(W1230-07-39) Nanocarriers’ Repartitioning of Drugs within the Blood Compartment as a Mechanism of Improving Pharmacokinetics and Efficacy

Purpose: Nanoscale drug carriers (e.g., liposomes, lipid nanoparticles) can improve pharmacokinetics (PK) and efficacy of drugs. A specific PK benefit of nanoparticles that is underutilized is the ability of nanoparticles to alter the partitioning of drug to compartments within the blood. Blood contains three compartments into which drugs can partition: 1) unbound in plasma’s water; 2) bound to plasma proteins; and 3) within red blood cells (RBCs). Partitioning of drugs into plasma proteins and RBCs can affect the pharmacokinetics, distribution, metabolism, and toxicity of drugs¹. Here, we use fingolimod as a model drug and test the ability of liposome nanoparticles to alter drug partitioning within blood and improve drug efficacy. Fingolimod is commonly used to treat multiple sclerosis, but has also shown promise for the treatment of stroke because it can improve barrier function of brain endothelial cells. However, fingolimod partitions strongly to RBCs (86% of drug in whole blood is in RBCs)². This limits its usefulness for treatment of stroke for 2 reasons:1) slow distribution to the brain (t_max in rat brain = 24-48 hours after IV administration)³ limits efficacy in rapidly-progressing conditions such as stroke, and 2) distribution to RBCs may cause RBC rupture/hemolysis, which is particularly dangerous in acute critical illnesses such as stroke. Here, we load fingolimod into liposomes and test partitioning in blood, and efficacy in a mouse model of neuroinflammation.

Methods: Liposomes were produced using the thin-film method. Liposomes were characterized by size and zeta potential (via DLS) and drug concentration (via HPLC). Partitioning of drug within blood was performed by incubating fingolimod (as free drug or in liposomes) with plasma or whole blood at 37 °C for 30 minutes. Liposomes, protein, and free drug were separated by size-exclusion chromatography (plasma studies), or centrifugation (whole blood studies). Drug concentration in different compartments was measured via LC-MS. A mouse model of neuroinflammation was induced via intra-striatal injection of TNFα. 16 hours after injury, mice were administered treatment. Brain edema was measured via radioactive albumin assay.

Results: Addition of anionic lipid to the liposome formulation improved drug loading and decreased drug leak [Figure 1]. We hypothesize this is due to electrostatic attraction between fingolimod (positively charged amine group) and the negatively charged lipids. Loading drug into liposomes dramatically reduced drug association with plasma proteins and RBCs [Figure 2]. Therefore, more of the drug is available in ‘free’ form. It is important to note that ‘free’ drug is still associated with liposomes and may not be available for distribution to tissue. Liposomes can be directed to specific tissue by coating the particle with targeting moieties (e.g., antibodies) that target the tissue of interest. Here we use anti-VCAM antibodies to target the brain⁴. Delivering fingolimod to the brain using anti-VCAM liposomes significantly reduced brain edema compared to untargeted liposomes and fingolimod free drug [Figure 3].

Conclusion: Nanoparticles can dramatically alter drug partitioning within the blood. For drugs highly associated with plasma proteins and RBCs, such as fingolimod, the use of nanoparticles can increase the amount of ‘free’ drug (although drug may still be encapsulated in the nanoparticle). The nanoparticle can then be targeted to the tissue of interest, allowing for more precise control of drug distribution. In the case of fingolimod, we were able to engineer nanoparticles for the treatment of stroke, and improved drug efficacy in a mouse model of neuroinflammation.

References: 1. Hinderling, P. H. Red blood cells: a neglected compartment in pharmacokinetics and pharmacodynamics. Pharmacol. Rev. 49, 279–295 (1997).
2. David, O. J., Kovarik, J. M. & Schmouder, R. L. Clinical pharmacokinetics of fingolimod. Clin. Pharmacokinet. 51, 15–28 (2012).
3. Meno-Tetang, G. M. L. et al. Physiologically based pharmacokinetic modeling of FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) in rats after oral and intravenous doses. Drug Metab. Dispos. 34, 1480–1487 (2006).
4. Marcos-Contreras, O. A. et al. Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier. Proc. Natl. Acad. Sci. U. S. A. 117, 3405–3414 (2020).

Figure 1. Addition of anionic lipid (Egg PG) into the liposome formulation improves fingolimod loading and reduces leak. A) Liposomes were made with varying amounts of Egg PG; size and polydispersity were measured via DLS. B) Drug loading and zeta potential were measured via HPLC and DLS. C) Drug leak from liposomes was measured under infinite dilution conditions.



Figure 2. Loading fingolimod into liposomes reduces drug partitioning into plasma proteins and RBCs. A) Schematic of plasma protein partitioning study. B) Fraction of drug associated with plasma proteins, when formulated as a free drug, drug-loaded liposomes, or drug-loaded liposomes coated with antibodies. Loading drug into liposomes dramatically reduces association with plasma proteins. C) Partitioning of fingolimod to the three blood compartments (RBC, plasma proteins, unbound in plasma water).



Figure 3. Fingolimod liposomes reduce brain edema in a mouse model of neuroinflammation. TNFα was injected into the mouse striatum to induce inflammation. Various treatments were administered 16 hours post-injury, and brain edema was measured by injecting radioactive albumin and measuring the amount radioactivity in the brain. Targeting fingolimod liposomes to the brain using anti-VCAM antibodies improved the efficacy of fingolimod in this disease context.