Rational Design, Characterization, and Therapeutic Evaluation of Niclosamide-Nafamostat Co-Crystal Systems for Oncology and SARS-CoV-2 Infection

1. Introduction

Cocrystal drug development is a rapidly evolving area of pharmaceutical research aimed at overcoming the limitations imposed by scientists, clinicians, and patients [1,2] for the treatment cancer, which is one of the leading causes of death worldwide [3,4]. Cocrystal is a powerful strategy in drug development to optimize the physicochemical and biopharmaceutical properties active pharmaceutical ingredients (APIs), which are poorly soluble compounds in Biopharmaceutics Classification System (BCS) classes II and IV [5,6,7]. By tailoring intermolecular interactions in the solid state without altering the covalent structure or primary pharmacology of the APIs can improve solubility, stability, and bioavailability by pharmaceutical cocrystals method. In similar, repurposing and reformulation of old drugs with newly recognized anticancer and antiviral activities, and it has become an attractive approach to accelerate therapeutic innovation. Niclosamide, an FDA-approved anthelmintic agent and several studies shown that niclosamide overcomes chemotherapy resistance across diverse tumor types, in part by modulating key signaling pathways, inducing apoptosis, and suppressing cancer stem cell–like phenotypes. Niclosamide has also been reported to enhance the efficacy of immunotherapies, including immune checkpoint blockades. Niclosamide has demonstrated by reshaping the tumor microenvironment and attenuating immune evasion mechanisms driven by cancer stem cells [8]. COVID-19 as a declaration for pandemic disease by the World Health Organization. Many individuals were continue to suffer from severe illness or death due to infection with COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [9,10,11]. Despite this promise, niclosamide has poor aqueous solubility and limited systemic exposure are considered major barriers to clinical translation in oncology.

Nafamostat mesylate (Nafamostat) is clinically used for the treatment of acute pancreatitis, acute respiratory distress, and disseminated intravascular coagulation as a serine protease inhibitor [12]. Niclosamide potently inhibits TMPRSS2-mediated priming of the viral spike protein and it blocks viral entry into human airway epithelial cells, it has attracted attention as a candidate therapeutic for SARS-CoV-2 infection [13]. Nafamostat distinctly reduces SARS-CoV-2 infection and associated lung pathology in cell and animal model at clinically achievable concentrations, making it a promising antiviral agent for hospitalized COVID 19 patients [14,15]. Additionally, nafamostat has shown anticancer and anti-inflammatory activities, enhancing the possibility of dual utility in lung cancer and virus-infected lung tissue. However, due to rapid hydrolysis and limited specificity and exposure constrain nafamostat therapeutic window and complicate sustained systemic treatment [16,17].

Pharmaceutical cocrystallization, defined as the formation of a single crystalline phase comprising two or more drug molecules held together by non-covalent interactions. It occurs in deferent way, such as hydrogen bonding, van der Waals forces, and π–π stacking, offers an attractive route to address these limitations [18,19]. Cocrystal synthesized different methods such as solution-based methods (e.g., solvent evaporation, cooling crystallization, spray drying) or solvent-free/mechanochemical approaches (e.g., neat or liquid-assisted grinding, hot-melt extrusion), enabling scalable and industrially relevant manufacture [20]. Current scenario, dual-API cocrystals demonstrates that combining two drugs in a single solid form can improve dissolution, control release, and potentially enable synergistic pharmacological effects with reduced dose- and formulation-related toxicity [21,22].

Having potent anticancer and antiviral profiles of niclosamide and nafamostat, as well as their recognized anti-inflammatory activities, co-engineer of dual drug of these agents as a strong rationale to overcome their solubility and bioavailability constraints and to enhance systemic exposure. In this research, we aim to design and develop a dual system of niclosamide–nafamostat cocrystal (NNC), potentially with suitable co-formers, to modulate the intermolecular packing and optimize key physicochemical parameters such as solubility, dissolution rate, and solid-state stability. Structural characterization of NNC, coupled with in vitro and in vivo evaluation of its anticancer and antiviral efficacy in human lung cell models, is expected to clarify whether this dual drug cocrystal, can deliver superior therapeutic performance compared with the individual and their physical mixtures. Eventually, our research strategy may support the development of an innovative solid dosage form capable of simultaneously targeting lung cancer progression and SARS CoV 2 infection while mitigating dose-limiting toxicity and pharmacokinetic shortcomings.

2. Materials and Methods

2.1. Materials

Niclosamide was purchased from Hengcheng Pharmaceuticals, Quinzhuhu, China. Nafamostat mesylate was purchased from Kukjeon, Hwaseong-si, Republic of Korea, and Sodium bicarbonate was obtained from Emmennar (Hyderabad, India). Phosphate-buffered saline (PBS), polyvinylpyrrolidone 30 (PVP 30), sodium bicarbonate, ethyl alcohol, methyl alcohol, acetonitrile, tetrahydrofuran, 2-propanol, and N, dimethylformamide were obtained from Sigma-Aldrich, USA. All the chemicals were used without further purification.

2.1. Noclosamide-Nafamostat Supramolecular Complex Preparation

Nafamostat was dissolved in distilled water at room temperature using a mechanical stirrer at 500 rpm. Sodium carbonate was dissolved in a separate vessel, and the clear solution was slowly added to the reaction vessels containing nafamostat with strong mechanical stirring. The reaction solution was filtered, and the filtrate was collected and stored in a reaction vessel. Niclosamide was dissolved in acetone in a separate vessel and filtered. Transfer the niclosamide solution to the Nafamostat vessel and heat the reaction vessel with the set temperature at 60 °C. The reaction vessels containing the nafamostat solution are stirred at room temperature at a speed of 4000 rpm for 1 h. After stirring, the solvent was removed by filtration through a vacuum pump and filter. The newly prepared cocrystals were dried under reduced pressure using a vacuum dryer for one day.

2.3. Nuclear Magnetic Resonance (¹H-NMR)

The ¹H-NMR samples were prepared using 10 mg of the cocrystal sample dissolved in DMSO-D6, followed by a filled-in NMR detection tube. The ¹H-NMR spectra were recorded using a 600 MHz nuclear magnetic resonance spectrophotometer (AVANCE III 600, Bruker, Massachusetts, USA).

2.4. Powder X-Ray Diffraction (PXRD)

PXRD data were recorded on a Rigaku SmartLab X-ray diffractometer (Tokyo, Japan) with Cu Kα radiation at 40 kV and 15 mA. The analysis was performed with a step size of 0.02° in the 2θ range from 4° to 45°.

2.5. Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) was performed using a NETZSCH DSC214 instrument (Selb, Germany) under a nitrogen gas atmosphere (40 mL/min). Each sample was sealed in a perforated aluminum pan, and analysis was performed over a temperature range from 0 °C to 300 °C at a heating rate of 10 °C/min.

2.6. Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra was analyzed using a Cary 640 FTIR spectrophotometer (Alginate Technology, Cary 600 Series FTIR spectrophotometer, California, USA). The Potassium Bromide (KBr) method was used to prepare the test samples, and KBr was used to form a thin pellet. The pellet was then placed under the sample holder of the FTIR spectrophotometer.

2.7. Cell Culture and IC₅₀ Determination

Breast cancer cell lines (MCF-7 and MDA-MB-231), pancreatic cancer cell lines (PANC-1 and MIA PaCa-2), and non-small lung cancer cell lines (A-549 and H-1299) were used in this study. Cells were cultured in DMEM containing 10% FBS and 1% antibiotics (Gibco), and cell lines were maintained at 37 °C and 5% CO₂ atmosphere. The IC₅₀ of NNC was measured using a cell viability assay. Six cell lines were used, and cell viability was measured using a CCK-8 assay kit (Abcam). Cells were seeded at 2-4 × 10³ cells/well in 96-well plates overnight. NNCs were treated for 48 h at concentrations of 0.3, 3, 30, 300, 3000, and 30000 ng/mL in triplicate. Cell viability was assayed following the manufacturer’s protocol, and the IC₅₀ was determined.

2.8. In Vivo Cytotoxicity Study

Fifteen female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were purchased from the Biotechnology Research Institute, Daejeon, Korea. The NOD/SCID mice were used for in vivo experiments, which allow for the engraftment and growth of human tumors. All animals received human care during housing and breeding in compliance with the Guide for Care and Use of Laboratory Animals published by the National Institute of Health. This test was conducted on animals at the National Cancer Center with the Animal Protection Act and the Act on Laboratory Animals. The study was performed according to a protocol approved by the ethics committee (NCC-21-702). Mice were randomly divided into three groups (n=5 each). The anticancer effectiveness was measured using an orthotropic model with PNAC-1 cells at a density of 1 × 10⁶/50 μl per subject. NNCs, at the concentrations of 15 mg/kg and 30 mg/kg, were dissolved in a vehicle containing 97.5% PBS and 2.5% PVP 30, administered for four weeks, and tumor mass and tumor burden were determined.

2.9. In Vivo Antivirus Efficacy

Vero cells (1.2 x 10⁴) were seeded per well in 384 tissue culture plates to assess antivirus efficacy. After 24 hours, the cells were treated with a compound prepared by serially diluting 2-fold in DMSO to a peak concentration of 50 µM at 10 points. Approximately 1 hour after compound treatment, the cells were infected with SARS-CoV-2 (0.0125 MOI) in a BSL3 facility and cultured at 37 °C for 24 hours. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized. Then, the cells were treated with the anti-SARS-CoV-2 nucleocapsid (N) primary antibody, followed by treatment with the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody and Hoechst 33342 to stain the cells. Fluorescence images of the infected cells were acquired using the high-throughput imaging analyzer Operetta (Perkin Elmer, Illinois, USA).

2.10. Statistical Analysis

Statistical analysis was performed using GraphPad Prism to determine significant difference. A student t-test was used to compare two groups with a significance threshold set at P<0.05.

3. Results and Discussion:

3.1. Proton Nuclear Magnetic Resonance (¹H NMR)

The combination of NNC was successfully prepared using a solution precipitation method. Upon complete solubilization of the components, intermolecular hydrogen-bonding interactions were established between the drug molecules, facilitating cocrystal formation. Structural confirmation was initially performed using proton nuclear magnetic resonance (¹H NMR) spectroscopy for the confirmation of chemical environments and characteristic shifts corresponding to niclosamide and nafamostat. The downfield shift of the hydroxyl proton to 13.92 ppm (Figure 1a, sup Figures S1–S3) confirms the formation of an O–H···N hydrogen bond, indicating that an aromatic nitrogen from the nafamostat is acting as the primary hydrogen-bond acceptor for the niclosamide hydroxyl group.

3.2. Fourier Transform Infrared Spectroscopy

Cocrystal formation was attained through Fourier-transform infrared (FTIR) spectroscopy for further structural confirmation of the cocrystal. The FTIR spectra revealed significant shifts in vibrational frequencies, indicating the formation of hydrogen-bonding interactions. Specifically, the characteristic O–H (phenolic) and N–H stretching vibrations of niclosamide, originally observed in the range of 3300–3400 cm⁻¹, shifted and broadened to 3252–3093 cm⁻¹ in the cocrystal, suggesting strong intermolecular interactions (Figure 1d). Additionally, the amide I band corresponding to C=O stretching was observed at approximately 1650 cm⁻¹. Changes in peaks around 889 cm⁻¹ further supported the involvement of niclosamide functional groups in hydrogen bonding with nafamostat. The presence of S=O and C–O stretching vibrations in the 1000–1200 cm⁻¹ region indicated the formation of a new molecular environment within the cocrystal. Peak shift changes at 1070 cm⁻¹ region due to the crystal lattice environment of Hydrogen bonding compared the carbonyl group.

3.3. Powder X-Ray Diffraction

Powder X-ray diffraction (PXRD) analysis provided definitive evidence for the formation of a new crystalline phase. Main diagnostic peaks for characteristics identification in 9.44°, 11.62°, 15.19°, 15.90°, 16.93°, 17.73°, 23.35°, 25.00°, 25.66° and 27.25°. The NNC cocrystal exhibited distinct diffraction peaks at 2θ values of 7.096°, 9.046°, 9.68°, 12.84°, 15.16°, 15.99°, 16.36°, 17.65°, 23.14°, and 26.14°, which were absent in the diffraction patterns of the individual components (Figure 1b). The appearance of new peaks, or shifts relative to those of pure niclosamide and nafamostat mesylate, supports the formation of a cocrystal. The overall diffraction pattern changes due to the creation of a new crystal lattice, and the patent also attributes certain peak shifts to the influence of non-stoichiometric hydrate forms in some comparisons. This confirms that the cocrystal is a new solid-state phase resulting from specific intermolecular interactions between the two drug molecules.

3.4. Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) analysis further supported cocrystal formation. Niclosamide and nafamostat exhibited endothermic melting peaks at 230.57 °C and 264.51 °C, respectively (Figure 1c). In contrast, the NNC cocrystal showed a single, distinct endothermic peak at 209.83 °C with a lower melting enthalpy (94.38 J/g), indicating altered intermolecular interactions and reduced lattice energy. This decrease in melting enthalpy suggests improved solubility and dissolution properties of the concrete system compared to the starting materials. Intermediate peaks in the 90–120 °C range can come from solid-state phase transitions, eutectic melting, or partial dissociation of the crystal lattice between niclosamide and nafamostate mesylate. A higher peak approaching multiple endothermic peaks at 92.9 and 209.83 °C is likely the main melting or decomposition-related event of the remaining crystalline fraction, indicating a complex thermal behavior rather than a simple physical mixture. Because pure niclosamide melts closer to 228 °C, a shift downward suggests crystal lattice weakening, impurity effects, or new intermolecular interactions with nafamostat mesylate.

3.5. In Vitro Cell Viability Assay

In vitro cytotoxicity of the NNC was evaluated in multiple cancer cell lines, including pancreatic, lung, and breast cancer lines. The cocrystal demonstrated superior growth inhibition compared to individual treatments (sup Figure S4). Notably, the highest inhibitory activity was observed in the H-1299 lung cancer cell line, indicating enhanced anticancer efficacy. The IC₅₀ value of the cocrystal was significantly lower than that of the individual drugs, reflecting increased potency. Cytotoxicity analysis revealed that nafamostat alone exhibited relatively low toxicity across tested cell lines, with a maximum growth inhibition of 22.66 ± 3.11% in lung adenocarcinoma cells (Figure 2). Niclosamide showed moderate activity, with 19.68 ± 0.42% inhibition in A-549 cells at a tenfold lower concentration than nafamostat, and an IC₅₀ value of 153.2 nM. In contrast, the NNC demonstrated enhanced efficacy, achieving 36.59 ± 4.39% inhibition in H-1299 cells at a low concentration of 30 nM, and 34.02 ± 3.12% inhibition in A-549 cells at 300 nM. However, limited activity was observed in MCF-7 and PANC-1 cells (sup Figure S5) under certain conditions, while MDA-MB cells showed increased proliferation (sup Figure S6) at higher concentrations. Nafamostat alone exhibited notable inhibition in MDA-MB cells at 30 µM.

3.6. In Vivo Tumor Growth Suppression Study

To further evaluate anticancer efficacy, the NNC was tested in an orthotopic pancreatic tumor mouse model. Treatment with NNC at doses of 15, 30, and 60 mg/kg resulted in significant tumor growth suppression compared to vehicle control. The NNC 15 group demonstrated nearly a twofold reduction in tumor weight, while the NNC 30 group showed a 1.74-fold reduction (Figure 3). Interestingly, higher doses did not result in improved efficacy, possibly due to reduced solubility and limited bioavailability at elevated concentrations. This observation suggests that optimal dosing is critical for maximizing therapeutic outcomes.

3.7. In Vivo Tumour Burden Study

Tumor burden analysis further supported these findings, with the NNC showing approximately 40-fold reduction compared to the control, and the NNC 30 group showing a 10-fold reduction (Figure 4). No significant changes in body weight were observed in the NNC-treated groups compared to the control, indicating good tolerability at lower doses. However, higher doses were associated with noticeable body weight changes, suggesting potential toxicity or reduced tolerability.

3.8. In Vivo Antivirus Efficacy

A smaller IC50 indicates superior efficacy, a larger CC50 indicates lower cytotoxicity, and a larger SI indicates superior inhibitory effect, considering toxicity. The IC50 values of the co-crystals were NNC=0.17 μM, which are approximately 45, 20, and 60 times higher than those of chloroquine 7.47 μM, remdesivir 3.48 μM, and lopinavir 10.3 μM, respectively; thus, they appear to have excellent efficacy in inhibiting viral infection (Figure 5). Although the CC50 values were similarly higher than the maximum concentration for both the NNC and the reference drug, the cell viability graph of the co-crystal showed a concentration-dependent decrease in cell survival rate starting from the lowest drug treatment concentration; this result is considered to indicate the possibility of cytotoxicity. The selectivity index (SI) value (Figure 6) of the cocrystal is approximately 290–310, which is about 15, 20, and 60 times higher than that of reference drugs chloroquine (SI=20.08), remdesivir (SI=14.35), and lopinavir (SI=4.85), respectively; therefore, it can be evaluated as a drug with good efficacy even when considering both the pharmacological effects and toxicity of the cocrystal simultaneously.

Overall, these results demonstrate that the NNC significantly enhances anticancer activity, particularly at lower doses, while maintaining acceptable safety profiles. NNC represents a promising strategy for improving the therapeutic performance of existing pharmaceutical agents. Research studies reflected that many anticancer drugs suffer from poor solubility and limited bioavailability, which restricts their clinical effectiveness [23]. Drug-drug cocrystal formation can enhance solubility and dissolution rates, leading to improved absorption and systemic availability. Furthermore, dual drugs with complementary mechanisms of action in a single cocrystal system may produce synergistic effects, resulting in enhanced antitumor efficacy. Therefore, the NNC presents an innovative approach for the treatment of cancer, particularly lung cancer, and warrants further investigation for clinical development.

4. Conclusions

Cocrystal is a successful drug development system, especially for improving the solubility and enhancing bioavailability. Niclosamide and nafomostate were a great combination of drug-drug cocrystal system for the treatment of cancer and anti-virus therapy, such as COVID 19. Significant results supported the evaluation and efficacy to prove the drug-drug candidate as a novel cocrystal and the data pathway for future development of NNC-based anticancer material in clinical patients. These findings could be helpful for patients with emergencies such as COVID-19, and more experiments and preclinical studies need to be done for further confirmation and to support patients’ compliance.