Search

Albumin accumulation in tumours could reflect a role of albumin in transport of endogenous nutrient cargos required for cellular growth and not just a suggested source of amino acids; a role driven by albumin engagement with its cognate cellular recycling neonatal Fc receptor. We investigate the hypothesis that albumin cellular recruitment is increased by higher human FcRn (hFcRn) expression in human cancer tissue that provides the mechanistic basis for exploitation in albumin-based drug designs engineered to optimise this process. Eight out of ten different human cancer tissue types screened for hFcRn expression by immunohistochemistry (310 samples) exhibited significantly higher hFcRn expression compared to healthy tissues. Accelerated tumour growth over 28 days in mice inoculated with hFcRn-expressing HT-29 human colorectal cancer cell xenografts, compared to CRISPR/Cas9 hFcRn-knockout HT-29, suggests a hFcRn-mediated tumour growth effect. Direct correlation between hFcRn expression and albumin recycling supports FcRn-mediated diversion of albumin from lysosomal degradation. Two-fold increase in accumulation of fluorescent labelled high-binding hFcRn albumin, compared to wild type albumin, in luciferase MDA-MB-231-Luc-D3H2LN breast cancer xenografts was shown. This work identifies overexpression of hFcRn in several human cancer types with mechanistic data suggesting FcRn-driven albumin recruitment for increased cellular growth that has the potential to be exploited with high FcRn-binding albumin variants for targeted therapies.

The human serotonin transporter is the primary target of several antidepressant drugs, and the importance of a primary, high affinity binding site (S1) for antidepressant binding is well documented. The existence of a lower affinity, secondary binding site (S2) has, however, been debated. Herein we report the synthesis of 3-position coupled imipramine ligands from clomipramine using a copper free Sonogashira reaction. Ligand design was inspired by results from docking and steered molecular dynamics simulations, and the ligands were utilized in a structure-activity relationship study of the positional relationship between the S1 and S2 sites. The computer simulations suggested that the S2 site does indeed exist although with lower affinity for imipramine than observed within the S1 site. Additionally, it was possible to dock the 3-linked imipramine analogues into positions which occupy the S1 and the S2 site simultaneously. The structure activity relationship study showed that the shortest ligands were the most potent, and mutations enlarging the proposed S2 site were found to affect the larger ligands positively, while the smaller ligands were mostly unaffected. The human serotonin transporter is the primary target of several antidepressant drugs, and the importance of a primary, high affinity binding site (S1) for antidepressant binding is well documented. The existence of a lower affinity, secondary binding site (S2) has, however, been debated. Herein we report the synthesis of 3-position coupled imipramine ligands from clomipramine using a copper free Sonogashira reaction. Ligand design was inspired by results from docking and steered molecular dynamics simulations, and the ligands were utilized in a structure–activity relationship study of the positional relationship between the S1 and S2 sites. The computer simulations suggested that the S2 site does indeed exist although with lower affinity for imipramine than observed within the S1 site. Additionally, it was possible to dock the 3-linked imipramine analogs into positions which occupy the S1 and the S2 site simultaneously. The structure activity relationship study showed that the shortest ligands were the most potent, and mutations enlarging the proposed S2 site were found to affect the larger ligands positively, while the smaller ligands were mostly unaffected.

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method. mNeonGreen or mCherry fusion to the N-terminal of the recombinant human albumin (rHA) variants null-binder (rHA NB), wild-type (rHA WT), high-binder I (rHA HBI), and high-binder II (rHA HBII) did not generally interfere with hFcRn interaction determined by Biolayer Interferometry. Co-localisation of the albumins with endosomal, but not lysosomal, markers was shown by confocal microscopy for high, but not low, hFcRn binders in a human microvascular endothelial hFcRn overexpressing cell line (HMEC-1 FcRn) suggestive of endosomal compartmentalisation. Furthermore, a cellular recycling assay revealed increased recycling of albumin fusions for the high binding variants (mNeonGreen WT; ~1, mNeonGreen HBI; 5.26-fold higher, and mNeonGreen HBII; 5.77-fold higher) in the hFcRn overexpressing cell line. In vivo experiments demonstrated a direct in vitro recycling/in vivo half-life correlation with a longer circulation for the mCherry fusions engineered with high hFcRn affinity that was highest with the HBII variant of 30.1 h compared to 18.2 h for the mCherry WT. This work gives the first direct evidence for an FcRn-driven endosomal cellular recycling pathway for recombinant albumin fusions that correlates with half-life extension controlled by the affinity to hFcRn; promoting a versatile method to tune the pharmacokinetics of albumin fusion-based therapeutics not met by current technologies.

The effectiveness of a drug is dependent on accumulation at the site of action at therapeutic levels, however, challenges such as rapid renal clearance, degradation or non-specific accumulation requires drug delivery enabling technologies. Albumin is a natural transport protein with multiple ligand binding sites, cellular receptor engagement, and a long circulatory half-life due to interaction with the recycling neonatal Fc receptor. Exploitation of these properties promotes albumin as an attractive candidate for half-life extension and targeted intracellular delivery of drugs attached by covalent conjugation, genetic fusions, association or ligand-mediated association. This review will give an overview of albumin-based products with focus on the natural biological properties and molecular interactions that can be harnessed for the design of a next-generation drug delivery platform.

We here report the synthesis of ethylene glycol N-interlinked imipramine dimers of various lengths from the tricyclic antidepressant desipramine via an amide coupling reaction followed by reduction with lithium aluminium hydride. The target molecules were found to be potent inhibitors of cellular viability while inducing cell type specific death mechanisms in three cancer cell lines including a highly chemoresistant Burkitt’s lymphoma cell line. Basic amine analogues were found to be important for increased potency. Imipramine and desipramine were also tested for apoptotic activity and were found to be much less active than the novel dimeric compounds. Imipramine dimers were only found to be moderate inhibitors of the human serotonin transporter (hSERT) having IC50 values in the micromolar region whilst the induction of cell death occurred independently of hSERT expression. These results demonstrate the potential of newly designed and synthesised imipramines derivatives for use against malignant cells, including those resistant to standard chemotherapy.