(M1130-07-42) Prevention of Lung Cancer Using Bio-Enhanced Sulfasalazine and Disulfiram Nano Self-Emulsifying Drug Delivery System Platform: Formulation Development, Pharmacokinetics, and Biodistribution Study

Purpose: Lung cancer is the second most commonly diagnosed malignancy and the foremost cause of cancer-related deaths, underscoring an urgent need for innovative and effective lung cancer prevention interventions. Previous investigations by our group have identified a unique lung cancer prevention efficacy using a repurposed combination of sulfasalazine (SAS) and disulfiram (DSF) ¹. However, the clinical translation of this combination has been constrained by the poor bioavailability of the drugs. To address this critical challenge, we have developed a unique and platform nano formulation strategy comprising oil-in-water (o/w) nano self-emulsifying drug delivery system (Nano-SEDDS) to enhance the solubility, absorption, and systemic bioavailability of SAS and DSF ^2,3. This pioneering platform technology holds significant potential for enhancing the preventive efficacy of the SAS-DSF drug combination and advancing the fields of lung cancer chemoprevention.

Methods: SAS and DSF were systematically developed and optimized using a design of experiments approach. The Nano-SEDDS formulations were subjected to detailed in vitro physicochemical characterization that consisted of globule size, polydispersity index, zeta potential, effect on pH, effect of dilution, drug content assay, and in vitro drug release study using the dialysis bag method. The formulations were also subjected to stability studies in compliance with the International Council for Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH) guidelines. Further, an in vivo pharmacokinetic and lung tissue biodistribution study was executed in a mouse model (analyzed using an in-house developed LC-MS-MS bioanalytical method) to confirm the hypothesis of bioavailability enhancement with the Nano-SEDDS formulation platform. All studies were conducted in triplicate (n=3). Data were presented as mean ± S.D. and analyzed using appropriate statistical tests.

Results: The Nano-SEDDS platform was developed using a 1-step mix method, making scalability and technology transfer lucrative. The formulation platform was optimized using a ternary phase diagram model to identify an optimum formulation composition consisting of a unique isotropic mixture of oil/s with surfactant/s- stabilizer/s at a specific weight ratio. The detailed in vitro formulation characterization of optimized SAS Nano-SEDDS and DSF Nano-SEDDS is presented in Figures 1 and 2, respectively. Overall, the optimal Nano-SEDDS presented low nanodroplet sizes ( < 200 nm) with uniform size distribution, and in vitro dissolution studies exhibited 4.5-fold (p < 0.01) and 3.75-fold (p < 0.01) increase in drug dissolution of SAS and DSF, respectively (compared to the free drug). The formulations were confirmed to be stable at room temperature in compliance with ICH stability guidelines. Further, the calculated in vivo pharmacokinetic and lung biodistribution parameters of SAS upon oral treatment with free drug SAS and SAS Nano-SEDDS at various doses in female A/J mice are reported in Table 1. The SAS Nano-SEDDS formulation, even at the lowest dose of 10 mg/kg, showed a 7.9-fold increase (p < 0.0001) in dose-normalized relative AUC in plasma and a 21.6-fold increase (p < 0.05) in dose-normalized relative AUC in lung tissue biodistribution compared with free SAS treatment at a very high control dose of 250 mg/kg.  Overall, the results corroborated that the Nano-SEDDS significantly increased the absorption of SAS in blood and lung tissue in a dose-dependent manner, allowing at least a 25-fold dose reduction. DSF is highly unstable in plasma, which impeded the drug quantification. Still, a significant enhancement in DSF drug solubilization (Figure 2) with the Nano-SEDDS platform provides potential proof-of-concept in overcoming the DSF drug solubility and bioavailability challenge.

Conclusion: Significant solubility and bioenhancement using the Nano-SEDDS platform were corroborated by in vitro dissolution studies, which confirmed significant enhancement in drug dissolution, and in vivo studies, which confirmed enhanced absorption and lung tissue biodistribution. Further, the SAS and DSF Nano-SEDDS mixed at various dose ratios for co-delivery will be tested for in vivo lung cancer prevention efficacy in a rodent model.

References: 1. Bagherpoor AJ, Shameem M, Luo X, Seelig D, Kassie F. Inhibition of lung adenocarcinoma by combinations of sulfasalazine (SAS) and disulfiram-copper (DSF-Cu) in cell line models and mice. Carcinogenesis. Jun 24 2023;44(4):291-303. doi:10.1093/carcin/bgad020
2. Desai P, Thakkar A, Ann D, Wang J, Prabhu S. Loratadine self-microemulsifying drug delivery systems (SMEDDS) in combination with sulforaphane for the synergistic chemoprevention of pancreatic cancer. Drug Deliv Transl Res. Jun 2019;9(3):641-651. doi:10.1007/s13346-019-00619-0
3. Desai P, Wang KZ, Ann D, Wang J, Prabhu S. Efficacy and Pharmacokinetic Considerations of Loratadine Nanoformulations and its Combinations for Pancreatic Cancer Chemoprevention. Pharm Res. Jan 2 2020;37(2):21. doi:10.1007/s11095-019-2737-

Acknowledgements: This study was supported by NIH/NCI grant (R01 CA231210-01A1) to F.K. The authors are thankful for the generous gift samples of a few excipients from Gattefosse Ltd., France, and Abitec Corp., USA. The in vivo pharmacokinetic and lung tissue biodistribution study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Minnesota, USA (Protocol # 2206-40135A). The authors declare that they have no conflict of interest.


Figure 1. SAS Nano-SEDDS Characterization.
(a) Images of preconcentrate and water diluted sample; (b) Various physicochemical parameters; (c) Particle size distribution plot; (d) In vitro drug dissolution studies using dialysis bag method. Statistics: Unpaired t-test; (e) Effect of pH and dilution.



Figure 2. DSF Nano-SEDDS Characterization.
(a) Images of preconcentrate and water diluted sample; (b) Various physicochemical parameters; (c) Particle size distribution plot; (d) In vitro drug dissolution studies using dialysis bag method. Statistics: Unpaired t-test; (e) Effect of pH and dilution.



Table 1. Comparison of in vivo parameters calculated upon a single oral dose of SAS free drug and SAS Nano-SEDDS at various doses in female A/J mice. Data presented as mean  SD (n=3).