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Appendix F: GPR65 ligands |
Target, Target Example Ligands/Ligan series, Comments (physchem properties/ pharmacology), References |
GPR65, Allosteric agonist, ZINC13684400, , Roth et al. Nature (2015) 527: 477), |
GPR65, Agonist, BTB09089, , Onozawa et al. E J Pharm 683 (2012) 325–331, Roth et al. Nature (2015) 527: 477), |
GPR65, Allosteric antagonist, ZINC62678696, , Roth et al. Nature (2015) 527: 477), |
Appendix G: Product-Specific In Vitro Cellular Assays and In Vivo Acute and Chronic Models for Advancement of GPR65 Research Target |
Lead and backup target criteria for advancement to IND‐enabling studies. Outlined chronic models are suggested based on the knowledge of the target to date, AbbVie reserves the right to test compounds in more than one chronic model. |
Attribute Activity/Assay Readouts/Success Criteria Comments |
Effects of test compound should be comparable to or superior to listed standards in each of the following assays or models |
In Vitro Screening Assay in addition to Heptares StaR protein assay for chemical matter identification |
1) AbbVie recommends the following in vitro screening assay based on internal experience and/or literature. 2) Criteria: Sosei Heptares to identify acceptable chemical matter based on data from a) Selectivity binding assays; b) Biochemical assays and c) Screening cell assay developed to demonstrate target modulation ba... |
Cellular Effects on cAMP Production, GPR65 OE cell line using either Baculovirus infection or stable cell line from single clone. Options to either purchase available commercial line or Abbvie GPR65 OE CHO line, • Dose dependent induction in cAMP levels at different pHs with GPR65 agonist., Appropriate experimental pos... |
GPR65 affinity‐based measurement based on competition study, GPR65 OE wild‐type and in membranes/whole cell, • Subject to suitable probe identified that can be custom radio / fluorescently labelled and shown to bind specifically to target in order to measure compound competition and affinity estimate, Option to outsour... |
Biophysical demonstration of a specific interaction with GPCR StaR protein confirmed versus unrelated GPCR, StaR protein‐based SPR or Thermostability assay, • SPR readout consistent with a bimolecular binding event or effect of compounds on thermostability of StaR protein |
Selectivity/specificity assays, Unrelated GPCR screening in cAMP format in an OE cell line(s) mimicking primary screen and/ or parental cell line functional test, • Appropriate experimental positive control included (forskolin for parental or GPCR agonist standard if in‐house assay), Limited insights into proton sensin... |
In Vitro Assays to understand mechanism of action |
i. AbbVie recommends the following in vitro assays to understand mechanism of action based on internal experience and literature. ii. Sosei Heptares would be required to demonstrate acceptable in vitro data package elucidating the mechanism of action of the target based on published literature. |
Cellular Effects on Autophagy, LC3‐GFP reporter assay in U937 cells, • Dose dependent increase in autophagy (as measured by reduction of LC3‐GFP after being normalized to cell confluency/YFP control • Demonstrate reduction of LC3‐GFP to positive control Torin1, Appropriate experimental positive control (Torin1; mTOR in... |
Cellular Effects on Bacterial Clearance, Bacterial clearance using bacterial killing assay with GFP‐E Coli in mouse BMDMs from WT versus GPR65 KO mice, • Dose dependent increase in bacterial killing in mouse BMDMs. • No loss of cell viability using live/dead dye staining by FACS at active doses of agonist. • Demonstrat... |
Cellular Effects on Cytokine Production, TNFa and IL‐6 production from LPS and peptidoglycan stimulation of BMDM from WT versus GPR65 KO mice, • Dose dependent reduction in TNF a and IL‐6 cytokine levels in culture supernatants • No loss of cell viability using live/dead dye staining by FACS at active doses of agonist.... |
In Vivo Acute Efficacy |
Criteria of robust and reproducible PK‐PD (exposure‐efficacy) relation demonstrated in at least one acute model with comparable efficacy to standard and demonstrate exposure covers the IC50/IC80 potency of the molecule for the target |
Acute LPS lung model, Measurement of neutrophil number, KC, TNF and IL‐6 cytokines in bronchoalveolar lavage (BAL) fluid after in vivo intratracheal administration of LPS, • Dose dependent reduction in neutrophil number, KC, TNF and IL‐6 cytokine levels in BAL fluids after intratracheal administration of LPS. • Demonst... |
Acute LPS challenge, Measurement of TNF and IL‐6 cytokines in serum/plasma after in vivo intra‐peritoneal administration of LPS, • Dose dependent reduction in TNF and IL‐6 cytokine levels serum/plasma 1‐2 hours post‐injection of LPS. • Demonstrate equal or greater inhibition to positive control (dexamethasone) • PK‐PD ... |
In Vivo chronic Efficacy (Criteria of robust and reproducible in vivo model with comparable efficacy to standard) |
i. Models are listed in the order of priority based on the knowledge of the target as of today |
Colitis, 7‐day Agonist CD40 Colitis, • Oral efficacy with reduction in inflammation as measured by histology. • Demonstrate equal or greater efficacy than positive control anti‐p40 Ab • Datasets to understand efficacy, level of target engagement and exposure response. • Dose response. • PK/PD understood to allow safety... |
Colitis, T cell transfer Model, • Oral efficacy with reduction endoscopy scores and histology. • Demonstrate equal or greater efficacy to positive control anti‐p40 Ab • Datasets to understand efficacy, level of target engagement and exposure response. • Dose response. • PK/PD understood to allow safety margin calculati... |
Colitis, Citrobacter rodentium model (prophylactic mode in C57BL6 mice WT or GPR65 KO) to confirm selectivity of the tool, • Oral efficacy with reduction in histology/endoscopy scores/reduced bacterial burden in colon tissue in C57BL6 WT mice, as well as reduced bacterial burdens and no activity in GPR65 KO mice to con... |
Colitis, Chronic DSS Model (6 week repeated low dose DSS; in C57BL6 mice WT or GPR65 KO) to confirm selectivity of the tool, • Oral efficacy with reduction in fibrosis endpoints • Equal or greater efficacy to positive control (to be defined upon model validation) • Datasets to understand efficacy, level of target engag... |
Appendix H: Stage 4a&b - CMC & Toxicology Workflow |
Appendix I: Nonclinical and CMC reports and documents to be included for an IND submission |
Nonclinical reports for an IND (small molecule) should cover, but not limited to, the following topics: |
Pharmacology |
Primary Pharmacology: in vitro, in vivo |
Secondary Pharmacology: off target binding assays for receptors, ion channels, enzymes and transporters |
Safety Pharmacology: CV (hERG, in vivo), CNS, respiratory |
Pharmacokinetics |
Bioanalytical method validation Absorption: cellular permeability, pharmacokinetics in nonclinical species, dose response, formulation effects (if necessary) |
Distribution: protein binding across species and concentration, red blood cell distribution, tissue distribution (optional) |
Metabolism: in vitro stability in liver microsomes/hepatocytes across species, CYP phenotyping, metabolite ID with proposed biotransformation pathway, in vivo metabolite profiles |
Excretion: definition of major clearance routes |
Drug‐Drug Interaction Potential: CYP inhibition (including TDI), CYP induction, interactions with transporters (inhibitor, substrate) |
Human pharmacokinetics predictions, including DDI potential at projected efficacious exposures |
Toxicology |
Single dose toxicity (optional) |
Repeat dose toxicity in rodent and non‐rodent |
Genotoxicity package: In vitro mutagenicity, in vitro chromosomal aberration, in vivo micronucleus |
CMC reports and documents for an IND (small molecule) should cover, but not limited to, the following topics: |
CMC |
Drug Substance Properties of the drug substance: Optical Isomerism, Physical State/Description, Melting Point, pKa, Solubility (specify conditions ‐ temp./pH), Hygroscopicity, Polymorphism |
Names and addresses of sites performing drug substance manufacture and testing (release and stability) |
Drug substance batch records which should include any testing done in‐process and at intermediates; ChemDraw file of synthetic scheme for GMP steps |
Any reports or batch records for manufacture of starting materials and CoAs for the starting materials (these are informational only) |
Documentation on how the GLP tox batch was manufactured (e.g., synthetic scheme or development report) so that a high‐level comparison can be made with the GMP batch |
Confirmation of structure: Information associated with Mass Spec, IR, NMR (H1 and C13), X‐Ray, UV (including specta) |
PGI assessment reports; Summaries of control strategies for Class 1‐2 impurities from starting materials and GMP synthesis steps; Option 4 control strategies for Class 1‐2 impurities; structures of any identified drug substance impurities if different from those specified in the drug substance |
Specifications |
Methods Validation information ‐ acceptance criteria, results, chromatgram overlays, linearity plots |
CoAs / results for clinical and non‐clinical lots |
Justification for each specification Reference Standard CoA or summary of results |
If details are not provided in the batch records, documentation describing all components of the drug substance package |
Stability Protocol(s) and details of study (e.g. packaging used for stability samples); Stability Data |
Drug Product |
List of all of the excipients used in the manufacture of the drug product; include quality of standard (e.g. USP/JP/Ph. Eu.), function and amount |
High level description of the manufacturing process. Names and addresses of sites performing drug product manufacture and testing (release and stability) |
Excipient amounts for an exemplary batch of drug product |
Detailed description of each step in the manufacturing process (including a flow diagram); diagram should include material inputs, process steps and process controls |
A list of all the critical steps in the manufacturing process |
Specification for each non‐compendial excipient, if applicable. |
Methods for evaluating each non‐compendial excipient, if applicable |
Method validation for each method for non‐compendial excipient, if applicable |
CoA for non‐compendial excipient, if applicable |
Attestation that there are not excipients of human or animal origin |
Listing of any novel excipients |
Drug Product Specifications |
Drug Product Methods |
Validation information ‐ acceptance criteria, results, chromatogram overlays, linearity plots |
CoAs / results for clinical and non‐clinical lots |
Characterization of impurities that are unique to the drug product; under what conditions are they formed |
Justification for each specification |
Reference Standard CoA or summary of results |
If details are not provided in the batch records, documentation describing all components of the drug product package |
Stability Protocol(s) and details of study (e.g. packaging used for stability samples); Stability Data |
Drug Product |
List of all of the excipients used in the manufacture of the drug product; include quality of standard (e.g. USP/JP/Ph. Eu.), function and amount |
High level description of the manufacturing process. |
Names and addresses of sites performing drug product manufacture and testing (release and stability) |
Excipient amounts for an exemplary batch of drug product |
Detailed description of each step in the manufacturing process (including a flow diagram); diagram should include material inputs, process steps and process controls |
A list of all the critical steps in the manufacturing process |
Specification for each non-compendial excipient, if applicable. |
Methods for evaluating each non-compendial excipient, if applicable |
Method validation for each method for non-compendial excipient, if applicable |
CoA for non-compendial excipient, if applicable |
Attestation that there are not excipients of human or animal origin |
Listing of any novel excipients |
Drug Product Specifications |
Drug Product Methods |
Validation information - acceptance criteria, results, chromatogram overlays, linearity plots |
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