Enhanced bioavailability and efficacy in antimalarial treatment through QbD approach enteric encased inclusion delivery

Abstract

Aim: In this study, we aimed to prepare enteric encapsulated spheroids containing inclusion complex using quality by design approach.

Methods: A Box–Behnken design was employed to determine effects of variables on selected responses. Risk assessment was conducted using Ishikawa fishbone diagram. A model with a p-value was less than 0.5 for being a significant error of model was determined based on significance ‘lack of fit’ value. Spheroids were formulated using the extrusion spheronization technique and were characterized using analytical techniques.

Results: In vitro release was performed in both acidic (pH 1.2) and simulated intestinal (pH 6.8) conditions. Permeability studies demonstrated tenfold enhancement compared with arteether. In vivo studies further validated increase of 51.8% oral bioavailability. Ex vivo studies revealed 3.4-fold enhancement in antimalarial activity compared with arteether.

Conclusion: These findings highlight effectiveness of inclusion complexation technique as a viable approach to enhance solubility and bioavailability for drugs with low aqueous solubility.

Graphical Abstract

Plain language summary

Article highlights.

• The enteric encapsulated spheroids of ART-cyclodextrin (CD) inclusion complex were prepared by extrusion spheronization using quality-by-design approach.

• A Box–Behnken design was consequently employed for investigation of impact on the response on selected variables.

• The preparation of spheroids was confirmed by x-ray diffraction, solubility studies, scanning electron microscopy, differential scanning calorimetry and flow properties. In vitro release studies confirmed that around 80% drug was released in 4.5 h which validated that the drug was released in intestine, the preferred absorption site.

• Flow property characterization confirmed free flowing nature of prepared spheroids. The permeability of ART containing formulations was investigated using the Franz diffusion cell technique.

• The concentration of ART-CD spheroids to pure ART in the pig’s intestine was compared. A nearly 7.1-fold, 6.8-fold, sevenfold, 4.6-fold increase in permeability compared with ART in its pure form was observed with SLNs, NLCs, SMEDDS, ART-CD inclusion complex respectively.

• For establishing plasma drug concentration-time profile of pure drug suspension, spheroids in enteric coated capsule shells were administered orally in rabbits. The pharmacokinetic parameters of the prepared formulations revealed that the absolute bioavailabilities of the formulations ART-CD spheroids (51.8%) were comparable with marketed formulation i.e. i.m. injection (43.73%). In order to compare the antimalarial efficacy of ART and ART-CD complx, IC₅₀ values against P. falciparum stain Pf3. The IC₅₀ value of ART and ART-CD complex were estimated to be 0.76 ng/ml and 0.733 ng/ ml in dimethylsulfoxide as solvent.

• The IC₅₀ value of ART-CD complex was almost equivalent to ART reflecting the acceptability of antimalarial activity of ART-CD complex drug.

• Therefore, it is concluded that present study have explored the feasibility to prepare oral formulation of ART with acceptable bioavailability. With benefits including higher acceptance, particularly among adolescents and female patients, enhanced compliance, lesser production cost and many dose-related difficulties, the suggested formulation may offer a new vista in the treatment of malaria. Application of various prevalent industrial approaches involving total quality management based on intergrative strategies like critically analysis, quality risk management, analytical method validation, formulation-by-design and ultimately quality by design in present study also provide a comprehensive solution to develop oral formulation of arteether with desired bioavailability at industrial scale.

1. Background

Recent developments in combination chemistry, parallel synthesis and high throughput screening have significantly increased the range of lipophilic compounds. The necessity of fixing delivery issues resulting from bioavailability concerns has motivated these initiatives. Remarkably, around half of recently created therapeutic molecules are undergoing clinical trials mainly because of unfavourable pharmacokinetic characteristics and roughly a third of these compounds show water insolubility [1]. The issue of low solubility gives rise to significant development hurdles, resulting in inadequate solubility levels for effective dosing and leading to compromised absorption and bioavailability. Also, the patient must contend with continuous high dose frequency, which affects the treatment expense [2,3].

α, β-arteether (ART) is a semisynthetic derivative of artemisinin, especially indicated for cerebral malaria and chloroquine-resistant Plasmodium falciparum malaria [4]. ART is more lipophilic as compare to other derivatives, which is possible advantages for its accumulation in brain tissues in patients with cerebral malaria and less toxic because its metabolites would be ethanol rather than methanol which avoid the problem of methanol toxicity [5,6]. But main problem arises with its low aqueous solubility, poor bioavailability and degradation in stomach. Approximately 40% drug gets degraded in stomach while aqueous solubility is only 17 μg/ml [7,8]. So, there is a need for enhancement in solubility and bioavailability of ART.

Drug absorption can be hindered by a number of variables, including poor water solubility and membrane permeability of drug [9]. When an active substance is delivered orally, initially it must show sufficient dissolution in gastric and/or intestinal media prior to permeation through the gastro-intestinal tract (GIT) membrane and reach systemic circulation. Thus, improving the solubility and dissolution rate of drugs showing limited aqueous solubility are of prime focus for the enhancement of oral bioavailability. Increasing the solubility and thus oral bioavailability persists as one of the most challenging feature of the formulation development especially for oral formulation development [10,11].

Techniques aimed at enhancing solubility encompass a range of approaches. Physically, modifications are employed to alter crystal habits, such as generating polymorphs, amorphous forms and cocrystallization, as well as reducing particle size via methods like nanosuspension and micronization. Additionally, drug dispersion within carriers, including solid dispersions, eutectic mixtures and solid solutions, contributes to solubility enhancement. Chemically, modifications involve pH adjustments, derivatization, buffer utilization, salt formation and complexation of the drug. Moreover, diverse methods like hydrotrophy, cryogenic techniques, supercritical fluid processes and the use of solubilizers, surfactants, novel excipients and cosolvents are employed to address solubility challenges [12–14].

The inclusion complexation has been utilized most frequently to increase aqueous solubility which in turn improve the dissolution rate as well as bioavailability of drugs with poor solubility. Cyclodextrins (CDs) are employed for complex formation to improve water solubility and drug stability [15].

Quality by design (QbD) is a more scientific, holistic, risk-based and proactive approach for pharmaceutical development that includes adequate feed forward and feed-back control measures. QbD incorporates quality into the product throughout development rather of simply testing for it, which aids in completely understanding the root-cause, i.e., critical process parameters and material attributes that effect the predetermined quality attributes [16]. Risk assessment identifies the possible risk factors to the product quality. Followed by multivariate experiments employing design of experiments (DOE) for process knowledge [17]. The DOE technique provides relationship between the CMAs/CPPs with the critical quality attributes (CQAs) and the design space (DS) is expanded. Plackett Burman, the most often used DOE, can screen the important factors among multiple input variables. The limitation of Plackett–Burman design include confounding of interactions between variables and difficult to establish due to a lack of degrees of freedom. Due to the fact that Box–Behnken design (BBD) can establish DS with fewer experimental runs than central composite design, it is a frequently employed optimization design.

The pharmaceutical market is dominated by oral dosage formulations as it possess remarkable merits viz., homogenous distribution in the GIT, enhancing absorption and lowering local GIT irritation risks. Pelletized/spheroids medication administration is becoming becoming increasingly popular in therapeutics because to its excellent flow qualities, restricted particle size range and low friability, which prevents dosage dumping. The technological advancements has opened up a new vista in the preparation and scalability of the spheroids/pelletized drug delivery [16,17].

The ultimate aim of this research work is improving the solubility and bioavailability of ART. QbD approach is applied for preparation of CD complex spheroids. Further, to prevent the drug from acid degradation the prepared ART-CD complex spheroids were encapsulated in enteric capsule shells.

2. Materials & methods

2.1. Materials

ART (Arteether ≥95% w/w, MW-312.4) obtained from Brooks Lab Ltd., Baddi, Himachal Pradesh, India as a gift sample and was utilized without further purification. β-CD (99.5% w/w, MW-1134.99) and polyvinyl pyrrolidone K30 (poly vinyl pyrrolidone K30) was procured from Sigma Aldrich Chemicals Pvt. Ltd, Bagalore, India. HPLC grade acetonitrile and methanol were obtained from Rankem, Gurugram, Haryana, India. Triple distilled Water was obtained by using the Millipore Milli-0 Plus water purification system. Match i.m. injection was used as marketed formulation. All the reagents and solvents employed were of analytical grade.

2.2. Molecular docking

The computational study was carried out utilizing the PyRx Virtual Screening software, which is often used to screen libraries of various active compounds against potential therapeutic targets. The PubChem database was used to download the 3D structure of active ligands arteether and β-CD [18], whereas PyRx Software program was used to minimize the energy of active ligand molecules. The 3D structure of Malarial proteins like, PDB ID: 5JWA were obtained from the PDB database and proteins were prepared by removing excess water content, het-atom, ligand molecule and polar hydrogen bond were added using BIOVIA-discovery studio 2021 software. Open babel and Autodock-vina wizard tools are in-builded in PyRx software, whereas Vina wizard is used to accomplishing docking of chosen prepared molecules and Open Babel is used to minimize the energy of active ligand molecules. Pymol was used to depict the molecular docking study and BIOVIA-discovery studio 2021 was utilized to investigate the 2D and 3D interaction, whereas, molecule with docking score with interaction type have been shown in Supplementary Table S6.

2.3. Preparation of complex enteric encapsulated spheroids

Spheroids were formulated by extrusion-spheronization process. Spheroids were prepared by using polyvinyl pyrrolidone K 30 as binder, sodium starch glycollate as disintegrant and microcrystalline cellulose (MCC or Avicel 101) as filler. As drug is slightly gummy so colloidal silica is also incorporated in the mixture. All the ingredients were accurately weighed and blended with required quantity of ART-CD complex [19,20]. Then ethanol was added to the blended ingredients so as to form wet mass or dough. When this dough was passed through extruder, cylindrical shaped extrudates were formed. The extrudates were collected in spheronizer for rounding the extrudates. The formed spheroids were optimized on the basis of spheronization time, shape, concentration of binder and disintegrant [21]. The prepared spheroids were encapsulated in enteric coated capsule shells to prevent the drug from acid degradation.

2.4. QbD based analysis for optimization development of spheroid

2.4.1. Defining quality target product profile & identification of CQAs

Quality target product profile (QTPP) is a primary step toward QbD-oriented development of ART-loaded spheroids and is defined as ‘the imminent glossary of the quality attributes of a drug product that will be achieved exemplary to ensure the desiderate quality, along with safety and efficacy of the drug product’. To meet the QTPP, various CQAs were allocated such as particle size, dissolution and drug entrapment. Supplementary Table S1 enlists the QTPP elements and CQAs for products/processes affecting the performance of ART-Spheroids.

2.5. Risk assessment

Supplementary Figure S1 demonstrates the Ishikawa fish bone diagram depicting the various causes that affect the CQAs of Spheroids formulations. The 6Ms probably affecting the CQAs like, machines, men, material (API & excipients), measurements, methods and milieu/environment were depicted by the fish-bone diagram. The risk assessment matrix was also constructed to observe the possible causes affecting the CQAs. The potential risks and corresponding causes were determined by construction of fishbone diagram [22]. Prior information, initial experimental data was utilized for failure mode and effect analysis in risk analysis of the factors of the preparation of spheroids. % cumulative release was selected as the CQA for spheroids [23].

2.6. BBD optimization study

BBD was chosen for aforementioned reasons as it require fewer experimental runs than central composite design. Based on the previous analysis of literature and brainstorming discussion among the research team, various critical factors that can affect the final quality parameters of spheroid were identified. Parameters such as stirring time, stirring speed, disintegration concentration and binder concentration were considered as critical one that can affect the final quality parameters [24].

Therefore, for optimizing the four factors at a time, four factors three levels BBD were applied. BBD was created using the Design Expert software version 13 (State-Ease Inc, Minneapolis, USA). Four critical factors i.e., stirring time, stirring speed, disintegration concentration and binder concentration were selected as independent variables and % dissolution were selected as responses or dependent variable. BBD for four factors gave 29 runs and shown in Supplementary Table S2. DOE not only employed in this study for determination of optimized value of the selected critical variables but it can also provide the correlation between the selected factors with that of dependent variables. The response surface methodology was employed for creating a nonlinear quadratic model which possibly depicts the relationship between the independent and dependent variables with model equation as given in Equation 1 [25].

$$\begin{array}{c}\text{Y}={\text{b}}_0+{\text{b}}_1\text{A}+{\text{b}}_2\text{B}+{\text{b}}_3\text{C}+{\text{b}}_4\text{D}+{\text{b}}_5\text{AB}+{\text{b}}_6\text{AC}+{\text{b}}_7\text{AD}\\ +{\text{b}}_8\text{BC}+{\text{b}}_9\text{BD}+{\text{b}}_{10}\text{CD}+{\text{b}}_{10}{\text{A}}^2+{\text{b}}_{11}{\text{B}}^2+{\text{b}}_{12}{\text{C}}^2+{\text{b}}_{13}{\text{D}}^2\end{array}$$ (1)

where Y is response, b₀ is the intercept and b₁-b₁₃ are regression coefficients of different factors and their quadratic polynomials.

The regression equation in Box–Behnken analyses, characterize the impact on responses due to the variables in linear, interactive and quadratic terms. Furthermore, the p-values associated with the regression coefficients demonstrated the importance of variables on responses. The coefficient of determination (R²) and analysis of variance (ANOVA) were used to assess the model’s suitability [26].

2.7. Physicochemical characterization of optimized formulation

Various physico-chemical parameters of spheroids like shape, spheroid size and size distribution, friability, drug content, flow properties, were assessed according to European Pharmacopoeia 7th Edn. while disintegration time and in vitro drug release are evaluated as follows.

2.8. Flow properties

Measurements of the tapped density, bulk density Carr’s index and Hausner’s ratio in triplicate using conventional techniques were used to examine the flow characteristics of enteric coated pellets.

2.8.1. Bulk density

Bulk density is characterized by the ratio of mass of powder to the volume of bulk. Particle form has a significant impact on bulk density; as particles grow more spherical, bulk density rises. The appropriately weighed pellets were introduced in graduated cylinder and the volume of the pellets was observed to measure the bulk density of the coated pellets.

2.8.2. Tapped density

The tapped density was ascertained by loading a graduated cylinder with a certain number of pellets and adjusting the cylinder in a mechanical tapper. The machine was run for a predetermined number of taps till the powder bed volume was at a minimum. The density of the minimal volume tapped was estimated using the weight of the pellets in the cylinder.

2.8.3. Hausner’s ratio

Hausner’s ratio gives the measurement of drug’s frictional resistance. It is determined as the ratio of tapped density to bulk density and reveals the flow characteristics of the pellets.

2.8.4. Carr’s index

Carr’s index is measured through bulk density and tapped density.

2.9. In vitro drug release

The dissolution of pellets filled in enteric coated capsule was studied utilizing the USP II paddle equipment. For the drug release of pellets, 900 ml of 0.1 N HCl was employed as dissolving medium for the first 2 h and 900 ml phosphate buffer pH 6.8 after that. Pellets (100 mg) were submerged in the dissolving media and kept at 37 ± 0.5°C while being agitated at 100 rpm. Aliquots of 5 ml were taken at regular intervals and replaced with an equivalent volume of dissolving media for sink conditions. Using a UV-spectrophotometer, the samples were evaluated spectrophotometrically at 254 nm for phosphate buffer.

2.10. Permeability studies

On the intestine of a pig, permeability was measured in triplicate using the Franz diffusion cell procedure. The medium was phosphate buffer (6.8 pH). The Franz cell’s donor chamber was augmented by an excess of pure drug and ART-CD inclusion complex spheroids. The analysis was carried out by taking 1 ml and replacing it with the equivalent quantity of fresh media at certain time intervals, namely 15, 30, 60, 90, 120, 240 and 360 min. The sample was properly diluted before being placed into the HPLC to test the medication present in the selection. The Franz diffusion cell was disassembled after 6 h and the skin (from the diffusion research) was carefully removed from the cell. The skin was swabbed with phosphate buffer. The procedure was repeated twice to ensure that no formulation residue remained on the skin’s surface. After cutting the skin into tiny pieces, it was kept in a pH 6.8 buffers to extract the drug contained within the skin. HPLC was used to determine the quantity of drug deposited in the skin after adequate dilution and filtration [27].

2.11. Ex vivo studies

The efficacy of pure ART and the CD-ART combination against malaria was also assessed. The adjusted parasitemias at various concentrations of the test compounds were normalized as a percentage of the DMSO control and plotted against the concentrations of the test compounds to derive IC₅₀ values using nonlinear regression and GraphPad Prism. This suggests that complex outperformed chloroquinine in terms of efficacy. At the CCMB in Hyderabad, antimalarial activity was tested on the Pf3D7 strain of P. falciparum [28].

2.12. In vivo studies

A total of 2.5–3 kg weighed male New Zealand rabbits were obtained from Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar. The rabbits were housed in animal houses with proper ventilation at 242°C and 50% relative humidity. Spheroids of inclusion complex encapsulated in enteric-coated capsules (Purecaps, Canton, Ohio, USA, acid-resistant capsule shells) were compared with ART aqueous solution (ART dispersed in water), IV arteether, IM arteether and enteric coated capsules filled with spheroids for enhancement in bioavailability [29]. All formulations were administered orally at a dose of 6 mg/kg. Microfuge tubes containing spray-dried EDTA were employed for blood collection from the marginal ear vein. The plasma was obtained after centrifuging the blood for 15 min at 2500 rpm, kept at 20°C and evaluated by HPLC.

Plasma was collected from marginal ear vein of the rabbit and stored in EDTA-free tubes. After 3 min vortexing with 0.4 ml acetonitrile, the sample was centrifuged at 25,000 rpm for 15 min at 10°C. The organic phase is then transferred to new tubes, autosampler vials and injected into the HPLC for analysis.

2.13. Pharmacokinetics & biostatistics

We generated arteether plasma concentration-time curve using pharmacokinetics and statistical analysis. Using obtained data, the maximal plasma concentration (C_max) and the required time to achieve C_max concentration (T_max) were visually examined. The trapezium approach was used to calculate the area under the concentration-time curve (AUC_0-t). Individual animal concentrations were plotted against time to determine C_max and T_max. The absolute bioavailability of ART aqueous solution (ART dispersed in water), IM arteether and enteric coated capsules filled with spheroids was estimated by dividing the AUC of IV arteether after difficult dosing by the AUC of IV arteether after suspension dosing. The statistical significance was signified by p < 0.01 [30,31].

The computational study was carried out utilizing the PyRx Virtual Screening software, which is often used to screen libraries of various active compounds against potential therapeutic targets. The PubChem database was used to download the 3D structure of active ligands arteether and β-CD, whereas PyRx Software program was used to minimize the energy of active ligand molecules [32]. The 3D structure of Malarial proteins like, PDB ID: 5JWA were obtained from the PDB database and proteins were prepared by removing excess water content, het-atom, ligand molecule and polar hydrogen bond were added using BIOVIA-discovery studio 2021 software. Open babel and Autodock-vina wizard tools are in-builded in PyRx software, whereas Vina wizard is used to accomplishing docking of chosen prepared molecules and Open Babel is used to minimize the energy of active ligand molecules. Pymol was used to depict the molecular docking study and BIOVIA-discovery studio 2021 was utilized to investigate the 2D and 3D interaction [33], whereas, molecule with docking score with interaction type have been shown in Supplementary Table S6.

The arteether was molecularly docked against malarial protein using PDB ID: 5JWA and dock score of -7.2 kcal/mol was obtained, whereas arteether having electron donating alkyl group and cyclohexane ring involved in hydrophobic Pi-alkyl interaction with PHE155 amino acid residues and substituted ring with ether bridge involve in hydrogen bonding with GLY245 amino acid residue. Other rings participating in the Vander wall interaction. The 2D and 3D structures of the ligand molecule arteether and their interactions with receptor sites have been demonstrated in (Table 1 & Figure 1).

Table 1.

Docking score and 2D constituent interaction with PBD id 5JWA.

S. No	2D interaction	Name of constituent	Docking score
1)		Arteether	-7.2
2)		β-CD	-6.9

CD: Cyclodextrin.

Figure 1.

Drug-Receptor interaction studies. (A) 3D and crystal structure of arteether against malarial receptor using PDB ID: 5JWA, (B) 3D and crystal structure of β-CD against malarial receptor PDB ID: 5JWA.

CD: Cyclodextrin.

The β-CD was docked against malarial protein using PDB ID: 5JWA and dock score of -6.9 kcal/mol was achieved, whereas β-CD having hydroxyl group in D-glucopyranose unit involve in hydrogen bonding with GLY471, SER472 and TRP469 amino acid residues and ether ring involve hydrogen bonding with GLY310, ALA308, PRO82. The 2D and 3D structures of the ligand molecule β-CD and their interactions with receptor sites have been demonstrated in (Supplementary Table S4 & Figure 1).

3. Results & discussion

3.1. Experimental design optimization & response surface methodology

BBD was applied for the optimization of selected critical factors such as stirring time, stirring speed, disintegration concentration and binder concentration in comparison to the selected response i.e., dissolution (%) BBD has been employed because it is an important optimization design that can help in the determination of the estimation relation between the independent and dependent variables as depicted in Supplementary Table S7 [34].

BBD in this study for four selected critical independent variables i.e., stirring time, stirring speed, disintegration concentration and binder concentration gave 29 runs and % dissolution was selected as the only response. Supplementary Table S5 depicts the value of response in comparison to various values of independent variables. The selected low and high value of the bender concentration (100 to 200 mg), disintegrant concentration (50 to 200 mg), stirring speed (500 to 900 rpm) and stirring time (5 to 9 min) were selected to get the desired runs.

3.2. Equation of responses in terms of coded factors

Suggested nonlinear quadratic model gave a coded equation for each response to depict the relationship between the possible independent variables and the response. The ‘+’ in the equation shows synergistic or positive effect while ‘-’ depict the negative effects. The coefficients are adjustment around the average based on various factors. The equation for the response i.e., % dissolution generated by BBD in this study has been shown as Equation 2.

$$\begin{array}{c}\%\text{ Dissolution}=+65.21+9.21\text{A}-2.34\text{B}+2.83\text{C}-0.9403\text{D}\\ -24.98\text{AB}+8.11\text{AC}+8.83\text{AD}+10.09\text{BC}-2.60\text{BD}+18.65\text{CD}\\ +1.50{\text{A}}^2-9.08{\text{B}}^2+0.4626{\text{C}}^2-2.75{\text{D}}^2\end{array}$$ (2)

3.3. ANOVA based statistical analysis

ANOVA was for analyzing the suitability of our model. Various ANOVA based statistical parameter was shown in Supplementary Table S5. The selection of model was done on the basis of analysis of various parameters such as f-value, p-value, R² value, predicted R² value, standard deviation (SD) value, adeq. Precision value is very important in significance determination of model. The p-value of the model should be less than 0.5 for being significant. Error of the model was determined on the basis of significance ‘lack of fit’ value [35]. A significant ‘lack of fit’ depict that the develop model does not have proper difference among the exploratory and predicted data points. The R² value represent that the how good the predicted data points fits into the experimental data points and the value of R² should be near to 1. The value of predicted R² should have difference of more than 0.2 for model to be significant. Adeq precison in the ANOVA measure the ration of signal and noise in the model and ratio of 4 or more should be necessary for being a model to be noise free. SD of the developed model should have less value for significance of the developed model [36].

As per the Supplementary Table S5, p-value and SD of the model was found to be less than 0.0001 and 7.92 respectively which showed the significance of model. The nonsignificant ‘lack of fit’ of developed model showed that the model have proper difference among the exploratory and predicted data points. Value of R² was found to be 0.9059 and have difference of more than 0.2 to that of predicted R² (0.4738). Adeq. Precision was found to be 13.480 whose value was found to be much more than 4. Thus, by analysing most of the statistical parameter it could be said that the developed model was significant in predicted the optimised value of independent variables (Supplementary Table S4) [37].

Figure 2A depict the normal plots of residuals for all the selected independent variables showing that data points are irregular distributed without much deviation from the best fit line. Closeness of data points to best fit line confirms the suitability of developed QbD model to optimize the selected independent variables.

Figure 2.

Residual and contour plots for optimization studies. (A & B) Normal plot of residuals and the predicted vs. actual plot which show how close the predicted data points to that of experimental data points (C) dimensional contour plot (D) Overlay plots showing the design and experimental space.

The intervariation, interdependence and co-dependance among the selected independent variables and their possible relationship with the response/dependent variable were shown by drawing the response surface diagram.

Figure 2B depict the 2D contour plot for the selected independent variable and dependent variable i.e., % dissolution.

After analysis of 2D contour plot, it was concluded that % dissolution (response) for the selected independent variables (i.e., stirring time, stirring speed, disintegration concentration and binder concentration) increases with some extent with increasing value but starts decreasing when the values of independent variables keep increasing.

3.4. Overlay plot & defining the DS

The developed model worked in an experimental domain which was created with the help of various ranges of independent variables (i.e., stirring time, stirring speed, disintegration concentration and binder concentration). But it is necessary to determine the DS which help in achieving the final quality profile for our formulation. Figure 2C depict the overlay plot showing the experimental area in gray color and DS which was important for achieving the final quality target profile in yellow region.

3.5. Point prediction & validation of developed model

The independent variables (i.e., stirring time, stirring speed, disintegration concentration and binder concentration) were optimized by utilizing point prediction feature of Design expert software as given in Supplementary Table S3 for achieving the quality target profile.

3.6. Particle size distribution

Supplementary Table S7 showed the particle size distribution data of the prepared ART spheroids showing % weight retained of spheroids in different sieves.

3.7. Shape of spheroids

When examined under a motic microscope, the shape of the spheroid was observed to be spherical as shown in Supplementary Figure S2. Pellips (characteristic of pellet shape) measured the spherical nature of spheroid. Pellets with pellips of 1.0 are deemed spherical and suitable for formulation development.

3.8. Size of spheroids

Size pellets was calculated by digital vernier calliper and the reported size is 0.7470 mm.

3.9. Bulk density

Hausner’s ratio and compressibility index were estimated utilizing bulk density and tapped density which aid in assessing the flow and compressibility of components. The difference in bulk density was negligible. The bulk density of spheroids was determined to be 0.72.

3.10. Tapped density

The difference in tapped density was negligible and was determined to be 0.84.

3.11. Carr’s index (%)

The compressibility study revealed 7.1 compressibility indexes indicating excellent flow of the prepared spheroids. As the compressibility index of the spheroids fell in the range ≤10% and concluded in the spheroids having excellent flowability.

3.12. Hausner’s ratio

Prepared spheroids were found to have a Hausner’s ratio of 1.14 showing satisfactory flow properties. Hausner’s ratios in range 1.12 and 1.18 exhibited good flow, whereas those with ratios above 1.35 indicated poor flow characteristics, according to USP 2000.

3.13. Angle of repose

The prepared spheroids were observed to have an angle of repose of 20. This is very good flow according to I.P.

3.14. Friability

Friability was found to be between 0.74% of spheroids, which is within the allowable range according to IP 2007. Friability testing revealed that the formulation was sufficiently hard for secure shipping.

3.15. In vitro drug release

The dissolution rate of the pure drug was very slow, owing to the poor solubility and hydrophobic nature of the drug. The increased apparent solubility of ART stemming from the interaction of the drug with βCD led to an evident improvement in the dissolution rate. As expected, spheroids filled in enteric-coated capsule shell were not disintegrated upon exposure to 0.1 N HCl for 2 h. But when exposed to phosphate buffer pH 6.8, disintegration was observed within 1 h. So this proved that the enteric coating was uniform and acid resistant enabling protection of the drug content from degradation in the acidic environment at the stomach. Furthermore, release in simulated intestinal fluid supported the hypothesis of increasing absorption of the drug. In vitro dissolution studies for the first 2 h in 0.1 N HCl revealed the acid resistance capacity of spheroids. The dissolution behavior of spheroids in phosphate buffer pH 6.8 revealed the drug release characteristics. The similarity factors (f2) between the couples ART, ART-CD spray dried complex and ART-CD spheroids filled in enteric-coated capsule shell were 7.29, 12.18 and 44.20, respectively, indicating that the dissolution profiles were significantly different from each other. The release of the drug was rapid up to some extent and after a certain time, it showed sustained release. The % cumulative drug release of ART, ART-CD, ART-CD spheroids showed that around 30% of the drug was released during 3 h and maximum drug release i.e. 96% which is mentioned in Figure 3A.

Figure 3.

Drug release profile and confirmation of arteether complexation in cyclodextrin. (A) Percentage cumulative drug release of arteether, ART-CD complex and ART-CD spheroids; (B) SEM Image of Inclusion complex.

CD: Cyclodextrin; SEM: Scanning electron microscope.

3.16. Morphology

Figure 3B revealed the surface topography of ART-CD complex spheroids visualized using scanning electron microscope (SEM). The film was prepared on an aluminium stub for samples preparation. Under an argon environment, the stubs were coated with gold to 200–500 thickness through gold sputter module in high vacuum evaporator. SEM camera (Jeol-1761, Cambridge, UK) was employed for sample scanning and photography.

3.17. Permeability studies

The Franz diffusion cell approach was used to investigate the permeability of prepared formulations of ART, which correlates enhanced bioavailability as a component of improved dissolvability. Absorption of drug via the intestine’s humid environment is limited by the diffusion limit through the lipophilic apical layer and the drug must be dissolved to maximize bioavailability [36]. Figure 4A compared the concentration of ART-CD to pure ART in the pig’s intestine.

Figure 4.

Permeation and bioavailability studies. (A) Concentration of ART diffused across the intestine of pig for freeze dried ART-CD ternary complex in comparison to ART pure drug, (B) Plasma drug concentration with time of various formulations.

CD: Cyclodextrin.

3.18. In vivo studies

Plasma drug concentration-time profile of pure drug suspension and enteric encapsulated spheroids containing arteether administered orally in rabbits are represented as Figure 4B. Table 2 represents various pharmacokinetic parameters of the same. The relative bioavailability of arteether spheroids was calculated in comparison to plain arteether suspension. It was concluded from the in vivo studies that enteric coated spheroids has better bioavailability than plain drug. An increase of 51.89% in absolute bioavailability was observed. Moreover, CDs are cyclic oligosaccharides that can enhance the bioavailability of drugs by improving their solubility and stability. CDs have a hydrophobic cavity that can encapsulate hydrophobic drugs, forming inclusion complexes that increase the solubility and stability of the drug. This increases the concentration of the drug in the bloodstream, leading to improved bioavailability [37].

Table 2.

Pharmacokinetic parameters of different drug delivery systems.

Parameters	ART	CD-spheroids	Intravenous injection	Intramuscular injection
Dose	6 mg/kg	6 mg/kg	1 mg/kg	150 mg/2 ml
Cmax	2.361879	412.0776	2292.009	134.3005
T1/2	11.13289	0.073242	0.44 h	0.466124
Vd	2.045708	0.000373	0.00647	2456.235
MRT	1.47919	1.01035	90.12881	1.025472
Total clearance	0.127326	0.003528	0.000154	3651.752
% Absolute bioavailability	0.9828%	51.89337	–	43.73363

CD: Cyclodextrin.

3.19. Antimalarial activity

The IC₅₀ values of ART and ART-CD were also determined. The fractional inhibitory concentration (FIC) was determined for both in each combination (FIC: concentration of a compound that caused 50% inhibition in the combination/concentration of the compound required for 50% inhibition when used alone). The FICs at different ratios for ART and ART-CD combinations were plotted using linear regression analysis to construct an isobologram using GraphPad Prism.

The IC₅₀ value of ART and ART-CD complex was estimated to be 0.76 ng/ml and 0.733 ng/ml equivalent to 3.4 ng/ml(calculated by molar ratios) in dimethylsulfoxide solvent respectively. The IC₅₀ value of ART-CD complex is almost equivalent to ART in dimethylsulfoxide solvent which showed the acceptability of antimalarial activity of ART-CD complex drug. The results in Figure 5 shows the experiments were repeated three-times for accuracy of result. Based on the IC₅₀ values, it appears that the inhibition rate of ART-CD complex was comparable for ART with pure drug [38]. This suggested that ART-CD complex may be effective in inhibiting the target. However, as mentioned earlier, it is important to consider other factors such as drug delivery and pharmacokinetics when evaluating the effectiveness of a drug.

Figure 5.

Mean % parasitemia for plain ART and ART-β-CD.

CD: Cyclodextrin.

4. Discussion

ART-CD inclusion complexes in a 1:1 molar ratio using the spray drying method were successfully prepared [39]. Spheroids of the ART-CD complex were made using the extrusion spheronization technique. The statistically optimized recipe was used to make the spheroids, which were then placed into enteric-coated capsule shells and evaluated for various characteristics such as size, shape, flow characteristics, disintegration time, release profile, etc. Angles of repose for the various formulations were compared with those of the formulation, which demonstrates that the formulation flow characteristics were good after pelletization [40]. The results of the weight variation test for spheroids were within the permitted ranges. Fourier Transformed Infrared (FT-IR) spectra showed their spectra were similar to the ones recorded for pure CD, strengthening the idea that ART was successfully included in their cavity. The morphologies, evaluated by SEM analysis, revealed important changes in the structure and shape of the binary lyophilized systems, proving that new compounds were formed. The recorded DSC curves showed that the complexation was complete in the case of ART-CD spheroids/ pellets drug delivery systems that presented a good mechanical resistance with a low friability and a weight variation within the compendial limits. A rapid disintegration was registered for both series of tables and it can be considered that the main purpose of this study was completely achieved, as disintegration is the CQA for tablets. The dissolution rates were satisfying and within the imposed pharmacopeial limits for prepared formulations [41]. As the performances were very similar for the formulation, the conclusion is that the pharmaco-technical properties present a higher dissolution rate. When compared with pure medication, the inclusion of complex permeability experiments is more favorable. This could be because the medication is trapped in the complex and all of it permeates through the skin of the gut. The produced spheroids filled in enteric-coated capsule shell pharmacokinetic experiments verified the improvement in oral bioavailability. 51.89 percent more of the bioavailability was made available. This is because, upon oral administration, only negligible quantities of intact CDs – due to their bulky and hydrophilic nature – are absorbed from the gastrointestinal system [42].

5. Conclusion

Arteether-β-CD inclusion complexes using the spray drying technique were transferred to spheroids which were then encapsulated in enteric coated capsules. SEM study of the morphologies showed significant changes in the structure and shape of the binary lyophilized systems, indicating the formation of novel compounds. In the instance of ART-β CD, the DSC curves revealed that the complexation was complete. The mechanical resistance of spheroids/pellets drug delivery systems was acceptable, with less friability and weight variation within compendial ranges. The dissolving rates for produced formulations were satisfactory and within the specified pharmacopeial limitations. Because the formulation results were so close, the conclusion is that the pharmaco-technical characteristics had a greater dissolving rate. Further the in vivo activities showed the spheroids showed maximum enhancement in oral bioavailability. The results of ex vivo studies confirm that the prepared complex spheroids showed more enhancement in antimalarial activity compared with pure arteether and chloroquinine. The findings revealed that complex spheroids could improve ART’s water solubility and bioavailability. The complex could be useful in the construction of innovative medical ART formulations, given the lack of ART applications. The combination of ART and CD could be a promising therapy option for malaria.

Funding Statement

The author thanks to the Indian Council of Medical Research (ICMR), New Delhi, for funding her work as a senior research fellow under the ICMR-SRF scheme (F.No. 53/2019/-ECD-II dated 19/07/2019).

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/20415990.2024.2377948

Author contributions

N Bajwa did the writing (original draft preparation). PA Singh, S Kumar, GC Arya did the writing (review and editing). A Baldi did the supervision. All authors have read and agreed to the published version of the manuscript.

Financial disclosure

The author thanks to the Indian Council of Medical Research (ICMR), New Delhi, for funding her work as a senior research fellow under the ICMR-SRF scheme (F.No. 53/2019/-ECD-II dated 19/07/2019). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The research work was approved by Institutional Animal Ethics Committee authorized IAEC (Approval number- MRSPTU/IAEC/2019/16).