Dose-dependent Target Engagement Research Articles

Dose-dependent target engagement of a clinical iTBS protocol: An interleaved TMS-fMRI study in healthy subjects

BackgroundIntermittent theta burst stimulation (iTBS) of the dorsolateral prefrontal cortex (DLPFC) is widely applied as therapeutic intervention in mental health, however understanding of its mechanisms is still incomplete. Prior MRI studies have mainly used offline iTBS or short sequences in concurrent TMS-fMRI. This study investigated a full 600 stimuli iTBS protocol using interleaved TMS-fMRI in comparison with two control conditions in healthy subjects. MethodsIn a crossover design, 18 participants underwent three sessions of interleaved iTBS-fMRI: 1) left DLPFC at 40% resting motor threshold (rMT) intensity, 2) left DLPFC at 80% rMT intensity, and 3) left primary motor cortex (M1) at 80% rMT intensity. We compared immediate blood-oxygen-level-dependent (BOLD) responses during interleaved iTBS-fMRI across these conditions including correlations between individual fMRI BOLD activation and iTBS induced electric field (E-field) strength at the target sites. ResultsWhole-brain analysis showed increased activation in several regions following iTBS. Specifically, left DLPFC, as well as bilateral M1, anterior cingulate cortex, and insula showed increased activation during 80% rMT left DLPFC stimulation. Increased BOLD activity in the left DLPFC was not observed with 40% rMT left DLPFC stimulation nor left M1 80% rMT iTBS, whereas activation in other regions was found to overlap between conditions. Of note, BOLD activation and E-field intensities were only correlated for M1 stimulation, but not for the DLPFC conditions. ConclusionsThe study showed dosage and target specific BOLD activation during interleaved TMS-fMRI with 600 stimuli iTBS in healthy subjects. Future studies may use our approach for demonstrating target engagement.

EMITT-1: Proof-of-mechanism immunopeptidome (ImPD) effects at target PK exposure, in a phase 1 study of GRWD5769 (a first-in-class inhibitor of Endoplasmic Reticulum Aminopeptidase 1 [ERAP1]) in patients with solid malignancies.

2589 Background: GRWD5769 is a first-in-class, orally bioavailable ERAP1 inhibitor that significantly modulates the peptide repertoire displayed on MHC-1 on tumor cells, driving novel T cell response and causing tumor cell killing in preclinical studies. This unique approach has potential to overcome central mechanisms of immune resistance by driving de-novo recognition of previously hidden tumor neoantigens and addressing T cell exhaustion. Here we report initial safety, pharmacokinetic (PK) and pharmacodynamic (PD) endpoints from Part A (monotherapy dose escalation) in the ongoing multicentre, modular phase I dose escalation study (ACTRN12623000108617). Methods: Patients with advanced refractory solid tumors are enrolled using a BOIN escalation design. GRWD5769 is administered BID on days 1-14 during 21-day cycles. The first cycle is the dose-limiting toxicity (DLT) observation period. The PK / PD relationship of GRWD5769 is being evaluated using mass spectrometry analysis of the ImPD. Tumor responses are evaluated by RECIST v1.1 / iRECIST. Results: As of 03 January 2024, a total of 12 patients had been treated with GRWD5769 in 3 dose cohorts (25 mg, 50 mg, 100 mg BID). On average patients have remained on treatment for 4 cycles (range 1-10) No Serious Adverse Reactions, DLTs, immune related AEs or deaths were reported. Two Gr 3 serious adverse events have occurred in 2 treated subjects (aspiration pneumonia and Urinary Tract Infection, neither attributable to GRWD5769). Gr 1+2 AEs were infrequent and manageable. PK analyses for the first 3 cohorts show dose proportional plasma levels, with Tmax at ~3 h and a ~8 h Thalf. The minimum biologically active dose (MBAD) is defined as an average steady state plasma concentration above the IC50 for ERAP1, which was achieved at the 100 mg BID dose level. PD data shows dose-dependent target engagement, with marked shifts in the ImPD. This is consistent with the expected mechanistic effects of ERAP1 inhibition seen in preclinical models and is the first demonstration that the human ImPD can be manipulated pharmacologically in cancer patients, potentially allowing T cells to recognise new targets on tumors. Dose escalation with GRWD5769 monotherapy continues to determine the recommended phase 2 dose (RP2D). GWRD5769 in combination with Immune Checkpoint Inhibition will be investigated in Part B now that the MBAD has been reached. Conclusions: GRWD5769 has been well tolerated at doses up to 100 mg BID. PK / PD data support dose-dependent target engagement of ERAP1. Proof of mechanism has been achieved for this first-in-class therapy, and this is the first demonstration that the human ImPD can be manipulated pharmacologically in cancer patients. Further PD analyses will explore shifts in the T cell repertoire and modulation of immune cell phenotypes. Clinical trial information: ACTRN12623000108617.

First-in-human study (FIH) of FS222, a next-generation tetravalent PD-L1/CD137 bispecific antibody: Safety, pharmacodynamics (PD), and antitumor activity in patients (pts) with advanced solid tumors including PD-1 refractory melanoma.

2505 Background: Most pts receiving immune checkpoint inhibitors do not respond to treatment or relapse. FS222 is a novel affinity optimized, tetravalent bispecific PD-L1/CD137 antibody. FS222’s structure allows for potent, PD-L1-dependent, CD137 activation across a wide range of dose levels, and is designed to provide selective CD137 agonism in the tumor. We present data from the ongoing, FIH, open-label, phase I trial of FS222 in advanced solid tumors. Methods: Pts with pretreated advanced solid tumors received increasing doses of FS222 in an accelerated dose titration and 3+3 design intravenously every 3 or 4 weeks (Q4W) until disease progression or unacceptable toxicity. The primary endpoint was safety. Secondary and exploratory endpoints included pharmacokinetics, PD and antitumor activity (by Response Evaluation Criteria in Solid Tumors [RECIST] 1.1). Results: As of data cut off (DCO) on 05Dec2023, a total of 104 pts had been treated across a range of doses and schedules in the FIH study (NCT04740424). We report interim results from the Q4W cohorts (N=90). Patients had a median age of 61 years (31-88 years) and had received a median of 2 (1-7) regimens of prior treatments. The median duration of exposure to FS222 was 82.5 days (24 – 529 days). The most common treatment-related AEs (TRAEs; >20% of pts) were increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT), pyrexia, thrombocytopenia, asthenia, and neutropenia. The most common TRAEs grade ≥3 (≥10% of pts) were increased AST (13.3%) and ALT (11.1%). The most common treatment related serious AEs (≥3 pts) were febrile neutropenia (5 pts, 5.6%); and pyrexia, cytokine release syndrome, increased ALT, increased AST (all in 3 pts, 3.3%). On-target FS222 pharmacology was confirmed by the presence of dose-dependent target engagement, significant peripheral CD8+ T cell modulation and increased tumor CD3+ CD8+ T cells at multiple dose levels. At the DCO, 20 (22.2%) pts remained on treatment. Objective responses (CR, PR) were observed in pts with melanoma, NSCLC, ovarian cancer, TNBC, liposarcoma and colon cancer, for an ORR of 15.7% with evidence of further enrichment by dose. In post-PD-1 treated metastatic/advanced cutaneous melanoma the ORR was 60% (9/15, all PRs - 7 confirmed) and the disease control rate was 86.7% (13/15). Conclusions: The novel PD-L1/CD137 bispecific antibody FS222 demonstrated PD activity across a broad range of doses. The safety profile was acceptable and manageable. Encouraging anti-tumor activity was observed, including in patients with PD-1 refractory cutaneous melanoma. Next steps include further dose optimization and further evaluation of FS222 in patients with melanoma and other tumor types. Clinical trial information: NCT04740424 .

Abstract B172: Potent anti-tumor activity of a selective and orally bioavailable reversible covalent CDK12 inhibitor

Abstract Cyclin-dependent kinase 12 (CDK12), in association with Cyclin K (CycK), regulates the elongation of transcription by modifying RNA polymerase II (RNAP II) through phosphorylation at Serine 2 (pS2) in the C-terminal domain, CDK12 plays an important role in maintaining genomic stability by modulating crucial cellular processes such as DNA damage response, splicing, and pre-mRNA processing. Overexpression of CDK12 has been observed in various tumor types, suggesting its potential oncogenic properties like other kinases involved in transcription. Given its significance in transcription and RNA processing, CDK12 has emerged as a promising therapeutic target for cancer treatment. We have identified a highly potent and selective reversible covalent inhibitor of CDK12, AU-22880, which exhibits significant anti-proliferative activity across various cancer cell lines. The covalent mode of action and on-target activity of AU-22880 were confirmed in a CDK12 target engagement assay and selective pS2 RNAPII inhibition respectively in a cellular context. Additionally, it has shown synergistic activity in combination with several standard-of-care agents in breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer cell lines. AU-22880 shows desirable ADME-PK properties and demonstrates excellent anti-tumor activity in xenograft models. In the HCC-70 TNBC xenograft model, AU-22880 demonstrated anti-tumor efficacy at well-tolerated doses. A pharmacokinetic-pharmacodynamic (PK-PD) study revealed dose-dependent target engagement, as evidenced by the inhibition of pS2 RNAPII and DNA damage repair (DDR) genes. Furthermore, AU-22880 exhibited good tolerability in rodents, dog, and monkeys at exposure levels that demonstrated efficacy. Efficacy of AU-22880 in combination with approved SOCs targeting DDR pathway are currently being investigated. The findings reported here highlight the promising therapeutic potential of AU-22880, both as a monotherapy and in combination with existing treatments for the treatment of cancers characterized by DDR dysregulation. Citation Format: Leena Khare, Ramulu Poddutoori, Subhendu Mukherjee, Samiulla DS, Devaraja TS, Sivapriya Marappan, Shilpa Nayak, Sasirekha Sivakumar, Lakshmi Kaza, Suraj Tgore, Amit Dhudashiya, Raghavendra NR, Bhargav Gunnepalli, Aravind A B, Amith A, Charamanna KB, Thomas Antony, Kavitha Nellore, Girish Daginakatte, Shekar Chelur, Sanjeev Giri, Murali Ramachandra, Susanta Samajdar. Potent anti-tumor activity of a selective and orally bioavailable reversible covalent CDK12 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B172.

Preliminary Results from a Phase 1 Dose Escalation Study of FHD-286, a Novel BRG1/BRM (SMARCA4/SMARCA2) Inhibitor, Administered As an Oral Monotherapy in Patients with Advanced Hematologic Malignancies

The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its adenosine triphosphate (ATP)-dependent chromatin remodeling activity, BAF complexes regulate the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. FHD-286 is a first-in-class, orally administered compound that potently and selectively inhibits the ATPase components of the BAF complexes, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). In preclinical models, acute myeloid leukemia (AML) cell lines were highly sensitive to BAF inhibition. FHD-286-C-002 (NCT04891757) is a multicenter, open-label, Phase 1 dose escalation study designed to determine the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose and/or the recommended phase 2 dose(s), as well as to evaluate the preliminary clinical activity of FHD-286, in patients with advanced hematologic malignancies. Patients with relapsed or refractory (R/R) AML or myelodysplastic syndrome (MDS) failing all available standard therapies were eligible to receive FHD-286 doses ranging from 2.5 mg once daily (QD) to 10 mg QD on a continuous dosing regimen. Patients had regularly scheduled assessments to evaluate safety, pharmacokinetics (PK), pharmacodynamics, preliminary clinical activity, and exploratory biomarkers. As of 02 Aug 2022, 40 patients with a median age of 65.5 years (range 25 to 84) with R/R AML (36 patients) or MDS (4 patients) had received at least 1 dose of FHD-286. 67.5% of patients had received ≥3 prior lines of therapy for AML/MDS/other antecedent hematologic disorder, with 22.5% having received ≥5 prior lines. 32.5% of patients had prior hematopoietic stem cell transplant. 65% of patients had adverse risk genetics by 2017 ELN recommendations. 85% of patients experienced a treatment-related adverse event (TRAE) of any grade; the most common (≥20% of patients) were dry mouth (27.5%), increased blood bilirubin (22.5%), and alanine aminotransferase (ALT) increased and rash (20% each). 50% of patients experienced a Grade ≥3 TRAE, the most common of which was increased blood bilirubin (12.5%); stomatitis, ALT increased, differentiation syndrome (DS), and hypocalcemia occurred in 7.5% (each). 2 DLTs were reported: Grade 3 hyperbilirubinemia in 1 patient receiving 5 mg QD and Grade 3 muscular weakness in 1 patient receiving 10 mg QD. Treatment-related DS of any grade was investigator-reported in 4 patients (10%). An independent adjudication committee determined that the rate of suspected DS was 15% (6 patients). 16 patients (15 AML; 1 MDS) had a best overall response of stable disease. Markers of myeloid differentiation with neutrophil recovery and reductions in bone marrow and/or peripheral blasts have been observed in a subset of patients in the study who had a broad range of cytogenetic backgrounds, including patients with enhancer-driven leukemias such as MECOM and KMT2A. Preliminary PK analysis indicates that FHD-286 appears to have a long half-life, at a minimum of >24 hours, and that plasma concentrations of FHD-286 increase with increasing dose. Exploratory flow cytometry assessment of markers of differentiation, hematopoietic stem cell identity, and apoptosis suggested dose-dependent target engagement. Additionally, exploratory sequencing analysis on bone marrow blasts revealed comprehensive impacts on AML-specific expression pathways and stemness genes in response to FHD-286. An exposure-response analysis using the percentage of peripheral blasts showed a trend toward lower blast count with higher FHD-286 area-under-the-curve. Enrollment into the single-agent dose escalation phase of the study is complete. Based on nonclinical, translational, and single-agent clinical data, FHD-286 in combination with decitabine or low-dose cytarabine is being evaluated in the combination dose escalation phase of the study.

Phase 1 dose escalation study of SL-172154 (SIRPα-Fc-CD40L) in platinum-resistant ovarian cancer.

5544 Background: SIRPα-Fc-CD40L is a hexameric, bi-functional fusion protein consisting of SIRPα linked to CD40L via an inert Fc linker. This molecule competitively inhibits CD47 to enhance tumor cell phagocytosis and activates CD40 to increase antigen processing and cross-presentation by antigen presenting cells (APCs) to CD8 T cells, thus bridging innate and adaptive immunity. Methods: This first-in-human, Phase 1 dose escalation study evaluated SL-172154 in patients (pts) with advanced platinum resistant ovarian, fallopian tube and primary peritoneal cancers (PROC). SL-172154 was administered intravenously across 5 dose levels (0.1, 0.3, 1.0, 3.0, 10mg/kg). Dose escalation followed a modified toxicity probability interval 2 design. Objectives included evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic (PD) parameters, and antitumor activity per RECIST 1.1. Results: As of 3 Jan 2023, 27 pts (median age 66 years [range 33-85]; median of 4 prior systemic therapies [range 2-9]) with ovarian (70%), fallopian tube (15%) or primary peritoneal (15%) cancer were dosed. Treatment-emergent AEs (>15%) regardless of grade (G) included infusion related reaction (IRR) (67%), fatigue (44%), nausea (33%), back pain (26%), constipation, diarrhea (both 22%), decreased appetite and pruritus (both 19%). There was one DLT of G3 ALT increased at 10mg/kg requiring dose interruption for resolution. A maximum tolerated dose was not reached. G3/4 treatment-related AEs (>1pt) were AST increased (G3) and lymphopenia (G4), each in 2 pts (7%); all were fully resolved with no dose modifications. There were no fatal AEs, no AEs that led to drug discontinuation and no events of cytokine release syndrome. The frequency of IRR events increased with increasing dose, and slowing the rate of infusion was utilized for mitigation. SL-172154 Cmax and AUC increased with dose with greater than proportional exposure noted at 3 and 10mg/kg potentially due to target saturation. These findings were supported by dose-dependent target engagement of CD47 and CD40 on CD4 T cells and B cells respectively, approaching 100% by 3 mg/kg. Rapid dose-dependent egress of CD40+ B cells was maximal at ≥3mg/kg. SL-172154 induced dose-dependent responses in IL-12, CXCL-8, CXCL-10, IL-10, CCL2, CCL20, and CCL22, which also approached a plateau at ≥3mg/kg. Analysis of peripheral and tumor immunophenotypes and anti-drug antibodies in response to SL-172154 is ongoing and will be presented. Best response was stable disease in 6/27 response evaluable (22%) pts. Conclusions: SL-172154 is well tolerated in heavily pretreated PROC pts. IRRs were readily manageable. Maximal CD47 and CD40 target engagement and CD40-dependent PD effects were observed with ≥3mg/kg SL-172154. 3mg/kg is a safe, tolerable, and pharmacologically active dose for evaluation in combination studies in pts with PROC. Clinical trial information: NCT04406623 .

352 (PB132) - Subasumstat, a first-in-class inhibitor of SUMO-activating enzyme, demonstrates dose-dependent target engagement and SUMOylation inhibition, leading to rapid activation of innate and adaptive immune responses in the dose escalation portion of a phase 1/2 clinical study

Background: Subasumstat (TAK-981), a small-molecule inhibitor of SUMOylation, promotes a type-1 interferon (IFN-1) response, leading to activation of the immune system with the potential to augment antitumor immunity. Previous preclinical data suggest that subasumstat enhances antigen cross-presentation and promotes T and NK cell-dependent antitumor responses in syngeneic mouse models. Here, we report pharmacodynamic (PD) data from the dose-escalation portion of a phase 1/2 clinical study (NCT03648372).

Selected Articles from This Issue

Kaposi sarcoma (KS) arises after Kaposi sarcoma herpesvirus infection and frequently develops in HIV-positive individuals. Ramaswami and colleagues conducted a clinical trial of pomalidomide, an oral immunomodulating thalidomide derivative, in patients with KS with or without HIV. The overall response rate was 71%, with 67% of HIV-positive patients and 80% of HIV-negative participants showing a response. Baseline IL-6 levels were higher in nonresponders compared with responders, although further assessment of IL-6 as a biomarker is warranted. Based on these results, the United Stated Food and Drug Administration granted accelerated approval to pomalidomide for the treatment KS.TIGIT is a novel target in the field of cancer immunotherapy. To explore the potential of anti-TIGIT immunotherapy, Mettu and colleagues conducted a phase 1a/b study to evaluate the safety, tolerability, and preliminary efficacy of the novel anti-TIGIT antibody etigilimab alone (phase 1a) and in combination with the anti-PD-1 nivolumab (phase 1b) in patients with refractory solid tumors. This first-in-human study showed etigilimab was well tolerated. Signs of anti-tumor activity were observed, with eight patients showing stable disease and one patient showing a partial response. Biomarker analyses demonstrated evidence of clear dose-dependent target engagement by etigilimab. These findings lay a foundation for future clinical trials targeting TIGIT.Detecting neuroendocrine prostate cancer (NEPC) in men with metastatic castration-resistant prostate cancer (mCRPC) has important prognostic and therapeutic implications but is challenging in clinical practice. To address this unmet need, Berchuck and colleagues developed a noninvasive biomarker, termed NEPC Risk Score, based on tissue-informed cell-free DNA (cfDNA) methylation analysis. Applying the NEPC Risk Score to cfDNA from two independent mCRPC cohorts discriminated between men with versus without NEPC with high accuracy. These data support the further clinical development of this cfDNA methylation-based biomarker to noninvasively identify men with NEPC who should be considered for platinum-based chemotherapy or clinical trials of NEPC-directed therapies.PD-1 blockade has provided benefit for some patients with advanced sarcomas. However, predicting response is not feasible currently. Furthermore, the relevance of hyperprogressive disease (HPD) is unclear. Klemen and colleagues performed a pooled analysis of prospective trials evaluating checkpoint inhibition in patients with sarcoma. In total, 134 patients were assessed, of which 16% had a complete or partial response, 36% had stable disease, 34% had progressive disease without HPD, and 11% with HPD. Patients with HPD and non-HPD progressive disease showed similarities in terms of disease subtype, clinical features, and genomic alterations. Interestingly, patients developing HPD had smaller tumors at baseline than patients who progressed without HPD. Additional clinical studies will be required to further understand HPD in patients with sarcoma.

Discovery and Preclinical Characterization of Usmarapride (SUVN-D4010): A Potent, Selective 5-HT4 Receptor Partial Agonist for the Treatment of Cognitive Deficits Associated with Alzheimer's Disease.

A series of oxadiazole derivatives were synthesized and evaluated as 5-hydroxytryptamine-4 receptor (5-HT4R) partial agonists for the treatment of cognitive deficits associated with Alzheimer's disease. Starting from a reported 5-HT4R antagonist, a systematic structure-activity relationship was conducted, which led to the discovery of potent and selective 5-HT4R partial agonist 1-isopropyl-3-{5-[1-(3-methoxypropyl) piperidin-4-yl]-[1,3,4]oxadiazol-2-yl}-1H-indazole oxalate (Usmarapride, 12l). It showed balanced physicochemical-pharmacokinetic properties with robust nonclinical efficacy in cognition models. It also showed disease-modifying potential, as it increased neuroprotective soluble amyloid precursor protein alpha levels, and dose-dependent target engagement and correlation of efficacy with oral exposures. Phase 1 clinical studies have been completed and projected efficacious concentration was achieved without any major safety concerns. Phase 2 enabling long-term safety studies have been completed with no concerns for further development.

Basmisanil, a highly selective GABAA-\u03b15 negative allosteric modulator: preclinical pharmacology and demonstration of functional target engagement in man

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Novel Non-Bile Acid FXR Agonist Tropifexor (LJN452) in Healthy Volunteers.

Tropifexor (LJN452) is a potent, orally available, non–bile acid farnesoid X receptor agonist under clinical development for chronic liver diseases. Here, we present results from a first‐in‐human study of tropifexor following single‐ and multiple‐ascending doses (SAD/MAD) and food effect substudy in healthy volunteers. The SAD study included 6 fasted cohorts receiving 10‐ to 3000‐µg tropifexor or placebo and 1 cohort receiving 300‐µg tropifexor with a high‐fat meal. The MAD study included 4 lean cohorts receiving 10 to 100 µg and 1 obese cohort receiving 30‐µg once‐daily doses or placebo for 14 days. Pharmacodynamic assessment of fibroblast growth factor 19 and fasting plasma lipids was performed after dosing. Overall, 95 volunteers received at least 1 tropifexor or placebo dose. Tropifexor was well tolerated up to 3000 µg and 100 µg in the SAD and MAD studies, respectively; however, 2 subjects discontinued the MAD study due to asymptomatic elevation of liver transaminases. At single doses, tropifexor showed a moderate rate of absorption (median time to maximum concentration, 4 hours), dose‐proportional increases in exposure, and elimination half‐life of 13.5 to 21.9 hours. When taken with food, tropifexor exposure increased by ∼60%. With multiple dosing, steady state was reached on day 4 with <2‐fold accumulation. Single and multiple doses showed dose‐dependent increases in fibroblast growth factor 19. No changes in serum lipids were observed in tropifexor‐ vs placebo‐treated obese subjects. In conclusion, tropifexor was well tolerated, had a pharmacokinetic profile suitable for once‐daily dosing and showed dose‐dependent target engagement without altering plasma lipids in healthy volunteers.

Results of a Clinical Trial of H3B-8800, a Splicing Modulator, in Patients with Myelodysplastic Syndromes (MDS), Acute Myeloid Leukemia (AML) or Chronic Myelomonocytic Leukemia (CMML)

Results of a Clinical Trial of H3B-8800, a Splicing Modulator, in Patients with Myelodysplastic Syndromes (MDS), Acute Myeloid Leukemia (AML) or Chronic Myelomonocytic Leukemia (CMML)

Abstract 3597: BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL)

Abstract DLBCL is the most common type of non-Hodgkin lymphoma. It is an aggressive and fast growing tumor with two major molecular subtypes: germinal center (GCB) and activated B-cell like (ABC). While the majority of patients are 60 years or older, DLBCL can occur at any age. Despite a cure rate of around 50% the need for novel therapies remains high, especially for relapsed/refractory DLBCL patients not eligible for stem cell transplant. DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. We recently discovered its role in AML differentiation (Sykes et al 2016, Cell) and we are investigating the novel DHODH inhibitor BAY 2402234 in an ongoing phase I study in myeloid malignancies (NCT03404726). Further screening of non-leukemia cancer types identified DLBCL as highly responsive to DHODH inhibition in preclinical studies. Here, we disclose for the first time the functional preclinical characterization of BAY 2402234 in DLBCL. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro it potently inhibits proliferation of DLBCL cell lines in the sub-nanomolar to low-nanomolar range. The anti-proliferative effects can be rescued by uridine supplementation which bypasses DHODH via the salvage pathway and demonstrates the on-target specificity of the inhibitor. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in subcutaneous models derived from patient-derived xenograft (PDX) and cell lines representing various DLBCL subtypes, including GCB and ABC. Dose dependent target engagement and drug exposure of BAY 2402234 could be observed by increases in plasma dihydroorotate levels and unbound plasma drug levels after treatment with the inhibitor. Based on preclinical data presented herein we plan to start clinical investigations of BAY 2402234 in patients with DLBCL in early 2019. Citation Format: Ashley Eheim, Sven Christian, Hanna Meyer, Detlef Stoeckigt, Claudia Merz, Katja Zimmermann, Marcus Bauser, Andrea Haegebarth, Steven Ferrara, David B. Sykes, David T. Scadden, Stefan Gradl, Andreas Janzer. BAY 2402234: Preclinical evaluation of a novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of diffuse large B-cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3597.

117. Biomarker Assessment of Dose Dependent Target Engagement of mGluR-2,3 Partial Agonist for Schizophrenia Treatment

There is a desperate need for novel drugs for treatment of schizophrenia. Glutamate is a prioritized target for drug development. Attempts to develop mGlu-R partial agonists were unsuccessful. Upon reviewing the data, we concluded that doses to achieve adequate target engagement were tested. This study tested the ability to an mGluR-2,3 partial agonist to inhibit the effects of a pharmacologic model of schizophrenia as reflected by fMRI.

F181. A Randomized, Single-Blind, Parallel-Group Study to Evaluate the Effects of TS-134 on Ketamine- Induced Bold Signals in Resting fMRI in Healthy Adult Subjects

Therapies targeting glutamatergic system for schizophrenia have yielded mixed results across studies to date, and there is a critical absence of validated target-engagement biomarkers. We evaluated the dose-dependent target engagement of TS-134, a metabotropic glutamate receptor 2/3 (mGlu2/3) agonist using ketamine-evoked pharmacoBOLD in healthy volunteers.

Preclinical characterization of substituted 6,7-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(5H)-one P2X7 receptor antagonists.

The synthesis, SAR, and preclinical characterization of a series of substituted 6,7-dihydro[1,2,4]triazolo[4,3]pyrazin-8(5H)-one P2X7 receptor antagonists are described. Optimized leads from this series comprise some of the most potent human P2X7R antagonists reported to date (IC50s<1nM). They also exhibit sufficient potency and oral bioavailability in rat to enable extensive in vivo profiling. Although many of the disclosed compounds are peripherally restricted, compound 11d is brain penetrant and upon oral administration demonstrated dose-dependent target engagement in rat hippocampus as determined by ex vivo receptor occupancy with radiotracer 5 (ED50=0.8mg/kg).

Abstract B161: Chemoproteomic profiling for target identification using chloroalkane derivatized compounds

Abstract Chemoproteomic technologies enable the quantitative and qualitative profiling of small molecule protein interactions. Here we report a new approach utilizing a chloroalkane (CA) capture tag which can be chemically attached to small molecules to enable the isolation of their respective protein targets through selective capture onto an immobilized HaloTag protein. We have combined our chemoproteomics workflow with multiplexed chemical labeling methods to enable the analysis of dose dependent target engagement studies in a single LC-MS/MS experiment. Here we demonstrate this workflow using BIRB796-CA, a modified kinase inhibitor. Target enrichment experiments were performed by incubating HEK293 cells with the BIRB-CA. Cells were then lysed and the BIRB-CA, together with the bound targets, was rapidly captured onto immobilized HaloTag protein. Unmodified compound was used to competitively elute putative interacting proteins. To study dose dependent effects on the protein interaction profile, cells were incubated with multiple mixtures of BIRB and BIRB-CA at varying relative concentrations. The eluted proteins were processed using SDS-PAGE and in-gel digestion. Digested material was labeled with Tandem Mass Tags (TMT) 10plex reagents and analyzed using nanoscale LC-MS/MS coupled with a Q Exactive mass spectrometer (Thermo). Protein identification was performed with MaxQuant (MaxQuant.org) and data validation and visualization was performed using Perseus (Perseus-framework.org). We tested this chemoproteomics workflow using the interaction of MAP kinases (MAPK) with BIRB796, an allosteric kinase inhibitor. Treatment of LPS simulated THP-1 cells with BIRB796 and BIRB-CA revealed minimal impact of the CA modification on the inhibition of TNFα secretion. The interaction of BIRB-CA with known BIRB targets MAPK13, MAPK12, MAPK9 and MAPK14 has been confirmed using label free quantitative proteomic profiling. We have optimized the TMT labeling method for minimal losses and sample processing steps. We are applying the TMT method to BIRB-CA and control pull downs and extending this workflow to include the BIRB/BIRB-CA titration samples. Here we will use post-acquisition data processing of the BIRB/BIRB-CA titration pull down data to assess compound binding characteristics with the inferred extension to compounds with unknown binding specificities. Citation Format: Michael Ford, Richard Jones, Ravi Amunugama, Danette Daniels, Rachel Ohana, Sergiy Levin, Thomas Kirkland, Marjeta Urh, Keith Wood. Chemoproteomic profiling for target identification using chloroalkane derivatized compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B161.

Multiplexing of receptor occupancy measurements for pharmacodynamic biomarker assessment of biopharmaceuticals

BackgroundReceptor occupancy (RO) assays measure drug target engagement, and are used as pharmacodynamic (PD) biomarkers. RO assays are commonly performed by flow cytometry and often require multiplexing for assessment of multiple PD biomarkers when specimen volumes are limited. We present multiplexed RO assays for an IGF1R‐EGFR bispecific antibody (Bs‐Ab) and a CTLA4‐Ig recombinant fusion protein to demonstrate key considerations for accurate RO assessment.MethodsRO in cynomolgus monkeys was determined in whole blood using flow cytometry. Free and total receptors were measured using anti‐receptor fluorescence‐labeled detection reagents, competitive and noncompetitive to drug, respectively.ResultsRO of IGF1R was examined as PD for Bs‐Ab, since IGF1R was expressed on blood cells. Multiplexed measurements of free and total IGF1R showed that IGF1R expression measured by total receptor was highly variable, impacting interpretation of free‐IGF1R. Normalization of free‐over‐total IGF1R measurements compensated for variability of receptor expression allowing for accurate RO assessment. RO of CTLA4‐Ig, a recombinant fusion protein targeting CD80 and CD86 receptors, was multiplexed to simultaneously measure target engagements for both receptors. Both RO methods demonstrated specificity of receptor measurements without cross‐reactivity to each other in multiplexed formats. RO methods were used for evaluation of PD activity of Bs‐Ab and CTLA4‐Ig in cynomolgus monkeys. In both cases, RO results showed dose‐dependent target engagement, corresponding well to the pharmacokinetics.ConclusionsMultiplexed RO methods allowed accurate assessment of PD activity for Bs‐Ab and CTLA4‐Ig, facilitating development of these biopharmaceuticals from preclinical to clinical stages. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.