1. Introduction
Orthopoxvirus infections pose a significant threat to global public health owing to their diverse transmission routes and severe clinical outcomes [
1]. Orthopoxviruses that infect humans include variola virus (VARV), monkeypox virus (MPXV), cowpox virus (CPXV), and vaccinia virus (VACV) [
2]. Smallpox, which has historically been one of the most lethal diseases, is caused by VARV infection [
2]. However, following a global eradication campaign in the 20th century, smallpox was declared fully eradicated; consequently, most of the current population lacks pre-existing immunity to the virus [
3]. Moreover, the ongoing potential threat of bioterrorism underscores the critical need for continual development of effective anti-smallpox therapeutics [
4], [
5].
Tecovirimat, a small-molecule antiviral identified by screening thousands of candidates for activity against VARV [
6], [
7], exerts its antiviral effect by targeting the VP37 protein, an essential mediator of envelope formation and viral shedding, to inhibit the formation of egress-competent orthopoxvirus particles [
8]. In 2005, its original developer, SIGA Technologies, Inc., submitted an investigational new drug (IND) application for the oral formulation of tecovirimat [
9]. Based on the human safety and pharmacokinetic (PK) data, phase II clinical trials were conducted under the animal rules [
10] using two animal infection models: MPXV-infected nonhuman primates (NHPs) and rabbitpox virus (RPXV)-infected rabbits [
11], [
12], [
13]. A phase III clinical trial conducted in 2015 demonstrated that tecovirimat exposure levels in humans following administration were several times higher than those observed in the two animal models [
8], [
11]. Ultimately, these findings supported the approval of the oral formulation of tecovirimat by the US Food and Drug Administration (FDA) for smallpox treatment under the brand name TPOXX® capsules [
14]. TPOXX® capsules were subsequently approved in the European Union (EU), the United Kingdom (UK), and Japan for the treatment of various orthopoxvirus infections, including MPXV, VARV, CPXV, and vaccinia-related complications following smallpox vaccination [
15]. Additionally, an intravenous (IV) tecovirimat formulation was developed to accommodate diverse patient populations, including patients with swallowing difficulties. Tecovirimat is currently the only approved drug for the treatment of mpox infection, although its therapeutic efficacy and potential application against MPXV warrant further investigation [
16], [
17].
Tecovirimat is classified as a biopharmaceutics classification system (BCS) class II drug that is characterized by poor solubility and high permeability, which limit its industrial development and therapeutic performance. To overcome the poor solubility of the active pharmaceutical ingredient (API), TPOXX® injection formulations incorporate a high concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD), substantially exceeding the dose limits for excipients set by the European Medicines Agency (EMA) and FDA [
18], [
19], [
20]. Moreover, high-dose HP-β-CD is linked with an elevated risk of hemolysis, kidney-related adverse effects, and impurity leaching into the solution [
19]. Consequently, TPOXX® injections are contraindicated in patients with severe renal impairment (creatinine clearance below 30 mL∙min
-1) [
21] and require storage at 2-8 °C [
19].
In this study, we developed a novel tecovirimat formulation suitable for industrial development. This formulation incorporated the two conventional pharmaceutical excipients HP-β-CD and meglumine (MEG) at dosages that strictly met regulatory requirements. Furthermore, we investigated the intermolecular interaction mechanisms of this novel formulation using multiple analytical techniques. Stability evaluations demonstrated robust stability under various stress conditions, with the formulation remaining stable for at least 90 days at room temperature. Additionally, safety profiles were comprehensively investigated by assessing hemolytic potential, allergenic responses, irritant effects, and repeated-dose toxicity. Collectively, the findings demonstrate that the new formulation (preferred formulation, lyophilized powder formulation 5 (LP5)) exhibited an excellent safety profile and underscore its potential as a novel formulation for industrial development.
2. Materials and methods
2.1. Materials
Vero cells and VACV (Tiantan strain) were owned by Beijing Institute of Biological Products Co., Ltd. (China). MPXV (WIBP-MPXV-001 strain) was owned by Wuhan Institute of Biological Products Co., Ltd. (China) [
22].
Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Thermo Fisher Scientific Inc. (China), Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Wuhan Pricella Biotechnology Co., Ltd. (China), CellTiter-Glo® was purchased from Promega (Beijing) Biotech Co., Ltd. (China), dimethyl sulfoxide (DMSO) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (China), saline was purchased from Zhejiang Tianrui Pharmaceutical Co., Ltd. (China), and ovalbumin was purchased from Beijing Psaitong Biotechnology Co., Ltd. (China).
HP-β-CD was purchased from Xi’an Deli Biochemical Co., Ltd. (China), MEG was purchased from Shanghai Pincheng Pharmaceutical Technology Co., Ltd. (China), sodium hydroxide (NaOH) was obtained from Hunan Er-Kang Pharmaceutical Co., Ltd. (China), and acetonitrile was obtained from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (China). Potassium dihydrogen phosphate, phosphoric acid, and ammonium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All reagents used were of analytical grade and used without further purification, except for acetonitrile, which was of high-performance liquid chromatography (HPLC) grade. Ultrapure and deionized water were produced in-house. The composition of solvents for HPLC was shown in Table S1 in Appendix A.
Hartley guinea pigs (male and female, 21-27 days old, 315.0-396.0 g) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). New Zealand white (NZW) rabbits (male and female, 90-120 days old, 2.65-2.95 kg) were obtained from Suzhou Huqiao Biotechnology Co., Ltd. (China). Cynomolgus monkeys (3.41-4.35 years old, 2.35-4.3 kg, individual body weights and ages are listed in Table S2 in Appendix A) were obtained from Guangxi Hechi Deheng Biotechnology Co., Ltd. (China). All animal experiments were approved by the Animal Care and Use Committee of the Institutional Animal Facility of Medicilon Puhui Pharmaceutical Technology (Shanghai) Co., Ltd. (China). All animals were treated humanely, and measures were taken to minimize their suffering. The approval protocol numbers were 02098-24008 (active systemic anaphylaxis (ASA) study), 02098-24009 (in vitro hemolysis study), 02098-24012 (vascular irritation study), and 02098-24007 (repeated-dose toxicity study).
2.2. Preparation and characterization of tecovirimat
Tecovirimat was synthesized according to previously published literature [
23]. Briefly, intermediate I (IM1) was prepared from cycloheptatriene (SM1) and maleic anhydride (SM2) via a Diels-Alder reaction using xylene as the solvent. Intermediate II (IM2) was produced by condensing methyl-4-(trifluoromethyl) benzoate (SM3) with 85% hydrazine hydrate (SM4). Subsequently, IM1 and IM2 were then reacted in ethanol (EtOH), with
N,
N-diisopropylethylamine (DIPEA) used as the catalyst, to yield crude tecovirimat. Purification of the crude product afforded tecovirimat monohydrate as the final product. The detailed synthetic procedure and characterization data are provided in Sections S1 and S2 and Figs. S1-S6 in Appendix A.
The chemical structure of tecovirimat was characterized using 1H nuclear magnetic resonance (NMR), 13C NMR, 19F NMR, Fourier transform infrared (FT-IR), and high-resolution mass spectrometry (HR-MS).
2.3. Preparation of tecovirimat formulations
HP-β-CD, MEG, and other excipients were weighed and dissolved in a suitable volume of water at room temperature. Tecovirimat was subsequently weighed and added to the solution, and the total volume was adjusted to 100 mL with water. The resulting mixture was stirred for up to 24 h.
Tecovirimat lyophilized powder formulations (LPs) were prepared as follows:
Step 1: Pre-freezing stage: The solution was cooled to −40 °C in 2 h and maintained at −40 °C for 6 h.
Step 2: When the cold trap temperature reached −45 °C, the vacuum pump was started to initiate evacuation.
Step 3: Once the vacuum level stabilized below 15 Pa, the temperature was increased from −20 to 35 °C as follows: ① Initially, the temperature was raised to −20 °C in 4 h and maintained at this temperature for 16 h; ② the temperature was raised to −10 °C in 4 h and maintained for 16 h; ③ the temperature was raised to 5 °C in 1 h and maintained for 13 h; and ④ finally, the temperature was raised to 35 °C in 2 h and maintained for 6 h.
We developed a series of formulations. To evaluate the physicochemical characteristics, stability, and safety of the novel formulations, we selected the LP5 for further investigation.
2.4. Characterization techniques
Several analytical techniques were employed to characterize the structure of LP5. Morphology was examined via scanning electron microscopy (SEM) using a VEGA COMPACT microscope (TESCAN, Czech Republic). X-ray powder diffraction (XRPD) patterns were acquired using an ARL X’TRA companion X-ray diffractometer (Thermo Fisher Scientific, USA) equipped with Cu Kα radiation. Operating conditions were set as follows: voltage = 45 kV, current = 40 mA, and scan rate = 10 (°)∙min-1 within a 2θ angle range of 3°-50°. FT-IR analysis was performed using a Nicolet Summit LITE FTIR spectrometer (Thermo Fisher Scientific) across the spectral range of 4000-400 cm−1.
2.5. Stability
Various stability tests were performed for up to 90 days to evaluate the stability of the LP5. LP5 was placed in glass vials in a stability chamber under the conditions listed in
Table 1, and the samples were analyzed at predetermined intervals. The evaluated parameters included appearance, pH, drug content, related substances, and moisture content. Drug content and related substances were determined via HPLC using a Thermo Scientific UltiMate 3000 LC instrument (USA) equipped with an Agilent Zorbax Eclipse AAA column (3.0 mm × 150 mm, 3.5 μm; USA); additional details are provided in Section S3 in Appendix A. Moisture content was measured using the Karl Fischer titration method.
2.6. In vitro antiviral activity and cytotoxicity assays
The
in vitro antiviral activity of the in-house-synthesized tecovirimat was assessed using the cytopathic effect (CPE) method. Vero cells were seeded into 96-well plates (5 × 10
4 cells per well). Tecovirimat was dissolved in DMSO, and the viral multiplicity of infection (MOI) was set at 0.05 for MPXV and 0.005 for VACV [
7], [
22], [
24]. Antiviral activity against VACV or MPXV and tecovirimat-induced cytotoxicity were assessed across eight compound concentrations, starting at 2 μmol∙L
-1 for VACV or 1 μmol∙L
-1 for MPXV, followed by three-fold serial dilutions. Cell viability data were collected at 4 days post-infection (dpi) for VACV and at 3 dpi for MPXV. Half-maximal effective concentration (EC
50) and half-maximal cytotoxic concentration (CC
50) values were calculated by fitting the data using GraphPad Prism 10.2.1 to generate dose-response curves.
2.7. Safety evaluation of the novel formulation
The safety of LP5 was evaluated using an ASA test in guinea pigs, an
in vitro hemolysis assay using rabbit erythrocytes, a vascular irritation experiment in rabbit ear veins, and a repeated-dose toxicity study in cynomolgus monkeys. All experiments were performed in accordance with the guidelines of the National Medical Products Administration (NMPA), China [
25], [
26]. In the repeated-dose toxicity study, LP5 was diluted with saline to a concentration of 10 mL∙kg
-1, and the excipient control was prepared with the same method used for the high-dose LP5 group to ensure comparability. In the other safety studies, LP5 was diluted with saline to a final concentration of 3.5 mg∙mL
-1, and the excipient control was diluted with the same volume of saline as LP5 to maintain consistency.
2.7.1. ASA study in guinea pigs
Thirty-two Hartley guinea pigs (16 males and 16 females) were randomly assigned to four groups: a negative control group (saline; 0.5 mL per animal), positive control group (ovalbumin; 2 mg per animal), high-dose formulation group (LP5; 1.75 mg per animal), and low-dose formulation group (LP5; 0.88 mg per animal). For sensitization, the guinea pigs received intraperitoneal injections three times at two-day intervals. Challenge experiments were performed either 14 (designated D19) or 21 days (designated D26) after the final sensitization: The animals were administered IV injections via the foot vein at twice the corresponding sensitization dose. At D19 and D26, two females and two males per group were tested. The animals were observed for 60 min after administration to monitor allergic reactions.
Table 2 summarizes the observation indices for the animal responses.
2.7.2. In vitro hemolysis study using rabbit erythrocytes
Briefly, 20 mL of blood was collected from the jugular vein of a rabbit into a triangular flask containing glass beads, which was agitated for 10 min to remove fibrinogen. Following this, approximately ten volumes of saline were added to the mixture, which was mixed thoroughly and centrifuged at 1500 r·min−1 for 10 min at room temperature. The supernatant was removed, and the washing procedure was repeated until the supernatant became colorless. Finally, the red blood cells (RBCs) were resuspended in saline to prepare a 2% (v/v) erythrocyte suspension.
Fourteen clean glass test tubes were prepared and divided into two groups, as listed in
Table 3. A 2% erythrocyte suspension, saline, deionized water, and test formulations were added sequentially. The tubes were incubated in a temperature-controlled incubator at 36.9-37.1 °C for 3 h, following which hemolytic activity was visually assessed and the tubes were photographed.
2.7.3. Vascular irritation study in rabbit ear veins
Eight NZW rabbits (four rabbits per group), housed individually, were randomly assigned to either a negative control (saline; 5 mL∙kg
-1) or LP5 group (5 mL∙kg
-1). The test formulation or control was administered intravenously via the left auricular vein, with the right auricular vein serving as a contralateral control. Treatments were administered as a 60-min infusion twice daily (BID) for 7 consecutive days, followed by a 14-day recovery period. Thirty minutes after the final administration (D7) and at the end of the recovery period (D21), signs of irritation (redness, swelling, and congestion) at the injection sites and surrounding tissues were visually assessed and graded according to the criteria in
Table 4. At 72 h after the final administration (D10), one male and one female from each group were randomly selected and euthanized, while the remaining animals were euthanized on D21. Vascular morphological changes were evaluated by histological examination.
2.7.4. Repeated-dose toxicity study in monkeys
Forty cynomolgus monkeys were randomly divided into four groups (
Table 5): an LP5 excipient control, low-dose LP5 (10 mg∙kg
-1∙d
-1), medium-dose LP5 (20 mg∙kg
-1∙d
-1), and high-dose LP5 group (40 mg∙kg
-1∙d
-1). The test articles were administered via the femoral vein as a 4-h IV BID for 14 consecutive days, followed by a 28-day recovery period.
Three females and three males from each group were euthanized at the end of the dosing period (D15), whereas the remaining animals (two females and two males per group) were euthanized at the end of the recovery period (D43). Evaluations included monitoring mortality, cage-side observations, body weight and body temperature recordings, food consumption assessments, clinical pathology (hematology, serum biochemistry, coagulation indices, and urinalysis), gross pathology, organ weight measurements, and histopathological examinations.
Toxicokinetic (TK) blood samples were collected from LP5-treated animals pre-dosing (0 h) and 2, 4, 6, 8, 12, 14, 16, 18, 20, and 24 h post-dosing on the first (D1) and last (D14) dosing days. Plasma tecovirimat concentrations were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The TK parameters were derived using a non-compartmental analysis (NCA).
3. Results
3.1. Synthesis of tecovirimat
Tecovirimat was synthesized according to the steps outlined in Section 2.2, achieving a total yield of 34.2%. Its chemical structure was confirmed via comprehensive characterization, including 1H NMR, 13C NMR, 19F NMR, FT-IR, and HR-MS data, as shown in Figs. S1-S6.
3.2. In vitro antiviral activity and cytotoxicity of tecovirimat
Tecovirimat exhibits broad and potent antiviral activity against orthopoxviruses [
24], [
27], [
28]. As presented in
Fig. 1, the EC
50 values of the in-house-synthesized tecovirimat against MPXV and VACV were 2.3 and 8.3 nmol∙L
-1, respectively. Under the same experimental conditions, tecovirimat exhibited a CC
50 exceeding 1 μmol∙L
-1 in Vero cells, indicating therapeutic indices exceeding 400 for MPXV and 120 for VACV.
3.3. Development of tecovirimat formulations
The poor solubility of tecovirimat has substantially hindered its industrial development [
29]. To overcome this, we developed novel injectable formulations of tecovirimat. Cyclodextrins are widely employed as pharmaceutical excipients; for instance, HP-β-CD is commonly incorporated into formulations for poorly soluble drugs, such as in the TPOXX® injection, in which high concentrations of HP-β-CD are used to dissolve tecovirimat [
21], [
30]. However, these high HP-β-CD doses are associated with safety risks, including nephrotoxicity and hemolysis [
31]. Li et al. [
29] used MEG and HP-β-CD to solubilize tecovirimat by forming a ternary complex. However, the resulting excipient levels exceeded regulatory limits. Furthermore, although tecovirimat is a weakly acidic compound and bases can promote its solubilization, they can also increase impurity levels and compromise formulation stability. To overcome these challenges, we systematically investigated various excipients, ultimately selecting MEG and NaOH as alkaline additives combined with a controlled dose of HP-β-CD [
32], [
33], [
34]. We optimized the excipient ratios to comply with regulatory limits and developed a series of formulations that significantly enhanced the solubility of tecovirimat, achieving a maximum solubility of 50 mg∙mL
-1 (
Table 6).
3.4. Characterization of LP5
The solution prepared according to formulation 5 was lyophilized to yield a well-formed powder (LP5). To investigate the structure of LP5, SEM was employed to characterize tecovirimat, the individual excipients (HP-β-CD and MEG), and LP5. SEM images revealed that tecovirimat appeared as hexagonal crystals, HP-β-CD as porous spheres, and MEG as needle-like crystals (
Fig. 2). Conversely, the morphology of the LP5 sample differed markedly from that of the individual components, appearing as irregular flaky solids, indicating successful formation of the inclusion complex.
Formation of the inclusion complexes was further confirmed through additional characterization. XRPD analysis revealed distinct diffraction peaks for tecovirimat, the excipient mixture (HP-β-CD, MEG, and NaOH mixed in the ratio specified in formulation 5), a physically mixed powder (MP; tecovirimat combined with HP-β-CD, MEG, and NaOH in the same ratio as formulation 5), and LP5. Tecovirimat exhibited sharp diffraction peaks at 2
θ = 6.63°, 9.02°, 12.96°, 16.24°, 16.66°, 18.11°, 21.02°, and 21.39°, confirming its crystalline structure (
Fig. 3). The excipient mixture exhibited a weak peak at 2
θ of 8.92°, and two broad peaks within the 2
θ of 5°-15° and 15°-25° ranges. Conversely, the XRPD pattern of MP reflected a simple superposition of the tecovirimat and excipient mixture peaks, whereas LP5 exhibited a distinct double-hump profile. This pattern further supports the formation of a new inclusion complex rather than mere physical blending.
FT-IR spectroscopy was employed to analyze the characteristic absorption peaks of tecovirimat, MEG, HP-β-CD, and LP5 (
Fig. 4). The results demonstrated that compared with the spectra of tecovirimat or MEG, the characteristic peaks in LP5 shifted and their intensities weakened. Specifically, the C=O absorption peak of the amide group underwent a red shift from 1718 cm
−1 in tecovirimat to 1684 cm
−1 in LP5, and the N-H stretching absorption peaks observed at 3469 cm
−1 (in tecovirimat) and 3331 cm
−1 (in MEG) were absent in LP5; instead, only a broad -OH band from HP-β-CD at ∼3398 cm
-1 was observed. Notably, absorption peaks in the fingerprint region of tecovirimat were not detected in LP5. Taken together, these data suggest that new intermolecular interactions were formed between the components of LP5, leading to complexation.
3.5. Stability of LP5
The stability of LP5 was evaluated through stress testing, accelerated testing, and long-term testing. Stress testing included exposure to high temperature (60 °C), high room humidity (RH; 92.5%), and photolytic conditions ((4500 ± 500) lux), with samples tested for up to 30 days. As shown in
Table 7, under high-temperature conditions, the drug content ranged from 95.8% to 96.5%, the maximum single impurity ranged from 0.012% to 0.026%, the total impurities ranged from 0.039% to 0.089%, the moisture content ranged from 0.088% to 0.180%, and the pH remained stable. Under the 92.5% RH condition, the drug content was 96.4%-96.9%, the maximum single impurity was 0.012%-0.018%, the total impurities were 0.039%-0.047%, and the moisture content was 0.088%-0.120%, with the pH remaining stable. Under the photolytic exposure, the drug content ranged from 95.9% to 97.6%, the maximum single impurity ranged from 0.011% to 0.018%, the total impurities ranged from 0.039% to 0.044%, the moisture content ranged from 0.088% to 0.110%, and the pH remained consistent and stable throughout the test period. Accelerated and long-term tests were conducted for up to 90 days. As summarized in
Table 8, in the accelerated test, the tecovirimat content ranged from 97.2% to 99.3%, the maximum single impurity ranged from 0.011% to 0.017%, the total impurities ranged from 0.028% to 0.052%, the moisture content ranged from 0.074% to 0.140%, and the pH remained stable without significant fluctuations. In the long-term test, the tecovirimat content was 97.9%-98.7%, the maximum single impurity was 0.012%-0.016%, the total impurities were 0.028%-0.036%, and the moisture content was 0.074%-0.100%, with no notable changes in pH. Collectively, these results indicated that the developed LP5 formulation exhibits excellent chemical and physical stability.
3.6. Safety evaluation of LP5
3.6.1. ASA study in guinea pigs
Following the final sensitization, challenge experiments were conducted either 14 (D19) or 21 days (D26). A double dose of the same test article used during sensitization (namely saline (negative control group), ovalbumin (positive control group), or LP5 (high- and low-dose LP5 groups)) was administered via IV injection into the foot vein. Within 8 min of the first (D19; two females and two males per group) or the second challenge (D26; two females and two males per group), the positive control group exhibited symptoms including restlessness, nose rubbing, piloerection, unsteady gait, and respiratory distress, ultimately resulting in death (severe positive allergic reaction). Conversely, no allergy-related symptoms were observed in either the LP5 or negative control groups, indicating that LP5 did not induce notable allergic reactions. Allergy grading results for each group are summarized in
Table 9. Furthermore, although not a primary endpoint of the ASA study, the body weights of surviving animals increased steadily across all groups throughout the study period (Fig. S7 in Appendix A).
3.6.2. In vitro hemolysis study in rabbit erythrocytes
After 3 h of incubation, hemolysis was observed only in the positive control group (A7 and B7), as shown in
Fig. 5. No hemolysis was detected in the negative control (A6 and B6), LP5 (B1-B5), or excipient control (A1-A5) groups. Overall, these findings indicate that LP5 exhibited no significant hemolytic activity.
3.6.3. Vascular irritation study in rabbit ear veins
Irritation at the injection sites was assessed 30 min after the final administration (D7) and at the end of the recovery period (D21), with irritation scores assigned to all surviving animals. As shown in
Table 10, no significant vascular irritation was observed at the injection sites in any of the groups. Additionally, histopathological analysis (Fig. S8 in Appendix A) revealed that 72 h after the final administration (D10), perivascular inflammatory cell infiltration, fibrosis, thrombosis, organization (fibrous tissue formation), and recanalization were present at the left ear vein administration sites in both the negative control and LP5 groups. These lesions were deemed as common background findings associated with IV administration and were not attributed to the test articles. By D21, these lesions, as well as perivascular inflammatory cell infiltration, fibrosis, thrombosis, organization, and recanalization, had resolved. Collectively, these results indicate that LP5 did not cause significant vascular irritation.
3.6.4. Repeated-dose toxicity study in cynomolgus monkeys
To comprehensively evaluate the potential toxic effects of LP5, we conducted a repeated-dose toxicity study in cynomolgus monkeys, encompassing multiple parameters. As shown in
Table 11,
Fig.6, and Figs. S9-S14 and Tables S3-S5 in Appendix A, no drug-related toxicities were observed.
The average recovery of tecovirimat concentrations in LP5 samples prepared at the start and end of the study ranged from 95.9% to 98.5%, with a relative standard deviation (RSD) ≤ 0.56%. No analyte was detected in the excipient control samples, which complied with the acceptance criteria.
Nearly all animals survived until their scheduled euthanasia. One male animal in the low-dose LP5 group (10 mg∙kg-1∙d-1) developed abnormal symptoms on D7, including reduced activity, lethargy, and convulsions, consequently requiring euthanasia followed by necropsy. Gross and histopathological examinations indicated that the immediate cause of death was respiratory failure induced by idiosyncratic pulmonary edema. We attributed this event to an idiosyncratic, severe stress response rather than drug-induced toxicity based on the following reasons: ① absence of dose dependency; ② lack of supporting drug-induced toxicological findings, such as hematological or serum biochemical indices and multi-organ histopathological lesions; and ③ presence of stress-associated pathological markers such as adrenal hypertrophy and thymic atrophy.
No significant differences in body weight or food consumption were observed between groups. Transient decreases in body weight (observed on D10), food intake (on D11), and body temperature (on D2) were observed across all groups (Fig. S9). Given the absence of dose dependency, lack of other related adverse effects (such as diarrhea), and subsequent rapid recovery of these parameters, these changes were attributed to a stress response associated with factors such as the infusion procedure and environmental conditions.
Clinical pathological examination revealed no test article-related toxicity. Specifically, although some fluctuations in eosinophil counts (absolute count (#EO) and relative proportion (%EO)) were observed in female animals in the high-dose LP5 group (
Fig. 6), these changes occurred before LP5 administration and were unrelated to the treatment. Additionally, slight changes in biochemical parameters (alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), globulin (GLO), glucose (GLU), Na, P, and total protein (TP)) were observed following drug administration but remained within the historical reference range (Fig. S10). No abnormal changes were observed in coagulation indices or urinalysis findings across all groups (Figs. S11 and S12).
Organ weight data for the animals at the end of the dosing period (D15) are summarized in Tables S3 and S4. In the high-dose LP5 group, male animals exhibited an approximately 40% increase in adrenal gland weight compared with the control group. However, as no histopathological changes were observed and there was no sex-related trend, this finding was considered unrelated to LP5 treatment. This isolated increase in adrenal gland weight in the high-dose males had no systemic impact on overall organ weights, and no significant changes in the weights of other organs were observed.
Histopathological examination revealed primary abnormalities across all groups, including thymic atrophy, lymphocyte depletion in the splenic white pulp (Fig. S13), perivascular hemorrhage at the injection site, local inflammatory responses, tissue damage with reparative changes, and vascular wall destruction and repair (Fig. S14). Comprehensive analysis indicated that thymic and splenic alterations were related to stress responses, whereas injection site changes were attributable to repeated mechanical stimulation during prolonged IV administration. Both were unrelated to LP5 administration. Notably, these lesions largely subsided after the 4-week recovery period.
TK parameters (listed in Table S5) revealed that tecovirimat plasma exposure in both male and female animals increased with increasing dose and demonstrated an approximately dose-proportional relationship. No significant sex-related differences were observed in plasma tecovirimat exposure between male and female animals, and no significant tecovirimat accumulation was observed across all dose groups.
In summary, the no-observed-adverse-effect level (NOAEL) for LP5 in this study was determined to be 40 mg∙kg-1∙d-1 (the highest tested dose). On D14, plasma area under the concentration-time curve (AUC(0−t)) values for tecovirimat in male and female animals were 96 453 and 82 201 ng·h∙mL-1, respectively.
4. Discussion
The emergence of a global mpox epidemic in mid-2022 prompted the World Health Organization to declare it a “Public Health Emergency of International Concern” in July 2022 and again in August 2024. Tecovirimat, as the first approved treatment for VARV infection and the only approved drug for MPXV infection, has been widely used in clinical practice. However, the efficacy of tecovirimat against human mpox remains unclear. The PALM007 trial (NCT05559099), a randomized placebo-controlled study conducted in the Democratic Republic of the Congo, reported that oral tecovirimat did not significantly shorten the time to lesion resolution in patients with clade I MPXV infections [
35]. Furthermore, subgroup analysis revealed that patients with MPXV may derive clinical benefit when treated earlier or in severe cases, which is consistent with previous reports [
36], [
37]. Notably, the PALM007 trial also revealed suboptimal drug exposure, with approximately 35% of patient samples failing to reach the minimum effective concentration [
35]. Consistent therapeutic exposure is paramount for the efficacy of antiviral agents. This variability in exposure is likely linked to the poor solubility of tecovirimat (BCS class II) [
19], which leads to inconsistent oral bioavailability. This challenge underscores the need for reformulation strategies such as IV administration to yield more reliable drug levels. Notably, the currently marketed tecovirimat injection is contraindicated in patients with severe renal impairment because the amount of the excipient HP-β-CD exceeds regulatory limits. Therefore, this study aimed to develop a novel, enhanced tecovirimat injection formulation suitable for patients with orthopoxviral infections, including those with severe renal impairment.
To this end, we first synthesized tecovirimat and characterized its structure using
1H NMR,
13C NMR,
19F NMR, FT-IR, and HR-MS. Following this, we evaluated the antiviral efficacy of the compound against various orthopoxviruses using a CPE protection assay, demonstrating that the resulting EC
50 values (8.3 nmol∙L
-1 for VACV and 2.3 nmol∙L
-1 for MPXV) were consistent with previously reported values (5-51 nmol∙L
-1 for VACV [
24], [
38], [
39] and 1-39 nmol∙L
-1 for MPXV [
24], [
40]). To improve the solubility of tecovirimat, we developed novel formulations incorporating HP-β-CD, MEG, and NaOH as excipients, which markedly enhanced the maximum solubility of tecovirimat to 50 mg∙mL
-1. We demonstrated that tecovirimat could only be completely dissolved when all three excipients were used in combination, within regulatory requirements; omitting any one of these excipients led to incomplete dissolution. SEM and XRPD analyses were used to characterize the LP5 to elucidate the underlying solubilization mechanisms, revealing that the crystalline characteristics of tecovirimat and MEG disappeared, indicating the formation of an amorphous inclusion complex. Consistent with this, FT-IR spectroscopy provided evidence of intermolecular interactions within this complex. Based on these findings and the relevant literature, we propose that the novel composite forms through the following mechanism: In the formulation, tecovirimat interacts with NaOH and is converted into a soluble sodium salt, thereby enhancing its aqueous solubility and facilitating the subsequent inclusion process. The hydrophobic portion of tecovirimat then enters the cavity of HP-β-CD, forming a soluble inclusion complex primarily stabilized by van der Waals forces and hydrophobic interactions. Similar findings have been reported in other studies: Phase solubility studies showed that the solubility of tecovirimat increased significantly with increasing HP-β-CD concentrations, supporting the formation of an inclusion complex [
41]. Moreover, MEG may function as a bridging component that further stabilizes the inclusion complex through hydrogen bonding and other noncovalent interactions [
29], [
32]. Notably, the dosage of HP-β-CD in the TPOXX® injection is 267 mg∙kg
-1∙d
-1, whereas in LP5 it is 100 mg∙kg
-1∙d
-1 (based on a 60 kg adult) [
21], which is well below the maximum EMA-permitted level of 200 mg∙kg
-1∙d
-1 [
18], [
19], [
20]. Furthermore, the dosages of both MEG and NaOH in LP5 are each 40 mg∙d
-1 for a 60 kg adult, which is below the FDA-stipulated limits of 75 mg∙d
-1 for MEG and 78 mg∙d
-1 for NaOH, confirming full compliance with regulatory standards [
20].
To further determine whether the LP5 formulation is suitable for industrial development, its stability was assessed. The results revealed that LP5 exhibited no significant changes in drug content, maximum single impurities, total impurities, moisture under high temperature, high humidity, and photolytic exposure. Accelerated and long-term stability tests indicated that following 90 days of storage, the key quality parameters remained within acceptable limits relative to their initial values. Taken together, these findings indicate that LP5 exhibits excellent stability and is suitable for storage at room temperature.
For antiviral drugs, a key criterion for efficacy is that the
in vivo exposure levels of the drug should be significantly higher than the
in vitro EC
50 [
42], [
43]. To characterize the exposure profile of LP5, we conducted a PK study in cynomolgus monkeys, a pharmacologically relevant species analogous to humans and the primary species employed in previous tecovirimat efficacy testing (data not shown). Comparative analysis of LP5 PK data against the
in vitro EC
50 values of tecovirimat for orthopoxviruses and published IV formulation data [
44] revealed that following LP5 administration, plasma tecovirimat concentrations remained above the
in vitro anti-orthopoxvirus EC
50 for at least 12 h. These concentrations substantially exceeded the levels required for effective
in vitro viral clearance. Furthermore, LP5 achieved exposure parameters comparable to those of IV tecovirimat at the identical dose. These PK/pharmacodynamic (PD) findings support the potential of LP5 to mitigate the suboptimal efficacy of oral tecovirimat by ensuring consistent and sustained therapeutic exposure.
Finally, we evaluated the safety of the formulation using ASA, hemolysis, vascular irritation, and repeated-dose toxicity studies. The ASA test revealed that LP5 did not induce allergic reactions in guinea pigs, while the in vitro hemolysis study showed no significant hemolytic risk at a clinical concentration of 3.5 mg∙mL-1. The vascular irritation test demonstrated that following seven days of administration, mild redness and swelling were observed at the injection sites in rabbits; however, this was primarily attributed to the injection procedure rather than the formulation itself, and the symptoms fully resolved within the 14-day recovery period. In the repeated-dose toxicity study, one fatality was recorded: A male cynomolgus monkey in the 10 mg∙kg-1 group that was euthanized on D7 owing to stress-induced pulmonary edema, leading to respiratory dysfunction. Comprehensive assessments of body weight, body temperature, food consumption, clinical pathology, and organ weight across all dosing groups revealed no LP5-induced toxic changes. Histopathological examination revealed no abnormalities, aside from stress responses in a few organs. At the injection site, reactions, such as fibrous hyperplasia and tissue edema, were observed that were attributed to mechanical stimulation associated with the administration procedure. However, these abnormalities resolved after the recovery period. Furthermore, no significant drug accumulation was observed following repeated dosing. Therefore, the study determined the NOAEL to be 40 mg∙kg-1∙d-1.
5. Conclusions
In summary, we developed a novel formulation that markedly enhanced the solubility of tecovirimat for industrial development. First, tecovirimat was synthesized and confirmed to exhibit broad-spectrum anti-orthopoxviral activity without any detectable cytotoxicity. Second, by incorporating HP-β-CD, MEG, and NaOH as excipients, the solubility of tecovirimat was substantially enhanced. Notably, all excipients complied with the relevant regulatory standards, and their specific ratios were critical for ensuring complete tecovirimat dissolution and formulation safety. Third, stability studies, including stress testing (high temperature, high humidity, and photolysis), as well as accelerated and long-term studies, demonstrated robust stability, supporting storage, transportation, and use at room temperature. Finally, based on the results from the ASA, hemolysis, vascular irritation, and repeated-dose toxicity studies, we concluded that LP5 exhibited an excellent efficacy and safety profile. Overall, this study developed a novel tecovirimat formulation with robust stability, high safety, and proven efficacy, making it suitable for clinical applications.
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