Ternary Polypeptide Nanoparticles with Improved Encapsulation, Sustained Release, and Enhanced In Vitro Efficacy of Carfilzomib.

PURPOSE: To develop a new nanoparticle formulation for a proteasome inhibitor Carfilzomib (CFZ) to improve its stability and efficacy for future in vivo applications. METHODS: CFZ-loaded ternary polypeptide nanoparticles (CFZ/tPNPs) were prepared by using heptakis(6-amino-6-deoxy)-ß-cyclodextrin(hepta-hydrochloride) (HaßCD) and azido-poly(ethylene glycol)-block-poly(L-glutamic acid sodium salt) (N3-PEG-PLE). The process involved ternary (hydrophobic/ionic/supramolecular) interactions in three steps: 1) CFZ was entrapped in the cavity of HaßCD by hydrophobic interaction, 2) the drug-cyclodextrin inclusion complexes were mixed with N3-PEG-PLE to form polyion complex nanoparticles, and 3) the nanoparticles were modified with fluorescent dyes (AFDye 647) for imaging and/or epithelial cell adhesion molecule (EpCAM) antibodies for cancer cell targeting. CFZ/tPNPs were characterized for particle size, surface charge, drug release, stability, intracellular uptake, proteasome inhibition, and in vitro cytotoxicity. RESULTS: tPNPs maintained an average particle size of 50 nm after CFZ entrapment, EpCAM conjugation, and freeze drying. tPNPs achieved high aqueous solubility of CFZ (>1 mg/mL), sustained drug release (t1/2 = 6.46 h), and EpCAM-mediated cell targeting, which resulted in increased intracellular drug accumulation, prolonged proteasome inhibition, and enhanced cytotoxicity of CFZ in drug-resistant DLD-1 colorectal cancer cells. CONCLUSIONS: tPNPs improved stability and efficacy of CFZ in vitro, and these results potentiate effective cancer treatment using CFZ/tPNPs in future vivo studies.

Carfilzomib-Loaded Ternary Polypeptide Nanoparticles Stabilized by Polycationic Complexation.

Carfilzomib (CFZ) is a second-generation proteasome inhibitor showing great efficacy in multiple myeloma treatment, yet its clinical applications for other diseases such as solid cancers are limited due to low aqueous solubility and poor biostability. Ternary polypeptide nanoparticles (tPNPs) are drug carriers that we previously reported to overcome these pharmaceutical limitations by entrapping CFZ in the core of the nanoparticles and protecting the drugs from degradation in biological media. However, preclinical studies revealed that tPNPs would require further improvement in particle stability to suppress initial burst drug release and thus achieve prolonged inhibition of proteasome activity with CFZ against tumor cells in vivo. In this study, CFZ-loaded tPNPs are stabilized by polycations which have varying pKa values and thus differently modulate nanoparticle stability in response to solution pH. Through polyion complexation, the polycations appeared to stabilize the core of tPNPs entrapping CFZ-cyclodextrin inclusion complexes while allowing for uniform particle size before and after freeze drying. Interestingly, CFZ-loaded tPNPs (CFZ/tPNPs) showed pH-dependent drug release kinetics, which accelerated CFZ release as solution acidity increased (pH < 6) without compromising particle stability at the physiological condition (pH 7.4). In vitro cytotoxicity and proteasome activity assays confirmed that tPNPs stabilized with cationic polymers improved bioactivity of CFZ against CFZ-resistant cancer cells, which would be greatly beneficial in combination with pH-dependent drug release for treatment of solid cancers with drug resistance and tumor microenvironment acidosis by using CFZ and other proteasome inhibitors.

Compatibility of calcium chloride with milrinone, epinephrine, vasopressin, and heparin via in vitro testing and simulated Y-site administration.

PURPOSE: The purpose of this study is to evaluate calcium chloride (CaCl) compatibility with commercially available and extemporaneously compounded milrinone, vasopressin, epinephrine, and heparin. This report describes 2 clinical scenarios in which patients experienced intravenous catheter precipitation when receiving multiple continuous infusions, including CaCl, and the results of an in vitro simulation of those scenarios. The hypothesis was that one or a combination of the medications would precipitate with CaCl. METHODS: CaCl compatibility was tested in 3 stages to simulate clinical situations where line precipitation occurred. Multiple tests were conducted in each stage to determine if precipitation had occurred, including visual assessment, absorbance measurement at 650 nm, and pH measurement. First, milrinone, vasopressin, epinephrine, and heparin were mixed pairwise with CaCl in a test tube. Second, the medications were mixed in different combinations deemed likely to precipitate. Finally, 5 medications were infused via simulated Y-site administration. Incompatibility was defined as observed crystals, haziness, or turbidity upon visual inspection or absorbance of greater than 0.01 absorbance unit (AU). All solutions were tested at time 0 and at 20, 60, 240, and 1,440 minutes. RESULTS: Across all tests, only a commercially available formulation of heparin 2 units/mL in 0.9% sodium chloride injection precipitated with CaCl, alone or in combination with other medications. Upon further review, it was found that this specific formulation of heparin contained a monohydrate and dibasic sodium phosphate buffer. CONCLUSION: CaCl only precipitated with a commercially available heparin formulation that contained a phosphate buffer. CaCl was deemed to be compatible with all other medications and formulations tested.

Effects of Organic Acids on Drug Release From Ternary Polypeptide Nanoparticles Entrapping Carfilzomib.

Carfilzomib (CFZ) is an FDA-approved proteasome inhibitor with antineoplastic properties against various cancers, yet its short blood retention time after intravenous injection (< 30 min) makes clinical applications limited to multiple myeloma. We previously developed ternary polypeptide nanoparticles (tPNPs) as a new nanoparticle formulation of CFZ to overcome these limitations. The formulation was prepared by polyion complexation between poly(ethylene glycol)-poly(L-glutamate) block copolymers (PEG-PLE) and CFZ-cyclodextrin (CD) inclusion complexes, where CDs were positively charged with 7 primary amines attached while PEG-PLE carried 100 carboxyl groups per polymer chain. Although tPNPs greatly improved biostability of CFZ, CFZ-loaded tPNPs (CFZ-tPNPs) still showed burst drug release and mediocre drug retention under physiological conditions. To address these issues, organic acids are tested as stabilizers in this study to improve particle stability and drug retention for tPNPs. Charge densities in the core of CFZ-tPNPs were optimized with selected organic acids such as citric acid (CA) and lactic acid (LA) at varying mixing ratios. Organic acids successfully maintained small particle size suitable for intravenous injection and drug delivery (diameters < 60 nm), improved CFZ solubility (> 1 mg/mL), allowed for lyophilization and easy reconstitution in various buffers, enhanced drug retention (> 60% post 24 h incubation), and suppressed burst drug release in the first 6 h following solubilization. These results demonstrate that organic acid stabilized tPNPs are useful as an injection formulation of CFZ, which may expand the utility of the proteasome inhibitor.