(M1130-04-20) Impact of Macrophages on the Dissolution of Long-Acting Injectable Suspensions

Purpose: Long-acting injectable (LAI) suspensions present a unique opportunity for the prolonged delivery of active pharmaceutical ingredients (APIs). These suspensions contain highly hydrophobic APIs of a desired particle size dispersed in a suitable dispersion medium. Upon subcutaneous or intramuscular administration, the drug product forms a depot, and the drug is released slowly in a controlled manner (1). The drug release from these suspensions is controlled by the particle size of the API, as per the Noyes-Whitney equation (2). In addition to particle size, literature reports suggest that the local inflammatory reaction at the injection site could also play a critical role on the dissolution kinetics (3,4). The inflammatory response can affect the in vivo dissolution and absorption of LAI suspensions through initial increase in the fluid volume at the site that can enhance the drug dissolution and later by the formation of a fibrous capsule around the depot which may slow down drug release. Another factor which is particularly important for LAIs is the recruitment of macrophages at the injection site. These macrophages may perform metabolism of the LAI prodrug (hydrolysis of the ester prodrug), converting them to a more soluble active metabolite and thereby enhancing drug dissolution and in vivo absorption. Accordingly, the purpose of this study was to investigate the impact of macrophages recruited at the injection site on the dissolution kinetics of the LAI suspensions.

Methods: Invega Trinza® and Aristada® containing prodrugs paliperidone palmitate and aripiprazole lauroxil were chosen as reference drug products and Q1Q2 equivalent formulations were prepared. Cell cytotoxicity was carried out to determine the working concentrations of LAI prodrugs without affecting cell viability. These in-house formulations at working concentration were incubated with human derived macrophage cells for three and five days. After incubation, the cells were washed, and metabolites (aripiprazole and paliperidone) were extracted using a solvent-solvent extraction method. Chromatographic separation of these metabolites was performed on a Waters® 15 cm HSS T3 column using a mobile phase of water: acetonitrile (10:90, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The separated metabolites were then determined using SYNAPT G2-Si mass spectrometer in a positive ion mode. Targeted ion MS² spectroscopy was utilized to confirm the metabolic conversion (aripiprazole lauroxil to aripiprazole and paliperidone palmitate to Paliperidone). To demonstrate the impact of the degree of inflammation, these suspensions were incubated with two different cell densities (2 X 10³ and 4 X 10³ cells) and the degree of metabolic conversion was determined. Lastly, the quantification of active metabolites generated at different time intervals was conducted to determine the degree of solubility enhancement by macrophages.

Results: The results demonstrated that the LAI suspensions (containing prodrugs aripiprazole lauroxil and paliperidone palmitate) tested at different concentrations were found to be safe at a working concentration of 900 µg/100 µl with cell viability greater than 100%. The chromatographic analysis performed on extracts of cells treated with LAI suspensions for three and five days showed that the developed method effectively separates the active metabolites aripiprazole and paliperidone with retention times at 3.61- and 4.25-min. Mass spectroscopy revealed that control groups I (untreated macrophage cells) and II (drug particles in growth media without macrophage cells), did not show ionization peaks for aripiprazole and paliperidone (Figure 2B & 2C). The ionization peaks of aripiprazole (m/z 448.15 parent peak) and paliperidone (m/z 427.21 parent peak) were present in the mass spectrum of the extracts from cells treated with in-house Q1Q2 equivalents, confirming that the macrophage metabolized the aripiprazole lauroxil and paliperidone palmitate prodrugs. The MS2 spectra showed the presence of the precursor ion peaks at 285.09 m/z for aripiprazole and 207.11 m/z for paliperidone, further confirming the metabolism. The amount of metabolite generated was dependent on the macrophage cell density. The peak intensity increased by 10-fold when the cell density was doubled from 2X 10³ to 4X10³, suggesting that degree of inflammation could impact the metabolic conversion and the subsequent in vivo dissolution and absorption. The solubility enhancement study demonstrated a gradual increase in the concentration of metabolites over time when the cells were treated with the LAI prodrug suspension. In contrast, no such increase in concentrations was observed when the LAI prodrug suspensions were incubated with PBS, confirming that the metabolic conversion by macrophages can enhance the dissolution and absorption of the LAI prodrugs.

Conclusion: In conclusion, these findings indicate that local inflammation at the administration site is a critical factor in determining the performance of LAI prodrug formulations. The macrophage infiltration and phagocytosis of the LAI prodrug depot can promote the dissolution and local conversion of the prodrug to the active metabolite, affecting the overall dissolution kinetics. The degree of inflammation alters metabolic conversion and drug release from LAI suspensions. This in vitro study provides novel insights into the mechanisms governing in vivo drug release from LAI formulations.

References: 1. Ho, Myoung Jin, Min Young Jeong, Hoe Taek Jeong, Min Seob Kim, Hyun Jin Park, Dong Yoon Kim, Hyo Chun Lee et al. "Effect of particle size on in vivo performances of long-acting injectable drug suspension." Journal of Controlled Release 341 (2022): 533-547.
2. Hattori, Yusuke, Yoshimasa Haruna, and Makoto Otsuka. "Dissolution process analysis using model-free Noyes–Whitney integral equation." Colloids and Surfaces B: Biointerfaces 102 (2013): 227-231.
3. Darville, Nicolas, Marjolein Van Heerden, Dirk Mariën, Marc De Meulder, Stefaan Rossenu, An Vermeulen, An Vynckier et al. "The effect of macrophage and angiogenesis inhibition on the drug release and absorption from an intramuscular sustained-release paliperidone palmitate suspension." Journal of controlled release 230 (2016): 95-108.
4. Darville, Nicolas, Marjolein Van Heerden, An Vynckier, Marc De Meulder, Patrick Sterkens, Pieter Annaert, and Guy Van den Mooter. "Intramuscular administration of paliperidone palmitate extended-release injectable microsuspension induces a subclinical inflammatory reaction modulating the pharmacokinetics in rats." Journal of pharmaceutical sciences 103, no. 7 (2014): 2072-2087.

Acknowledgements: Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this abstract. This work was financially supported through the Pfizer Distinguished Chair of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Connecticut.


Figure1: Pictorial representation of: (A) local inflammatory reaction and granuloma formation around a depot at the injection site upon subcutaneous/intramuscular administration of LAI suspensions; and (B) mechanism of uptake of LAI suspension particles and metabolism of the prodrug to a more soluble active metabolite.


Figure 2: (A) Experimental workflow to study the role of macrophages in the metabolism of long-acting injectable (LAI) suspensions; (B and C) Mass spectra of control groups I (untreated macrophage cells) and II (drug particles in growth media without macrophage cells) with no ionization peaks for aripiprazole and paliperidone; Mass spectra of (D) aripiprazole (precursor ion m/z 448.1599) and (E) paliperidone (precursor ion m/z 427.2119) obtained after incubating human macrophages with LAI suspensions of prodrugs aripiprazole lauroxil and paliperidone palmitate, respectively, for three and five days; (F) Targeted ion mass spectrum (MS2) for aripiprazole with characteristic ion signature peak at m/z 285.0950; and (G) Targeted ion mass spectrum (MS2) for paliperidone with characteristic ion signature peak at m/z 207.112. (All data are presented as mean ± SD, n = 3)


Figure 3: Mass spectra of (A) aripiprazole (precursor ion m/z 448.1599) and (B) paliperidone (precursor ion m/z 427.2119) obtained after incubating human macrophages at two different cell densities (2 x E3 and 4 x E3) with long-acting injectable suspensions containing prodrugs aripiprazole lauroxil and paliperidone palmitate; (C) graph representing a 10-fold increase in peak intensities of aripiprazole (m/z 448.1599) and paliperidone (m/z 427.2119) when the cell density of human macrophages was increased from 2 x E3 to 4 x E3 cells; and (D) graph representing increase in solubility of aripiprazole and paliperidone at different time intervals when incubated with human derived macrophages. (All data are presented as mean ± SD, n = 3)