Fatty acid-based solid lipid nanoparticles and their effects on metabolism and permeability of drugs

Patel, Rahulkumar (2023) Fatty acid-based solid lipid nanoparticles and their effects on metabolism and permeability of drugs. (PhD thesis), Kingston University, .


Conclusions from previously reported studies have revealed that many commonly used pharmaceutical excipients, known to be pharmacologically inert, show effects on drug transporters and/or metabolic enzymes. Thus, the pharmacokinetics (absorption, distribution, metabolism, and elimination) of active pharmaceutical ingredients are possibly altered because of their transport and metabolism modulation from the incorporated excipients. Excipients such as surfactants, polymers, fatty acids, and solvents have been reported previously as CYP450 inhibitors. Based on all the stated outcomes, the most potent inhibitors were found to be surfactants and the least effective were organic solvents. However, there are many factors that can influence the inhibition of CYP450, for instance type of excipient, concentration of excipient, type of CYP450 isoenzyme, incubation condition, etc. Such evidence will be very useful in dosage form design, so that the right formulation can be designed to maximize drug availability, especially for poorly bioavailable drugs. The aim of this study is to identify the most potent fatty acids that can inhibit CYP450 enzymes and produce solid-lipid nanoparticles (SLNs) to minimize metabolism and increase drug permeability without harmful side effects. The high-performance Liquid Chromatography-Mass Spectrometric (LCMS) methods were developed for (i) simultaneous quantification of omeprazole, dextromethorphan, verapamil, and propranolol (IS) (ii) individual quantification of testosterone. The separation for both methods were achieved on Poroshell 120 EC-C18 (150 x 4.6 mm) using gradient and isocratic approaches. These analytical methods were successfully used for screening of fatty acids, pre-formulation, and formulation studies. Microsomal assay with rat-liver microsomes was used for screening of fatty acids based on their inhibitory effect on various CYP450s. The most potent inhibitor (stearic acid) was then incorporated in three different solid-lipid nanoparticles formulations (SLNs). The pre-formulation and formulation studies were systematically optimized to produce SLNs. These SLNs were characterized by SEM, zetasizer, DSC, dialysis membrane, and HPLC. Moreover, metabolic assay, permeability assay and cytotoxicity were also studied to investigate the effect of stearic acid, present in SLNs, on CYP450s enzymes and caco-2 cell permeability. The results of the LCMS method validation revealed that the coefficient of determination (R2) values for all probe drugs were greater than 0.99. Furthermore, all the analytes showed highest recovery and lowest interday/intraday values. The effect of fatty acids on CYP450s activity was evaluated using rat-liver microsomes. The percentage of probe drugs recovered in the presence of heptadecanoic acid, myristic acid, pentadecanoic acid, and stearic acid were found to be higher than the control study. Stearic acid was the most potent inhibitor out of all the excipients with an IC50 value ³ 30 µM for CYP3A4, 3A5, 2C8, 2C19, and 2D6. At approximately 200 µM of stearic acid, nearly 100% of the compounds were recovered. Based on the screening phase data, three solid-lipid nanoparticles formulations were produced using stearic acid as an oil phase. Results indicated that testosterone loaded SLNs were spherical in shape with an average particle of 278.29 nm diameter with 55% entrapment efficiency. Moreover, microsomal and permeability assays revealed that the presence of stearic acid in testosterone decreased metabolism by CYP450s and increased permeability via caco-2 layer. Approximately, 54% of testosterone was recovered from the metabolic assay with CLint of 0.04 µL/min/mg and apparent permeability was successfully improved with Papp value of 1.53x10-6 cm/sec. On the other hand, verapamil loaded SLNs were spherical in shape with an average particle size of 300.30 nm but with a better entrapment efficiency of 77%. The formulation demonstrated burst release with nearly 20% drug release in under 0.5 hours, followed by sustained release for 23.5. In contrast to testosterone, metabolic assay revealed poor inhibition for CYPs with net recovery of 2.53% and CLint of 0.014 µL/min/mg for verapamil. However, there was significant improvement in the apparent permeability of verapamil (Papp = 2.39x10-6 cm/sec) when compared to control (Papp = 9.00 x10-6 cm/sec). The third formulation, dextromethorphan loaded SLNs was unsuccessful because of its chemical nature and water solubility. Several strategies were used to prepare dextromethorphan-SLNs such as double emulsion, solvent evaporation, and convention hot emulsion method. The affinity of lipophilic drugs for lipids makes them simple to integrate into SLN. While hydrophilic drugs (such as dextromethorphan) are more readily partitioned in the water phase during the production process, they are more difficult to incorporate into the hydrophobic matrix and therefore, further investigation is required for this formulation. The cell cytotoxicity study further confirms the safety of all the ingredients at different concentrations present in various formulations. Thus, stearic acid based SLNs are a potential formulation approach which can inhibit CYP450 enzymes and improve intestinal permeability of various drugs and therefore, requires further investigation to improve the formulation with various approaches.

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