Predicting Long-Term Stability of an Oral Delivered Antibody Drug Product with Accelerated Stability Assessment Program Modeling.

The oral delivery of protein therapeutics offers numerous advantages for patients but also presents significant challenges in terms of development. Currently, there is limited knowledge available regarding the stability and shelf life of orally delivered protein therapeutics. In this study, a comprehensive assessment of the stability of an orally delivered solid dosage variable domain of heavy-chain antibody (VHH antibody) drug product was conducted. Four stability related quality attributes that undergo change as a result of thermal and humidity stress were identified. Subsequently, these attributes were modeled using an accelerated stability approach facilitated by ASAPprime software. To the best of our knowledge, this is the first time that this approach has been reported for an antibody drug product. We observed overall good model quality and accurate predictions regarding the protein stability during storage. Notably, we discovered that protein aggregation, formed through a degradation pathway, requires additional adjustments to the modeling method. In summary, the ASAP approach demonstrated promising results in predicting the stability of this complex solid-state protein formulation. This study sheds light on the stability and shelf life of orally delivered protein therapeutics, addressing an important knowledge gap in the field.

Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking ß-sheets across the VHH protein, thereby preventing ß-sheet-ß-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

Cyclodextrin Reduces Intravenous Toxicity of a Model Compound.

Solubilization of new chemical entities for toxicity assessment must use excipients that do not negatively impact drug pharmacokinetics and toxicology. In this study, we investigated the tolerability of a model freebase compound, GDC-0152, solubilized by pH adjustment with succinic acid and complexation with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to enable intravenous use. Solubility, critical micelle concentration, and association constant with HP-ß-CD were determined. Blood compatibility and potential for hemolysis were assessed in vitro. Local tolerability was assessed after intravenous and subcutaneous injections in rats. A pharmacokinetic study was conducted in rats after intravenous bolus administration. GDC-0152 exhibited pH-dependent solubility that was influenced by self-association. The presence of succinic acid increased solubility in a concentration-dependent manner. HP-ß-CD alone also increased solubility, but the extent of solubility enhancement was significantly lower than succinic acid alone. Inclusion of HP-ß-CD in the solution of GDC-0152 improved blood compatibility, reduced hemolytic potential by ∼20-fold in vitro, and increased the maximum tolerated dose to 80 mg/kg.

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model.

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.

Lead identification of novel and selective TYK2 inhibitors.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.

Lead optimization of a 4-aminopyridine benzamide scaffold to identify potent, selective, and orally bioavailable TYK2 inhibitors.

Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.