A First-in-Class TRPA1 Antagonist Overcomes Toxicity Hurdles to Become Cough Candidate

Genentech’s GDC-6599 is the first oral TRP Ankyrin 1 (TRPA1) antagonist to reach Ph. IIa (NCT05660850) for chronic cough after preclinical studies and a Ph. I trial showed it was well-tolerated, in contrast to prior molecules. The transient receptor potential (TRP) family of ion channels has been the subject of intensive drug discovery efforts due to their critical role in the development and progression of pain, itch, and respiratory conditions. In the Ph. IIa trial, GDC-6599 will be evaluated for its efficacy and safety in patients with chronic cough (persistent cough > 8 weeks). The challenges and promise of modulating another ion channel target in chronic cough, P2X3, has been recently discussed here. Previous TRPA1 antagonists have struggled in development, often due to PK and toxicity issues. For example, Genentech’s previous TRPA1 antagonist, GDC-0334 (Figure 1), was terminated in Ph. I (NCT03381144) due to toxicity findings in preclinical models. GDC-6599 offers a notable case study in addressing several unexpected toxicities while pursuing a novel target through mechanistic investigation.

Figure 1. Examples of advanced TRPA1 antagonists

TRP Ion Channel Targets Have Moved From TRPV1 to TRPA1

Companies including Merck and Janssen initially pursued TRP vanilloid 1 (TRPV1) (e.g., Neurogen/Merck’s MK-2295, Figure 1) as an analgesic target because the ion channels appear to play a critical role in pain sensation in preclinical studies, and targeting the channels might not come with the significant side effects, such as abuse potential, associated with commonly used opioid analgesics. However, several TRPV1 antagonists failed in clinical trials due to thermoregulatory side effects, such as hyperthermia and hypothermia, as well as an increased heat pain threshold that could lead to burn injuries. Attention appears to have shifted to antagonizing TRPA1 because while it plays a prominent role in mediating pain, it is not involved in regulating body temperature, possibly escaping the hyperthermia and related side effects associated with TRPV1 antagonists.

TRPA1 is Abundantly Expressed in Pain Neurons and May Exacerbate Inflammation

TRPA1 is abundantly expressed in sensory neurons for pain where it acts as a sensor of stress and tissue damage. When activated, the channel triggers vasodilation and immune cell recruitment to inflamed sites through the release of inflammatory neurotransmitters and neuropeptides including neurokinin, substance P, and calcitonin gene-regulating protein (CGRP), exacerbating inflammation and potentially leading to the development of chronic inflammatory diseases like asthma. In asthma, TRPA1 plays a key role in airway hyper-responsiveness (AHR) and smooth muscle contraction triggered by sensory neurons. Both of these phenomena can lead to an inflammatory cascade that ultimately causes increased mucus secretion, coughing, and shallow breathing. Genetic ablation of TRPA1 in mice inhibited allergen-induced airway leukocyte infiltration, which suggests that antagonizing TRPA1 could reduce airway inflammation and cough symptoms. Hence, TRPA1 is seen as a promising pharmacological target for treating asthma and other allergic inflammatory conditions.  

Toxicity Tripped Up Previous TRPA1 Antagonists

To date, no clinical TRPA1 modulators have been approved, although multiple companies have attempted to develop one. Hydra Biosciences, a Cambridge, Mass. biotech company founded in 2001, was among the first to launch a TRPA1 drug discovery program. The company teamed up with Boehringer Ingelheim to advance HX-100 (structure not disclosed) into clinical trials to treat allergic asthma and painful diabetic neuropathy. A Ph. I trial was completed in 2016, and the molecule was expected to proceed to Ph. II, but was discontinued due to dose-related skin and musculoskeletal adverse events (details undisclosed). LY3526318 (HX-260), a later molecule discovered by Hydra Biosciences and acquired by Lilly in 2018, is an orally bioavailable, potent, and selective TRPA1 antagonist that is currently in Ph. II (NCT05986292) to evaluate its efficacy in chronic pain. A Ph. I trial (NCT04183283) showed that the molecule was safe, well tolerated, and effective in modulating TRPA1. Several other hcompanies have taken TRPA1 antagonists forward with limited success (e.g., Glenmark/Ichnos: GRC17536/ISC 17536 (undisclosed), endpoints not met in Ph. IIa for pain, Cubist/Hydra, CB-189625 (undisclosed), for acute pain but likely discontinued, Orion, ODM-108 (undisclosed, negative allosteric modulator), Ph. I, terminated due to “complex PK”). Genentech recently ended the clinical development of GDC-0334 earlier than anticipated in Ph. I (NCT03381144) due to toxicity findings in preclinical studies conducted in parallel with the trial. However, it’s unclear whether the toxicities observed with GDC-0334 were off or on target.

A Next-Generation TRPA1 Antagonist Arises from a Novel Chemotype from Amgen

The discovery of GDC-6599 started from Genentech's first-generation TRPA1 antagonist GDC-0334 (Figure 2). 

Figure 2. High-level overview of the journey from GDC-0334 to GDC-6599.

GDC-0334 showed high permeability and low clearance in preclinical PK studies, but has poor solubility and requires an amorphous formulation for adequate exposure  (Table 1). In addition, the molecule carried a DDI risk, and researchers observed unfavorable neurological findings in dogs and cynomolgus monkeys. After clinical development of GDC-0334 was terminated, Genentech sought to identify structurally distinct TRPA1 antagonists devoid of the liabilities associated with GDC-0334. The purinone oxadiazole in vivo tool AM-0902 (Amgen) is a potent and selective TRPA1 antagonist that was discovered via HTS on human and rat TRPA1 in a FLIPR calcium imaging assay followed by SAR optimization. Genentech used the molecule as a starting point for the discovery of second-generation TRPA1 antagonists  (Table 1).

Table 1. First-generation candidate GDC-0334 carried a DDI risk and neurological toxicities in higher species. GNE-0230 is significantly more potent than its starting point, but shows an unusual anti-coagulation side effect. - = not reported. * PK data for AM-0902

Ligand-based optimization of the core and linker led to the identification of GNE-9159, which was 5-fold more cell potent with improved metabolic stability compared with AM-0902. It was stated that the fluorine substitution at the benzylic carbon played a key role in improving potency and metabolic stability. Moreover, the 2-oxo-oxadiazole GNE-9159 was more synthetically tractable via alkylation chemistry compared to oxadiazole AM-0902, which allowed for the rapid exploration of different linker systems and changes to the phenyl moiety. 

An Unexpected Coagulopathy Side Effect Identified in Higher Species

In 7-day repeat-dose toxicity studies of GNE-9159, the molecule was tolerated in rats up to 1000 mg/kg/day, but it was not tolerated in cynomolgus monkeys at all doses tested (>8× AUC0-24) (toxicity not specified), and the compound formed reactive metabolites in human liver microsomes. The molecule was also flagged as positive in the GSH trapping assay, suggesting it had a potential liver toxicity issue. Furthermore, GNE-9159 caused dose-dependent coagulopathy (impaired clotting) in cynomolgus monkeys. GNE-9159 also led to the development of vacuolation in the retina and renal tubular degeneration and inflammation in the monkeys. These were serious adverse events that the Genentech team needed to understand and avoid to make progress.

TRPA1 Antagonist-Caused Coagulopathy Explained by Off-Target Activity on Vitamin K-Dependent Clotting Factors

Experiments with GNE-9159 on the blood clotting cascade showed that the molecule inhibited vitamin K-dependent clotting factors (e.g., FII, FIX, FX, and Protein C), but not non-vitamin K-dependent clotting factors (e.g., FV, FXI). Researchers suspected GNE-9159 or its metabolites may be inhibiting enzymes involved in the vitamin K redox cycle, such as GGCX or VKOR, the target of well-established anticoagulant, warfarin (Figure 3).

Figure 3. The vitamin K redox cycle and the mechanism of action of warfarin. Warfarin inhibits VKOR, which is essential for vitamin K turnover. Vitamin K hydroquinone is involved in a key step of the activation of clotting factors like Factor X, in which a glutamate residue is carboxylated.

Rigidification of the linker in GNE-9159 led to the more potent GNE-0230 having an improved window against coagulation, but anticoagulation was still an issue. Fortunately, unlike GNE-9159, GNE-0230 did not cause retinal changes in cynomolgus monkeys, though histopathology at all doses still revealed renal tubular neuropathy characterized by basophilia and individual cell necrosis. While GNE-0230 improved safety over GNE-9159, coagulopathy and renal toxicities had not been adequately addressed, so progression of GNE-0230 was discontinued. 

Metabolites Drive Anticoagulant Activity

MetID studies of GNE-0230 revealed that the molecule formed a hypoxanthine metabolite (M8) with dicarbonyl motifs similar to warfarin and related anticoagulants (Figure 4). GNE-9159 produced similar hypoxanthine-derived metabolites. Significant levels of circulating M8 in cynomolgus monkey plasma was detected at all doses of GNE-0230 and time points, and M8 accumulated with repeating doses, which was expected to translate to humans according to the MetID data and thus could not be ignored. These findings led to the suspicion that these metabolites were the cause of the undesired anticoagulation activity. 

Figure 4. MetID studies for GNE-0230 and GNE-9159 revealed significant levels of metabolites arising from oxidation, which was suspected to be mediated by AO. Metabolites like M8 and M16 show a conspicious similarity to the tricarbonyl-containing warfarin class of anticoagulants.

The Smoking Gun: Dogs Don’t Have Liver Aldehyde Oxidase Activity

It was hypothesized that aldehyde oxidase (AO) was the driver of this metabolism because nitrogen-containing heteroaromatic rings such as hypoxanthine are susceptible to AO activity, and these metabolites weren’t formed in dogs (Table 2), which do not have liver aldehyde oxidase activity. The team therefore started to optimize against AO-mediated metabolism.

Table 2. % relative the two major metabolites M7 and M8 in different species hepatocytes reveals a conspicuous stability of GNE-0230 in dogs.

Mitigating Aldehyde Oxidase Activity Resolves Coagulation Issue

To prevent the formation of anticoagulant metabolites by AO, medicinal chemists tested multiple structural changes to block the position of oxidation (Table 3). 

Table 3. Optimization of GNE-0230 into GDC-6599. HH = human hepatocytes, CH = cyno hepatocytes. hTRPA1 was determined via a calcium influx dose−response assay on the CHO cell line.

Blocking a potential site of AO metabolism at C-2 of the hypoxanthine moiety by installing an amine (compound A), replacing the hydrogen with deuterium (compound B), or installing a methyl group at C-2 (GDC-6599) led to a reduction in AO metabolism.

GDC-6599 was associated with good metabolic stability and minimal prolongation of coagulation parameters and was selected as a candidate for further characterization.

GDC-6599 is a Selective TRPA1 Antagonist with Favorable Preclinical Activities

GDC-6599 demonstrated a favorable pharmacokinetic profile in different species, as well as high potency, solubility, permeability, and metabolic stability (Table 4). It also was selective for TRPA1 over other TRP ion channels, hERG, and other targets. In addition, the molecule demonstrated minimal prolongation of prothrombin time (PT) in cynomolgus monkeys and no clinical or histopathological findings related to coagulopathy up to 50 mg/kg. 

Table 4. Characteristics of GDC-6599

A Guinea Pig Efficacy Model Translates to Humans

Allyl isothiocyanate and cinnamaldehyde activate TRPA1 receptors to different extents. Cinnamaldehyde induces cough that can be partially reversed by TRPA1 antagonists. When AITC is applied to skin, it induces TRPA1-mediated inflammation and pain. GDC-6599 dose-dependently reduced cinnamaldehyde-induced cough in guinea pigs, which unlike rats and mice have a similar cough reflex to humans. GDC-6599 also showed dose-dependent activity in an AITC-induced dermal blood flow study in guinea pigs, which was important since this AITC-induced dermal blood flow study would also be used to assess target engagement in humans (see below). 

GDC-6599 is Well-Tolerated in Healthy Volunteers, Shows PK/PD Translation, and is Now in Ph. IIa for Cough

The safety and PK of GDC-6599 was investigated in a Ph. I Single Ascending Dose (SAD) and Multiple Ascending Dose (MAD) dose escalation study (NCT not reported) in 71 healthy volunteers. GDC-6599 was tolerated in both SAD and MAD doses for up to 8 days, and all reported adverse events were mild; no AEs led to treatment discontinuation. While taste effects have been observed with mice, so far no taste issues have been disclosed with GDC-6599, which in contrast were quickly reported with P2X3 inhibitors in chronic cough. GDC-6599 displayed a dose-proportional PK, while the half-life was dose-independent and averaged ~8 hours. Furthermore, GDC-6599 showed a PK/PD correlation in AITC-induced dermal blood flow suggesting target engagement in humans, which is consistent with preclinical animal model data in guinea pigs, confirming that the guinea pig AITC model translates for TRPA1 modulators.  (An analogous test utilizing cinnamaldehyde was conducted in an earlier clinical trial to assess LY3526318’s PK/PD correlation). This is notable as translation between humans and rodents is often challenging, especially for TRPA1 in which the sequence homology between human TRPA1 and rat TRPA1 is <80%. GDC-6599 is currently undergoing a randomized, multicenter Ph. IIa study (NCT05660850) to evaluate its efficacy, safety, PK, and PD effects in 80 patients with chronic refractory cough (CRC) associated with non-atopic asthma, atopic asthma, chronic obstructive pulmonary diseases (COPD), COPD with chronic bronchitis, or unexplained chronic cough (UCC). The clinical advancement of the molecule is a culmination of a significant scientific journey involving novel target biology, challenging pharmacology, and strategic medicinal chemistry. 

Chemical Synthesis

The patent med. chem. synthesis of GDC-6599 started with 4-chlorobenzaldehyde. Grignard addition of allyl magnesium chloride to the aldehyde, followed by epoxidation and acid-catalyzed cyclization afforded the 3-hydroxytetrahydrofuran intermediate. The hydroxyl group was converted to a nitrile via a mesyl intermediate, followed by conversion into an amidoxime and the subsequent formation of the 1,2,4-oxadiazole intermediate (Figure 5). 

Figure 5. Synthesis of 1,2,4-oxadiazole intermediate

The hypoxanthine intermediate was synthesized in parallel starting from 4,6-dichloro-2-methylpyrimidin-5-amine. After methylation, chlorine displacement with ammonia and subsequent condensation utilizing triethyl orthoformate afforded the hypoxanthine intermediate. Alkylation of this intermediate with the 1,2,4-oxadiazole intermediate yielded the final product, GDC-6599 (Figure 6).

Figure 6. Synthesis of GDC-6599

Relevant Patent

Oxadiazole transient receptor potential channel inhibitors. US20190284179A1 (GDC-6599 corresponds to analog 8).

Updated 3/5/2024: Link to new publication on GDC-6599 discovery added.