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ii. Readout: mRNA or protein levels of disease relevant cytokine and chemokines (including but not limited to TNF-a, IL6 and CXCL10) |
iii. Criteria: Compound-induced modulation of mRNA or protein levels of measured chemokines and cytokines |
2. Cellular Mechanism of Action (MoA) |
a. Nuclear translocation assay |
i. Thp1, RAW264.7, or another myeloid cell type are stimulated with TLR7, 8, or 9 ligand. |
ii. Readout: nuclear translocation is assessed by immunofluorescence or other equivalent technique |
iii. Criteria: Compounds induced reduction in IRF5 nuclear translocation |
3. Biophysical Mechanism of Action (MoA) |
a. AUC / SEC-MALS assay |
i. Assays are utilized as orthogonal biophysical methods to further demonstrate the compound effects on IRF5 dimerization. |
ii. Readout: Determination of percent monomer and dimer |
iii. Criteria: Shifting of equilibrium towards the monomeric form |
4. Biophysics |
a. IRF5 Binding Assay |
i. Binding assessed by SPR, GCI, and/or MST |
ii. Readout: Binding to IRF5 protein |
iii. Criteria: measurable Kd |
5. Structural Enablement |
a. NanoDSF (to prioritize compounds for co-crystallization studies) |
b. Perform crystallization and co-crystal-structure studies |
c. Perform protein NMR studies using appropriately labelled protein |
Determination that the Stage 1 Activities have been completed is the responsibility of the JGC. |
Stage 1 (Lead Generation to Lead Optimization) Deliverables: |
Compounds from two lead chemical series that demonstrate in vitro activity, chemical properties, and SAR that meet the criteria outlined in Table 1 below. Deliverables to include analyzed data summaries on all compounds meeting lead optimization (LO) criteria for each activity performed in this stage and as requested b... |
Table 1. Stage 1 Pre-specified Criteria for Lead Optimization |
Activity / Assay |
Chemical Series and SAR driven design |
Chemical Series |
Synthetic Tractability |
Structure Activity Relationships |
Structural Biology |
Physicochemical Properties |
Intellectual Property |
In Vitro Pharmacology |
Target IRF5 (in biochemical dimerization assay) |
Off Target IRF family members |
Binding assays |
Cell based assays |
Cellular Selectivity |
Cellular target occupancy |
In Vitro ADME |
r/mu/hu microsomal &/or hepatocyte stability |
Plasma Protein Binding (r/mu/hu) |
CYP Inhibition Panel |
Thermodynamic Solubility (pH 7.4) |
Cellular Permeability |
In Vivo Pharmacokinetics |
Rodent PK |
In Vitro Safety Pharmacology |
hERG |
In Vivo Efficacy |
Acute Rodent PK/PD relationship (refer to Appendix A: In Vivo Acute Efficacy section) |
Stage 2: Lead Optimization to Candidate Nomination Activities |
Aim: To optimize lead candidates from two (2) distinct chemical series and demonstrate in vivo activity in relevant chronic disease model(s) and acceptable safety profile to support nomination of a Development Candidate |
As further described below, the activities to be performed in Stage 2 include the following: |
fulfilling the desired in vitro pharmacology |
target engagement in relevant human cell/tissue assays, |
in vivo efficacy, |
DMPK |
safety profile |
CMC |
All Stage 2 Activities are the responsibility of Hotspot Therapeutics unless noted otherwise below. |
The prioritized and elaborated two (2) lead chemical series will be subjected to further optimization. Both functional activity and target engagement of compounds will be monitored, with the emphasis on relevant assay(s) that correlate to PK/PD understanding. In addition to functional activity on the human variant of t... |
Co-crystallization efforts are currently underway. If X-ray structures of IRF5 with compounds bound are obtained, they will be used to guide the design of compounds for synthesis. Well-established medicinal chemistry considerations will be used to ensure good developability properties of compounds, consistent with oral... |
The DMPK properties of compounds with appropriate characteristics for this stage of development will be continually measured and optimized to support further progression toward Candidate Nomination / Selection. Properties such as solubility, lipophilicity, metabolic stability, clearance, volume of distribution and brai... |
The advanced lead molecules from both chemical series, if possible, and minimally one chemical series, will be tested in in vitro and in vivo studies to confirm target engagement (biomarker), drug distribution profile and efficacy in in vivo animal model studies as listed in Appendix A. Studies and endpoints will be al... |
1. In vitro evaluation |
Confirm pharmacology in relevant human cell / tissue-based assays and demonstrate evidence of in vitro target engagement and pathway modulation. |
Identify target-engagement biomarkers (e.g., gene transcription / phosphorylation profiles) suitable for future clinical validation/development as determined by the JGC. Biomarker development and validation work will progress beyond the end of candidate selection. Biomarkers anticipated to be reproducible, show dose de... |
2. In vivo evaluation |
Compounds will undergo an acute PD assessment via orally dosing test compounds followed by subcutaneously dosed R848 and measurement of changes in cytokines, including TNFα, IL-6, and IL-12 at 1-2 hours post-stimulation. |
At Stage 2, in vivo efficacy will be determined through evaluation of compounds in the rodent collagen-induced arthritis (CIA) model or other relevant model as agreed by the JGC. |
Confirm distribution of drug to the target site of action. |
Confirm PD activity of lead molecule(s) in a relevant acute/sub-chronic in vivo efficacy model(s). |
Demonstrate in vivo target engagement / modulation of pathway specific biomarkers. |
Determine a PK/PD relationship between unbound drug concentrations at the target and relevant acute/sub-acute/chronic efficacy endpoints to support human dose predictions. |
Develop a physiological based pharmacokinetic (PBPK) model to support human PK prediction. |
The best compound or compounds as determined by the JGC, will undergo additional in vitro and in vivo toxicological profiling with anticipation that in vivo safety may only be assessed for such compounds. |
1. In vitro safety assessment |
The lead molecules will be evaluated in assays to assess hERG inhibition and other off-target pharmacology, cellular toxicity (e.g., hepatotoxicity), and in vitro genotoxicity (AMES and micronucleus) conducted by Hotspot Therapeutics. |
2. Mechanistic safety |
If needed based on preliminary findings, an assessment of mechanistic safety may also be undertaken. Any proposed de-risking studies will be discussed and agreed upon by the JGC, who will also determine when and who will perform additional work. |
CMC |
1. Fit-for-purpose route selection/development will be conducted at Hotspot Therapeutics to provide a batch to support preclinical studies including high dose oral PK studies, additional mechanistic safety/PD studies and solid form characterization. |
2. Initial solid form characterization (XRPD, TGA, DSC, DVS, microscopy), biopharmaceutics screening (to include biorelevant solubility and chemical stability) as well as preclinical formulation development (for early in vivo and toxicology studies) will be conducted on this batch by Hotspot Therapeutics. |
Determination that the Stage 2 Activities have been completed is the responsibility of the JGC. |
Stage 2 (Lead Optimization to Candidate Nomination) Deliverables: |
Prioritized leads, as approved by the JGC, from at least one and ideally two (2) distinct chemical series that demonstrate in vitro and in vivo activity that meet the criteria outlined in Table 2 below. For clarity, the minimum number of lead molecules is 2 total. The goal but not necessarily the expectation, will be t... |
For clarity, Hotspot Therapeutics will deliver to AbbVie any data from compounds that do not meet the candidate nomination criteria from Stage 2, upon request. Optimization of additional compounds from each chemical series will be done in parallel with the goal of identifying additional chemical matter fulfilling crite... |
Table 2. Stage 2 Pre-specified criteria for Nomination of a Development Candidate |
Inclusive of meeting Stage 1 prespecified criteria listed in Table 1. |
Activity / Assay |
In vitro |
Target IRF5 (in biochemical dimerization assay) |
Off Target IRF family members |
Cell based assays |
Cellular Selectivity |
In vivo |
Acute in vivo model: R848 model |
Short term efficacy model: CIA or other relevant model |
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