Industry Perspective on the Pharmacokinetic and Absorption, Distribution, Metabolism, and Excretion Characterization of Heterobifunctional Protein Degraders.

Targeted protein degraders (TPDs), specifically the bifunctional protein degraders discussed in this manuscript, consist of two linked ligands for a protein of interest and an E3 ligase, resulting in molecules that largely violate accepted physicochemical limits (e.g., Lipinski's Rule of Five) for oral bioavailability. In 2021, the IQ Consortium Degrader DMPK/ADME Working Group undertook a survey of 18 IQ member and nonmember companies working on degraders to understand whether the characterization and optimization of these molecules were different from any other beyond the Rule of Five (bRo5) compounds. Additionally, the working group sought to identify pharmacokinetic (PK)/absorption, distribution, metabolism, and excretion (ADME) areas in need of further evaluation and where additional tools could aid in more rapid advancement of TPDs to patients. The survey revealed that although TPDs reside in a challenging bRo5 physicochemical space, most respondents focus their efforts on oral delivery. Physicochemical properties required for oral bioavailability were generally consistent across the companies surveyed. Many of the member companies used modified assays to address challenging degrader properties (e.g., solubility, nonspecific binding), but only half indicated that they modified their drug discovery workflows. The survey also suggested the need for further scientific investigation in the areas of central nervous system penetration, active transport, renal elimination, lymphatic absorption, in silico/machine learning, and human pharmacokinetic prediction. Based on the survey results, the Degrader DMPK/ADME Working Group concluded that TPD evaluation does not fundamentally differ from other bRo5 compounds but requires some modification compared with traditional small molecules and proposes a generic workflow for PK/ADME evaluation of bifunctional TPDs. SIGNIFICANCE STATEMENT: Based on an industry survey, this article provides an understanding of the current state of absorption, distribution, metabolism, and excretion science pertaining to characterizing and optimizing targeted protein degraders, specifically bifunctional protein degraders, based upon responses by 18 IQ consortium members and non-members developing targeted protein degraders. Additionally, this article puts into context the differences / similarities in methods and strategies utilized for heterobifunctional protein degraders compared to other beyond Rule of Five molecules and conventional small molecule drugs.

Discovery and Characterisation of Highly Cooperative FAK-Degrading PROTACs.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

Optimization of a Series of RIPK2 PROTACs.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.

GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice.

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB-POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB-POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB-POZ domain. The compound has good solubility (128 µg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

In Vivo Half-Life Extension of BMP1/TLL Metalloproteinase Inhibitors Using Small-Molecule Human Serum Albumin Binders.

Reducing the required frequence of drug dosing can improve the adherence of patients to chronic treatments. Hence, drugs with longer in vivo half-lives are highly desirable. One of the most promising approaches to extend the in vivo half-life of drugs is conjugation to human serum albumin (HSA). In this work, we describe the use of AlbuBinder 1, a small-molecule noncovalent HSA binder, to extend the in vivo half-life and pharmacology of small-molecule BMP1/TLL inhibitors in humanized mice (HSA KI/KI). A series of conjugates of AlbuBinder 1 with BMP1/TLL inhibitors were prepared. In particular, conjugate c showed good solubility and a half-life extension of >20-fold versus the parent molecule in the HSA KI/KI mice, reaching half-lives of >48 h with maintained maximal inhibition of plasma BMP1/TLL. The same conjugate showed a half-life of only 3 h in the wild-type mice, suggesting that the half-life extension was principally due to specific interactions with HSA. It is envisioned that conjugation to AlbuBinder 1 should be applicable to a wide range of small molecule or peptide drugs with short half-lives. In this context, AlbuBinders represent a viable alternative to existing half-life extension technologies.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.

Targeted protein degradation in vivo with Proteolysis Targeting Chimeras: Current status and future considerations.

Proteolysis Targeting Chimeras (PROTACs) are a rapidly expanding new therapeutic modality inducing selective protein degradation and offering the potential of a differentiated pharmacological profile across multiple therapeutic areas. As the repertoire of protein targets and E3 ligases available for incorporation into PROTACs continues to grow, understanding the drug- and system-dependent parameters for PROTACs will be critical for achieving tissue/cell specific pharmacology. The review discusses the current knowledge and future direction of in vivo PROTAC study evaluation. The importance of establishing the quantitative relationship between loss of protein target and biological function in vivo, coupled with building mechanistic PK/PD and ultimately PBPK/PD models, is emphasised with the aim to aid translation from preclinical to clinical space.

Identification and Optimization of Novel Small c-Abl Kinase Activators Using Fragment and HTS Methodologies.

Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.

Rapid Bioavailability and Disposition protocol: A novel higher throughput approach to assess pharmacokinetics and steady-state brain distribution with reduced animal usage.

Besides routine pharmacokinetic (PK) parameters, unbound brain-to-blood concentration ratio (Kp,uu) is an index particularly crucial in drug discovery for central nervous system (CNS) indications. Despite advantages of Kp,uu from steady state after constant intravenous (i.v.) infusion compared with one- or multiple time points after transient dosing, it is seldom obtained for compound optimization in early phase of CNS drug discovery due to requirement of prerequisite PK data to inform the study design. Here, we designed a novel rat in vivo PK protocol, dubbed as Rapid Bioavailability and Disposition (RBD), which combined oral (p.o.) dosing and i.v. infusion to obtain steady-state brain penetration, along with blood clearance, oral exposure and oral bioavailability for each discovery compound, within a 24 hour in-life experiment and only a few (e.g., 3) animals. Protocol validity was verified through simulations with a range of PK parameters in compartmental models as well as data comparison for nine compounds with distinct PK profiles. PK parameters (Kp,brain, CLb and oral AUC) measured from the RBD protocol for all compounds, were within two-fold and/or statistically similar to those derived from conventional i.v./p.o. crossover PK studies. Our data clearly indicates that the RBD protocol offers reliable and reproducible data over a wide range of PK properties, with reduced turnaround time and animal usage.

First administration of the Fc-attenuated anti-ß amyloid antibody GSK933776 to patients with mild Alzheimer's disease: a randomized, placebo-controlled study.

OBJECTIVE: To assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the Fc-inactivated anti-ß amyloid (Aß) monoclonal antibody (mAb) GSK933776 in patients with mild Alzheimer's disease (AD) or mild cognitive impairment (MCI). METHODS: This was a two-part, single blind, placebo-controlled, first-time-in-human (FTIH) study of single (n = 18) and repeat dose (n = 32) intravenous GSK933776 0.001-6 mg/kg (ClinicalTrials.gov: NCT00459550). Additional safety data from an open-label, uncontrolled, single dose study of intravenous GSK933776 1-6 mg/kg (n = 18) are included (ClinicalTrials.gov: NCT01424436). RESULTS: There were no cases of amyloid-related imaging abnormalities-edema (ARIA-E) or -hemorrhage (ARIA-H) after GSK933776 administration in both studies. Three patients across the two studies developed anti-GSK933776 antibodies. Plasma GSK933776 half-life (t1/2) was 10-15 days after repeat dosing. After each of three administrations of GSK933776, plasma levels of total Aß42 and Aß increased whereas plasma levels of free Aß decreased dose dependently; no changes were observed for placebo. For total Aß42 the peak:trough ratio was ≤2 at doses ≥3 mg/kg; for total Aß the ratio was ≤2 at 6 mg/kg. CSF concentrations of Aß showed increases from baseline to week 12 for Aß X-38 (week 12:baseline ratio: 1.65; 95%CI: 1.38, 1.93) and Aß X-42 (week 12:baseline ratio: 1.18; 95%CI: 1.06, 1.30) for values pooled across doses. CONCLUSION: In this FTIH study the Fc-inactivated anti-Aß mAb GSK933776 engaged its target in plasma and CSF without causing brain ARIA-E/H in patients with mild AD or MCI. TRIAL REGISTRATION: ClinicalTrials.gov NCT00459550.

Pharmacokinetic and pharmacodynamic profiling of a P2X7 receptor allosteric modulator GSK1482160 in healthy human subjects.

AIMS: This paper describes findings from the first-in-human study for GSK1482160, an orally available allosteric P2X7 receptor modulator. The study aimed to assess the pharmacokinetics (PK), pharmacodynamics (PD), safety and tolerability of the compound in healthy subjects. METHODS: Escalating single doses of up to 1 g were administered to healthy subjects in a single-blind and placebo-controlled fashion. Safety, tolerability, blood drug concentrations and ex vivo Il-1ß production in blood were evaluated. RESULTS: Drug concentration peaked within 3.5 h of dosing under fasting conditions and declined thereafter with a relatively short half-life of less than 4.5 h. Exposure was proportional to dose with between subject variability of less than 60%. A PK/PD model quantified Il-1ß as a function of drug exposure. The model allowed simulation of in vivo pharmacology for various untested dose levels and regimens. Furthermore, the mechanistic model supported the hypothesis that the compound reduces the efficacy of ATP at the P2X7 receptor without affecting its affinity. No major safety or tolerability concerns were identified in this small study (n = 29), except for one case of asymptomatic accelerated idioventricular rhythm at the top dose. CONCLUSION: The model-based approach maximized analysis power by integrating all biomarker data and revealed mechanistic insight into the pharmacology of P2X7 modulation by GSK1482160. Simulations by this model ultimately led to the discontinuation of the development of this compound. The therapeutic relevance of the P2X7 receptor remains to be tested in patients. The mechanistic-model-based approach can be applied widely to drug development.

Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans.

The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [(3)H]flumazenil binding or [(11)C]flumazenil small-animal positron emission tomography (microPET) in rats, [(123)I]iomazenil gamma-scintigraphy in rhesus monkeys, and [(11)C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.

Contribution of specific binding to the central benzodiazepine site to the brain concentrations of two novel benzodiazepine site ligands.

The in vivo occupancy of brain benzodiazepine binding sites by compounds A and B was measured using a [(3)H]Ro 15-1788 binding assay and related to plasma and brain drug concentrations. The plasma concentration associated with 50% occupancy was higher for compound A than compound B (73 and 3.7 nM, respectively), however, there was little difference in the brain concentrations required (73 and 63 nM). Both compounds showed a non-linear relationship between plasma and brain concentrations such that above brain concentrations of approximately 100 nM increasing plasma concentrations did not result in a concomitant increase in brain concentrations. This is consistent with brain concentrations being dependent on a saturable compartment which was postulated to be the benzodiazepine binding site-containing GABA(A) receptors. This hypothesis was tested in alpha1H101R mice, in which the alpha1 subunit of the GABA(A) receptor is rendered insensitive to benzodiazepine binding resulting in an approximate 50% reduction in the total benzodiazepine-containing GABA(A) receptor population. It was shown that the Occ(50) brain concentrations in the alpha1H101R animals was lower (17 nM) than in wild type mice (63 nM), as was the plateau concentration in the brain (105 and 195 nM, respectively). These data suggest measured concentrations of compounds A and B in brain tissue are dependent on receptor expression with a minimal contribution from unbound and non-specifically bound compound.

Comparison of lorazepam [7-chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one] occupancy of rat brain gamma-aminobutyric acid(A) receptors measured using in vivo [3H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) binding and [11C]flumazenil micro-positron emission tomography.

The occupancy by lorazepam of the benzodiazepine binding site of rat brain GABA(A) receptors was compared when measured using either in vivo binding of [(3)H]flumazenil (8-fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) in terminal studies or [(11)C]flumazenil binding in anesthetized animals assessed using a small animal positron emission tomography (PET) scanner (micro-PET). In addition, as a bridging study, lorazepam occupancy was measured using [(3)H]flumazenil in vivo binding in rats anesthetized and dosed under micro-PET conditions. Plasma lorazepam concentrations were also determined, and for each occupancy method, the concentration required to produce 50% occupancy (EC(50)) was calculated because this parameter is independent of the route of lorazepam administration. For the in vivo binding assay, lorazepam was dosed orally (0.1-10 mg/kg), whereas for the micro-PET study, lorazepam was given via the i.v. route as a low dose (0.75 mg/kg bolus) and then a high dose (0.5 mg/kg bolus then 0.2 mg/ml infusion). The lorazepam plasma EC(50) in the [(11)C]flumazenil micro-PET study was 96 ng/ml [95% confidence intervals (CIs) = 74-124 ng/ml], which was very similar to the [(3)H]flumazenil micro-PET simulation study (94 ng/ml; 95% CI = 63-139 ng/ml), which in turn was comparable with the [(3)H]flumazenil in vivo binding study (134 ng/ml; 95% CI = 119-151 ng/ml). These data clearly show that despite the differences in dosing (i.v. in anesthetized versus orally in conscious rats) and detection (in vivo dynamic PET images versus ex vivo measurements in filtered and washed brain homogenates), [(11)C]flumazenil micro-PET produces results similar to [(3)H]flumazenil in vivo binding.

An evaluation of a low-density DNA microarray using cytochrome P450 inducers.

The aim of this study was to validate a low-density DNA microarray "Rat HepatoChip", which contains 59 genes from a range of potential toxic markers and drug metabolism-related genes. Liver mRNA was isolated from rats dosed with six different chemicals, dexamethasone, troleandomycin, miconazole, clotrimazole, and methylclofanapate, which are all known to induce different cytochrome P450 genes, and isoniazid, which does not cause histopathological changes. Replicate microarrays were used to measure the variability in the chips and in the process. The average variability in signal between different chips observed in triplicate experiments was 33% ranging from 21 to 39% depending on genes. We also demonstrated a strong correlation between the liver histopathology and the gene expression profiles indicating that the gene expression profile reflects histopathological changes. These results suggest that the Rat HepatoChip microarray may provide a fast and effective tool for assessing the toxicity profile of developmental drug candidates during the drug discovery process.