A deep eutectic-based, self-emulsifying subcutaneous depot system for apomorphine therapy in Parkinson's disease.

Apomorphine, a dopamine agonist, is a highly effective therapeutic to prevent intermittent off episodes in advanced Parkinson's disease. However, its short systemic half-life necessitates three injections per day. Such a frequent dosing regimen imposes a significant compliance challenge, especially given the nature of the disease. Here, we report a deep eutectic-based formulation that slows the release of apomorphine after subcutaneous injection and extends its pharmacokinetics to convert the current three-injections-a-day therapy into an every-other-day therapy. The formulation comprises a homogeneous mixture of a deep eutectic solvent choline-geranate, a cosolvent n-methyl-pyrrolidone, a stabilizer polyethylene glycol, and water, which spontaneously emulsifies into a microemulsion upon injection in the subcutaneous space, thereby entrapping apomorphine and significantly slowing its release. Ex vivo studies with gels and rat skin demonstrate this self-emulsification process as the mechanism of action for sustained release. In vivo pharmacokinetics studies in rats and pigs further confirmed the extended release and improvement over the clinical comparator Apokyn. In vivo pharmacokinetics, supported by a pharmacokinetic simulation, demonstrate that the deep eutectic formulation reported here allows the maintenance of the therapeutic drug concentration in plasma in humans with a dosing regimen of approximately three injections per week compared to the current clinical practice of three injections per day.

Locoregional drug delivery for cancer therapy: Preclinical progress and clinical translation.

Systemic drug delivery is the current clinically preferred route for cancer therapy. However, challenges associated with tumor localization and off-tumor toxic effects limit the clinical effectiveness of this route. Locoregional drug delivery is an emerging viable alternative to systemic therapies. With the improvement in real-time imaging technologies and tools for direct access to tumor lesions, the clinical applicability of locoregional drug delivery is becoming more prominent. Theoretically, locoregional treatments can bypass challenges faced by systemic drug delivery. Preclinically, locoregional delivery of drugs has demonstrated enhanced therapeutic efficacy with limited off-target effects while still yielding an abscopal effect. Clinically, an array of locoregional strategies is under investigation for the delivery of drugs ranging in target and size. Locoregional tumor treatment strategies can be classified into two main categories: 1) direct drug infusion via injection or implanted port and 2) extended drug elution via injected or implanted depot. The number of studies investigating locoregional drug delivery strategies for cancer treatment is rising exponentially, in both preclinical and clinical settings, with some approaches approved for clinical use. Here, we highlight key preclinical advances and the clinical relevance of such locoregional delivery strategies in the treatment of cancer. Furthermore, we critically analyze 949 clinical trials involving locoregional drug delivery and discuss emerging trends.

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow.

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.

Strategies to improve the EPR effect: A mechanistic perspective and clinical translation.

Many efforts have been made to achieve targeted delivery of anticancer drugs to enhance their efficacy and to reduce their adverse effects. These efforts include the development of nanomedicines as they can selectively penetrate through tumor blood vessels through the enhanced permeability and retention (EPR) effect. The EPR effect was first proposed by Maeda and co-workers in 1986, and since then various types of nanoparticles have been developed to take advantage of the phenomenon with regards to drug delivery. However, the EPR effect has been found to be highly variable and thus unreliable due to the complex tumor microenvironment. Various physical and pharmacological strategies have been explored to overcome this challenge. Here, we review key advances and emerging concepts of such EPR-enhancing strategies. Furthermore, we analyze 723 clinical trials of nanoparticles with EPR enhancers and discuss their clinical translation.

Product-Specific Impact of Viscosity Modulating Formulation Excipients During Ultra-High Concentration Biotherapeutics Drug Product Development.

Developing ultra-high concentration biotherapeutics drug products can be challenging due to increased viscosity, processing, and stability issues. Excipients used to alleviate these concerns are traditionally evaluated at lower protein concentrations. This study investigates whether classically known modulators of stability and viscosity at low (<50 mg/mL) to high (>50 - 150 mg/mL) protein concentrations are beneficial in ultra-high (>150 mg/mL) concentration protein formulations and drug products. This study evaluates the effect of arginine monohydrochloride, proline, and lysine monohydrochloride on viscosity and concentratability at different high and ultra-high protein concentrations using a monoclonal antibody, mAbN, formulation as a candidate protein system. The effect of excipients on the viscosity and concentratability (rate and extent) was different at high versus ultra-high protein concentrations. These results highlight that classical excipients in literature known to modulate protein interactions at low protein concentrations and reduce viscosity at high protein concentrations may need to be evaluated at target protein concentrations in a product-specific manner while developing ultra-high concentration biologics drug products.

Modulation of Gastrointestinal Mucus Properties with Ionic Liquids for Drug Delivery.

The mucus barrier lining the gastrointestinal tract poses a significant barrier to the oral delivery of macromolecular drugs. Successful approaches to overcoming this barrier have primarily focused on reducing drug and carrier interactions with mucus or disrupting the mucus layer directly. Choline-based ionic liquids (ILs) such as choline geranate and choline glycolate (CGLY) have recently been shown to be effective in enhancing the intestinal absorption of macromolecules such as insulin and immunoglobulin (IgG), respectively. Herein, the use of choline-based ILs as mucus-modulating agents for safely improving drug penetration through mucus is described. Choline-based ILs significantly increase the diffusion rates of cationic dextrans through mucin solution. Choline-maleic acid (CMLC 2:1) enhances the diffusion of 4 kDa cationic dextran in mucin solution by more than fourfold when compared to phosphate-buffered saline control. Choline-based ILs also reduce mucus viscosity without significantly impacting the native mucus gel structure. In vitro studies in a mucus-secreting coculture model with Caco-2 and HT29MTX-E12 cells further demonstrate the effectiveness of ILs in improving transport of cationic molecules in the presence of secreted mucus. This work demonstrates the potential for choline-based ionic liquids to be used as nondestructive mucus-modulating agents for enabling enhanced oral delivery of macromolecular drugs.

Toward Biotherapeutics Formulation Composition Engineering using Site-Identification by Ligand Competitive Saturation (SILCS).

Formulation of protein-based therapeutics employ advanced formulation and analytical technologies for screening various parameters such as buffer, pH, and excipients. At a molecular level, physico-chemical properties of a protein formulation depend on self-interaction between protein molecules, protein-solvent and protein-excipient interactions. This work describes a novel in silico approach, SILCS-Biologics, for structure-based modeling of protein formulations. SILCS Biologics is based on the Site-Identification by Ligand Competitive Saturation (SILCS) technology and enables modeling of interactions among different components of a formulation at an atomistic level while accounting for protein flexibility. It predicts potential hotspot regions on the protein surface for protein-protein and protein-excipient interactions. Here we apply SILCS-Biologics on a Fab domain of a monoclonal antibody (mAbN) to model Fab-Fab interactions and interactions with three amino acid excipients, namely, arginine HCl, proline and lysine HCl. Experiments on 100 mg/ml formulations of mAbN showed that arginine increased, lysine reduced, and proline did not impact viscosity. We use SILCS-Biologics modeling to explore a structure-based hypothesis for the viscosity modulating effect of these excipients. Current efforts are aimed at further validation of this novel computational framework and expanding the scope to model full mAb and other protein therapeutics.

Microfluidic continuum sorting of sub-populations of tumor cells via surface antibody expression levels.

The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

Ultra-Specific Isolation of Circulating Tumor Cells Enables Rare-Cell RNA Profiling.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.