Size optimization of carfilzomib nanocrystals for systemic delivery to solid tumors

The ubiquitin-proteasome system (UPS) has emerged as a promising therapeutic target for cancer chemotherapy in the past two decades. The UPS is responsible for controlled degradation of misfolded and numerous other proteins involved in cell growth, survival and division, and plays an essential role in maintaining cellular homeostasis [1]. The inhibition of proteasomal activity results in accumulation of polyubiquitinated proteins, causing significant endoplasmic reticulum stress, cell cycle arrest and cell death [2,3]. Cancer cells generally synthesize proteins more rapidly than normal cells owing to their accelerated proliferation. As such, cancer cells become more responsive to proteasome inhibition than normal cells, providing a manageable therapeutic window that kills cancer cells but spares normal cells [4,5].

Carfilzomib (CFZ) is a second-generation proteasome inhibitor with a tetrapeptide backbone. The epoxyketone pharmacophore of CFZ binds covalently to the catalytic threonine residue of the proteasome catalytic subunits (preferentially β5, inhibiting the chymotrypsin-like (CT-L) activity) [6,7]. CFZ was approved by the US FDA for the treatment of multiple myeloma patients refractory to the first generation agent bortezomib [8]. However, due to its extremely poor aqueous solubility [5,9], CFZ is formulated with 50-fold weight excess of the solubilizing agent Captisol® (sulfobutylether-β-cyclodextrin) in the marketed formulation (Kyprolis®), creating a high excipient burden. Moreover, CFZ undergoes extensive extra-hepatic and hepatic metabolism mediated mainly by peptidases and microsomal epoxide hydrolase [10,11], resulting in a half-life shorter than 1 h after intravenous (IV) administration [[12], [13], [14]]. The instability of CFZ in vivo likely contributes to limited distribution of CFZ and insufficient proteasomal inhibition in solid tumors, thereby minimal therapeutic benefits in solid cancer patients [15].

To tackle these pharmaceutical and pharmacokinetic challenges and enable systemic delivery of CFZ to solid tumors, several delivery strategies have been pursued [15]. CFZ has been loaded in nanoparticulate formulations, such as liposomes [5], polymeric micelles [16,17], tethered polymer nanoassemblies [18], polylactic-co-glycolic acid nanoparticles [9], polypeptide nanoparticles [11] or lipid nanodiscs [14]. These systems have facilitated CFZ dissolution in aqueous medium, improved CFZ metabolic stability and in vitro cytotoxicity against tumor cells, or prolonged its half-life in vivo. However, they still suffer from poor drug loading capacity, which necessitates the use of large amount of excipients per dose [9], and/or circulation instability, which offset the benefits of nanoformulations, bringing little contribution to solid tumor therapy [16].

We have previously reported that albumin-coated nanocrystals (NC) may address these issues [19,20]. Consisting of ∼90% drug and ∼ 10% surface stabilizers, NC can reduce biological burden of excipients. Serving as a surface stabilizer and dysopsonin, albumin contributes to reducing the particle size during the NC production and suppressing non-specific adsorption of serum proteins in the systemic circulation [21,22]. Moreover, growing evidence suggests that the surface-bound albumin can mediate nanoparticle endocytosis via albumin-binding proteins overexpressed on peritumoral endothelial cells and many cancer cells, facilitating the distribution of nanoparticles in tumors [19,20]. As a result, albumin-coated paclitaxel NC showed higher antitumor efficacy than the equivalent dose of Abraxane, the commercial benchmark formulation of paclitaxel [20].

With this premise, we have produced an albumin-coated CFZ nanocrystals (CFZ-NCs) for solid tumor therapy and shown that the NC formulation improved the metabolic stability, cellular interactions and in vivo antitumor efficacy of CFZ in a mouse model of breast cancer [12]. However, the improvement of the antitumor efficacy by CFZ-NCs was not as pronounced as observed with paclitaxel NC, possibly attributable to the relatively large size of CFZ-NCs [12]. The large size is unfavorable for systemic CFZ delivery to solid tumors as it does not only reduce the particle extravasation and cellular uptake in tumors but also increases drug accumulation in the filtering organs of the reticuloendothelial system (RES), where the viability of residing immune cells may decrease upon the prolonged exposure to CFZ, thus posing a safety concern.

Therefore, we aim to reduce the size of CFZ-NCs and further improve the antitumor efficacy and safety of systemic CFZ delivery for solid tumor therapy. We hypothesize that size reduction will decrease the RES distribution of CFZ-NCs and selectively increase their uptake by tumor cells. CFZ-NCs were prepared by the crystallization-in-medium method [20], where CFZ crystals are formed in the presence of Pluronic F127 as a stabilizer and further coated with human serum albumin. To reduce the CFZ-NC size, several processing parameters were varied during the NC preparation. The composition, physical stability, and cytotoxicity of the size-optimized CFZ-NCs were evaluated in comparison with the larger counterpart produced by the previously reported method. The tolerability, RES organ uptake, antitumor effect, and biodistribution of the original- and size-optimized CFZ-NCs were evaluated and compared to Captisol®-solubilized CFZ, analogous to the commercial formulation (Kyprolis®), in a murine 4T1 orthotopic breast cancer model.

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