https://orcid.org/0009-0009-9512-3727
Abstract
This research aims to prepare a superior amorphous solid dispersion (ASD) formulation via solvent method for the oral delivery of Niclosamide to enhance oral bioavailability and absorption.
Phase solubility tests were conducted to select an optimal carrier combination for Niclosamide solid dispersion (SD). The Niclosamide ASD was synthesized using a solvent rotary evaporation method and characterized through in vitro dissolution tests, differential scanning calorimetry (DSC), modulated DSC, powder X-ray diffraction, and scanning electron microscopy. In vivo oral pharmacokinetic studies were conducted in Sprague Dawley rats at a dose of 50 mg/kg.
PEG6000 and poloxamer 188 combination was selected as an optimal carrier. ASD named ASD-5 was successfully prepared, encompassing a 25% drug loading, Niclosamide within ASD-5 remains amorphous and stable. 75% of Niclosamide in ASD-5 rapidly dissolved in 5 min, while below 5% of pure Niclosamide and physical mixture dissolved in a longer time. Amorphization of Niclosamide in ASD-5 notably contributes to its dissolution rate and extent. Furthermore, among reported Niclosamide SD formulations with drug loading above 25%, ASD-5 demonstrated the highest bioavailability, showing a 2.33-fold increase in plasma exposure and bioavailability compared to pure Niclosamide.
Amorphous ASD-5 prepared by the solvent method has higher drug loading and is scalable in pre-clinical stage preparation. Due to using PEG6000 and poloxamer 188 combination, ASD-5 had the highest bioavailability among reported Niclosamide SDs with a drug load exceeding 25%. Also, ASD-5 presented a simplified preparation procedure compared to other reported Niclosamide SD.
Introduction
Niclosamide received World Health Organization and Food and Drug Administration (FDA) approval in 1960 as a molluscicide and has been employed as an oral anthelmintic for treating parasitic worm infections across various nations, including France, Germany, and the UK. Classified as a salicylamide-class drug, it is particularly effective against tapeworm infections [1]. Recent years have seen burgeoning interest in repurposing Niclosamide to address a range of diseases, marking notable progress. Recognized as a potent inhibitor of signal transducer and activator of transcription 3 (STAT3) in HeLa cells, Niclosamide displays anti-cancer capabilities and inhibits DNA replication in Vero E6 cells [2]. In 2018, Chen et al. highlighted Niclosamide’s potential in clinical applications against cancer, bacterial and viral infections, and metabolic disorders [3]. Moreover, its application has extended into clinical trials for conditions such as familial adenomatous polyposis and Coronavirus disease 2019 (COVID-19) [4]. Despite its broad utility, Niclosamide’s minimal water solubility (5.7 μg/ml) and the requirement for substantial oral doses for various treatments pose significant challenges. For instance, it is orally administered as (2000 mg/day) cestode parasites (tapeworms) [5]. In the clinical trial NCT02532114, the daily oral dose reaches 1500 mg/day [6]. These limitations necessitate the development of a formulation that enhances its absorption, bioavailability, and drug-loading capability.
To address these issues, numerous Niclosamide formulation strategies have been explored, including salt formulations [7], cocrystals [8], prodrugs [9], nanosuspensions [10], lipid emulsions [11], and micelles [12]. Among these, amorphous solid dispersion (ASD) stands out as an advantageous formulation strategy to improve the bioavailability of drugs with poor water solubility, dispersing the drug within one or more solid matrices, typically polymers and surfactants. The mechanism is to increase the dissolution rate and solubility of poorly soluble drugs through the double action of highly dispersing drugs at matrices and changing the crystal form of drugs from crystal to amorphous [13, 14]. The significance of ASD is well documented, with several FDA-approved drugs adopting this technology [15].
Various technologies, including the hot-melt extrusion method, solvent method, and hot melt fusion merge for solid dispersion (SD) preparation. The solvent-based approach to preparing SDs has the capability to attain a high level of molecular mixing uniformity, yielding greatly improved characteristics of poorly soluble compounds. Thus, the solvent-based method is widely used. In 2022, Niclosamide ASD was explored by solvent vacuum evaporation method with hydroxyethyl cellulose as a carrier [1]. In 2023, Gupta et al. prepared a similar formulation using a kneading method with cyclodextrins by adding solvent water and methanol [4]. Despite these advancements, therapeutic needs remain unmet due to operational complexities, low drug loading, and suboptimal dissolution rate and extent in Niclosamide ASD preparation.
This study aims to overcome these limitations by developing a novel Niclosamide ASD with enhanced drug loading, dissolution rate and extent, and bioavailability. The proposed formulation uses a simple solvent method adaptable to both rotary evaporation and spray drying techniques. The effectiveness of this formulation was evaluated through various analytical techniques, including in vitro dissolution, differential scanning calorimetry (DSC), powder X-ray diffraction (p-XRD), scanning electron microscopy (SEM), and oral pharmacokinetic (PK) studies in Sprague Dawley (SD) rats.
Materials and methods
Materials
Niclosamide was purchased from bidepharm.com. and purity is higher than 99.99%. Sodium carboxymethyl cellulose (CMC-Na) was purchased from Sigma-Aldrich (product from USA). PEG 6000 was purchased from Thermo Scientific (Germany). Poloxamer 188 (P188) was purchased from Rhawn (China). Bile salt was purchased from Macklin (China). The polymer Poly (1-vinypyrrolidone-co-vinyl acetate), copolymer 4:6 (PVP–VA46) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd (China). HPMC was purchased from Alfa Chemical Co., Ltd (China). Mannitol was purchased from Beijing Innochem Technology Co., Ltd (China). Simulated intestinal fluid was purchased from Shanghai Maiji Biotechnology Co., Ltd (China). All the other reagents and chemicals used were of analytical grade and distilled water was used throughout the study.
UV-visible spectrophotometric method for Niclosamide quantitative analysis
First, the detection wavelength of Niclosamide was determined. Niclosamide powder was weighed and dissolved in methanol. Subsequently, a solution was prepared using serial dilution to achieve concentrations of 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, and 200 μg/ml. All samples were scanned using a UV and visible light spectrometer (SpectraMax M5, Shanghai) in the range of 200–500 nm. The wavelength of 330 nm was selected for Niclosamide quantification as shown in Fig. 1A. Following the quantitative measurement of each sample, a standard curve for Niclosamide was plotted for further analysis, as shown in Fig. 1B. Samples from the experiments were analysed in triplicate.
Nicolsamide quantifications: (A) UV scan from 200 to 500 nm; (B) standard curve using UV 330 nm absorption.
Phase solubility studies
To determine the most suitable carriers for Niclosamide ASD, phase solubility studies were conducted. Eight carriers (HPMC, PEG 6000, CMC-Na, P188, PEG 6000/P188, PVP-VA46, HPMC/mannitol, and CMC-Na/mannitol) were assessed including single carrier and combined carriers. The thermodynamic equilibrium solubility of Niclosamide was determined following a previously published protocol and research methodology [1, 10, 16].
Carriers were weighed and prepared in a 1% (m/v) concentration using distilled water. Subsequently, 1 ml of each prepared solution was transferred to a 24-well plate. An excess amount of Niclosamide was added to the wells containing carriers, and the plate was then shaken at 300 rpm for 24 h at 37 °C. Post-equilibration, these samples were centrifuged at 25 °C for 5 min at 10 000 rpm. The absorption of the carrier solution was measured at a wavelength of 330 nm. The drug concentration in the supernatant of the drug and carrier mixture was determined by subtracting the absorption value of the carrier solution from that of the drug and carrier mixture solution.
The methodology for studying phase solubility for increasing concentrations of PEG 6000 and P 188 (1%, 5%, 10% m/v) was identical to the above-mentioned procedure.
Preparation of solid dispersions by solvent rotary evaporation method
The ASD-5, an ASD of Niclosamide, was prepared using the solvent rotary evaporation method. Initially, 100 mg of Niclosamide was accurately weighed and dissolved in 5 ml of ethanol. Separately, PEG 6000 (50 mg) and P 188 (250 mg) were weighed and dissolved in 5 ml of methanol. Both solutions were vortexed individually for 10 min. Subsequently, they underwent ultrasonication in a water bath heated at 40–45 °C for 5 min until clear solutions were obtained.
The Niclosamide solution and the carrier solution were thoroughly mixed. This mixed solution was then placed in a rotary evaporator to remove methanol and ethanol from the solution. The evaporator was operated at a speed of 170 rpm, while the water bath temperature was maintained at 37 °C. Once the solvents were removed, which was left at room temperature overnight. Ultimately, approximately 400 mg of the resulting fully dried crisp solid ASD-5 was ground in mortar for 30 min and sieved through a #60 mesh. The product was then stored in a desiccator at room temperature for subsequent use.
p-XRD studies
The p-XRD pattern of Niclosamide pure drug, ASD, and two kinds of carriers PEG 6000 and P 188 were recorded with a radiation source of Cu Kα (λ = 45 kV, 40 mA) anode of powder X-ray diffractometer (D8 Advance, Bruker AXS GmbH). The Niclosamide and ASD-5 were scanned between 5 and 50° with a 0.5 s/ step scan rate at an increment of 0.017°. PEG 6000 and P 188 were scanned from 3 to 90°, with a 0.5 s/ step scan rate at an increment of 0.017°.
DSC and modulated DSC studies
DSC/Modulated DSC (mDSC) analysis was performed with a DSC Instrument of TA (the DSC Instrument brand name Thermo Analyzer). An appropriate amount of a sample (1–3 mg for DSC and 7.66 mg for mDSC) was placed into an aluminium pan with pinhole and heated with the parameters in Table 1. DSC sample was heated from 25 °C to 300 °C at a heating rate of 10 °C/min. Dry N2 gas at a flow rate of 50 ml/min was used to purge the DSC equipment during the measurement. mDSC sample was heated in the DSC from −30 °C to 300 °C by Modulated Heat Only method. The underlying heating rate was 2 °C/min, the modulation period was 120 s, and the temperature amplitude of modulation was ±0.64 °C. Dry N2 gas at a flow rate of 50 ml/min was used to purge the DSC equipment during the measurement. The data was analysed using TRIOS (the software accompanying the DSC Instrument Thermo Analyzer).
Parameters of DSC analysis.
| Instrument | Sample pan | Procedure for DSC | Procedure for mDSC | Purge gas | Flow rate |
|---|---|---|---|---|---|
| TA, Discovery DSC 250 | Aluminum, pin-holed | Equilibrate 25 °C Ramp 10 °C/min to 300 °C | Data Off: Equilibrate −30 °C Modulate Template 0.64 °C for 120.0 s Isothermal 10.0 min Data On: Ramp 2 °C/min to 300 °C | N2 | 50 ml/min |
| Instrument | Sample pan | Procedure for DSC | Procedure for mDSC | Purge gas | Flow rate |
|---|---|---|---|---|---|
| TA, Discovery DSC 250 | Aluminum, pin-holed | Equilibrate 25 °C Ramp 10 °C/min to 300 °C | Data Off: Equilibrate −30 °C Modulate Template 0.64 °C for 120.0 s Isothermal 10.0 min Data On: Ramp 2 °C/min to 300 °C | N2 | 50 ml/min |
Parameters of DSC analysis.
| Instrument | Sample pan | Procedure for DSC | Procedure for mDSC | Purge gas | Flow rate |
|---|---|---|---|---|---|
| TA, Discovery DSC 250 | Aluminum, pin-holed | Equilibrate 25 °C Ramp 10 °C/min to 300 °C | Data Off: Equilibrate −30 °C Modulate Template 0.64 °C for 120.0 s Isothermal 10.0 min Data On: Ramp 2 °C/min to 300 °C | N2 | 50 ml/min |
| Instrument | Sample pan | Procedure for DSC | Procedure for mDSC | Purge gas | Flow rate |
|---|---|---|---|---|---|
| TA, Discovery DSC 250 | Aluminum, pin-holed | Equilibrate 25 °C Ramp 10 °C/min to 300 °C | Data Off: Equilibrate −30 °C Modulate Template 0.64 °C for 120.0 s Isothermal 10.0 min Data On: Ramp 2 °C/min to 300 °C | N2 | 50 ml/min |
Scanning electron microscopy
P188, PEG 6000, Niclosamide, and ASD-5 were examined by SEM (GEMINISEM 500, China). The powders were placed on a brass stub using double-sided adhesive tape and then metallized with gold by Ar plasma.
In vitro dissolution tests
In vitro dissolution tests were conducted for Niclosamide, ASD-5, physical mixture (Niclosamide, PEG 6000, and P188). The dissolution medium consisted of simulated intestinal fluid with a pH of 6.8 and 0.6% bile salt to imitate the natural intestinal environment [17]. In total, 3.0 mg Niclosamide, equivalent ASD-5, and physical mixture (consistent with weight ratio of ASD-5) were accurately weighed. These weighed samples were individually added to 250 ml standard sample bottles. Subsequently, 100 ml of the dissolution medium was added to each bottle and placed in a shaker at 100 rpm and 37 °C.
Aliquots from the dissolution solution were extracted at 5, 10, 15, 30, and 45-min intervals. At each time point, the withdrawn dissolution solution was replenished with an equal volume of fresh 37 °C medium. The collected samples were then centrifuged at 25 °C for 5 min at 10 000 rpm. 100 μl volume of supernatant from each time point was transferred in triplicate to a 96-well plate. Subsequently, the 96-well plate was subjected to ultraviolet light (UV) and visible light spectrometry to determine the Niclosamide content at 330 nm.
Oral pharmacokinetic studies in SD rat
The objective of this study is to determine the pharmacokinetic profiles of pure Niclosamide and ASD-5 following oral administration in male SD rats. The experiment is conducted in Pharmaron (Beijing, China). The study protocol was approved and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines at Pharmaron. (IACUC Protocol Number PK-R-06012023, approved date: March 24, 2023). These animals were maintained on the standard pellet diet and water as per the standard laboratory conditions. All animals for per oral (PO) administration will be fasted overnight and fed after 4 h of administration. These SD rats are divided into two groups, Niclosamide group and ASD-5 group, three male rats per group, and the age at the day of dosing is 6–8 weeks. Pure Niclosamide and ASD-5 were administered at a Niclosamide dose of 50 mg/kg by oral gavage, respectively. The dosing samples are both suspensions of 0.5% Carboxymethyl Cellulose Sodium in water containing 5 mg/ml Niclosamide, and dose volume is 10 ml/kg. Blood collection for each rat at each time point is approximately 0.2 ml. Blood samples collected were centrifuged at 4000g for 10 min at 4 °C, to get plasma. The blood collection time points are 0.25, 0.5, 1, 2, 4, 8, and 24 h post dose.
Pharmacokinetic data analysis
Niclosamide concentrations of plasma samples were determined using liquid chromatography-tandem mass spectrometry. WinNonlin (PhoenixTM, version 8.3) software is employed for pharmacokinetic parameters (T1/2, Cmax, Tmax, area under curve [AUC]last, and AUCinf) calculations from the plasma concentration versus time data. The pharmacokinetic data will be described using descriptive statistics such as mean and standard deviation. The relative bioavailability (%) was calculated using the equation:
Results and discussions
Carrier selection using phase solubility studies
Hydrophilic polymers and sugars are commonly utilized drug carriers for producing SDs, enhancing the absorption of poorly water-soluble drugs in the gastrointestinal tract [18]. Phase solubility serves as a valuable method to screen select optimal carriers and is widely employed in current practices [19–21]. Here, we investigated eight promising single carriers and combinations, seeking the most suitable carrier and their combination to prepare an optimal Niclosamide ASD. As depicted in Fig. 2, the solubility of pure Niclosamide as a control in water was 5.7 μg/ml. Hydroxy propyl methylcellulose (HPMC) and CMC-Na slightly increase the solubility of Niclosamide, and adding mannitol has no improvement. The solubilization effect of PVP-VA46 was equal to those mentioned two carriers. While PEG 6000 or P188 improves the solubility of Niclosamide significantly. P188 was better than PEG 6000, P188 increased the Niclosamide solubility to 30.7 μg/ml, and PEG 6000 increased it to 25.3 μg/ml. According to the “Like Dissolves Like” principle, this structural similarity contributes to their strong affinity for each other, as shown in Fig. 3. The combination of PEG 6000 and P 188 was equal to P188. Then Niclosamide solubility in aqueous solution in different amounts (1%, 5%, and 10%, w/w) of PEG 6000 and P188 was studied. The result indicated a near-linear increase in Niclosamide solubility with rising concentrations of PEG 6000 and P188 respectively, as shown in Fig. 4. However, P188 always exhibited better solubilization ability compared to PEG 6000. The phenomenon is similar to that of Albendazole SD research [22]. The possible reason is that P188 has better wettability than PEG 6000 due to the physical-chemical properties of P188. Therefore, P188 was a promising choice to prepare Niclosamide ASD for solubility increase. However, in our research, we found that ASD with P188 as the carrier is viscous, which makes it difficult to prepare ASD non-sticky powder. Flowability and homogeneity are key characteristics of successful SDs. Hence, a certain amount of PEG 6000 was added to the Niclosamide ASD to achieve better homogeneity and mobility, which is beneficial to large- and small-scale production operations. Also, PEG 6000 is a common carrier for SD and has excellent solubility in many kinds of solvent, indicating it is suitable for the solvent method. And PEG 6000 due to its higher molecular weight leads to more drugs in the molecularly dispersed form [23]. During the preparation of Niclosamide SD, PEG 6000 is expected to achieve a synergistic effect with P188. Consequently, the combined PEG 6000 and P188 were selected to prepare ternary Niclosamide ASD.
Phase solubility result of Niclosamide in 1% (w/w) aqueous solution of eight kinds of carriers and carrier combinations. Solubility was shown as mean ± SD, (n = 3).
Structure of Niclosamide, P188, and PEG6000.
Niclosamide solubility in increasing % concentration (w/w) of PEG 6000 and P 188 aqueous solution. Solubility was shown as mean ± SD, (n = 3).
Amorphous solid dispersion of ASD-5 preparation
Given the low melting point and good water solubility of PEG 6000, it was used to prepare Nifedipine ASD by hot fusion method and solvent method. However, some authors indicated the solvent method produces a better dissolution rate than the fusion method based on their research work [22, 23]. Similarly, the SD study for the poorly water-soluble drug Ritonavir indicated that the ritonavir SD via solvent method exhibited a nine-fold increase in bioavailability compared to that obtained through the melt method [24]. The fusion method achieves a well-distributed drug-carrier blend, albeit with limited homogeneity. In contrast, the solvent method excels in achieving molecular-level mixing of the drug into the carrier, resulting in the most homogeneous SDs, as previously documented.
Hence, the hot fusion method is not ideal for making Niclosamide ASD. In 2021, Jara et al. used a hot melt-extrusion technique to create Niclosamide ASD with 35% drug loading, doubling the bioavailability [6]. However, they processed the drug and carriers at 180 °C in large machines, which isn’t suitable for pre-clinical stages where only a few grams are needed. As a result, the solvent method was used to prepare Niclosamide ASDs with PEG 6000 and P188 as carriers. This method can make Niclosamide ASD with as little as milligrams of the drug, fitting the pre-formulation stage and allowing for scale-up using the spray-dry method.
Drug loading remains a major challenge for Niclosamide SDs. Both the type of carrier and the solvent used in preparation can affect drug loading and drug solubility. Based on our findings, we developed ASD-5 with 25% drug loading using a composition of Niclosamide: PEG 6000: P188 = 2:1:5. This was prepared using the solvent rotary evaporation method in less than an hour. This method is simpler compared to the kneading method used by Gupta et al., where methanol and water (1:1) were used for 24 h [4]. In our method, methanol and ethanol solvents fully dissolved Niclosamide in PEG 6000 and P188, creating a Niclosamide solution, and both solvents were easily removed. As for physical appearance, ASD-5 appears to be an easy handle, fine non-sticky powder. The ASD-5 drug loading of 25% was improved in comparison with the previously reported 20% and 9.6% drug loading in Niclosamide ASD formulations via solvent method [1, 4].
Powder X-ray diffraction
Figure 5 illustrates the powder X-ray diffractograms for Niclosamide, PEG 6000, P 188, and ASD-5. The diffraction spectrum of pure Niclosamide exhibits high-intensity peaks at 2θ values of 6.68°, 13.04°, 13.78°, 17.26°, 19.84°, 22.21°, 23.42°, 24.92°, 25.69°, 26.28°, and 26.78°, indicating the presence of a crystalline form of Niclosamide, which is aligned with previous reports PEG 6000 shows intensity peaks at 2θ of 19.10° and 23.26°, while P 188 exhibits two high-intensity peaks at 2θ of 19.10° and 23.26°. ASD-5 displays two prominent peaks at 2θ of 23.26° and 19.10°. Comparison of the carrier and original drug patterns reveals that these two peaks primarily originate from PEG 6000 and P 188, respectively, with no significant Niclosamide crystal peaks observed in ASD-5. These findings indicate the absence of Niclosamide crystalline structure in ASD-5, supporting the formation of amorphous Niclosamide in the SD. As in the previous report, an amorphous or metastable form would shortly dissolve due to its higher internal energy and greater molecular motion, which enhanced the thermodynamic properties compared to crystalline materials [22]. The phenomenon was observed in our dissolution tests. Pure Niclosamide showed slow drug release, with a maximum release of less than 5%, highlighting its limited solubility (5.7 μg/ml) and poor wettability over the same time period. In contrast, ASD-5 demonstrated a significant improvement, releasing about 75% of the drug within the first 5 min. The amorphization of Niclosamide and combined PEG6000 and poloxamer 188 in ASD-5 are responsible for the enhanced dissolution.
p-XRD analysis of Niclosamide, P 188, PEG 6000, and ASD-5.
DSC and modulated DSC
A DSC study was conducted to further elucidate the crystallization profile of pure Niclosamide, ASD-5, and carriers PEG 6000 and P 188. As depicted in Fig. 6, the results revealed the characteristic melting peak of Niclosamide at 231.99 °C, indicative of distinct crystalline structures in pure Niclosamide. The melting peaks of PEG 6000 and P 188 were observed at 64.37 °C and 57.57 °C, respectively, confirming their crystalline nature. In contrast, ASD-5 exhibited a single melting peak at 57.16 °C, suggesting the absence of crystalline Niclosamide in ASD-5. This observation supports the transition of Niclosamide from its crystalline form to an amorphous state within the ASD. Consistent with published reports, which indicate that Niclosamide cannot independently form an amorphous solid but can do so when dissolved in a polymer [25, 26]. Our findings align with these documented phenomena. Niclosamide itself is a crystalline state but formulated as SD, it can effectively disperse in PEG 6000 and P188 as an amorphous state. In summary, the DSC analyses further underscore the elimination of Niclosamide characteristic crystalline peaks in ASD-5.
DSC thermogram of Niclosamide, P 188, PEG 6000, and ASD-5.
Moreover, the amorphous form is a thermodynamically unstable state and usually converts back to the more stable crystalline form. The mDSC study was conducted to evaluate the stability of ASD-5. The mDSC analysis (Fig. 7) showed the ASD-5 Tg is as high as 133.45 °C. The empirical rule is that amorphous will transform to a glass state above the glass transition temperature (Tg). While storage at 50 °C lower than Tg will prevent the conversion of amorphous to crystalline [14, 27]. So, ASD-5 will be apparently stable at a storage temperature of 4 °C, which is much lower than 83.45 °C (50 °C lower than Tg of ASD-5).
mDSC thermogram of ASD-5.
Scanning electron microscopy
SEM analysis of Niclosamide, PEG 6000, P 188, and ASD-5 is depicted in Fig. 8. These images of Niclosamide showcased a crystalline morphology with a sharp surface at 400× and 1000× magnifications. The images of P 188 at 400× and PEG 6000 at 200× also revealed crystalline particles and pieces with sharped surfaces. The images of ASD-5 at 100× and 200× displayed a smooth surface and soft appearance, indicating Niclosamide dispersed in an amorphous state.
SEM microphotographs. (a) Niclosamide 400×, (b) Niclosamide 1000×, (c) P 188 400×, (d) PEG 6000 200×, (e) amorphous solid dispersion ASD-5 (Niclosamide: PEG 6000:P 188 = 2:1:5), 100×, (f) amorphous solid dispersion ASD-5 (Niclosamide: PEG 6000:P 188 = 2:1:5), 200×.
In vitro dissolution tests
The dissolution and drug release in the intestinal are critical determinants affecting its absorption and bioavailability, thus significantly impacting both drug efficacy and toxicity [28]. Niclosamide is a BCSII and weak acidic drug, it remains mostly un-ionized in the stomach [6]. The dissolution and absorption of Niclosamide primarily occur in the small intestine. Thus, in vitro dissolution profiles of ASD-5, original Niclosamide, and their physical mixture were assessed in simulated intestinal fluid (pH 6.8) with 0.6% bile salt to mimic the natural intestinal milieu. Figure 9 illustrates the time-dependent cumulative drug release percentages for Niclosamide, its physical mixture (matching ASD-5’s composition), and ASD-5. The results highlight that pure Niclosamide exhibited slow drug release, with a maximum release below 5%, underscoring its limited solubility (5.7 μg/ml) and wettability. In contrast, ASD-5 showed a marked improvement, releasing approximately 75% of the drug within the first 5 min and rapidly stabilizing thereafter. The result indicated Niclosamide ASD effectively enhanced the dissolution rate and extent of pure Niclosamide. The physical mixture’s dissolution profile closely mirrored that of pure Niclosamide, indicating that ASD-5’s enhanced dissolution is attributed to its ASD formulation, rather than the solubilizing effects of the carriers (PEG 6000 and poloxamer 188). No decline in drug release percentage during dissolution suggests no crystalline formation in the solution. PEG 6000 likely inhibits drug precipitation, enabling enhanced dissolution, as seen in previous Nifedipine studies [23].
In vitro dissolution profiles of Niclosamide, amorphous solid dispersion ASD-5 (Niclosamide: PEG 6000:P 188 = 2:1:5), physical mixture.
In summary, ASD-5 demonstrated a significantly superior dissolution profile compared to both the pure Niclosamide and its physical mixture with PEG 6000 and poloxamer 188 at the same ratio found in ASD-5. The amorphous state of Niclosamide in ASD-5 contributes to the high dissolution rate observed due to the minimal energy required for disrupting the crystalline lattice structure during dissolution. Previous research evidenced the contribution of amorphization to drug dissolution [6, 22], and our study provides another case for this.
Oral pharmacokinetic studies in SD rat
Pharmacokinetic studies comparing pure Niclosamide and ASD-5 were conducted in healthy male SD rats at an oral dose of 50 mg/kg. The PK profile, depicted in Fig. 10, and key pharmacokinetic parameters, listed in Table 2 (including Cmax, Tmax, AUClast, and AUCinf), revealed that ASD-5 afforded significantly higher plasma exposure than pure Niclosamide. Specifically, ASD-5 achieved a Cmax of 909 ng/ml compared to 279 ng/ml for pure Niclosamide, indicating a 3.26-fold increase and significantly improved absorption. The Tmax values for ASD-5 and pure Niclosamide were 0.417 h and 0.83 h, respectively, suggesting that the ASD formulation accelerates Niclosamide absorption. These in vivo results are in line with the in vitro dissolution findings, showing rapid drug release and subsequent enhanced absorption. ASD-5 exhibited an AUClast of 2334 ngh/ml versus 1005 ngh/ml for pure Niclosamide, and an AUCinf of 2387 ngh/ml compared to 1024 ngh/ml, respectively, marking substantial improvements in drug exposure. The relative bioavailability of ASD-5 compared to pure Niclosamide was calculated to be 233%. In previous reported research, Jara et al. developed Niclosamide ASD achieving two-fold bioavailability improvement, with 35% drug loading [6] and Jara et al. also a Niclosamide nanoparticle capsule achieving 2.6-fold bioavailability improvement, with 23% drug loading using hot-melt extrusion [15]; Gupta et al. developed Niclosamide ASD achieving 1.69-fold bioavailability improvement, with 9.6% drug loading [4]; Bhanushali et al. also developed Niclosamide ASD achieving 4.4-fold bioavailability improvement, with 20% drug loading [1]. Among those works, the drug loading above 25%, ASD-5 showed the highest bioavailability, which is a 2.33-fold increase in bioavailability compared to pure Niclosamide.
Various PK parameters after oral administration of Niclosamide and ASD-5 in SD rats.
| Parameter | Unit | Niclosamide | ASD-5 | Fold-increase (ASD-5 vs. Niclosamide) |
|---|---|---|---|---|
| Tmax | h | 0.83 ± 0.29 | 0.417 ± 0.144 | NA |
| Cmax | ng/ml | 279 ± 142 | 909 ± 150 | 3.26 |
| AUClast | h*ng/ml | 1005 ± 463 | 2334 ± 321 | 2.32 |
| AUCInf | h*ng/ml | 1024 ± 455 | 2387 ± 243 | 2.33 |
| Parameter | Unit | Niclosamide | ASD-5 | Fold-increase (ASD-5 vs. Niclosamide) |
|---|---|---|---|---|
| Tmax | h | 0.83 ± 0.29 | 0.417 ± 0.144 | NA |
| Cmax | ng/ml | 279 ± 142 | 909 ± 150 | 3.26 |
| AUClast | h*ng/ml | 1005 ± 463 | 2334 ± 321 | 2.32 |
| AUCInf | h*ng/ml | 1024 ± 455 | 2387 ± 243 | 2.33 |
Tmax, time to reach maximum plasma concentration; Cmax, maximum plasma concentration; AUC, area under the curve.
Various PK parameters after oral administration of Niclosamide and ASD-5 in SD rats.
| Parameter | Unit | Niclosamide | ASD-5 | Fold-increase (ASD-5 vs. Niclosamide) |
|---|---|---|---|---|
| Tmax | h | 0.83 ± 0.29 | 0.417 ± 0.144 | NA |
| Cmax | ng/ml | 279 ± 142 | 909 ± 150 | 3.26 |
| AUClast | h*ng/ml | 1005 ± 463 | 2334 ± 321 | 2.32 |
| AUCInf | h*ng/ml | 1024 ± 455 | 2387 ± 243 | 2.33 |
| Parameter | Unit | Niclosamide | ASD-5 | Fold-increase (ASD-5 vs. Niclosamide) |
|---|---|---|---|---|
| Tmax | h | 0.83 ± 0.29 | 0.417 ± 0.144 | NA |
| Cmax | ng/ml | 279 ± 142 | 909 ± 150 | 3.26 |
| AUClast | h*ng/ml | 1005 ± 463 | 2334 ± 321 | 2.32 |
| AUCInf | h*ng/ml | 1024 ± 455 | 2387 ± 243 | 2.33 |
Tmax, time to reach maximum plasma concentration; Cmax, maximum plasma concentration; AUC, area under the curve.
The oral pharmacokinetic analysis of Niclosamide and ASD-5 in SD rats (mean ± SD, N = 3) at 50 mg/kg. Time (h) vs log mean plasma concentration (ng/ml) profile of Niclosamide and ASD-5.
Conclusion
The ASD of Niclosamide (ASD-5) was successfully developed using a simple, scalable solvent method with a PEG 6000 and P188 combination as the carrier. ASD-5 (Niclosamide: PEG 6000: P 188 = 2:1:5), achieved a 25% drug loading. Characterization through p-XRD, SEM, DSC, and modulated DSC confirmed the transformation of Niclosamide from a crystalline to an amorphous state in ASD-5 and remains amorphously stable. Notably, dissolution studies showed that this formulation significantly improved the dissolution rate and extent of Niclosamide compared to its crystalline form, primarily due to the amorphization of Niclosamide in ASD-5. Additionally, by all reported SDs of Niclosamide with drug loading above 25%, (i) ASD-5 showed the highest bioavailability, with a 2.33-fold increase in plasma exposure and bioavailability compared to pure Niclosamide. (ii) ASD-5 demonstrated a simplified preparation procedure compared to other reported Niclosamide SD.
Acknowledgements
The authors truly appreciate the Global Health Drug Discovery Institute (GHDDI) for its management, financial support, and research facilities. The sample test support of p-XRD studies from the Tsinghua University equipment platform is gratefully acknowledged, DSC/mDSC sample analysis and rat oral PK studies from Pharmaron (Beijing, China) are gratefully acknowledged.
Author contributions
Jing Li: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Resources; Writing—Original Draft; Writing—Review & Editing; Visualization. Zhiyang Zou: Conceptualization; Methodology; Formal analysis; Resources; Writing—Review & Editing; Supervision; Project administration. Yu Chang: Investigation.
Conflict of interest
None declared.
Funding
This work was supported in whole by the Bill & Melinda Gates Foundation Grant no. (INV-008249). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.
Data availability
All the data was available online.
