The synthesis of IDD-1040 was performed as detailed in our previous study (25). Following synthesis, IDD-1040 (100 µg) was stored under a nitrogen atmosphere at −18°C until subjected to analysis. Liquid chromatography-mass spectrometry (LC/MS)-grade acetonitrile (J.T. Baker; Thermo Fisher Scientific, Inc.) and LC/MS-grade methanol and water (Bio-Lab Ltd.) were utilized for the analytical experiments. The high-performance liquid chromatography (HPLC) cartridge employed was LiChroCART^® 250-4 LiChrospher^® 100 RP-18 (5 µm), while a LiChroCART^® 4-4 LiChrospher^® 100 RP-18 (5 µm) guard column was used to safeguard the integrity of the analytical column (both from Merck KGaA).

High Performance Liquid Chromatography (HPLC) system (Thermo Fisher Scientific, Inc.) included an Accela pump with a degasser module, an Accela autosampler and an Accela photodiode-array (PDA) detector. To record the mass spectrum, the HPLC system was connected to a TSQ Quantum Access Max triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific, Inc.) via a heated electrospray ionization (H-ESI) interface. Data acquisition and processing were performed using Xcalibur^™ software version 4.3 (Thermo Fisher Scientific, Inc.).

Optimal chromatographic separation was achieved via the use of an isocratic mobile phase consisting of a binary solvent mixture of methanol:water, 82:18, v/v, at a constant flow rate of 1.0 ml/min, with a total run time of 7 min. The chromatographic oven temperature was maintained at 40°C, while the autosampler tray temperature was carefully controlled at 5°C. Samples with a volume of 5 µl were introduced into the column via a partial loop injection system, and the injection needle was thoroughly cleansed with 1 ml methanol:water, 80:20, v/v, between successive injections to prevent cross-contamination. The initial investigation of the optimal UV detection wavelength was conducted using the PDA detector within a spectral range of 200–300 nm. Final quantification was carried out at a fixed wavelength of 225 nm.

The mass spectrometer was configured to operate in positive ionization mode. The key operational parameters included an ionization spray voltage of 3.0 kV, a skimmer offset of 0 V and a capillary transfer tube temperature of 350°C. The nitrogen sheath gas and auxiliary gas flow rates were set at 35 and 30 l/min, respectively. The vaporizing temperature in the H-ESI source was maintained at a consistent 250°C, and a rapid scan time of 0.1 sec was employed.

Stock solution A (1.0 mg/ml) was prepared by dissolving 10.0 mg IDD-1040 in acetonitrile in a 10.0-ml volumetric flask. Following this, stock solution B (0.1 mg/ml), was generated by diluting 1.0 ml stock solution A with acetonitrile in a 10.0-ml volumetric flask. These stock solutions were stored in a freezer at −20°C. To construct the linear calibration curve, eight standards were created, at concentrations of 10, 20, 50, 100, 200, 500, 750 and 1,000 µg/ml. To ensure accuracy, the calibration samples were meticulously prepared in duplicate and analyzed in duplicate as well.

To initiate the experiment, cold tubulin polymerization buffer was prepared as follows. A 750-µl volume of general tubulin buffer was combined with a 250-µl volume of tubulin glycerol buffer, and 10 µl GTP stock solution (100 mM) was added to the mixture. The general tubulin buffer was warmed to room temperature to facilitate tubulin ligand dilution in the next step.

Tubulin (100 µl) was rapidly thawed and diluted with 210 µl tubulin polymerization buffer to achieve a final concentration of 3 mg/ml.

The following samples were prepared: General tubulin buffer (negative control); PTX at a final concentration of 100 µM (positive control), as specified in the kit; IDD-1040 at a final concentration of 100 µM; and IDD-1040 at a final concentration of 500 µM. Then, 30 µl of each sample was added to 270 µl diluted tubulin.

The spectrophotometric analysis was conducted with an Evolution 300 spectrophotometer (Thermo Fisher Scientific, Inc.) equipped with a controlled heating system. Cuvettes with a volume of 750 µl were used. The samples were incubated at 37°C, and absorbance readings were taken at 340 nm for a total duration of 10 min, for 60 cycles of 10 sec each.

The in vivo experiment was performed by Pharmaseed Ltd., a good laboratory practice (GLP)-certified laboratory, following the guidelines of the Organisation for Economic Co-operation and Development Principles of GLP [C(97)186/final]. Animal handling was performed in accordance with the guidelines of the National Institutes of Health and the Association for Assessment and Accreditation of Laboratory Animal Care. The study was performed after approval by the Israel Board for Animal Experiments (approval no. GB06/68708), in compliance with the Israel Animal Welfare Act. The formulation used for in vivo studies consisted of 6 mg/ml of the active compound in solution containing 527 mg Cremophor EL (polyethoxylated castor oil surfactant) and 49.7% (v/v) dehydrated alcohol. Before administration, the solution was diluted to 0.3–1.2 mg/ml.

In the study, 39 CD-1 nude mice were divided into 13 groups (n=3/group), with each group representing a different time point. Mice were anesthetized with inhalational isoflurane, with induction using 3–4% isoflurane in oxygen and maintenance using 1–2% isoflurane in oxygen prior to dosing. Then, an intravenous bolus of 50 mg/kg IDD-1040 (5 mg/ml solution, 10 ml/kg dose volume) was administered via the tail vein. Blood samples were collected at time points of 0 h (pre-dose), 5 min, and 1, 3, 6, 8, 10, 12, 24, 48, 72, 120 and 168 h post-dose from the tail vein. A sparse sampling schedule was used in which 3 mice were tested at each time point and two samples were collected from each mouse. The mice were anesthetized by isoflurane inhalation prior to blood withdrawal. The blood samples were transferred to a heparinized tube and centrifuged at 7,500 × g at 4°C for 10 min. The resulting plasma was separated and kept frozen at −80°C prior to analysis. The plasma samples were analyzed for detection of the prodrug IDD-1040 and its PTX hydrolysis product. Pharmacokinetic parameters (Tables I and II) were computed from the data using PK Solutions-2 software, version 2 (Summit Research Services). The clearance (Cl) and volume of distribution (Vd) for IDD-1040 were calculated as follows: Cl=dose/area under the concentration-time curve) and Vd=Cl/ke (elimination rate constant).

Development of IDD-1040 formulations: A formulation development experiment was conducted to address the poor aqueous solubility of IDD-1040. Initially, the solubility of IDD-1040 was assessed in various excipients suitable for parenteral administration, including dimethylacetamide (DMA), propylene glycol (PG), polyethylene glycol 400 (PEG 400), and ethanol. Subsequently, different formulations were prepared using these excipients, along with cosolvents, surfactants, and cyclodextrin. The excipients and their precise quantities for each formulation type are detailed in Tables III and IV.

One-way ANOVA followed by Tukey's HSD post-hoc test was employed to analyze the results of in vivo pharmacokinetic study using SPSS version 29 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

The purity of IDD-1040 used in the present study was assessed using HPLC and LC-MS analyses. A single symmetric eluting peak is evident in the chromatogram, with a retention time of 5.39 min (Fig. 2). The R² value of 0.9901 indicates excellent linearity, confirming the method's reliability for quantifying IDD-1040 within the tested concentration range. ESI-MS analysis revealed the presence of the sodiated stable adduct ion [M+Na]⁺ as the prominent peak, at an m/z value of 1,064 Da. Additionally, another, weaker peak, corresponding to the protonated IDD-1040 conjugate [M+H]⁺, is present, with a m/z value of 1,042 Da (Fig. 3).

Female mice were intravenously dosed with a bolus of 50 mg/kg IDD-1040, and blood samples were collected at 13 time points, ranging from 0 h to 7 days. Plasma from these blood samples was analyzed to determine the concentration of the pro-drug IDD-1040 and its hydrolysis product, PTX, at each time point. Given the extended observation period, encompassing the metabolism and elimination of IDD-1040, the concentrations spanned six orders of magnitude. Consequently, a natural log-common log (ln-log) conversion was applied to facilitate the visualization of all the data collectively (Fig. 4A). Three distinct phases can be observed. First, the initial 1 h following administration is the distribution phase of the drug. Second, between 1 and 10 h post-injection, the drug undergoes a phase characterized by both metabolism and elimination. Third, the final phase is the terminal elimination phase, extending to 5 days. A parallel pattern is observed when the ln-log transformation of concentration data for the hydrolysis product PTX is examined. Up to 10 h, a primary phase marked by an increase in PTX concentration in the blood is evident, followed by a terminal elimination phase extending up to 5 days following the administration of IDD-1040 (Fig. 4B).

The line graphs in Fig. 4C and D show the initial and terminal phases of the pharmacokinetics of IDD-1040. Both phases are shown as exponential decay curves from which corresponding rate constants (k values) can be determined. Parallel representations of the PTX profiles are presented in Fig. 4E and F. Pharmacokinetic parameters were computed from the data using PK Solutions-2 software version 2 and are presented in Tables I and II.

As indicated by the area under the curve (AUC) values in Table I and the calculated using the corresponding pharmacokinetic equations, the clearance for IDD-1040 is 1.689 l/h.kg, while the volume of distribution is 1.93 l/kg. This distribution exceeds the volume of systemic blood by >30-fold, which suggests extensive tissue distribution beyond the bloodstream. Utilizing these parameters, a half-life of 1.14 h was calculated for IDD-1040 in the central elimination phase, which is notably shorter than the terminal elimination half-life of 8.64 h.

Given that PTX is derived from the administration of IDD-1040, it is not feasible to estimate its clearance and volume of distribution using the data obtained (Table II). A notable observation arises when the data for IDD-1040 and PTX are compared: The AUC of IDD-1040 is >14-fold higher than that of PTX. This suggests that the conversion of IDD-1040 into its metabolite occurs slowly, and hints at the possibility that some of the biological activity previously observed for IDD-040 may be attributed to the prodrug itself.

While the data on the half-life of PTX in the literature vary and are challenging to compare against (28,29), it is generally noted that PTX exhibits a significantly shorter half-life than IDD-1040. Consequently, it is evident that the extended half-life of IDD-1040 observed in the current study was associated with the continuous release of PTX by the prodrug IDD-1040.

The results of the in vitro tubulin polymerization assay used to evaluate the MoA of IDD-1040 are presented in Fig. 5. The assay was conducted using PTX as a positive control, along with IDD-1040 at two different concentrations. The results demonstrated that PTX at a concentration of 10 µM increased tubulin polymerization by ~41.8% compared with that of the negative control. In comparison, IDD-1040 at a concentration of 50 µM increased tubulin polymerization by only ~9.1% compared with that of the negative control. The tubulin-binding characteristics exhibited by IDD-1040 appear to differ from those of PTX, suggesting a different MoA. The results of this assay provide valuable insights into the MoA of IDD-1040 and its interaction with tubulin, highlighting its potential as an anticancer agent with distinctive properties.

The development of an effective formulation for a therapeutic agent with low aqueous solubility, such as IDD-1040, is a critical aspect of drug delivery, as it can markedly improve the pharmacokinetics and therapeutic efficacy of the drug. An aim of the present study was to improve the formulation of IDD-1040 to address the limitations associated with its current formulation, which comprises a vehicle primarily composed of Cremophor EL and dehydrated alcohol. As the aqueous solubility of PTX is very low, Cremophor EL was included in the formulation to increase the solubility of PTX. However, it is toxic and causes serious side effects (17), such as hypersensitivity, myelosupression, nephrotoxicity and neurotoxicity.

As aforementioned, IDD-1040 has low aqueous solubility and lacks ionizable functionalities, which renders pH adjustment ineffective for increasing its solubility; therefore, it is crucial to find another means of improving its delivery. Using the current formulation, the maximal concentration that resulted in a clear solution was 0.4 mg/ml; higher drug concentrations were milky. In an attempt to achieve a suitable parenteral formulation for IDD-1040, a formulation development experiment was conducted. In the first stage, the solubility of IDD-1040 in excipients suitable for parenteral administration was tested. IDD-1040 was found to be soluble in dimethylacetamide (DMA; 100 mg/g), propylene glycol (PG; 16 mg/g), polyethylene glycol 400 (PEG 400; 10 mg/g) and ethanol (20 mg/g). This profile differs from that reported in the literature for PTX, which is considered to be insoluble (<0.1 mg/ml) in PG and PEG 400 (30). The solvents found to solubilize the drug, together with cosolvents, surfactants and cyclodextrin, were then used to prepare and test several different IDD-1040 formulations. These were diluted with either water or Lipofundin (a commercial parenteral nutrition nanoemulsion). A total of 31 formulations diluted with water were found to be clear after preparation (Table III). These included 1–3 mg/g formulations using solvents and cyclodextrin. Formulations with higher drug concentrations (4–10 mg/g) containing the surfactant Tween^™ 80 were also clear and exhibited a 25-fold increase in solubility compared with that of the reference formulation. When the drug was solubilized in solvent mixtures followed by dilution with Lipofundin instead of water, higher drug concentrations could be achieved (12 mg/g), resulting in visually homogeneous formulations (Table IV) with a high drug content. These were obtained without the use of surfactants and, overall, had fewer excipients compared with the formulations diluted with water.

Based on these results, the following are the key findings and their implications for the development of IDD-1040 formulations.

The investigation began with an assessment of the solubility of IDD-1040 in various excipients suitable for parenteral administration. Notably, appreciable solubility was observed in DMA, PG, PEG 400 and ethanol. This solubility profile differs slightly from that of PTX, its parent compound, which is generally considered to be insoluble in PG and PEG 400. Understanding these solubility characteristics is crucial for formulating an effective delivery system.

In the subsequent stages of the present study, to develop a suitable parenteral formulation, various agents that had the potential to enhance the solubility of IDD-1040, including co-solvents, surfactants and cyclodextrin, were explored.

It was observed that the formulations containing IDD-1040 in combination with the solvents DMA, PG, PEG 400 and ethanol resulted in clear solutions at drug concentrations of 1–3 mg/g. This suggests that co-solvent systems have the potential to increase the solubility of IDD-1040, which presents a method for improving the formulations.

The addition of Tween 80, a surfactant, resulted in an increase in IDD-1040 solubility in certain formulations, particularly at higher drug concentrations (4–10 mg/g). This increase in solubility indicates that this as a promising strategy for the development of formulations with a higher drug content and enhanced therapeutic efficacy.

Cyclodextrin-based formulations also warrant consideration, as they yielded clear solutions at certain drug concentrations. The use of cyclodextrin may offer an alternative route for the improvement of IDD-1040 formulations.

Notably, when the drug was solubilized in solvent mixtures and diluted with Lipofundin, higher drug concentrations (12 mg/g) could be achieved, resulting in visually homogeneous formulations. This approach is particularly appealing, as it achieves high levels of drug content without the need for additional surfactants and can reduce the amount of excipient required.

The findings for the formulations prepared in the present study have important clinical implications. The low solubility and toxic nature of Cremophor EL, which is included in the current Taxol^® formulation, have been associated with serious side effects. The successful development of alternative IDD-1040 formulations that have improved solubility and do not contain Cremophore EL should mitigate these side effects and increase patient safety. Furthermore, the ability to achieve higher drug concentrations in formulations is crucial for optimizing the therapeutic effects of IDD-1040. This should enable the reduction of dosing volumes and improve convenience of administration for patients.

In the present comprehensive study, the compound IDD-1040, formed through the conjugation of lipoic acid and PTX, was investigated. The study has provided valuable insights into the analytical, pharmacokinetic and pharmacological aspects of IDD-1040, which aid in understanding the improved antitumor efficacy it has previously demonstrated compared with PTX alone. Pharmacokinetic analysis using PK Solutions-2 software revealed key parameters that characterize the behavior of IDD-1040 in vivo. When intravenously administered to mice, IDD-1040 exhibited a total clearance of 1.689 l/h.kg, a volume of distribution of 1.93 l/kg, an average half-life of 1.14 h, and a terminal half-life of 8.64 h. Particularly noteworthy is the markedly (>14-fold) higher AUC of IDD-1040 compared with that of PTX, indicating a slower conversion of IDD-1040 to its metabolites. This suggests that a considerable proportion of the antitumor activity of IDD-1040 can be attributed to the prodrug. The substantial volume of distribution observed for IDD-1040 reflects efficient absorption into tissues in the secondary compartment, possibly contributing to its enhanced antitumor effects. Furthermore, the results revealed a notably extended half-life of PTX when delivered via IDD-1040, indicating sustained release from the prodrug, which should offer a prolonged therapeutic effect and contribute to its enhanced therapeutic potential. To gain further insights into the MoA of IDD-1040, an in vitro tubulin polymerization assay was conducted, in which IDD-1040 was compared with PTX. This assay indicated that tubulin-binding by IDD-1040 is much lower than that by PTX, suggesting a unique MoA for the prodrug and highlighting its potential as an innovative anticancer agent.

Despite the extremely lipophilic nature of IDD-1040, its formulation with solvent mixtures and Lipofundin provided a reasonably high drug concentration (12 mg/g) with few excipients and no surfactant, indicating its potential for future use. Certain formulations explored show promise in improving the solubility and formulation of IDD-1040 and provide a foundation for further research aimed at optimizing the drug delivery of IDD-1040 and enhancing its therapeutic outcomes while minimizing toxicity concerns. In future studies, it is essential to assess the stability, pharmacokinetics and pharmacodynamics of these IDD-1040 formulations to determine their suitability for clinical use. Additionally, the compatibility of these formulations with different routes of administration requires investigation to explore their versatility for addressing various clinical scenarios.

IDD-1040 represents a marked advancement in cancer therapy, offering improved pharmacokinetics, enhanced anticancer efficacy and reduced side effects compared with conventional PTX formulations. The extended circulation, efficient tissue distribution, reduced metabolite formation and unique MoA of IDD-1040, as revealed through the in vitro tubulin polymerization assay, underscore the importance of further investigations into the MoA and therapeutic potential of IDD-1040. These findings strongly support the promise of IDD-1040 as a candidate for cancer treatment. Given its potential to make a substantial impact in the field of oncology, its advancement into clinical trials is highly recommended. Future studies should focus on optimizing the formulation, stability and clinical application of IDD-1040 to harness its full therapeutic potential.