, Pogodin PV, Poroikov VV. Prediction of the Biological Activity Spectra of Organic Compounds Using the Pass Online Web Resource. Chem Heterocycl Compd. 2014;50(3):444–57. https://doi.org/10.1007/s10593-014-1496-1.
Yang C, Di P, Fu J, Xiong H, Jing Q, Ren G, Tang Y, Zheng W, Liu G, Ren F. Improving the physicochemical properties of bicalutamide by complex formation with bovine serum albumin. Eur J Pharm Sci. 2017;106:381–92. https://doi.org/10.1016/j.ejps.2017.05.059.
Pasaribu YP, Fadlan A, Fatmawati S, Ersam T. Biological Activity Evaluation and In Silico Studies of Polyprenylated Benzophenones from Garcinia celebica. Biomedicines. 2021;9(11):1654. https://doi.org/10.3390/biomedicines9111654.
Moinuddin SM, Ruan S, Huang Y, Gao Q, Shi Q, Cai B, Cai T. Facile formation of co-amorphous atenolol and hydrochlorothiazide mixtures via cryogenic-milling: enhanced physical stability, dissolution and pharmacokinetic profile. Int J Pharm 2017, 532 (1), 393–400 DOI: https://doi.org/10.1016/j.ijpharm.2017.09.020.
KS NS, Dengale SJ, Mutalik S, Bhat K. Raloxifene HCl–Quercetin Co-amorphous System: Preparation, Characterization, and Investigation of its Behavior in Phosphate Buffer. Drug development industrial pharmacy 2022, (just-accepted), 1–34 DOI: https://doi.org/10.1080/03639045.2022.2104308.
Löbmann K, Grohganz H, Laitinen R, Strachan C, Rades T. Amino acids as co-amorphous stabilizers for poorly water soluble drugs–Part 1: Preparation, stability and dissolution enhancement. Eur J Pharm Biopharm. 2013;85(3):873–81. https://doi.org/10.1016/j.ejpb.2013.03.014.
Pešić N, Dapčević A, Ivković B, Kachrimanis K, Mitrić M, Ibrić S, Medarević D. Potential application of low molecular weight excipients for amorphization and dissolution enhancement of carvedilol. Int J Pharm. 2021;608:121033. https://doi.org/10.1016/j.ijpharm.2021.121033.
Laitinen R, Löbmann K, Strachan CJ, Grohganz H, Rades T. Emerging trends in the stabilization of amorphous drugs. Int J Pharm. 2013;453(1):65–79. https://doi.org/10.1016/j.ijpharm.2012.04.066.
Fael HD, Levent A. Indomethacin co-amorphous drug-drug systems with improved solubility, supersaturation, dissolution rate and physical stability. Int J Pharm. 2021;600:120448. https://doi.org/10.1016/j.ijpharm.2021.120448.
Dengale SJ, Ranjan OP, Hussen SS, Krishna B, Musmade PB, Shenoy GG, Bhat K. Preparation and characterization of co-amorphous Ritonavir–Indomethacin systems by solvent evaporation technique: Improved dissolution behavior and physical stability without evidence of intermolecular interactions. Eur J Pharm Sci. 2014;62:57–64. https://doi.org/10.1016/j.ejps.2014.05.015.
M’bitsi-Ibouily GC, Marimuthu T, Kumar P, Choonara YE, du Toit LC, Pradeep P, Modi G, Pillay V. Synthesis, characterisation and in vitro permeation, dissolution and cytotoxic evaluation of ruthenium (ii)-liganded sulpiride and amino alcohol. Sci Rep. 2019;9(1):1–18. https://doi.org/10.1038/s41598-019-40538-1.
Gollavilli H, Hegde AR, Managuli RS, Bhaskar KV, Dengale SJ, Reddy MS, Kalthur G, Mutalik S. Naringin nano-ethosomal novel sunscreen creams: Development and performance evaluation. Colloids Surf B: biointerfaces. 2020;193:111122. https://doi.org/10.1016/j.colsurfb.2020.111122.
Mohamed GG, Soliman MH. Synthesis, spectroscopic and thermal characterization of sulpiride complexes of iron, manganese, copper, cobalt, nickel, and zinc salts. Antibacterial and antifungal activity. Spectrochimica acta part A: molecular biomolecular spectroscopy. 2010;76(3–4):341–7. https://doi.org/10.1016/j.saa.2010.03.016.
Colombo M, Minussi C, Orthmann S, Staufenbiel S, Bodmeier R. Preparation of amorphous indomethacin nanoparticles by aqueous wet bead milling and in situ measurement of their increased saturation solubility. Eur J Pharm Biopharm. 2018;125:159–68. https://doi.org/10.1016/j.ejpb.2018.01.013.
Zheng K, Gao S, Chen M, Li A, Wu W, Qian S, Pang Q. Color tuning of an active pharmaceutical ingredient through cocrystallization: a case study of a metronidazole–pyrogallol cocrystal. CrystEngComm. 2020;22(8):1404–13. https://doi.org/10.1039/C9CE01726G.
M’bitsi-Ibouily G, Marimuthu T, Kumar P, Choonara Y, Toit L, Pradeep P, Modi G, Pillay V. Synthesis, Characterisation and In Vitro Permeation, Dissolution and Cytotoxic Evaluation of Ruthenium(II)-Liganded Sulpiride and Amino Alcohol. Sci Rep. 2019;9:4146. https://doi.org/10.1038/s41598-019-40538-1.
Malgarim Cordenonsi L, Sponchiado R, Chagas Campanharo S, Garcia C, Raffin R, Schapoval E, STUDY OF FLAVONOIDS PRESENT IN POMELO. (Citrus Maxima) BY DSC, UV-VIS, IR, 1H AND 13C NMR AND MS. Drug Anal Res. 2017;1:31–7. https://doi.org/10.22456/2527-2616.74097.
Informatics P. ChemDraw 20.0. 2020.
Simoben C, Robaa D, Chakrabarti A, Schmidtkunz K, Marek M, Lancelot J, Kannan S, Melesina J, Shaik T, Pierce R, Romier C, Jung M, Sippl W. A Novel Class of Schistosoma mansoni Histone Deacetylase 8 (HDAC8) Inhibitors Identified by Structure-Based Virtual Screening and In Vitro Testing. Molecules. 2018;23(3):566. https://doi.org/10.3390/molecules23030566.
Pinheiro Barros MC, Sousa Lima MA, Braz-Filho R, Rocha Silveira E. 1H and 13C NMR assignments of abietane diterpenes from Aegiphila lhotzkyana. Magn Reson Chem. 2003;41(9):731–4. https://doi.org/10.1002/mrc.1236.
Celiz G, Suarez SA, Arias A, Molina J, Brondino CD, Doctorovich F. Synthesis, structural elucidation and antiradical activity of a copper (II) naringenin complex. Biometals. 2019;32(4):595–610. https://doi.org/10.1007/s10534-019-00187-3.
Kalam MA, Alshamsan A, Alkholief M, Alsarra IA, Ali R, Haq N, Anwer MK, Shakeel FJA. o., Solubility measurement and various solubility parameters of glipizide in different neat solvents. ACS Omega 2020, 5 (3), 1708–1716 DOI: https://doi.org/10.1021/acsomega.9b04004.
Yoda O, Kuriyama I, Odajima AJ. J. o. A. P., RDF analysis for the degree of interchain ordering in amorphous solid polyethylene. J Appl Phys. 1978;49(11):5468–72. https://doi.org/10.1063/1.324516.
Srolovitz D, Egami T, Vitek VJPRB. Radial distribution function and structural relaxation in amorphous solids. Phys Rev B. 1981;24(12):6936. https://doi.org/10.1103/PhysRevB.24.6936.
Yang Y, Tang W, Liu S, Han D, Liu Y, Gong JJJ. o. M. L., Solubility of benzoin in three binary solvent mixtures and investigation of intermolecular interactions by molecular dynamic simulation. J Mol Liq. 2017;243:472–83. https://doi.org/10.1016/j.molliq.2017.07.125.
Li B, Wang Y, Feng Y, Yuan D, Xu R, Jiang C, Xiao X, Lu S. J. I. J. o. P., Design and molecular insights of drug-active metabolite based co-amorphous formulation: A case study of toltrazuril-ponazuril co-amorphous. Int J Pharm. 2022;615:121475. https://doi.org/10.1016/j.ijpharm.2022.121475.
Li B, Hu Y, Guo Y, Xu R, Fang X, Xiao X, Jiang C, Lu SJ. J. o. P. S., Coamorphous system of Florfenicol-Oxymatrine for improving the solubility and dissolution rate of Florfenicol: Preparation, characterization and molecular dynamics simulation. J Pharm Sci. 2021;110(6):2544–54. https://doi.org/10.1016/j.xphs.2021.02.005.
Russo MG, Baldoni HA, Dávila YA, Brusau EV, Ellena JA, Narda GE. J. T. J. o. P. C. B., Rational Design of a Famotidine–Ibuprofen Coamorphous System: An Experimental and Theoretical Study. J Phys Chem B. 2018;122(37):8772–82. https://doi.org/10.1021/acs.jpcb.8b06105.
Terban MW, Billinge SJJCR. Structural analysis of molecular materials using the pair distribution function. Chem Rev. 2021;122(1):1208–72. https://doi.org/10.1021/acs.chemrev.1c00237.
Fawcett RWJ. C. t. r., A radial distribution function analysis of an amorphous calcium phosphate with calcium to phosphorus molar ratio of 1.42. Calcif Tissue Res. 1973;13(1):319–25. https://doi.org/10.1007/bf02015422.
Lai M, Zhang X, Fang FJ. N. r. l., Crystal orientation effect on the subsurface deformation of monocrystalline germanium in nanometric cutting. Nanoscale Res Lett. 2017;12(1):1–10. https://doi.org/10.1186/s11671-017-2047-3.
Arulraj RA, Gajjela R, Chauthe SK, Bagadi M, Mathur AJC. Correction to: High-Throughput GC-FID Method for the Determination of Residual Solvents in Early-Phase Drug Discovery Samples. 2022, 85 (7), 677–7 DOI: https://doi.org/10.1007/s10337-022-04157-9.
Zhu S, Gao H, Babu S, Garad S. Co-amorphous formation of high-dose zwitterionic compounds with amino acids to improve solubility and enable parenteral delivery. Mol Pharm. 2018;15(1):97–107. https://doi.org/10.1021/acs.molpharmaceut.7b00738.
Liu W, Pan H, Zhang C, Zhao L, Zhao R, Zhu Y, Pan W. Developments in methods for measuring the intestinal absorption of nanoparticle-bound drugs. Int J Mol Sci. 2016;17(7):1171. https://doi.org/10.3390/ijms17071171.
Xu Y, Shrestha N, Préat V, Beloqui A. An overview of in vitro, ex vivo and in vivo models for studying the transport of drugs across intestinal barriers. Adv Drug Deliv Rev. 2021;175:113795. https://doi.org/10.1016/j.addr.2021.05.005.
Baluom M, Friedman M, Rubinstein A. Improved intestinal absorption of sulpiride in rats with synchronized oral delivery systems. J Controlled Release. 2001;70(1–2):139–47. https://doi.org/10.1016/s0168-3659(00)00337-0.
Gurunath S, Nanjwade BK, Patil P. Oral bioavailability and intestinal absorption of candesartan cilexetil: Role of naringin as P-glycoprotein inhibitor. Drug Dev industrial Pharm. 2015;41(1):170–6. https://doi.org/10.3109/03639045.2013.850716.
Choi J-S, Shin S-C. Enhanced paclitaxel bioavailability after oral coadministration of paclitaxel prodrug with naringin to rats. Int J Pharm. 2005;292(1–2):149–56. https://doi.org/10.1016/j.ejpb.2003.11.002.
Shirasaka Y, Li Y, Shibue Y, Kuraoka E, Spahn-Langguth H, Kato Y, Langguth P, Tamai I. Concentration-dependent effect of naringin on intestinal absorption of β1-adrenoceptor antagonist talinolol mediated by P-glycoprotein and organic anion transporting polypeptide (Oatp). Pharm Res. 2009;26(3):560–7. https://doi.org/10.1007/s11095-008-9771-4.
Yang T, Ferrill L, Gallant L, McGillicuddy S, Fernandes T, Schields N, Bai S. Verapamil and riluzole cocktail liposomes overcome pharmacoresistance by inhibiting P-glycoprotein in brain endothelial and astrocyte cells: A potent approach to treat amyotrophic lateral sclerosis. Eur J Pharm Sci. 2018;120:30–9. https://doi.org/10.1016/j.ejps.2018.04.026.
Snyder S, Murundi S, Crawford L, Putnam D. Enabling P-glycoprotein inhibition in multidrug resistant cancer through the reverse targeting of a quinidine-PEG conjugate. J Controlled Release. 2020;317:291–9. https://doi.org/10.1016/j.jconrel.2019.11.027.
Li L, Yao Q-q, Xu S-y, Hu H-h, Shen Q, Tian Y, Pan L-y, Zhou H, Lu C, Yu L. -s., Cyclosporin A affects the bioavailability of ginkgolic acids via inhibition of P-gp and BCRP. Eur J Pharm Biopharm. 2014;88(3):759–67. https://doi.org/10.1016/j.ejpb.2014.06.012.
Garg A, Bhalala K, Tomar DS. In-situ single pass intestinal permeability and pharmacokinetic study of developed Lumefantrine loaded solid lipid nanoparticles. Int J Pharm. 2017;516(1–2):120–30. https://doi.org/10.1016/j.ijpharm.2016.10.064.
Mahdizadeh SJ, Thomas M, Eriksson LA. Reconstruction of the Fas-Based Death-Inducing Signaling Complex (DISC) Using a Protein–Protein Docking Meta-Approach. J Chem Inf Model. 2021;61(7):3543–58. https://doi.org/10.1007/s10337-022-04157-9.

GA.png
Graphical abstract
Supplementoryfile.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

A study of monophasic supramolecular formulation of Sulpiride with P-glycoprotein efflux inhibitor to enhance solubility and intestinal permeability with molecular modeling insights

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Analytical method development

2.2 Preparation of co-amorphous system (CAM)

3. Solid state characterization

3.1 Thermogravimetric analysis (TGA)

3.2 Differential scanning calorimetry (DSC)

3.3 Powdered X-ray diffraction (PXRD)

3.4 Polarized light microscopy (PLM)

3.5 total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy

3.6 Nuclear magnetic resonance (NMR) Spectroscopy

3.7 Scanning Electron Microscope (SEM)

3.8 Quantum mechanics contingent molecular dynamics study

3.9 Residual solvents analysis by GC-FID

3.10 Saturation solubility

3.11 In vitro dissolution

3.12 Ex-vivo permeability

3.13 In-silico biological activity studies

3.14 Physical stability

3.15 Statistical analysis

4. Results and Discussion

4.1 Solid state characterization of CAM

4.2 TGA

4.3 DSC

4.4 PXRD

4.5 PLM

4.6 ATR-FTIR

4.7 NMR

4.8 SEM

4.9 Quantum Mechanics contingent Molecular Dynamics

4.9.1 Miscibility analysis

4.9.2 Molecular interaction investigation by RDF analysis

4.9.3 Hydrogen-bond interactions and π-π interactions quantitative simulations

4.10 Residual solvents analysis by GC-FID

4.11 Equilibrium solubility

4.12 In vitro dissolution

4.13 Permeability study

4.14 In-silico biological activity studies

4.15 Physical stability

5. Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1