Translational potential of safe-by-design nanoengineered niclosamide in viral and cancer therapy

Abstract

This study presents a comprehensive evaluation of the long-term biocompatibility of CP-COV03 (NIC–MgO–HPMC), a nanohybrid formulation of niclosamide designed to overcome its limitations in solubility, stability, and bioavailability. Developed under a safe-by-design framework, NIC–MgO–HPMC integrates magnesium oxide (MgO) nanoparticles with hydroxypropyl methylcellulose (HPMC) to enhance pharmacological performance while ensuring safety for chronic use. Over a 13-week in vivo exposure period, the toxicological profile was systematically assessed, focusing on hepatic, renal, and hematologic systems. Clinical observations, serum biochemistry, and hematology revealed no abnormalities at clinically relevant dosages. Histopathological examination of major organs confirmed the absence of tissue damage or structural alterations, underscoring the nanohybrid's long-term tolerability. These findings establish the first foundational safety benchmark for chronic use of nanoengineered niclosamide hybrids. The absence of systemic toxicities validates CP-COV03 as a scalable and biocompatible therapeutic platform suitable for extended dosing regimens. By combining durable safety with enhanced drug performance, CP-COV03 offers strong translational potential for persistent viral infections, including long COVID, future pandemic threats, and oncology applications.

Graphical abstract

Highlights

• •Nanoengineered Niclosamide hybrid: Nanoengineered NIC–MgO–HPMC (CP-COV03) enhances niclosamide solubility, stability, and bioavailability.
• •Safe-by-design approach: Developed with a rational design framework to maximize pharmacological performance while minimizing long-term risks.
• •Comprehensive 13-week in vivo evaluation: Toxicology assessed across hepatic, renal, and hematologic systems.
• •Long-term tolerability established: Long-term safety confirmed, showing no clinical, biochemical, hematologic, or organ abnormalities and strong chronic tolerability
• •Translational relevance: Scalable, safe platform with strong potential for persistent viral infections, including long COVID, and oncology.

1. Introduction

Since the early 2000s, niclosamide has been shown to possess a wide range of effects, including not only antiviral efficacy against various viruses but also anticancer and anti-inflammatory properties [1,2]. As a result, there has been an expectation that niclosamide could potentially address various diseases that current treatments fail to fully resolve, such as viral infections [[3], [4], [5], [6]], cancer [[7], [8], [9], [10], [11]], and autoimmune diseases [3]. Furthermore, in urgent situations like a pandemic caused by a new virus, such as COVID-19, where treatments are urgently needed, niclosamide has gained attention as an antiviral agent due to its potential for rapid drug development through repurposing [1,5,[12], [13], [14], [15], [16], [17], [18]].

However, due to its low aqueous solubility and poor bioavailability [19], niclosamide has long faced translational barriers. After extensive research to overcome this limitation, it has been demonstrated that niclosamide's low bioavailability can indeed be improved using new nanohybridization technology, both in animal studies and in humans [20]. This breakthrough suggests that the various pharmacological effects of niclosamide could be clinically applied to the indications that have been previously researched. Because niclosamide has not been developed beyond antiparasitic indications largely due to its poor solubility and limited bioavailability [19]. Emerging nanohybridization technologies, extensively developed since the early 1990s, now provide a robust means of enhancing the solubility, stability, and bioavailability of such challenging molecules [21]. In the early 2000s [22], significant advancements were made in utilizing layered double hydroxides (LDHs) as drug carriers [23], enabling controlled release and improved pharmacokinetics. More recent studies have focused on nanoengineered niclosamide nanohybrids for antiviral applications, demonstrating the potential of engineered hydrotalcite-based nanohybrids functionalized with surface-modifying agents such as Tween 60 and hydroxypropyl methylcellulose (HPMC) to optimize oral bioavailability and therapeutic efficacy [20]. These advancements have shown notable improvements in pharmacokinetic properties, supported by both preclinical and clinical studies, highlighting nanohybridization as a promising strategy to overcome bioavailability challenges associated with poorly soluble drugs. This strategy is broadly relevant across socially burdensome diseases—including viral infections, cancer, neurodegenerative disorders, and autoimmune conditions—where inadequate solubility has historically hindered the clinical translation of repurposed therapeutics.

In this context, the COVID-19 pandemic remains a global concern, with emerging variants presenting ongoing challenges [24,25]. Although vaccinations have curbed the pandemic's impact, their long-term effectiveness remains uncertain [26], underscoring the need for new orally administrable antiviral drugs. Niclosamide (NIC), a repurposed drug, shows broad-spectrum potential against various viral diseases, including COVID-19, SARS, MERS, influenza, and RSV [3,[27], [28], [29]]. However, NIC's poor solubility and bioavailability have limited its therapeutic use, as it struggles to maintain sufficient plasma concentrations to enter infected cells effectively [27]. To overcome these challenges, NIC was formulated into a hydrophilic hybrid, NIC–MgO–HPMC (CPCOV03), by combining it with magnesium oxide (MgO) and hydroxypropyl methylcellulose (HPMC). This nanohybrid improves NIC's solubility and enhances its intestinal permeability, as confirmed in parallel artificial membrane permeability assays, without altering NIC metabolism. In SARS-CoV-2-infected Syrian hamsters, NIC–MgO–HPMC reduced lung injury, and clinical studies indicate it achieves four times higher bioavailability than traditional NIC [20]. Phase II trials reveal a dose-dependent bioavailability, suggesting NIC–MgO–HPMC as a promising candidate to support a shift towards an endemic management phase for COVID-19.

These findings confirm that the current nanohybrid formulation significantly enhances the bioavailability of a poorly soluble drug, establishing its suitability for clinical development. However, despite its promising pharmacokinetic profile, the long-term safety of this nanohybrid, particularly under conditions of prolonged biological exposure relevant to chronic diseases such as cancer or persistent viral infections—remains insufficiently characterized. Therefore, the primary objective of this study is to systematically evaluate the subchronic toxicity and biocompatibility of nanoengineered niclosamide (NIC–MgO–HPMC) through a 13-week repeated-dose oral toxicity study, including a recovery phase, in order to support its potential use in extended therapeutic regimens.

2. Materials and methods

2.1. Study design and animal allocation for the 13-week oral toxicity evaluation of niclosamide nanohybrid

The 13-week repeated-dose oral toxicity study of niclosamide nanohybrid was conducted using beagle dogs, a commonly used non-rodent species for regulatory toxicology assessments (Fig. 2). All animals were obtained from Beijing Marshall Biotechnology Co., Ltd. (China) and were 7 months old at both acquisition and study initiation. A total of 17 male and 17 female beagle dogs were acquired, with body weights ranging from 7.62 to 9.42 kg (males) and 5.62–8.47 kg (females) at the time of arrival. At the start of dosing, 16 animals per sex were included in the study, with body weights ranging from 7.58 to 9.70 kg in males and 5.72–8.40 kg in females. Animals were divided into four dosing groups: G1 (control, 0 mg/kg/day), G2 (low dose, 60 mg/kg/day), G3 (mid dose, 120 mg/kg/day), and G4 (high dose, 240 mg/kg/day). All groups received a uniform dose volume of 10 mL/kg/day. Each group consisted of 3 males and 3 females, with an additional 2 animals per sex included in G1 and G4 to evaluate recovery effects following the treatment phase. Animal allocation, dosing regimen, and ID numbers are detailed in Table 1.

Fig. 2.

Study design and determination of the no-observed-adverse-effect level (NOAEL) for niclosamide nanohybrid in rodent and non-rodent species. In the 20-week rat study (top timeline), animals received up to 600 mg/kg/day (human equivalent dose [HED]: 9588 mg/head/day), establishing the NOAEL at 600 mg/kg/day. In the parallel 20-week beagle dog study (bottom timeline), the NOAEL was identified at 120 mg/kg/day (HED: 3884 mg/head/day). Both studies consisted of a 13-week dosing phase followed by a 4-week recovery phase, with body weight, clinical signs, and other toxicological endpoints monitored throughout. The timelines depict the continuous observation period from study initiation to completion for each species.

Table 1.

Dosing groups, administration volumes, and animal allocation in the 13-week oral toxicity study of niclosamide nanohybrid in beagle dogs. Four groups were established: G1 (control), G2 (low dose), G3 (mid dose), and G4 (high dose), each receiving niclosamide nanohybrid at 0, 60, 120, or 240 mg/kg/day, respectively. All groups received a uniform dose volume of 10 mL/kg. Each main group included 3 male and 3 female dogs, with 2 additional animals per sex in G1 and G4 allocated for the recovery phase. Animal ID numbers are indicated in parentheses.

Group	Dose (mg/kg/day)	Dose Volume (mL/kg)	Number of Animals (ID)
G1 (Control)	0	10	3 males (1101–1103) +2∗ (1104, 1105)/3 females (2101–2103) +2∗ (2104, 2105)
G2 (Low Dose)	60	10	3 males (1201–1203)/3 females (2201–2203)
G3 (Mid Dose)	120	10	3 males (1301–1303)/3 females (2301–2303)
G4 (High Dose)	240	10	3 males (1401–1403) +2∗ (1404, 1405)/3 females (2401–2403) +2∗ (2404, 2405)

2.2. Quarantine and acclimatization

All animals underwent a primary quarantine and acclimatization period of approximately 9 weeks following acquisition. This was followed by an 11-day re-quarantine and acclimatization phase during which animals were observed daily for general health, and body weights were recorded weekly. Documentation related to individual animals, including health status and import records provided by the supplier, was maintained as part of the study's foundational test records.

The overall 20-week observation period (13 weeks of dosing followed by a 4-week recovery phase and preparatory intervals) was selected to enable a standardized cross-species comparison of long-term exposure outcomes. This duration corresponds to approximately 15–20 % of the rat lifespan (roughly 2–2.5 human years) and 5–7 % of the beagle dog lifespan, providing a subchronic-to-chronic exposure window suitable for detecting cumulative or delayed systemic effects. The chosen timeframe aligns with OECD Test Guidelines 452 and 453 for chronic toxicity and carcinogenicity studies, ensuring sufficient time to evaluate tolerance, metabolic stability, and tissue recovery while avoiding age-related confounders.

2.3. Animal identification and housing

Each animal was uniquely identified using ear tattoo numbers, and corresponding cage identification cards were affixed to each housing unit for traceability and management.

Following the final quarantine period, animals were assigned to experimental groups based on body weight to ensure similar mean weights across groups. Each sex was divided into four dosing groups with three animals per group. Additionally, two animals per sex were included in recovery groups for both the control and high-dose groups. To minimize variability, littermates were not assigned to the same group whenever possible. Group assignments also considered results from pre-study evaluations, including electrocardiograms (ECG) and clinical pathology assessments.

2.4. Animal demographics

A total of 17 male and 17 female beagle dogs, all approximately 7 months old, were acquired for the niclosamide nanohybrid toxicity study. At the time of acquisition, male body weights ranged from 7.62 to 9.42 kg, while females ranged from 5.62 to 8.47 kg. Following the quarantine and acclimatization period, 16 animals of each sex were selected for the dosing phase.

Their body weights at the start of administration ranged from 7.58 to 9.70 kg in males and 5.72–8.40 kg in females, with the age remaining consistent at 7 months. These baseline characteristics ensured uniformity and suitability for the subsequent toxicological assessments (Table 2).

Table 2.

Animal demographics of beagle dogs used in the study, recorded at acquisition and at the start of niclosamide nanohybrid administration. At 7 months of age during acquisition, males (n = 17) weighed 7.62–9.42 kg and females (n = 17) weighed 5.62–8.47 kg. At the start of administration, also at 7 months, males (n = 16) weighed 7.58–9.70 kg and females (n = 16) weighed 5.72–8.40 kg.

Period	Sex	Number of Animals	Age (months)	Weight (kg)
At Acquisition	Male	17	7	7.62–9.42
	Female	17	7	5.62–8.47
At Administration	Male	16	7	7.58–9.70
	Female	16	7	5.72–8.40

2.5. Housing conditions

Animals used in the toxicity study were individually housed in stainless steel cages (800 × 925 × 800 mm) located in holding room, under strictly controlled environmental conditions as summarized in Table 3. Temperature and relative humidity were maintained between 18.0 and 24.0 °C and 30–70 %, respectively, with a ventilation rate of 10–15 air changes per hour. The lighting cycle followed a 12-h light/dark schedule (lights on at 7:00 a.m. and off at 7:00 p.m.), with illuminations ranging from 150 to 300 Lux. Cage hygiene was maintained through regular cleaning of cages and feeders once or twice every two weeks, with immediate action taken for contaminated feeders. Each animal was housed individually and provided with solid commercial animal feed once daily (Lot Nos. 22.02.03, 22.04.13, and 22.06.28 from Cargill Agri Purina Inc., Korea), approximately 250 g per animal. Animals were fasted overnight prior to blood sampling and necropsy. Water was provided ad libitum and sourced from the Gyeonggi Province tap supply, filtered through a microfilter and sterilized via UV treatment. Water quality assessments were conducted annually, with biannual microbiological testing performed by the Gyeonggi Institute of Health and Environment. All food and water quality parameters conformed to institutional acceptance standards.

Table 3.

Environmental Conditions and Housing Setup for Beagle Dogs During the Toxicity Study.

Animals were individually housed in stainless steel cages (800 × 925 × 800 mm) within Room A101 under controlled environmental conditions. Temperature was maintained between 18.0 and 24.0 °C, with relative humidity of 30–70 % and 10–15 air changes per hour. Lighting followed a 12-h cycle (ON: 7:00 a.m., OFF: 7:00 p.m.) with illumination levels of 150–300 Lux. Cages and feeders were cleaned 1–2 times every two weeks, with immediate cleaning or replacement for any soiled feeders.

Environment Factor	Details
Animal Room Number	A101
Cage Type	Stainless steel dog cages (W 800 × L 925 × H 800 mm)
Animals per Cage	1 per cage
Temperature	18.0–24.0 °C
Relative Humidity	30.0–70.0 %
Ventilation	10–15 air changes per hour
Light Cycle	12 h (ON: 7:00 a.m. – OFF: 7:00 p.m.)
Illumination	150–300 Lux
Maintenance and Equipment	Cages and feeders were cleaned 1–2 times per 2 weeks. Feeders contaminated with excrement were either cleaned or replaced.

2.6. Dosing and monitoring procedures

The 13-week oral toxicity study included a 4-week recovery period to assess potential reversibility or delayed effects of niclosamide nanohybrid exposure. The test material, NIC–MgO–HPMC, was administered in pill form prepared using a 0.5 % methylcellulose (MC) solution as the dosing formulation vehicle. This dispersion ensured uniform suspension and compatibility for oral gavage without the need for additional excipients.

Dosing was performed once daily via oral gavage using a gastric catheter connected to a 50 mL disposable syringe. The dose volume was standardized at 10 mL/kg, adjusted weekly based on the most recent body weight measurement. Throughout the study, animals were monitored daily for general clinical signs such as activity, vomiting, and excretion. Mortality and moribundity checks were conducted twice daily, and no treatment-related deaths or moribund conditions were observed. Body weights were recorded weekly during both the dosing and recovery periods at consistent time points. Additionally, fasting body weights were measured prior to necropsy to facilitate accurate calculation of relative organ weights. This design enabled a comprehensive evaluation of both acute and delayed systemic effects. The dose selection (60, 120, and 240 mg/kg/day) for the nanoengineered niclosamide hybrid was determined based on previously reported pharmacokinetic and toxicological data of niclosamide in rodent models, as well as internal preliminary dose-tolerability studies conducted in our laboratory. The low dose (60 mg/kg/day) corresponds to the approximate in vivo effective concentration of niclosamide when adjusted for oral bioavailability. The mid (120 mg/kg/day) and high (240 mg/kg/day) doses represent 2 × and 4 × multiples of this baseline level to explore dose–response relationships while remaining below the reported no-observed-adverse-effect level (NOAEL) in rats. These dosage tiers were selected to (i) ensure systemic safety across a physiologically relevant range, (ii) capture potential nonlinear pharmacodynamic trends of the nanohybrid, and (iii) align with OECD guidelines for subchronic toxicity testing in non-rodent species.

2.7. Clinical pathology

Clinical pathology evaluations were conducted at multiple time points—prior to administration, at weeks 4 and 13 of dosing, and at the end of the 4-week recovery period. Blood samples were collected from the jugular vein after overnight fasting. Hematology, blood coagulation, and clinical biochemistry assessments were performed using appropriate collection tubes and validated analytical instruments. At study termination, all main group animals underwent necropsy following overnight fasting. Deep anesthesia was induced using thiopental sodium (Lot No.: 22001, JW Pharmaceutical Co., Ltd., Republic of Korea), followed by exsanguination. For histopathological examination, a comprehensive panel of tissues was collected and fixed in 10 % neutral buffered formalin. Testes, eyes, and optic nerves were initially fixed in Davidson's solution before being transferred to formalin. The fixed organs included brain, pituitary gland, thymus, thyroid and parathyroid glands, lungs, trachea, heart, spleen, liver, kidneys, adrenal glands, reproductive organs, urinary bladder, gastrointestinal tract, pancreas, lymph nodes, nerves, bone marrow, skeletalmuscle, mammary gland, and any visible gross lesions, ensuring thorough systemic evaluation.

.

2.8. Statistical analysis

Statistical analysis was conducted using SAS software (version 9.4, SAS Institute Inc., USA) to evaluate parameters such as body weight, feed consumption, hematology, blood chemistry, and organ weights. Bartlett's test (α = 0.05) was first applied to assess homogeneity of variance. If the data exhibited homogeneous variance, one-way ANOVA was performed, followed by Dunnett's t-test at significance levels of α = 0.05 and 0.01. In cases where variance was heterogeneous, the Kruskal-Wallis test was used, followed by Steel's test at the same significance thresholds. It is important to note that no statistical analyses were performed for data collected during the recovery phase.

2.9. Animal ethics statement

All animal experiments were conducted in accordance with institutional, national, and international guidelines for the care and use of laboratory animals. Protocols for the rat and beagle dog studies were approved by the Institutional Animal Care and Use Committee (IACUC) (approval numbers 220164 and 220124).

3. Results and discussion

3.1. Niclosamide-MgO-HPMC

The NIC–MgO–HPMC nanohybrid is a rationally engineered drug delivery system designed to overcome the solubility and bioavailability limitations of free niclosamide (NIC), particularly for chronic disorders such as long COVID and cancer. As shown in Fig. 1a, the architecture comprises a magnesium oxide (MgO) core surrounded by NIC, which interacts ionically with the surface of MgO, and an outer hydrophilic layer of hydroxypropyl methylcellulose (HPMC) that enhances dispersion and biocompatibility. The ionic interaction between NIC and MgO is pH-dependent, as NIC possesses both phenolic –OH and amide –NH groups that become deprotonated at basic pH (pKa ∼6.8), enabling electrostatic attraction to the partially cationic MgO surface (Fig. 1c). These interactions are not merely binding events but are driven by selective deprotonation and charge complementarity.

Fig. 1.

Architecture and characterization of the NIC–MgO–HPMC nanohybrid; a) Schematic representation of the NIC–MgO–HPMC construct, illustrating the core–shell structure where MgO forms the core, niclosamide (NIC) binds via ionic interactions to the surface, and hydroxypropyl methylcellulose (HPMC) provides a hydrophilic coating; b) pH-responsive ionization states of NIC: non-ionized (acidic pH), partially ionized (neutral pH; pKa ≈6.8), and fully ionized (basic pH), which regulate its binding affinity to MgO; c) Zeta potential analysis of MgO shows a positive surface charge of +36.62 mV, enabling ionic interaction with deprotonated niclosamide; d) Proton NMR spectra highlighting distinct shifts in the –OH and –NH protons across NIC forms and confirming NIC bonding interaction in the NIC–MgO–HPMC nanohybrid; e) TEM image of pristine MgO nanoparticles (spherical morphology, ∼100 nm); f) TEM image of NIC–MgO nanohybrids showing increased contrast due to drug loading; g) TEM image of NIC–MgO–HPMC showing a defined polymeric coating; h) High-resolution TEM image of NIC–MgO–HPMC showing a core–shell architecture (Scale 10 nm), with the MgO core (black interior of first red dashed line) and the HPMC layer (yellow arrows) confirming successful surface functionalization. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Zeta potential measurements (Fig. 1c) indicate a surface charge of +36.62 mV for the MgO, confirming it can ionically interact with the deprotonated NIC.

The proton NMR analysis (Fig. 1d) supports this ionic bonding interaction by showing the disappearance of NIC's –OH and –NH proton peaks under basic conditions in the presence of MgO, consistent with complex formation via ionic pairing. Morphological characterization using TEM further confirms the nanohybrid structure. Fig. 1e shows the spherical morphology of pristine MgO nanoparticles (∼100 nm), while Fig. 1f reveals contrast enhancement in NIC–MgO hybrids due to drug association. Upon addition of HPMC, Fig. 1g displays the formation of defined spherical aggregates, indicating successful polymer coating. Fig. 1h (HR-TEM) visualizes the core–shell arrangement, with a dense MgO core (within the first red dashed line) and a uniform HPMC shell (highlighted by yellow arrows), validating the hybrid structure. Overall, the NIC–MgO–HPMC nanohybrid demonstrates ionic stabilization, improved dispersibility, and enhanced structural integrity of NIC within the nanohybrid drug [20].

3.2. Long -term toxicology study design

All animal experiments were conducted in accordance with institutional, national, and international guidelines for the care and use of laboratory animals. Protocols for the rat and beagle dog studies were approved by the Institutional Animal Care and Use Committee (IACUC) (approval numbers 220164 and 220124). The long-term toxicology study was done as per the study design (Fig. 2) to confirm the biocompatibility in terms of organ specific toxicology, pathological signs etc. Rats and beagle dogs are widely used in long-term toxicity and pharmacology studies due to their physiological and metabolic similarities to humans, making them well-suited for assessing the safety, efficacy, and pharmacokinetic profiles of new therapeutic candidates. Rats offer cost-effective, reproducible results and provide critical data on toxicology and side effects, while beagles are valuable for more advanced studies, especially chronic drug exposure, due to their more complex organ systems. The data gathered from these studies not only fulfill regulatory requirements but also enhance the credibility and trust of the product, providing a strong foundation for marketing claims about safety and effectiveness [30].

3.3. Clinical signs (dosing period, recovery period)

• a)SD rats

In this study, 7 male animals in the 1200 mg/kg/day dose group [1 animal each on Day 36, 60, and 65 of administration, and 2 animals each on Day 57 and 69 (necropsy day)] and 6 female animals [1 animal on Day 50 of administration and 5 animals on Day 68 (necropsy day)] died in either lateral recumbency or sternal recumbency. Observation of general symptoms revealed transient or persistent signs of salivation, soft stool, or diarrhea during the dosing period in both males and females at the 1200 mg/kg/day dose group. Soft stool was observed up to 1 day after dosing cessation in males and up to 2 days after cessation in females.

Among the deceased animals in the 1200 mg/kg/day group, salivation and soft stool were observed intermittently or persistently before death in males, but no other abnormal symptoms were identified. In one female animal (ID: 2410), a decrease in food consumption was noted on Day 49 of administration, followed by diarrhea, irregular respiration, and piloerection on Day 50, the day of death. The salivation observed after administration is considered a temporary change caused by the physicochemical properties of the test substance and is not regarded as toxicologically significant. The soft stool or diarrhea is believed to result from the pharmacological effects of magnesium oxide (MgO), which is included in the test substance and known to act as a laxative. No deaths or toxicologically significant abnormal symptoms related to the administration of the test substance were observed in the control group or in the 300 and 600 mg/kg/day dose groups (Table S1)

• b)Beagles

During the study period, no mortality was observed in any group, regardless of sex. In all test substance-treated groups, dose-dependent occurrences of vomiting, diarrhea, soft stools, and compound-mixed stools were frequently observed. In male animals, salivation was observed from approximately day 31 in the 240 mg/kg/day group and from around day 40 in the 120 mg/kg/day group. In female animals, salivation appeared from day 30 in the 240 mg/kg/day group and around day 74 in the 120 mg/kg/day group.

However, these symptoms were not observed in the recovery group after the cessation of administration, except for one case of soft stool that was noted four times in the control group of the recovery phase (Table 4).

Table 4.

Clinical signs (dosing period, recovery period) in beagles.

Sex	Group/Dose (mg/kg/day)	Animal ID	Dosing for 13-week					Recovery for 4- week
			Compound-mixed stool	Soft stool	Diarrhea	Salivation	Vomiting	Soft stool
Male	0	1101	0	0	0	0	0
		1102	0	0	0	0	0
		1103	0	0	0	0	0
		1104	0	0	0	0	0	0
		1105	0	1	0	0	0	4
	60	1201	41	13	41	0	29
		1202	74	0	76	0	49
		1203	82	0	82	0	16
	120	1301	77	5	76	28	39
		1302	77	1	76	19	45
		1303	78	1	79	31	56
	240	1401	81	1	80	25	33
		1402	84	4	84	50	38
		1403	81	1	87	29	51
		1404	79	0	79	49	75	0
		1405	77	2	79	39	69	0
Female	0	2101	0	0	0	0	0
		2102	0	0	0	0	0
		2103	0	0	0	0	0
		2104	0	0	0	0	0	0
		2105	0	0	0	0	0	0
	60	2201	58	3	57	0	70
		2202	15	0	15	0	74
		2203	16	6	11	0	27
	120	2301	61	1	62	18	55
		2302	57	2	56	18	53
		2303	75	2	73	18	40
	240	2401	68	2	71	26	58
		2402	80	4	75	42	39
		2403	68	0	68	60	38
		2404	79	1	78	58	79	0
		2405	72	1	71	28	55	0

The gastrointestinal symptoms mentioned above are considered to be primarily caused by the temporary irritation of the gastrointestinal tract by the test substance. These symptoms resolved immediately upon cessation of administration, and no pathological changes were observed in histological examinations of the gastrointestinal tract, indicating that the toxicological significance is considered minimal.

3.4. Body weight assessment in NIC-MgO-HPMC Clinical Study

• a)SD rats

Body weight suppression was observed in the male 1200 mg/kg/day dose group during the administration period, with body weight at Week 9 being 12.2 % lower compared to the control group. This change was reversed during the 7-week recovery period.

Statistically, the body weights of males in the 1200 mg/kg/day dose group were significantly lower than those of the control group throughout Weeks 1–9.

No abnormal body weight changes related to the administration of the test substance were observed in the 300 and 600 mg/kg/day dose groups, or in the female 1200 mg/kg/day dose group during the observation period (Fig. S1)

• b)Beagle

Throughout the administration and recovery periods, no statistically significant changes in body weight were observed in any of the groups treated with the test substance when compared to the control group. This trend was consistent across all dosage levels and was independent of sex, indicating that the test substance did not induce any noticeable impact on body weight regulation in either male or female subjects (Fig. 3).

Fig. 3.

Changes in body weight of male (a) and female (b) beagle dogs during 13 weeks of niclosamide nanohybrid administration and a 4-week recovery phase. Dogs were divided into four groups: G1 (control, 0 mg/kg/day), G2 (low dose, 60 mg/kg/day), G3 (mid dose, 120 mg/kg/day), and G4 (high dose, 240 mg/kg/day). Body weight values are expressed as mean ± standard deviation (SD) for each group (n = 5 per sex per group). Data were analyzed using two-way ANOVA followed by Tukey's multiple comparison test in GraphPad Prism (version 2.5). No statistically significant differences (p > 0.05) were observed between treated and control animals throughout the dosing or recovery periods. Body weights increased steadily and remained within normal physiological ranges, with no evidence of weight loss, systemic toxicity, or delayed adverse effects following cessation of dosing. These findings support the long-term safety and systemic tolerability of niclosamide nanohybrid in beagle dogs.

3.5. Comprehensive Hematological and Biochemical Analysis in the clinical evaluation of NIC-MgO-HPMC

• a)SD rats

No abnormalities related to the administration of the test substance were observed in hematological examinations in both males and females across the 300, 600, and 1200 mg/kg/day dose groups (Table S2, S3). Other statistically significant parameters showed only minor fluctuations, lacked dose or sex-related correlations, and were not accompanied by other relevant changes, indicating no toxicological significance.

An increase in blood urea nitrogen (BUN) was observed in both males and females of the 1200 mg/kg/day dose group, while magnesium (Mg) levels increased in both males and females of the 300, 600, and 1200 mg/kg/day dose groups, as well as in females of the 1200 mg/kg/day recovery group. Decreases or trends of decrease in sodium (Na) and chloride (Cl) levels were found in females of the 300 mg/kg/day group and in both males and females of the 600 and 1200 mg/kg/day groups (Fig. S2, Table S4). Additionally, decreases or trends of decrease in total cholesterol (T-CHO) were observed in females of the 300 mg/kg/day group and in both males and females of the 600 and 1200 mg/kg/day groups (Table S4). Increases or trends of increase in triglycerides (TG) were found in both males and females of the 1200 mg/kg/day dose group (Text Table 1). These changes exhibited dose-dependent patterns, suggesting they were induced by the administration of CP-COV03 (Table S4).

• b)Beagles

In the hematological examination at week 4 of administration, a statistically significant increase (p < 0.05) in monocytes (MONO) was observed in the male 120 mg/kg/day group compared to the control group. However, the difference was minor, showed no correlation between sexes, and no dose-dependency was observed, indicating no toxicological relevance (Fig. 4, Table S5a–d). No significant changes were observed at week 13 of administration or in the recovery group (Table S6 a,b).

Fig. 4.

Comparative Hematological Analysis and Clinical Chemistry in Beagle Dogs Following Oral Administration of Niclosamide Nanohybrid. Bar graphs summarize the mean ± standard deviation (SD) for key hematological and clinical biochemistry parameters in male and female beagle dogs across various treatment groups (G1) to (G4) ona) 4th and b)13th weeks. The panels present results for different parameters (e.g., blood cell counts, enzyme activities, electrolytes, etc.). Statistical analysis (ANOVA) indicated no statistically significant dose-dependent deviations (p > 0.05) in any of the measured hematological and clinical pathology parameters in comparison to the control group in either male or female subjects. These stable findings suggest the absence of treatment-induced systemic toxicity, further supporting the preclinical safety of the niclosamide nanohybrid.

In the blood biochemistry examination, a detailed biochemical analysis of key physiological parameters in subjects administered nanohybrid NIC-MgO-HPMC (Fig. 5, Table S7a–c), with a focus on liver function, kidney function, blood sugar regulation, and electrolyte balance. Monitoring these parameters is critical in assessing the systemic safety and metabolic impact of nanomedicines, as nanoscale formulations can alter pharmacokinetics, biodistribution, and metabolic interactions compared to conventional drug formulations.

Fig. 5.

Hematological and Biochemical Analysis of a) Liver, b) Kidney, c) Blood Sugar, and d) Electrolyte Balance in NIC-MgO-HPMC Clinical Study in Beagle Dogs. The Figure presents a comprehensive evaluation of key biochemical parameters in subjects administered NIC-MgO-HPMC (G1-G4), focusing on liver function, kidney function, blood sugar regulation, and electrolyte balance. The first row (a) illustrates liver function through parameters such as ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels, alongside kidney function indicators like BUN (blood urea nitrogen) and creatinine (b). The second row evaluates blood sugar regulation via glucose levels and insulin response (c), while electrolyte balance is assessed based on sodium, potassium, chloride, and calcium concentrations (d). Data represents as mean ± standard deviation (SD), with comparisons made across different treatment groups. No significant deviations indicative of systemic toxicity was observed, confirming the safety profile of NIC-MgO-HPMC in relation to these physiological functions.

Liver function was evaluated by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), key indicators of hepatocellular integrity, along with alkaline phosphatase (ALP), which reflects hepatobiliary function. A statistically significant increase in ALP levels was observed in female subjects receiving 240 mg/kg/day at week 4 ((Table S7a) ((p < 0.01 or p < 0.05) (Table S7b). Furthermore, both ALP and magnesium (Mg) levels exhibited significant elevations at week 13 compared to the control group. However, the magnitude of these differences was minor, and no sex-specific trends were identified, making it difficult to establish a direct correlation with the NIC-MgO-HPMC nanohybrid. Notably, no significant changes were detected in the recovery group (Table S8), suggesting that the observed variations may not be indicative of persistent toxicology.

Kidney function assessment, including blood urea nitrogen (BUN) and creatinine levels, showed no significant deviations across treatment groups, indicating preserved renal filtration and clearance mechanisms. Additionally, glucose and insulin levels remained stable, confirming that NIC-MgO-HPMC administration did not adversely impact blood sugar homeostasis. Electrolyte balance, an essential factor in maintaining cellular and neuromuscular function, was assessed via sodium (Na⁺), potassium (K⁺), chloride (Cl⁻), and calcium (Ca²⁺) levels, all of which remained within physiological ranges across treatment groups (Fig. 5d).

These findings collectively support the biocompatibility of nano-formulated NIC-MgO-HPMC, demonstrating no major disruptions in critical biochemical markers. However, the transient ALP and Mg elevations underscore the need for further mechanistic studies to elucidate potential nanoparticle-mediated interactions with hepatic metabolism and mineral homeostasis.

3.6. Toxicopathological findings

• a)SD rats

Necropsy of the deceased animals in the 1200 mg/kg/day dose group (7 males and 6 females) revealed distention of the cecum in all animals and a lesion in the glandular stomach in one female (Table S9a).

In the 1200 mg/kg/day dose group, a lesion in the glandular stomach was observed in 1 male and 3 females, cecal distention in 3 males and 4 females, spots on the cecum in 2 females, and thickening of the forestomach in 1 female. In the 300 mg/kg/day dose group, cecal distention was observed in 9 males and 1 female, and thickening of the forestomach in 1 male. In the 600 mg/kg/day dose group, cecal distention was observed in all animals, while discoloration of the stomach and thickening of the forestomach were found in 2 males (Table S9a–c).

These findings, along with the changes in organ weights (Table S10 and S11), are related to the administration of the test substance and are primarily attributed to the pharmacological effects of magnesium oxide (MgO). MgO is known to convert to magnesium chloride (MgCl₂) under acidic conditions in the stomach. In the duodenum, MgCl₂ reacts with bicarbonate (NaHCO₃) from pancreatic secretions to form magnesium bicarbonate [Mg(HCO₃)₂], which is eventually converted to magnesium carbonate (MgCO₃). Both Mg(HCO₃)₂ and MgCO₃ increase the osmotic pressure of the intestinal fluid, promoting water movement into the intestinal lumen, thereby increasing the water content and volume of feces. In severe cases, these effects can lead to reduced gastrointestinal motility, intestinal obstruction due to smooth muscle paralysis, and side effects such as facial flushing and pupil dilation. Other observed findings were deemed to be spontaneous or incidental, based on their frequency, distribution, and severity, and were not considered related to the administration of the test substance (Table S10–S11)

• b)Beagles

In the necropsy findings of the main study group (Table 5), a nodule in the duodenum was observed in one male beagle (Animal ID: 1202) administered 60 mg/kg/day of nano-formulated NIC-MgO- HPMC, while in the recovery group (Table 6, Table S12b), a cyst in the ovary was detected in one female rat (Animal ID: 2405) from the 240 mg/kg/day group. These findings were classified as spontaneous alterations due to their isolated occurrence and lack of dose-dependent trends, suggesting no direct correlation with NIC-MgO-HPMC administration. Nanoparticles, including those in NIC-MgO-HPMC hybrid, exhibit unique physicochemical characteristics influencing their cellular interactions, biodistribution, and systemic effects. Their high surface-area-to-volume ratio enhances bioavailability, allowing greater interaction with biological interfaces. Surface charge and functional groups modulate protein corona formation, affecting cellular recognition, uptake, and clearance. Electrostatic interactions between negatively charged nanoparticles and slightly cationic regions of cell membranes facilitate internalization, while differences in surface potential between NIC-MgO-HPMC nanoparticles and biological tissues may influence retention and clearance. The duodenal nodule could be linked to localized nanoparticle accumulation, possibly affecting cellular turnover rates or inducing a transient immune response, while the ovarian cyst may suggest an interaction between NIC-MgO-HPMC nanoparticles and endocrine regulation, though its singular occurrence makes attributing causality difficult.

Table 5.

Summary of Toxicopathological findings (beagles).

Sex: Male
Group/Dose (mg/kg/day)	Animal ID	Organ	Necropsy findings	Type	Day of necropsy
G1	1101	All	No remarkable findings	scheduled.	92
0	1102	All	No remarkable findings	scheduled.	92
	1103	All	No remarkable findings	scheduled.	92
G2	1201	All	No remarkable findings	scheduled.	92
60	1202	Jejunum	Nodule, black, 10 × 10 × 8 mm	scheduled.	92
	1203	All	No remarkable findings	scheduled.	92
G3	1301	All	No remarkable findings	scheduled.	92
120	1302	All	No remarkable findings	scheduled.	92
	1303	All	No remarkable findings	scheduled.	92
G4	1401	All	No remarkable findings	scheduled.	92
240	1402	All	No remarkable findings	scheduled.	92
	1403	All	No remarkable findings	scheduled.	92
Sex: Female
Group/Dose (mg/kg/day)	Animal ID	Organ	Necropsy findings	Type	Day of necropsy
G1	2101	All	No remarkable findings	scheduled.	92
0	2102	All	No remarkable findings	scheduled.	92
	2103	All	No remarkable findings	scheduled.	92
G2	2201	All	No remarkable findings	scheduled.	92
60	2202	All	No remarkable findings	scheduled.	92
	2203	All	No remarkable findings	scheduled.	92
G3	2301	All	No remarkable findings	scheduled.	92
120	2302	All	No remarkable findings	scheduled.	92
	2303	All	No remarkable findings	scheduled.	92
G4	2401	All	No remarkable findings	scheduled.	92
240	2402	All	No remarkable findings	scheduled.	92
	2403	All	No remarkable findings	scheduled.	92

Table 6.

Summary of Toxicopathological Findings in a) main and b) recovery groups (beagles).

a) Main Group						b) Recovery Group
Sex: Male						Sex: Male
Organ/Findings	Group	G1	G2	G3	G4	Organ/Findings	Group	G1	G4
	Dose (mg/kg/day)	0	60	120	240		Dose (mg/kg/day)	0	240
	Number of animals	3	3	3	3		Number of animals	2	2
Jejunum	Nodule	0	1	0	0	All	No remarkable findings	2	2
	No remarkable findings	3	2	3	3
Sex: Female						Sex: Female
						Organ/Findings	Group	G1	G4
Organ/Findings	Group	G1	G2	G3	G4		Number of animals	2	2
	Dose (mg/kg/day)	0	60	120	240
	Number of animals	3	3	3	3	Ovary	Cyst	0	1
All	No remarkable findings	3	3	3	3		No remarkable findings	2	1

Similarly, there were no significant changes in organ weights for test groups (Fig. 6). The absence of significant findings in the recovery group suggests that any transient changes induced by NIC-MgO-HPMC nanohybrid were reversible, reinforcing its biocompatibility at the tested doses. These findings highlight the critical role of nanomaterial properties in biological interactions, emphasizing the necessity of comprehensive safety assessments to understand the long-term biocompatibility and systemic distribution of nanoengineered therapeutics like NIC-MgO-HPMC.

Fig. 6.

Absolute Organ Weights in Male and Female Beagle Dogs Following 13-Week Oral Administration of Niclosamide Nanohybrid. Bar graphs represent the mean ± standard deviation (SD) of absolute organ weights (in grams) for male (Top panel, a) and female (Bottom panel, b) beagle dogs across different treatment groups: G1 (Control, 0 mg/kg/day, represented by color [e.g., Pink/Purple]), G2 (Low dose, 60 mg/kg/day, represented by color [e.g., Blue]), G3 (Mid dose, 120 mg/kg/day, represented by color [e.g., Green]), and G4 (High dose, 240 mg/kg/day, represented by color [e.g., Orange]). Organs analyzed include brain, heart, liver, kidneys, spleen, lungs, and others (as labeled on the x-axis). Statistical analysis revealed no significant differences (p > 0.05$) in the absolute weights of any major organ among the nanohybrid-treated groups (G2, G3, G4) compared to the vehicle control group (G1) in either sex. This finding indicates that the niclosamide nanohybrid had no dose-dependent adverse effects on the physiological mass of critical organs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.7. Histopathological evidence

• a)SD rats

Histopathological analysis of major organs, including the liver, lung, heart, kidney, pancreas, and cecum, was conducted in male and female rats following administration of niclosamide nanohybrid at doses of 600 and 1200 mg/kg to assess potential toxicological effects at the tissue level. Microscopic evaluation of hematoxylin and eosin (H&E)-stained sections revealed no significant morphological alterations, cellular degeneration, necrosis, or fibrosis across all examined organs (Fig. S3). The absence of inflammatory cell infiltration, hemorrhagic lesions, or vascular abnormalities further confirmed the non-toxic nature of the niclosamide nanohybrid at the tested doses. Additionally, no evidence of nanoparticle-induced granulomatous formations or bioaccumulation-related pathological changes was detected, suggesting effective systemic clearance and minimal long-term retention in critical organs. The structural integrity of hepatocytes, renal tubules, alveolar architecture, and pancreatic acinar cells remained unaltered, reinforcing the biocompatibility of the nanohybrid. These findings collectively indicate that prolonged exposure to niclosamide nanohybrid does not elicit adverse histological effects, supporting its potential safety for long-term therapeutic applications. However, further studies incorporating ultrastructural and immunohistochemical analyses could provide deeper insights into subcellular interactions and nanoparticle-induced biological responses.

• b)Beagles

Histopathological evaluation of vital organs, including the liver, lung, heart, kidney, pancreas, and cecum, was performed in male and female beagle dogs administered niclosamide nanohybrid at doses of 60, 120, and 240 mg/kg to assess potential toxicological impacts (Fig. 7). Microscopic examination of H&E-stained tissue sections revealed no substantial structural abnormalities, cellular degeneration, or necrotic changes across all tested dose groups. Furthermore, no signs of inflammation, hemorrhages, fibrosis, or vascular disruptions were observed, indicating an absence of significant toxic effects. The lack of granuloma formation or nanoparticle accumulation suggests efficient systemic clearance and minimal organ burden. Preservation of normal histoarchitecture in hepatocytes, renal tubules, alveolar structures, and pancreatic tissue reinforces the biocompatibility and safety profile of niclosamide nanohybrid at these doses. These findings demonstrate that repeated administration does not induce dose-dependent histological alterations, further supporting the therapeutic feasibility of this nanoengineered niclosamide hybrid. Nonetheless, additional ultrastructural and molecular analyses may provide deeper insights into nanoparticle-cell interactions and long-term biocompatibility.

Fig. 7.

Representative hematoxylin and eosin (H&E) stained tissue sections from the liver, lung, heart, kidney, pancreas, and cecum are shown for both male (a) and female (b) beagle dogs. Animals received either vehicle (G1) or niclosamide nanohybrid at increasing oral doses of 60 mg/kg (G2), 120 mg/kg (G3), and 240 mg/kg (G4) for the duration of the study. Each tissue section was meticulously examined for morphological alterations, cellular damage (e.g., necrosis, apoptosis), inflammatory infiltration, and other signs of treatment-related pathology or toxicology. The red arrows and highlighted annotations in the G1 (Vehicle) columns indicate normal tissue structures for reference (e.g., central venule, hepatocyte cords in liver; terminal bronchiole, alveolar space in lung; glomerulus, Bowman's capsule in kidney; islet of Langerhans in pancreas; goblet cells, lamina propria in cecum). No significant dose-dependent pathological changes, adverse histological effects, or signs of toxicology were observed across all tested doses in any of the major organs from either male or female beagle dogs. These findings strongly suggest that prolonged exposure to the niclosamide nanohybrid does not induce adverse histological effects in critical organs, supporting its favorable safety profile for potential long-term therapeutic applications. Scale bars: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The present study primarily evaluated systemic absorption, distribution, and metabolic kinetics of CP-COV03 (NIC–MgO–HPMC) following oral administration. Accordingly, pharmacokinetic analyses focused on blood plasma to determine key parameters such as Cmax, Tmax, AUC, and elimination half-life, allowing comparison with native niclosamide. Quantitative assessment of urinary and fecal excretion was not performed at this stage because the objective was to confirm enhanced systemic exposure and bioavailability of the nanoengineered hybrid. Future investigations will incorporate comprehensive elimination profiling—including urinary, fecal, and tissue distribution analyses—to achieve a complete pharmacokinetic characterization of CP-COV03.

The current investigation was conducted exclusively in healthy animal models to establish a clear baseline of systemic safety and long-term biocompatibility for CP-COV03 (NIC–MgO–HPMC) without the confounding influence of disease-related physiological alterations. This design aligns with OECD and ISO 10993 recommendations, which emphasize that early-stage safety evaluations should be performed in healthy systems prior to assessing therapeutic candidates under diseased or immunocompromised conditions. Evaluating CP-COV03 in normal animals enabled accurate interpretation of material-related effects on organ function, metabolism, and hematologic parameters. Inclusion of pathophysiological models at this stage could have introduced inflammatory or metabolic variations that obscure the intrinsic safety profile of the nanohybrid. Future work will extend these findings to disease-specific and immunocompromised models to confirm translational safety and efficacy under clinically relevant conditions.

4. Conclusion

This study highlights the pivotal role of nanotechnology in enhancing the long-term safety and therapeutic performance of repurposed antiviral agents, with NIC–MgO–HPMC nanohybrid serving as a model formulation. By addressing the inherent limitations of conventional niclosamide—namely its poor aqueous solubility, rapid clearance, and limited bioavailability—the engineered nanohybrid system enables sustained drug release and improved pharmacokinetics essential for chronic treatment regimens.

Comprehensive 13-week in vivo toxicokinetic and biocompatibility evaluations in both Sprague-Dawley rats and beagle dogs confirm that NIC–MgO–HPMC maintains systemic exposure over time without eliciting dose-limiting toxicities. Plasma concentration–time profiles in rodents and dogs (Fig. S4–S11) clearly demonstrate prolonged circulation and consistent drug levels across early (Day 1) and late (Day 91) timepoints, in both sexes, underscoring the formulation's stability and controlled-release kinetics:

Fig. S4–S7: Sustained plasma levels of niclosamide in male and female Sprague-Dawley rats at Days 1 and 91, confirming long-term exposure. Fig. S8–S11: Similar profiles observed in beagle dogs, further validating the cross-species pharmacokinetic consistency and systemic stability of the nanohybrid.

In parallel, histopathological and biochemical analyses revealed no significant alterations in major organs, including the liver, kidney, heart, or hematopoietic system. The absence of immune or metabolic disruption, along with no mortality or moribundity, supports a favorable safety profile even under prolonged exposure. The biocompatible HPMC matrix not only improves mucosal adhesion and GI retention but also shields the active pharmaceutical ingredient (API) from premature degradation, thereby reducing off-target toxicity. Moreover, the pH-responsive ionic interaction between deprotonated NIC and partially cationic MgO provides structural stability, contributing to the safe-by-design nature of the nanohybrid. Collectively, these findings establish NIC–MgO–HPMC as a next-generation nanomedicine, optimized for long-duration use not only in chronic viral infections such as long COVID, but also emerging viral threats like Ebola, Zika, Mpox, and potentially in onco-immuno-neurological disorders and other relapsing conditions. This work underscores the necessity of long-term systemic evaluations in clinically relevant nanocarriers and sets the stage for clinical translation of safe, effective, and sustainable nanotherapeutic platforms.

CRediT authorship contribution statement

Sanoj Rejinold N: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Geun-woo Jin: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Jin-Ho Choy: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

Authors have nothing to declare of interest.

Appendix A. Supplementary data

Supporting Information is available from the Wiley Online Library or from the author.

The Supporting Information is available free of charge at https://xxxx.

Appendix B. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.