Purpose: A site-specific, delayed-release crosslinked bio-polymeric dual-biotic system containing amoxicillin and the probiotic Lactobacillus acidophilus (DBioS) has been extensively evaluated in this study to overcome the therapeutic issue of probiotic killing due to the incorrect administration in relation to the antibiotic product.
Methods: The DBioS comprised of a crosslinked denatured ovalbumin protective matrix encapsulation with glyceryl-monostearate that was thereafter incorporated with amoxicillin in a gastro-resistant capsule. In vitro and ex vivo release studies, structural and thermal characterization as well as microbial stability studies have been undertaken on the DBioS. In vivo analyses utilizing a Large White pig model were also undertaken with commercial products used as a comparison. Lactobacillus acidophilus cell counts in intestinal fluid were determined through luminescence. Plasma amoxicillin concentrations were determined through UPLC. A Level-A IVIVC was thereafter formed. The reactional profile and crosslinking mechanism of ovalbumin and genipin was also elucidated using molecular mechanic energy relationships (MMER) in a vacuum system by exploring the spatial disposition of different concentrations of genipin with respect to ovalbumin with ovalbumin/genipin ratios of 1:1, 1:5 and 1:10.
Results: The FTIR analysis of the non-crosslinked ovalbumin displayed a peak at 1634 cm-1 attributed to C=O of the amide I band, a peak at 1444 cm-1 due to the bending vibration of N-H molecules of the amide II band and a peak at 3282 cm-1 attributed to O-H stretching vibrations, N-H extension vibrations and intermolecular H-bonds that occur in protein structures. Evaluation of the crosslinked ovalbumin system revealed a significant increase in transmittance of the 2105 cm-1 band due to the crosslinking of the free amide groups in the ovalbumin structure leading to decreased N-H stretching. Other significant changed were determined at the 1274 cm-1 band representative of a mixed vibration of CO-N and N-H. DSC analysis the non-crosslinked ovalbumin matrix determined an endothermic peak at 82.27°C indicative of albumin denaturation without degradation, an endothermic peak of 218.64°C representative of the initial phase of protein degradation and an exothermic peak at 290.05°C noting protein decomposition. Comparison to the crosslinked ovalbumin determined a shift in the endothermic denaturation peak to 106.40°C with an increased initial endothermic degradation and exothermic decomposition peak at 221.77°C and 294.96°C respectively. In vitro probiotic release profiles in simulated gastrointestinal fluid determined the success of the DBioS to protect and deliver 108-109 viable bacteria required to exert functional health benefits to a patient (Figure 1a). A similar result was determined in aspirated porcine gastrointestinal media (Figure 1b).
In vivo evaluation of the DBioS determined an initial lag in amoxicillin absorption attributed to gastric-emptying (Figure 2). The subsequent release of the capsule contents resulted in a rapid absorption of amoxicillin to a maximum plasma concentration of ~3.40 µg/ml after ~2.65 h (Fig. 7a). The resultant rapid elimination of amoxicillin (2.884 µg/ml at 4 h to 0.964 3.4 µg/ml at 12 h), to a concentration undetectable by UPLC at 18 h, was also documented due to the short plasma half-life of amoxicillin (22). Maximum Lactobacillus viability (~455% baseline viability) noted 6 h after oral administration (Figure 2). Concurrent administration of the commercial products revealed a 75% decrease in bacterial colony-forming units compared to the controls analyzed. A level A in vitro-in vivo correlation (IVIVC) was also established with 96.96% predictability of amoxicillin release ascertained.
Molecular mechanics analysis revealed energetic paradigms emerging out of the crosslinking of ovalbumin (OVA) by genipin (GNP) as depicted in Figure 3. These molecular simulations are in corroboration with the observed experimental results after the addition the crosslinker. Additionally, the spectral analysis corroborated with the mechanistic simulation in terms of functional groups involved in the crosslinking reaction which further supported the in silico-in vitro correlation.
Conclusion: The DBioS in this study proved effective in delivering amoxicillin and protecting and delivery L. acidophilus probiotics. This was achieved through extensive in vitro, ex vivo, in vivo and in silico evaluation of the DBioS. The results of this study therefore provide insight into current microbiome research to identify, classify and use functional healthy bacteria to develop novel probiotic delivery technologies.
Sunaina Indermun– Post-Doctoral Research Fellow, University of the Witwatersrand, Johannesburg, Gauteng
Yahya Choonara– Chairman and Head I Pharmacy and Pharmacology, University of the Witwatersrand, JHB, Gauteng
Sandy van Vuuren– Professor, University of the Witwatersrand, Johannesburg, Gauteng
Pradeep Kumar– Senior Researcher: Wits Advanced Drug Delivery Platform, University of the Witwatersrand, JHB, Gauteng
Lisa du Toit– Senior Researcher : Wits Advanced Drug Delivery Platform, University of the Witwatersrand, JHB, Gauteng
Viness Pillay– Director: Wits Advanced Drug Delivery Platform, University of the Witwatersrand, JHB, Gauteng