(W0930-03-15) Oral Delivery of Probiotics Using Delayed-Release Capsules: Formulation and Dosage Form Considerations

Purpose: In this work, we aim to formulate a new oral dosage form of probiotics using delayed-release capsules. Several research articles have shown that commercial capsules leak acid during pH-shift dissolution studies and acid-uptake tests.^1–3 For this purpose, dried powders of Lactobacillus acidophilus prepared by thin-film freezing technology (TFF) and ACGcaps^TM HX from ACG capsules were employed.

Methods: Delayed-release capsules (ACGcaps^TM HX) and standard HPMC capsules (ACG North America, Piscataway, NJ) were utilized. The 15 mm side-by-side diffusion cells (PermeGear, Hellertown, PA, USA) were employed to evaluate the capsule shell properties of ACGcaps^TM HX (Figure 1A). The capsules were cut and used as a membrane to evaluate the diffusion/permeation of caffeine (a model small molecule) and 1.0 µm fluorescent microparticles (Thermo Fisher Scientific Inc., Waltham, MA) (Figure 1B). The following solutions were prepared for the diffusion assays: (A) 0.1 N HCl, (B) caffeine solution at 5 mg/mL or the microparticles in 0.1 N HCl, and (C) FD&C Red 40 at 1 mg/mL in 0.1 N HCl. The donor cell was filled with 4 mL of solution A, 1 mL of solution B, and 100 µL of solution C. In contrast, the receptor cell was filled with 5 mL of solution A. Using TFF, two formulations of dry powders of L. acidophilus were prepared, with and without the enteric polymer hydroxypropyl methylcellulose acetate succinate (HPMC-AS), abbreviated as LAB-HPMCAS and LAB, respectively. LAB comprised 15% (w/v) sucrose plus 5% (w/v) maltodextrin, while LAB-HPMCAS was prepared with 20% (w/v) sucrose plus 2% (w/v) HPMC-AS. These powders were encapsulated, as shown in Figure 2A, and placed in 100 mL of 0.1 N HCl with 0.9% sodium chloride. The dissolution tests were performed in a Hanson SR8-Plus apparatus (Hanson Research Co., Chatsworth, CA) using the 200 mL vessels, with a paddle speed of 100 rpm and temperature set to 37°C. The capsules were removed from the sinker after 30 min and cut open. The content was resuspended in MRS broth for serial dilution. Live bacteria were enumerated by counting the bacterial colonies on MRS agar plates after two days of anaerobic incubation at 37°C and reported as colony-forming units (CFU) per mL.

Results: In the experiment using side-by-side diffusion cells, ACGcaps^TM HX’s shells were used as a semipermeable membrane surrounded by 0.1 N HCl on both sides to confirm the delayed-release property of the capsules. For caffeine, a small molecule, a two-phase diffusion profile was seen, slow in the first 30 min but faster after 30 min (Figure 1C). In the case of the 1.0 µm fluorescent microparticles, they were detected in the receiver cell until after 33 ± 8 min. These results confirmed the delayed-release property of the ACGcaps^TM HX. The ACGcaps^TM HX’s ability to protect L. acidophilus, a probiotic bacterium, in TFF powders in a simulated gastric fluid was then tested. ACGcaps^TM HX were filled with LAB or LAB-HPMCAS formulations (Figure 2A) and placed in dissolution vessels filled with 0.1 N HCl. After 30 min, the capsules were swollen, but their overall shape was retained (Figure 2B). Surprisingly, there were no viable bacteria recovered from the capsules when the LAB was encapsulated in the delayed-release capsule (Table 1). Bacteria in the LAB-HPMCAS formulation showed improved viability (Table 1). In the capsule-in-capsule setting, i.e., TFF bacterial powders were filled in one ACG HX capsule, which was then placed into another larger ACGcaps^TM HX capsule, bacterial viability loss was minimized to less than 1 log reduction for the LAB-HPMCAS formulation (Table 1).

Conclusion: The combination of thin-film freeze-drying of L. acidophilus in a formulation containing the acid-resistant HPMC-AS polymer and the capsule-in-capsule delayed release design can minimize bacterial viability loss in simulated gastric fluid. This new dosage form has the potential for oral delivery of probiotics.

References: 1. Al-Tabakha, M. M., Arida, A. I., Fahelelbom, K. M. S., Sadek, B. & Abu Jarad, R. A. Performances of New Generation of Delayed Release Capsules. JYP 7, 36–44 (2014).
2. Fu, M., Al-Gousous, J., Blechar, J. A. & Langguth, P. Enteric Hard Capsules for Targeting the Small Intestine: Positive Correlation between In Vitro Disintegration and Dissolution Times. Pharmaceutics 12, 123 (2020).
3. Jara, M. O. et al. Oral Delivery of Niclosamide as an Amorphous Solid Dispersion That Generates Amorphous Nanoparticles during Dissolution. Pharmaceutics 14, 2568 (2022).

Acknowledgements: We would like to thank Anita Solanki from ACG Associated Capsules (Canada) Ltd. We also would like to thank Jnanadeva Bhat and Manali Dalvi from ACG Scitech Centre (Mumbai, India). Finally, we would like to thank Angela Ren from the College of Pharmacy, University of Texas at Austin for facilitating side-by-side diffusion cell experiments. Declaration of Interest: ROW and ZC report financial support provided by TFF Pharmaceuticals, Inc. ZC reports a relationship with TFF Pharmaceuticals, Inc. that includes: equity or stocks and funding. ROW reports a relationship with TFF Pharmaceuticals, Inc. that includes: consulting or advisory, equity or stocks, and funding. HX reports a relationship with TFF Pharmaceuticals, Inc. that includes: consulting or advisory.

Figure 1. (A) Picture of the side-by-side diffusion cells. FD&C Red 40 was employed for visualization purposes. (B) ACGcapsTM HX were cut and used as a membrane. (C) Caffeine concentration in the receptor cell over time. (D) microspheres permeation over time.

Table 1. Viability reduction (CFU/mL) of L. acidophilus powders filled into different capsule delivery systems. Data are ± mean S.D. (n = 3).

Figure 2. (A) Capsule-in-capsule delivery system with encapsulated TFF powder. (B) Delayed-release capsules after 30 min in 0.1 N HCl.