(T1430-07-40) Preformulation Studies on Two Novel Indole-2-Carboxamides Highly Potent Against Nontuberculous Mycobacteria

Purpose: Nontuberculous mycobacteria (NTM) pathogens particularly infect patients with structural lung disorders, such as chronic obstructive pulmonary disease or cystic fibrosis (1). Our laboratory has developed several novel indole-2-carboxamides that are highly potent against a wide panel of NTM pathogens. This preformulation studies aimed to determine the solid-state properties of two lead (compounds N2 and N21) active pharmaceutical ingredients (APIs) that will be crucial during their formulation development.

Methods: An Ultra-Performance Liquid Chromatography (UPLC) method was developed for the quantitation of both N2 and N21 and validated as per the USP guidelines. A Waters Acquity UPLC system equipped with PDA detector and BEH C18 (150 mm x 2.1 mm x 1.7 µm) column was used. The mobile phase consisted of acetonitrile: 0.1% (v/v) aqueous formic acid in a ratio 70:30 (v/v) at a 0.3 mL/min flow rate. Analysis was carried out at the λ_max of 295 nm for N2 and 301 nm for N21. This method was used to determine the octanol/water partition coefficient (LogP), the aqueous equilibrium solubility, and the permeability of N2 and N21 via caco-2 cell monolayer studies. Solid-state properties of both N2 and N21 were characterized using Scanning Electron Microscopy (SEM), and Thermal Analyses (Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Hot-stage Microscopy),

Results: The standard curves both N2 and N21 were linear in the concentration range of 0.078-10 0 µg/mL with r² ≥ 0.999. The method was specific for N2 and N21, the retention time for N2 was 3.4 ±0.05 min and 3.8 ± 0.05 min for N21, respectively. Within-day (n=4) and day-to-day (n=6) analytical precision for both N2 and N21 were within the recommended acceptance criteria for the analytical methods for bias of less than or equal to 10% (2). The accuracy (n=9, each QC level) of the method was within 98-103%. The LogP value for N2 and N21 was 4.88 ± 0.02 and 5.76 ± 0.07, respectively. The aqueous equilibrium solubility of N2 was 0.16 ± 0.02 µg/mL, and of N21 was 0.17 ± 0.03 µg/mL at 37^◦C respectively. The percent extent of absorption (%fa) values were >95% for N2 and ~100% for N21 indicating the high permeability of these APIs (3). The SEM analysis revealed that N2 is cylindrical in shape with particle size between a few microns to 200 µm (Figure 1A). N21 is irregular in shape with particle sizes ranging from a few microns to 100 µm (Figure 1B). The DSC thermogram of N2 showed a sharp endothermic peak at 233^◦C and XRD data confirmed the crystalline nature of this API. Thermogravimetric analysis revealed no significant weight loss for N2 and N21 over the temperature ranges. However, the DSC thermogram of N21 as shown in Figure 2 showed a sharp endothermic peak at 139^◦C immediately followed by an exothermic peak at 143^◦C, and a second endothermic peak at 168^◦C. Hot-stage microscopy of N21 was performed from 20-190^◦C at a controlled heating rate to visually observe the effect of heating on N21. Both melting events at 139^◦C and 168^◦C are visually confirmed by the Hot-stage microscope. However, the exothermic peak at 143^◦C was not able to be detected during hot-stage microscopy. Variable temperature X-ray crystallography (VTXRD) were used to confirm this exothermic event. VTXRD results shown in Figure 3 revealed that until 140^◦C there was no change in crystal structure of N21. At 140^◦C, some new peaks started to emerge, and some of the original peaks of N21 disappeared indicating that there is a possibility of formation of another polymorph of N21.

Conclusion: The solid-state characterization, UPLC method development and validation, and preformulation studies were performed for two novel APIs that are active against a wide panel of NTM pathogens. N2 and N21 both observed to be crystalline and lipophilic. N2 exhibited a sharp melting peak at 233^◦C. Variable temperature XRD results along with DSC and hot-stage microscopic studies revealed the existence of at least two polymorphs of N21. The solubility and permeability studies revealed that both N2 and N21 belongs to BSC class II. The outcomes of these studies will be crucial during the subsequent formulation development of both N2 and N21.

References: 1. Lapinel NC, Jolley SE, Ali J, Welsh DA. Prevalence of non-tuberculous mycobacteria in HIV-infected patients admitted to hospital with pneumonia. The International Journal of Tuberculosis and Lung Disease. 2019 Apr 1;23(4):491-7.
2. USP, Biological Assay Validation, USP 38 (USP, 2010).
3. Volpe DA, Faustino PJ, Ciavarella AB, Asafu-Adjaye EB, Ellison CD, Yu LX, Hussain AS. Classification of drug permeability with a Caco-2 cell monolayer assay. Clinical Research and Regulatory Affairs. 2007 Jan 1;24(1):39-47.

Acknowledgments: The authors are grateful for the Creighton University Department of Chemistry and Biochemistry for the use of the 400 MHz Bruker NMR. We would like to thank Dr. Raj Suryanarayanan, College of Pharmacy, University of Minnesota for XRD analysis.
Funding and other Disclosure: The authors gratefully thank the National Institutes of Health (R15 AI164443 and P20 GM139762-01) for funding this project.
Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work and there has been no significant financial support for this work that could have influenced its outcome.

Figure 1: Scanning electron microscopy image of (A) N2 and (B) N21.



Figure 2: Differential scanning calorimetry thermogram of N21.



Figure 3: Variable temperature X-ray crystallographic analysis of N21 from room temperature to 170 ◦C.