Trigriluzole (FC-4157) is a third-generation prodrug of riluzole, which is known to upregulate the excitatory amino acid transporter (EAAT)-2 and lower extracellular glutamic acid levels.
In 1995, riluzole was approved by the FDA for the treatment of amyotrophic lateral sclerosis (ALS). In this setting, riluzole has been shown to extend patient life by 4-6 months. Riluzole has also been evaluated as a treatment for spinocerebellar ataxia, a somewhat rare degenerative genetic condition characterized by extremity, eye movement, and speech coordination issues. However, perhaps one of the most interesting findings with regards to this drug was its activity against metabotropic glutamate receptor 1 (GRM1)-expressing tumors (e.g., melanomas) in preclinical mouse xenograft studies.
This led to the undertaking of a phase 0 study that evaluated riluzole in doubly refractive melanoma patients over the course of 2 weeks. The variable results obtained in that study highlighted the need for a prodrug that would address many of the pharmacokinetic issues with riluzole. Research to address those issues led to the identification of trigriluzole (FC-4157, BHV-4157), a tripeptide-functionalized prodrug of riluzole that had improved PK properties. This compound is now being evaluated in combination with the anti-PD-1 monoclonal antibody nivolumab in a phase Ib clinical trial (NCT03229278) being undertaken by Ann Silk, MD, and colleagues at Rutgers Cancer Institute of New Jersey.
“We are very excited about this trial, as we believe trigriluzole could improve the effectiveness of immunotherapies like the checkpoint inhibitors by altering the normally immunosuppressive tumor microenvironment,” Silk said.
Riluzole & Murine Melanoma
On Dec. 12, 1995, the FDA approved riluzole as the first treatment for ALS, a neurodegenerative disorder that is often fatal within 5 years from time of diagnosis. Riluzole exerts these pharmacological effects by virtue of its ability to reduce synaptic glutamate levels, which reduces glutamate-associated neuroexcitation and toxicity. Subsequent to that approval, in 2007, a paper was published by Chen et al. (Cancer Res 2007; 67(5);2298) which showed that the overexpression of GRM1 played an important role in the onset of melanoma in mice.
Ectopic expression of GRM1 in melanocytes induced the development of melanoma, which suggested that the glutamate pathway is implicated in malignant transformation. The researchers in that study showed that the melanoma cells released elevated levels of glutamate, thereby implicating a possible autocrine loop. Indeed, when human melanoma cells that expressed GRM1 were treated with riluzole or GRM1 antagonists such as BAY36-7620 or LY367385, both extracellular glutamate levels and cell proliferation were suppressed.
Studies performed by that laboratory showed the treatment of murine melanoma lines with a GRM1 agonist such as L-quisqualate resulted in activation of mitogen-activated protein kinase (MAPK). In prior studies, constitutively activated MAPK, in specific, extracellular signal-regulated kinase (ERK), has been noted in human melanoma cell lines and tissue samples. However, in those studies, it was the V600E BRAF-activating mutation which served as the activator for ERK. In their studies, Chen et al. showed that activation of the MAPK pathway can occur in GRM1-expressing melanoma cells via treatment with GRM1 agonists, even in the absence of a V600E BRAF-activating mutation.
In this same article, preclinical mouse xenograft models were performed with human melanoma cell lines. In these studies, dosing with riluzole either PO or IV was shown to inhibit the tumor growth of the GRM1-expressing tumors by 50 percent relative to those of the control group. Those results, when taken with the fact that more than 60 percent of the tested human melanoma samples tested displayed ectopic expression of GRM1, provided the rationale for a phase 0 study for riluzole in refractory stage III or IV melanoma patients.
Phase 0 Melanoma Trial
In 2009, Yip et al. performed a phase 0 trial which evaluated the use of riluzole in patients with resectable stage III or IV melanoma (Clin Cancer Res 2009;15(11); 3896 ). The dosage level utilized in this study (200 mg/day) was obtained from previous maximum recommended daily dose data derived in the treatment of ALS patients, thus obviating the need for a dose-determining phase I trial in melanoma patients.
Additionally, the duration of treatment—2 weeks—was chosen because this value was approximately 7x the half-life of riluzole after it reaches its steady state, and in the aforementioned preclinical mouse xenograft studies, 2 weeks of dosing was shown to produce a measurable effect in tumor reduction.
In this phase 0 trial, enrolled patients were initially subjected to a pretreatment biopsy. Once tissue samples and other pre-treatment metrics were obtained, the participants were dosed with 100 mg riluzole p.o. BID for 2 weeks. Following completion of the riluzole regimen, post-treatment metrics were taken and the tumors were resected. The resection samples were then subjected to analysis to measure ERK and phosphatidylinositol-3-kinase (PI3K)/AKT levels using Western blotting techniques. These results were compared to those obtained from pretreatment samples to assess riluzole's effect.
In addition to these analyses, tumor sizes were assessed radiologically pre- and post-treatment using CT. Also, PET was performed using 18F 2-fluorodeoxyglucose (FDG) prior to and after treatment with riluzole to assess patients' tumor metabolic activities to assess the effect of GRM1 inhibition.
Twelve patients were enrolled in this study and, of these, 11 were able to successfully complete their therapy for inclusion. The lone patient that was removed from the study was due to grade 3 level dizziness, which was resolved upon dosing cessation.
On post-treatment PET/CT scans, four out of 12 patients demonstrated both a significant decrease in their FDG-PET intensity and a significant decrease in the phosphorylated ERK and/or AKT in their post-treatment tumor samples. Clinical benefit (reduction in pain, etc.) was noted in patients after riluzole treatment. Tumors from all 12 patients expressed GRM1.
The variable responses noted in the patients were, in part, thought to arise from riluzole's pharmacokinetic properties. “Riluzole is metabolized by CYP1A2 in the liver, and a substantial portion of the compound is eliminated by a significant first pass effect. In this case, it was thought that the differing results obtained by the patients in this study could be a result of varying systemic exposures for riluzole, potentially due to different levels of CYP1A2 expression in the patients,” Silk explained.
Prodrug Search Yields Trigriluzole
In order to circumvent the potential pharmacological issues, several different prodrugs (i.e., compounds which are taken and then undergo metabolic reaction in vivo to afford the active drug) were synthesized. Among the issues that the investigators sought to address were to reduce the first pass effect by minimizing the CYP1A2 metabolism, increase solubility, metabolize to riluzole once in the bloodstream; and extend the time of maximum concentration (Tmax) for riluzole to possibly permit QD dosing. These favorable properties would also be expected to mitigate the unfavorable liver enzyme elevation occasionally seen with riluzole treatment.
First-generation prodrugs for riluzole were created by functionalizing the exocyclic primary amine with a single amino acid. While these new analogs did show improved PK properties and efficacy in mouse xenograft models, interspecies variability and chemical stability issues necessitated the continued search for optimized analogs.
Next, second-generation prodrugs were synthesized which consisted of dipeptide-functionalized riluzole derivatives. These compounds also showed improved PK properties. In addition, mouse xenograft studies with a human melanoma cell line showed that, for one analog, similar efficacy to riluzole was obtained at a dosage that was one-third of the parent compound (on a molar basis). This particular compound, FC-3423, however, had other limitations; it showed sub-micromolar IC50 values for hERG, the potassium channel associated with cardiac arrhythmias and QT elongation in vivo.
As a consequence of this unwanted and potentially fatal activity, third-generation riluzole analogs were synthesized and evaluated. From these efforts, investigators identified FC-4157 (trigriluzole), a tripeptide-based riluzole prodrug as a strong candidate.
One early study in mice compared PK data for the presence of riluzole from direct dosing (7.5 mg/kg, PO) and from trigriluzole dosing (5.0 mg/kg, PO). This study showed that the Tmax was increased four-fold for trigriluzole dosing (direct dosing-0.5 hr vs. trigriluzole dosing-2.0 hrs). Additionally, the systemic bioavailability (%F) was increased for riluzole formed upon trigriluzole dosing (direct dosing-18% vs. trigriluzole dosing-56%). When evaluating the dose-adjusted AUC (area under the time-concentration curve), there was an approximately two-fold increase for trigriluzole dosing (119 vs. 65 ng-h/ml/kg/mg).
In vivo, trigriluzole has been demonstrated to be absorbed via the gut Peptide transporter PepT1, a transporter of di- and tripeptides, as the glycine-sarcosine portion of the tripeptide moiety serves as a recognition motif. The use of this transporter is advantageous, and reasonable cross-species homology has been noted. Additionally, this same transporter has been utilized for known drugs such as the antiviral valacyclovir. Evidence for the efficiency of this absorption was shown by the fact that less than 2 percent of trigriluzole remained in the rat alimentary canal after 30 minutes post dosing (5 mg/kg, PO). Uptake by PepT1 is likely the reason why trigriluzole will not be subject to the fasting requirement before and after dosing that accompanies treatment with riluzole itself.
The release of riluzole from trigriluzole appears to be mediated by exo-peptidases in vivo. Dosing with the terminal peptidase inhibitors amastatin and bestatin drastically reduced the formation of riluzole in rat plasma (95% and 85% prodrug remaining, respectively, after 2 hours).
To assess the efficacy of this prodrug in an in vivo setting, a study was performed using a C8161 human melanoma mouse xenograft model. That study showed that trigriluzole (1.7 mg/kg) had greater potency when compared to a 10-fold higher dose of riluzole (7.5 mg/kg), thus providing further proof of concept for the prodrug approach.
GRM1-Mediated Tumor Immune Evasion
In addition to its effect on MAPK signaling, GRM1 may also play an important role in the ability of tumors to evade T cell-mediated immune destruction. Immortalized mouse melanoma cells that express GRM1 (MASS20) were shown to have increased production of two cytokines—C-C motif chemokine ligand 2 (CCL2) and macrophage colony-stimulating factor (M-CSF).
When explaining these findings, Silk noted: “These cytokines serve to both recruit (CCL2) and polarize (M-CSF) the tumor-associated macrophages (TAM) and increase the M2 macrophage fraction, thus facilitating immune evasion and allowing tumor progression.”
MASS20-based tumors in C57BL6 mice were shown to be inhibited by treatment with a macrophage-targeting agent (liposomal clodronate (LC)) relative to the untreated control mice. Flow cytometry analyses of tumor cells showed that the LC-dosed mice had depleted macrophage (by 90%) and increased CD8+ T-cell and CD335+ natural killer cell levels relative to their control counterparts.
“The data from this and other experiments in our laboratories would seem to suggest that there is a link between GRM1-signal transduction and TAM-induced suppression of effector T cells in melanoma,” Silk explained. “We conclude that GRM1-induced suppression of tumor immunity occurs through the recruitment and polarization of M2 macrophages in the tumor microenvironment.”
Trigriluzole + Anti-PD-1 Antibody
To investigate the role of glutamate signaling in TME-mediated immunity, a MASS20, GRM1-positive mouse allograft immunocompetent model was used to evaluate the use of trigriluzole plus a mouse anti-PD-1 monoclonal antibody.
In this study, both the riluzole+anti-PD-1 antibody and trigriluzole + anti-PD-1 antibody mice tumors were smaller compared to all other mice in the study (controls, riluzole, trigriluzole, anti-PD-1 antibody). Although the reduction in tumor outgrowth was comparable for the riluzole + anti-PD-1 and trigriluzole + anti-PD-1 treated mice, the dosage level for trigriluzole was approximately 10 percent that of riluzole on a molar basis.
Phase Ib Human Studies of Trigriluzole
When asked about the next steps for this compound, Silk replied “Initially, we will be performing a phase Ib dose escalation study evaluating the use of trigriluzole in combination with the anti-PD-1 antibody nivolumab.
“Although the initial part of our study is utilizing nivolumab, we do have plans to evaluate the maximum tolerated dose of trigriluzole in combination with pembrolizumab in a small expansion cohort,” she added.
“The primary endpoint for this study will be the safety profile and maximum tolerated dose, while the secondary endpoints include response rate and overall survival,” Silk elaborated. When discussing the regimen in this study, she noted, “There will be a 2-week lead-in of trigriluzole dosing prior to beginning the antibody immunotherapy. This allows us to evaluate the safety of trigriluzole monotherapy and collect tissue for correlative analyses.”
While discussing plans for this trial, Silk noted, “We plan to evaluate this combination therapeutic approach in melanoma and possible other solid tumors in which GRM1 is a target, like breast cancer.”
“We also plan to have a cohort of melanoma patients that are PD-1 inhibitor refractory—so that we can see if trigriluzole can ‘rescue’ patients who did not respond to anti-PD-1 therapy because of an overly suppressive tumor microenvironment,” she continued. “We also plan to have a cohort of PD-1 inhibitor-naïve melanoma patients, so that we can evaluate whether trigriluzole + PD-1 inhibitor is more effective than the PD-1 inhibitor alone in previously untreated patients.”
Currently, enrollment on the dose escalation trial is ongoing.
Richard Simoneaux is a contributing writer.