Target Engagement Biomarkers Research Articles

The angiotensin (1–7) glycopeptide PNA5 improves cognition in a chronic progressive mouse model of Parkinson's disease through modulation of neuroinflammation

Cognitive decline in Parkinson's Disease (PD) is a prevalent and undertreated aspect of disease. Currently, no therapeutics adequately improve this aspect of disease. It has been previously shown that MAS receptor agonism via the glycosylated Angiotensin (1–7) peptide, PNA5, effectively reduces cognitive decline in models of vascular contributions to cognitive impairment and dementia (VCID). PNA5 has a brain/plasma ratio of 0.255 indicating good brain penetration. The goal of the present study was to determine if (1) systemic administration of PNA5 rescued cognitive decline in a mouse model of PD, and (2) if improvements in cognitive status could be correlated with changes to histopathological or blood plasma-based changes. Mice over-expressing human, wild-type α-synuclein (αSyn) under the Thy1 promoter (Thy1-αSyn mice, “line 61”) were used as a model of PD with cognitive decline. Thy1-αSyn mice were treated with a systemic dose of PNA5, or saline (1 mg/kg/day) beginning at 4 months of age and underwent behavioral testing at 6 months, compared to WT. Subsequently, mice brains were analyzed for changes to brain pathology, and blood plasma was examined with a Multiplex Immunoassay for peripheral cytokine changes. Treatment with PNA5 reversed cognitive dysfunction measured by Novel Object Recognition and spontaneous alteration in a Y-maze in Thy1-αSyn mice. PNA5 treatment was specific to cognitive deficits, as fine-motor disturbances were unchanged. Enhanced cognition was associated with decreases in hippocampal inflammation and reductions in circulating levels of Macrophage Induced Protein (MIP-1β). Additionally, neuronal loss was blunted within the CA3 hippocampal region of PNA5-treated αsyn mice. These data reveal that PNA5 treatment reduces cognitive dysfunction in a mouse model of PD. These changes are associated with decreased MIP-1β levels in plasma identifying a candidate biomarker for target engagement. Thus, PNA5 treatment could potentially fill the therapeutic gap for cognitive decline in PD.

Alzheimer's Disease: Combination Therapies and Clinical Trials for Combination Therapy Development.

Alzheimer's disease (AD) is a complex multifaceted disease. Recently approved anti-amyloid monoclonal antibodies slow disease progression by approximately 30%, and combination therapy appears necessary to prevent the onset of AD or produce greater slowing of cognitive and functional decline. Combination therapies may address core features, non-specific co-pathology commonly occurring in patients with AD (e.g., inflammation), or non-AD pathologies that may co-occur with AD (e.g., α-synuclein). Combination therapies may be advanced through co-development of more than one new molecular entity or through add-on strategies including an approved agent plus a new molecular entity. Addressing add-on combination therapy is currently urgent since patients on anti-amyloid monoclonal antibodies may be included in clinical trials for experimental agents. Phase 1 information must be generated for each agent in combination drug development. Phase 2 and Phase 3 of add-on therapies may contrast the new molecular entity, the approved agent as standard of care, and the combination. More complex development programs including standard or modified combinatorial designs are required for co-development of two or more new molecular entities. Biomarkers are markedly affected by anti-amyloid monoclonal antibodies, and these effects must be anticipated in add-on trials. Examining target engagement biomarkers and comparing the magnitude and sequence of biomarker changes in those receiving more than one therapy, compared with those on monotherapy, may be informative. Using network-based medicine approaches, computational strategies may identify rational combinations using disease and drug effect network mapping.

A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B

BackgroundCirculating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of cccDNA and may help to evaluate HBV treatments activity. Different research assays have been proposed and, although 2 PCR-based research use only (RUO) investigational assays (IA) have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays. MethodsHBV RNA producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP-domain (Y63F) of the polymerase, and the ε-loop of the pgRNA (mutation A1G). ResultsThe clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virions-like particles and only a minority in small extracellular vescicles (sEV). Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29 secreted HBV RNAs start at the 5' end of pgRNA and pgRNA derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from 1 flask. ConclusionWe generated a clonal cell line that produces high amounts of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assays calibration. Impact and ImplicationsSeveral investigational assays and 2 research use only (RUO) assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of cccDNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNAs quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (e.g., pgRNA, HBx transcripts). This new RNA standard will serve for the calibration of all PCR-based assays for circulating HBV RNA quantification to evaluate in a non-invasive manner the size of the transcriptionally active cccDNA pool and the activity of novel strategies aiming at curing HBV infection.

Utilization of fluid-based biomarkers as endpoints in disease-modifying clinical trials for Alzheimer’s disease: a systematic review

BackgroundClinical trials in Alzheimer’s disease (AD) had high failure rates for several reasons, including the lack of biological endpoints. Fluid-based biomarkers may present a solution to measure biologically relevant endpoints. It is currently unclear to what extent fluid-based biomarkers are applied to support drug development.MethodsWe systematically reviewed 272 trials (clinicaltrials.gov) with disease-modifying therapies starting between 01–01-2017 and 01–01-2024 and identified which CSF and/or blood-based biomarker endpoints were used per purpose and trial type.ResultsWe found that 44% (N = 121) of the trials employed fluid-based biomarker endpoints among which the CSF ATN biomarkers (Aβ (42/40), p/tTau) were used most frequently. In blood, inflammatory cytokines, NFL, and pTau were most frequently employed. Blood- and CSF-based biomarkers were used approximately equally. Target engagement biomarkers were used in 26% (N = 72) of the trials, mainly in drugs targeting inflammation and amyloid. Lack of target engagement markers is most prominent in synaptic plasticity/neuroprotection, neurotransmitter receptor, vasculature, epigenetic regulators, proteostasis and, gut-brain axis targeting drugs. Positive biomarker results did not always translate to cognitive effects, most commonly the small significant reductions in CSF tau isoforms that were seen following anti-Tau treatments. On the other hand, the positive anti-amyloid trials results on cognitive function were supported by clear effect in most fluid markers.ConclusionsAs the field moves towards primary prevention, we expect an increase in the use of fluid-based biomarkers to determine disease modification. Use of blood-based biomarkers will rapidly increase, but CSF markers remain important to determine brain-specific treatment effects. With improving techniques, new biomarkers can be found to diversify the possibilities in measuring treatment effects and target engagement. It remains important to interpret biomarker results in the context of the trial and be aware of the performance of the biomarker. Diversifying biomarkers could aid in the development of surrogacy biomarkers for different drug targets.

Apilimod dimesylate in C9orf72 amyotrophic lateral sclerosis: a randomized phase 2a clinical trial.

Apilimod dimesylate is a first-in-class phosphoinositide kinase, FYVE-type zinc finger-containing (PIKfyve) inhibitor with a favourable clinical safety profile and has demonstrated activity in preclinical C9orf72 and TDP-43 amyotrophic lateral sclerosis (ALS) models. In this ALS clinical trial, the safety, tolerability, CNS penetrance and modulation of pharmacodynamic target engagement biomarkers were evaluated. This phase 2a, randomized, double-blind, placebo-controlled, biomarker-end-point clinical trial was conducted in four US centres (ClinicalTrials.gov NCT05163886). Participants with C9orf72 repeat expansions were randomly assigned (2:1) to receive twice-daily oral treatment with 125 mg apilimod dimesylate capsules or matching placebo for 12 weeks, followed by a 12-week open-label extension. Safety was measured as the occurrence of treatment-emergent or serious adverse events attributable to the study drug and tolerability at trial completion or treatment over 12 weeks. Changes from baseline in plasma and CSF and concentrations of apilimod dimesylate and its active metabolites and of pharmacodynamic biomarkers of PIKfyve inhibition [soluble glycoprotein nonmetastatic melanoma protein B (sGPNMB) upregulation] and disease-specific CNS target engagement [poly(GP)] were measured. Between 16 December 2021 and 7 July 2022, 15 eligible participants were enrolled. There were no drug-related serious adverse events reported in the trial. Fourteen (93%) participants completed the double-blind period with 99% dose compliance [n = 9 (90%) apilimod dimesylate; n = 5 (100%) placebo]. At Week 12, apilimod dimesylate was measurable in CSF at 1.63 ng/ml [standard deviation (SD): 0.937]. At Week 12, apilimod dimesylate increased plasma sGPNMB by >2.5-fold (P < 0.001), indicating PIKfyve inhibition, and lowered CSF poly(GP) protein levels by 73% (P < 0.001), indicating CNS tissue-level proof of mechanism. Apilimod dimesylate met prespecified key safety and biomarker end-points in this phase 2a trial and demonstrated CNS penetrance and pharmacodynamic target engagement. Apilimod dimesylate was observed to result in the greatest reduction in CSF poly(GP) levels observed to date in C9orf72 clinical trials.

The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

EEG band power and phase‐amplitude coupling in patients with Dravet syndrome

AbstractObjectiveDravet syndrome (DS) is an epileptic encephalopathy caused by haploinsufficiency of the SCN1A gene. SCN1A gene deficiency limits the firing rates of fast‐spiking inhibitory interneurons, which should reflect in abnormal aggregate network oscillatory electroencephalography (EEG) activity that can be measured by spectral power and phase‐amplitude coupling (PAC) analysis. In this retrospective pilot study, we tested whether spectral EEG frequency band power and PAC metrics distinguish children with DS from age‐matched controls, an early step toward establishing EEG markers of target engagement by gene or drug therapy.MethodsEEG data were collected from patients with DS (N = 6) and age‐matched control pediatric participants (N = 11) and analyzed for cumulative spectral power and PAC and classification capacity of these metrics, by logistic regression analysis. For this initial spectral and PAC analysis, we focused on sleep EEG, where myogenic artifact is minimal and where δ–γ and θ–γ coupling is otherwise expected to be robust.ResultsCumulative δ (1– &lt;4 Hz) and θ (4–7 Hz) power was significantly reduced in the DS group, compared with age‐matched controls (p = 0.001 and p = 0.02, respectively). The δ power was a stronger classifier of separating DS from controls than θ power, with 87% and 83% accuracy, respectively. The γ power trended toward significant reduction (p = 0.08) in the DS group. We found significantly lower PAC between 1–2 Hz phase and 63–80 Hz amplitude in patients with DS compared with the age‐matched controls (p = 0.003), with 78% classification accuracy between groups for PAC.InterpretationIn this pilot study assessing EEG patterns during sleep, we found lower δ–θ power and PAC in patients with DS versus controls, which may reflect abnormal aggregate macroscale network communication patterns resulting from SCN1A deficiency. These measures may be useful metrics of therapeutic target engagement, particularly if the therapy restores the underlying DS pathophysiology. The sorting capacity of these metrics distinguished patients with DS from patients without DS and may in turn facilitate near‐future development of disease and therapy target engagement biomarkers in this syndrome.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3233.

Abstract 520: circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition

Abstract DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. DHX9 can bind specifically to inverted repeat Alu elements, preventing back-splicing events that lead to circular RNA and exon skipping of the linear transcript. Upon DHX9 inhibition, a robust induction of Alu-mediated circular RNA is observed. For example, circBRIP1 is an Alu-mediated circular RNA that is robustly elevated upon DHX9 loss, with no observed change to levels of linear BRIP1. Here we describe data demonstrating circBRIP1 as a DHX9 specific target engagement pharmacodynamic (PD) biomarker and its potential utility as a non-invasive clinical PD biomarker. We have previously demonstrated that DHX9 inhibition by novel small molecule inhibitors is efficacious in microsatellite instable (MSI) colorectal cancer (CRC) with defective mismatch repair (dMMR), both in vitro and in vivo. DHX9 inhibition in the HCT116 dMMR CRC cell line results in a robust dose dependent induction of the Alu-mediated circular RNA, circBRIP1, as early as 6 hours after compound treatment. The development of lead series DHX9 inhibitors exhibited a positive correlation between circBRIP1 EC50 and DHX9 biochemical activity, as well as proliferation inhibition in a dMMR CRC cell line. However, dose dependent induction of circBRIP1 following DHX9 inhibition occurs with similar potencies across DHX9i sensitive and insensitive CRC cell lines. This suggests that elevation of circBRIP1 is a unique proximal target engagement PD biomarker of DHX9 inhibition, but not a predictor of DHX9 sensitivity. Importantly, induction of circBRIP1 is observed intratumorally in human xenograft in vivo efficacy studies in mice. Induction in each individual tumor correlates with observed tool compound exposure, providing evidence of a predictable PK/PD relationship. We also observe dose dependent circBRIP1 induction in mouse peripheral blood mononuclear cells (PBMC). We have extended these studies to primary human whole blood, where we see robust dose dependent induction of circBRIP1 after ex vivo treatment with DHX9 inhibitor for 24 hours. Altogether, these studies show evidence for the utility of circBRIP1 as a non-invasive target engagement biomarker for DHX9 inhibitors in the clinic. Citation Format: David Brennan, Jennifer Castro, Matthew H. Daniels, Monique Laidlaw, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 520.

Abstract 3366: FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models

Abstract The formation of poly(ADP-ribose) (PAR) chains from NAD+ precursors by PARP enzymes is a characteristic posttranslational modification during DNA damage repair. PAR chains function as docking platforms for DNA repair proteins that are required to resolve DNA lesions. Equally important is the subsequent removal of PAR chains from protein substrates to conclude the repair process and restore genomic integrity. Poly(ADP-ribose) glycohydrolase (PARG) is the key enzyme in this process and has recently garnered significant attention as a novel cancer target. FoRx-06-428 is a novel, orally bioavailable and highly potent, low molecular weight PARG inhibitor. It binds to the catalytic domain of human PARG with high affinity, as evidenced by surface plasmon resonance (SPR), and displays potent inhibition of enzymatic activity. FoRx-06-428 shows selective reduction of viability in a panel of cancer cell lines highlighting therapeutic potential in multiple cancer indications. Pharmacological inhibition of PARG leads to accumulation of PAR chains and is indicative of target engagement, accompanied by modulation of DNA damage response markers, such as phosphorylation of replication protein A (pRPA) and phosphorylation of H2AX (γH2AX). Compelling anti-tumor efficacy as single agent was observed in several xenograft models (breast and gastric cancer) with excellent tolerability. Dose-dependent accumulation of PAR chains was demonstrated in vivo and constitutes a tractable target engagement biomarker for clinical exploration. Sensitivity to PARG inhibition is partly driven by distinct genomic instabilities, such as defects in homologous recombination (HR) repair, however the activity of PARG inhibitors is also influenced by aberrations in other cancer-relevant pathways. This report represents the first disclosure of the pharmacological characterization of FoRx-06-428 and highlights the value of PARG inhibition as a novel targeted approach to treat DNA repair-deficient cancers. Citation Format: Olivier Querolle, Ulrich Lücking, Luca Iacovino, Alena Freudenmann, Giacomo Rossetti, Marina Gysin, Serena Bologna, Vasilis Dionellis, Marta Malattia, Alessandro Potenza, Nicolas Bocquet, Irena Konstantinova, Andreas Goutopoulos, Sotirios Sotiriou, Thanos Halazonetis, Tarig Bashir, Frank Zenke. FoRx-06-428 is a novel PARG inhibitor with potent anti-tumor efficacy in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3366.

Biomarker analysis from the phase 2b randomized placebo-controlled trial of riociguat in early diffuse cutaneous systemic sclerosis.

To examine disease and target engagement biomarkers in the RISE-SSc trial of riociguat in early diffuse cutaneous systemic sclerosis and their potential to predict the response to treatment. Patients were randomized to riociguat (n = 60) or placebo (n = 61) for 52 weeks. Skin biopsies and plasma/serum samples were obtained at baseline and week 14. Plasma cyclic guanosine monophosphate (cGMP) was assessed using radio-immunoassay. Alpha smooth muscle actin (αSMA) and skin thickness were determined by immunohistochemistry, mRNA markers of fibrosis by qRT-PCR in skin biopsies, and serum CXC motif chemokine ligand 4 (CXCL-4) and soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1) by enzyme-linked immunosorbent assay. By week 14, cGMP increased by 94 ± 78% with riociguat and 10 ± 39% with placebo (p < 0.001, riociguat vs placebo). Serum sPECAM-1 and CXCL-4 decreased with riociguat vs placebo (p = 0.004 and p = 0.008, respectively). There were no differences in skin collagen markers between the 2 groups. Higher baseline serum sPECAM-1 or the detection of αSMA-positive cells in baseline skin biopsies were associated with a larger reduction of modified Rodnan skin score from baseline at week 52 with riociguat vs placebo (interaction P-values 0.004 and 0.02, respectively). Plasma cGMP increased with riociguat, suggesting engagement with the nitric oxide-soluble guanylate cyclase-cGMP pathway. Riociguat was associated with a significant reduction in sPECAM-1 (an angiogenic biomarker) vs placebo. Elevated sPECAM-1 and the presence of αSMA-positive skin cells may help to identify patients who could benefit from riociguat in terms of skin fibrosis. Clinicaltrials.gov, NCT02283762.

Use of biomarkers in clinical trials and future developments that will help identify novel biomarkers.

Engineering new solutions for therapeutic benefit in Amyotrophic Lateral Sclerosis (ALS) has proved a difficult task to accomplish. This is largely the reflection of complexities at multiple levels, that require solutions to improve cost-effectiveness and outcomes. The main obstacle related to the condition's clinical heterogeneity, chiefly the broad difference in survival observed among ALS patients, imposes large populations studies and long follow-up to evaluate any efficacy. The emerging solution is composite clinical and biological parameters enabling prognostic stratification into homogeneous phenotypes for more affordable studies. From a therapeutic development perspective, the choice of a medicinal product requires the availability of treatment-specific biomarkers of target engagement to identify off-target effects based on the compound's putative modality of action. More importantly, there are no established biomarkers of treatment response that can complement clinical outcome measures and support futility and end of treatment analyses of efficacy. Ultimately the onus rests on the development of biomarkers encompassing the unmet needs of clinical trial design, from inclusion to efficacy. These readouts of the pathological process may be used in combination with clinical and paraclinical outcome measured, significantly reducing the time and financial burden of clinical studies. Progress towards a biomarker-driven clinical trial design in ALS has been possible thanks to the accurate detection of neurofilaments and of other immunological mediators in biological fluids with the disease progression, a step change enabling the testing of novel therapeutic agents in a new clinical trial setting. However, further progress remains to be made to find treatment specific target engagement biomarkers along with readouts of treatment response that can be reliably applied to all emerging therapies and clinical studies. Here we will cover the basic notions of biomarker development in ALS clinical trials, the most crucial unanswered questions and the unmet needs in the ALS biomarkers space.

The citrate transporter SLC13A5 as a therapeutic target for kidney disease: evidence from Mendelian randomization to inform drug development

BackgroundSolute carrier family 13 member 5 (SLC13A5) is a Na+-coupled citrate co-transporter that mediates entry of extracellular citrate into the cytosol. SLC13A5 inhibition has been proposed as a target for reducing progression of kidney disease. The aim of this study was to leverage the Mendelian randomization paradigm to gain insight into the effects of SLC13A5 inhibition in humans, towards prioritizing and informing clinical development efforts.MethodsThe primary Mendelian randomization analyses investigated the effect of SLC13A5 inhibition on measures of kidney function, including creatinine and cystatin C-based measures of estimated glomerular filtration rate (creatinine-eGFR and cystatin C-eGFR), blood urea nitrogen (BUN), urine albumin-creatinine ratio (uACR), and risk of chronic kidney disease and microalbuminuria. Secondary analyses included a paired plasma and urine metabolome-wide association study, investigation of secondary traits related to SLC13A5 biology, a phenome-wide association study (PheWAS), and a proteome-wide association study. All analyses were compared to the effect of genetically predicted plasma citrate levels using variants selected from across the genome, and statistical sensitivity analyses robust to the inclusion of pleiotropic variants were also performed. Data were obtained from large-scale genetic consortia and biobanks, with sample sizes ranging from 5023 to 1,320,016 individuals.ResultsWe found evidence of associations between genetically proxied SLC13A5 inhibition and higher creatinine-eGFR (p = 0.002), cystatin C-eGFR (p = 0.005), and lower BUN (p = 3 × 10−4). Statistical sensitivity analyses robust to the inclusion of pleiotropic variants suggested that these effects may be a consequence of higher plasma citrate levels. There was no strong evidence of associations of genetically proxied SLC13A5 inhibition with uACR or risk of CKD or microalbuminuria. Secondary analyses identified evidence of associations with higher plasma calcium levels (p = 6 × 10−13) and lower fasting glucose (p = 0.02). PheWAS did not identify any safety concerns.ConclusionsThis Mendelian randomization analysis provides human-centric insight to guide clinical development of an SLC13A5 inhibitor. We identify plasma calcium and citrate as biologically plausible biomarkers of target engagement, and plasma citrate as a potential biomarker of mechanism of action. Our human genetic evidence corroborates evidence from various animal models to support effects of SLC13A5 inhibition on improving kidney function.

Abstract B037: A pharmacodynamic pilot study of DS8201a in patients with HER2 expressing advanced solid tumors

Abstract Background: DS-8201a (Trastuzumab (T) deruxtecan(D)) is an antibody-drug conjugate consisting of the HER2-targeting antibody T covalently bound via a lysosomal protease-cleavable linker to the novel topoisomerase 1 (Top1) inhibitor D with activity in tumors with low HER2 IHC scores of 1+ or 2+. The primary purpose of this study is to investigate the pharmacodynamic (PD) mechanisms underlying the activity of DS-8201a in advanced solid tumors, including those with low HER2 expression. Methods: This multicenter pilot study enrolled patients (pts) with advanced solid tumors expressing HER2 as defined by CLIA-certified labs i.e., a HER2 IHC score of 1+ or greater or ERBB2 amplifications (amp) or mutations (mut). DS-8201a was administered at 5.4 mg/kg intravenously once every 3 weeks, in 21-day cycles (C). PD biomarkers of Top1 target engagement by D payload, consequent DNA damage and repair (DDR), and immune microenvironment changes were analyzed in mandatory tumor biopsies collected at baseline, post-dose C1 and pre-dose C3. Blood samples for biomarker analyses were taken at several time points. Safety and overall response were also evaluated. Results: As of June 16, 2023, a total of 48 pts have been enrolled with diverse HER2 status distribution: 11 IHC 1+, 20 IHC 2+, 6 IHC 3+, 7 ERBB2 amp, 3 ERBB2 mut, and 1 with both IHC 1+ and ERBB2 amp. Median number of prior lines of systemic therapy was 3.5 (range 1-13) and 11 pts received prior Top1 inhibitor therapy. Grade (Gr) 3 or higher treatment-related adverse events (trAEs) were reported in 12 pts (25%). The most common Gr3/4 trAEs were myelosuppression. There were 7 pts (15%) with Gr2 and 1 pt (2%) with Gr3 pneumonitis. One pt with Gr2 pneumonitis was retreated after trAE resolution with steroid therapy and continued for another 9 Cs prior to progression. Of the evaluable patients, 1 pt (2%) had complete response (cervical, HER2 2+), 6 pts (13%) had confirmed partial response (PR) (uterine, 2+; ovarian, 2+; bladder, ERBB2 mut; uterine carcinosarcoma, 2+; 2 head and neck cancers, 1+ and 2+), 4 pts (8%) had unconfirmed PR (breast, 1+; extramammary Paget’s disease, 2+; pancreas, ERBB2 amp; uterine, 2+), and 17 pts (35%) had stable disease as their best response. Median progression free survival was 3.4 months (range 0.5-17.2). Top1 target engagement and induction of nuclear biomarkers of DDR (RAD51, pNBS1) were observed in more than half of the paired biopsies tested to date. DDR biomarker response was associated with both HER2 1+ and 2+ status. PD analyses are currently ongoing. Conclusion: DS-8201a demonstrated responses in various tumor types outside of breast cancer with HER2 low/ERBB2 alteration status. Preliminary PD data demonstrated expected Top1 target engagement and DDR induction, the extent of which is possibly independent of the degree of HER 2 staining. Analyses are currently underway to further define the effect of DS-8201a in advanced solid tumors. Citation Format: Sarah J Shin, Geraldine O'Sullivan-Coyne, Elizabeth K Lee, Arjun Mittra, Cora N Sternberg, Mary Jane Ong, Deborah Wilsker, Katherine Ferry-Galow, Jared Foster, Lamin Juwara, Naoko Takebe, Cheryl A Pickett, Ralph Parchment, James Doroshow, Alice Chen. A pharmacodynamic pilot study of DS8201a in patients with HER2 expressing advanced solid tumors [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 B037.

Abstract A044: The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer

Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [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 A044.

Development of KER-012, a modified ActRIIB ligand trap, for the treatment of pulmonary arterial hypertension

Abstract Background Pulmonary Arterial Hypertension (PAH) is a debilitating disorder characterized by elevated pulmonary vascular resistance (PVR) due to severe constriction obliteration of pulmonary vessels (Tielemans 2019). The pathology of PAH is driven by endothelial dysfunction and vascular remodeling that leads to increased wall thickening and resistance to blood flow. Dysregulated TGF-β, specifically SMAD signaling, is a key molecular defect in proliferative thickening of pulmonary arterioles in PAH leading to increased arterial pressure and compensatory right ventricular hypertrophy, ultimately resulting in decompensated right heart failure. Increased levels of activin A, a TGF-β superfamily ligand, have been found in PAH patients, and in the lungs of hypoxia-induced pulmonary hypertensive mice (Yndestad 2009). KER-012 is an investigational modified ActRIIB ligand trap designed to bind and inhibit activin A, activin B, GDF8 and GDF11 to maximally inhibit SMAD2/3 (pro proliferative), while permitting SMAD1/5/9 (anti-proliferative) signaling. This profile is expected to rebalance defective SMAD signaling in PAH and potentially delay or reverse disease progression. Aim To evaluate safety and tolerability of KER-012 in healthy post-menopausal women (PMW) and present the planned design of a Phase 2 trial in participants with PAH. Methods A two-part Phase 1 study has been conducted with KER-012 in healthy PMW. Participants received either placebo or a single dose of KER-012 ranging between 0.75 and 5.0 mg/kg in Part 1 or multiple doses between 0.75 and 4.5 mg/kg in Part 2 subcutaneously once every 4 weeks over a 12-week period. Safety, PK and biomarkers of activin target engagement were assessed. Results KER-012 was generally well tolerated at dose levels up to 5 mg/kg as a single dose and at dose levels up to 4.5 mg/kg administered Q4W for a total of 3 doses (Table 1). Maximal target engagement of KER-012, as demonstrated by changes in biomarkers of activin inhibition, namely follicle stimulating hormone (FSH) and bone specific alkaline phosphatase (BSAP), was observed in this study. As previous studies with activin receptor type II ligand traps have shown a potentially dose-limiting increase in erythropoiesis, changes in red blood cells (RBC) were closely monitored in this study. KER-012 did not elicit any changes in hemoglobin or RBC count in the Phase 1 study (Figure 1). Summary KER-012 was generally well tolerated in healthy participants. The observed biomarker changes are consistent with inhibition of activins, suggestive of a balance shift in favor of signaling via the anti-proliferative SMAD 1/5/9 pathway. Based on this mechanism, administration of KER-012 could potentially result in reverse vascular remodeling and clinical cardiopulmonary improvements in patients with PAH, without targeting erythropoietic pathways. Given this profile, KER-012 is being advanced for development in patients with PAH in an upcoming Ph2 clinical trial (Figure 2).

FRI546 VRDN-001, A Full Antagonist Antibody To IGF-1 Receptor In Development For Thyroid Eye Disease (TED): Pharmacodynamic Responses In Healthy Volunteers And Patients With Active TED

Abstract Disclosure: K. Foster: Employee; Self; Viridian Therapeutics Inc. B.A. Dickinson: Employee; Self; Viridian Therapeutics Inc. R.M. Summerfelt: Employee; Self; Viridian Therapeutics Inc. A. She: Employee; Self; Viridian Therapeutics Inc. V. Bedian: Employee; Self; Viridian Therapeutics Inc. B. Katz: Employee; Self; Viridian Therapeutics Inc. Introduction: VRDN-001, a full antagonist antibody to IGF-1 receptor (IGF-1R), is in development for the treatment of thyroid eye disease (TED). Clinical and preclinical evidence demonstrate IGF-1R antagonism reduces the inflammation and proptosis that occur in TED. We provide pharmacodynamic (PD) results in healthy volunteers and TED patients from our ongoing phase 1/2/3 clinical trial (NCT05176639) evaluating VRDN-001. Methods: Healthy volunteers (HVs) and patients with active, moderate-to-severe TED were randomized 3:1 to receive 2 intravenous infusions 3 weeks apart (i.e., at week 0 and week 3) of either VRDN-001 (at 3-20 mg/kg) or placebo. PD parameters (IGF-1 serum levels) were assessed through 7 weeks. Results: Thirteen HVs received either placebo (n=3) or VRDN-001, 3 mg/kg (n=3), 10 mg/kg (n=3), or 20 mg/kg (n=4). Additionally, 16 TED patients received either placebo (n=4), 10 mg/kg of VRDN-001 (n=6), or 20 mg/kg of VRDN-001 (n=6). Baseline characteristics were similar across all TED cohorts. All VRDN-001 doses (3-20 mg/kg) elicited rapid, sustained, and similar increases in IGF-1 serum levels, a biomarker for target engagement and IGF-1R inhibition. In HVs receiving VRDN-001 (n=10), IGF-1 serum levels increased within a day of the first infusion for all doses, reaching 4-6-fold above baseline; levels continued to increase after the second infusion, ultimately reaching 4-9-fold above baseline. In TED patients receiving VRDN-001 (n=12), IGF-1 serum levels increased for both doses 2-6-fold above baseline after the first infusion, and further increased after the second infusion, ultimately reaching levels 4-9-fold above baseline. No increases in IGF-1 serum levels occurred for placebo. Conclusions: Two infusions of VRDN-001 in healthy volunteers as well as TED patients elicited rapid and sustained increases in IGF-1 serum levels that were similar across groups, indicating maximal target engagement at all doses tested. Results from an additional ongoing cohort of 3 mg/kg in TED patients will further inform optimal VRDN-001 dosing and potential treatment regimens to balance efficacy and safety and minimize treatment burden in TED. Presentation: Friday, June 16, 2023

TRPA1 Antagonist LY3526318 Inhibits the Cinnamaldehyde-Evoked Dermal Blood Flow Increase: Translational Proof of Pharmacology.

Transient receptor potential Ankyrin 1 (TRPA1) is an ion channel expressed by sensory neurons, where it mediates pain signaling. Consequently, it has emerged as a promising target for novel analgesics, yet, to date, no TRPA1 antagonists have been approved for clinical use. In the present translational study, we utilized dermal blood flow changes evoked by TRPA1 agonist cinnamaldehyde as a target engagement biomarker to investigate the in vivo pharmacology of LY3526318, a novel TRPA1 antagonist. In rats, LY3526318 (1, 3, and 10 mg/kg, p.o.) dose-dependently reduced the cutaneous vasodilation typically observed following topical application of 10% v/v cinnamaldehyde. The inhibition was significant at the site of cinnamaldehyde application and also when including an adjacent area of skin. Similarly, in a cohort of 16 healthy human volunteers, LY3526318 administration (10, 30, and 100 mg, p.o.) dose-dependently reduced the elevated blood flow surrounding the site of 10% v/v cinnamaldehyde application, with a trend toward inhibition at the site of application. Comparisons between both species reveal that the effects of LY3526318 on the cinnamaldehyde-induced dermal blood flow are greater in rats relative to humans, even when adjusting for cross-species differences in potency of the compound at TRPA1. Exposure-response relationships suggest that a greater magnitude response may be observed in humans if higher antagonist concentrations could be achieved. Taken together, these results demonstrate that cinnamaldehyde-evoked changes in dermal blood flow can be utilized as a target engagement biomarker for TRPA1 activity and that LY3526318 antagonizes the ion channel both in rats and humans.

Local neuroanatomical and tract-based proxies of optimal subcallosal cingulate deep brain stimulation

BackgroundDeep brain stimulation of the subcallosal cingulate area (SCC-DBS) is a promising neuromodulatory therapy for treatment-resistant depression (TRD). Biomarkers of optimal target engagement are needed to guide surgical targeting and stimulation parameter selection and to reduce variance in clinical outcome. Objective/HypothesisWe aimed to characterize the relationship between stimulation location, white matter tract engagement, and clinical outcome in a large (n = 60) TRD cohort treated with SCC-DBS. A smaller cohort (n = 22) of SCC-DBS patients with differing primary indications (bipolar disorder/anorexia nervosa) was utilized as an out-of-sample validation cohort. MethodsVolumes of tissue activated (VTAs) were constructed in standard space using high-resolution structural MRI and individual stimulation parameters. VTA-based probabilistic stimulation maps (PSMs) were generated to elucidate voxelwise spatial patterns of efficacious stimulation. A whole-brain tractogram derived from Human Connectome Project diffusion-weighted MRI data was seeded with VTA pairs, and white matter streamlines whose overlap with VTAs related to outcome (‘discriminative’ streamlines; Puncorrected < 0.05) were identified using t-tests. Linear modelling was used to interrogate the potential clinical relevance of VTA overlap with specific structures. ResultsPSMs varied by hemisphere: high-value left-sided voxels were located more anterosuperiorly and squarely in the lateral white matter, while the equivalent right-sided voxels fell more posteroinferiorly and involved a greater proportion of grey matter. Positive discriminative streamlines localized to the bilateral (but primarily left) cingulum bundle, forceps minor/rostrum of corpus callosum, and bilateral uncinate fasciculus. Conversely, negative discriminative streamlines mostly belonged to the right cingulum bundle and bilateral uncinate fasciculus. The best performing linear model, which utilized information about VTA volume overlap with each of the positive discriminative streamline bundles as well as the negative discriminative elements of the right cingulum bundle, explained significant variance in clinical improvement in the primary TRD cohort (R = 0.46, P < 0.001) and survived repeated 10-fold cross-validation (R = 0.50, P = 0.040). This model was also able to predict outcome in the out-of-sample validation cohort (R = 0.43, P = 0.047). Conclusion(s)These findings reinforce prior indications of the importance of white matter engagement to SCC-DBS treatment success while providing new insights that could inform surgical targeting and stimulation parameter selection decisions.

749-P: Selection of BI 456906 as a Dual GCGR/GLP-1R Agonist Based on In Vitro Potency and In Vivo Target Engagement Biomarkers

Glucagon (GCG) and glucagon-like peptide 1 (GLP-1) are peptide hormones that pharmacologically regulate bodyweight by increasing energy expenditure and reducing energy intake, respectively. Several dual (GCG receptor [GCGR]/GLP-1 receptor [GLP-1R]) agonistic peptides are in clinical development for obesity. We describe preclinical data leading to selection of BI 456906 as a dual agonist. Functional potencies of the compounds were determined in CHO-K1 cells stably expressing human GCGR and GLP-1R. EC50 for GCGR mediated cAMP increase were 0.52 nM for BI 456906, 0.92 nM for BI 456908 and 0.44 nM for BI 456987. EC50 for GLP-1R engagement were 0.33 nM, 0.61 nM and 1.43 nM, respectively. Upon acute, single dosing to lean mice, engagement of the GCGR and GLP-1R was determined by testing for improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma FGF-21 and liver nicotinamide N-methyltransferase mRNA expression (100 nmol/kg), respectively. Specificity of the biomarkers was confirmed using the selective GLP-1R agonist semaglutide and GLP-1R knockout mice. Bodyweight and glucose lowering (HbA1c) efficacies were investigated in subchronic dosing studies in diet-induced obese (DIO) and diabetic (db/db) mice, respectively. In DIO mice, BI 456906 (30 nmol/kg qd), BI 456908 (30 nmol/kg qd) and BI 456897 (10 nmol/kg qd) achieved bodyweight lowering from baseline of 25%, 27%, and 26%, respectively. In db/db mice, BI 456906 and BI 456908 (10 and 20 nmol/kg qd) substantially lowered HbA1c (0.4-0.6%); no clear effect was observed for BI 456897 (3 and 7 nmol/kg qd). As a balanced GCGR/GLP-1R agonist with robust in vivo efficacy BI 456906 (30 nmol/kg qd) was further characterized, and superior bodyweight lowering (32% vs 27%; p&amp;lt;0.05) was seen versus a maximally effective semaglutide dose (100 nmol/kg qd). This was attributed to an increase in energy expenditure. The selected candidate, BI 456906, is currently in clinical development in people with obesity and NASH. Disclosure R.Augustin: None. L.Thomas: Employee; Boehringer Ingelheim Pharma GmbH&amp;Co.KG. T.Zimmermann: Employee; Boehringer Ingelheim Pharma GmbH&amp;Co.KG. E.Simon: Employee; Boehringer Ingelheim Pharma GmbH&amp;Co.KG. W.Rist: None. I.Uphues: Employee; Boehringer Ingelheim Pharma GmbH&amp;Co.KG. W.Reindl: None. T.Klein: Employee; Boehringer Ingelheim Pharma GmbH&amp;Co.KG. H.Neubauer: None. Funding Zealand Pharma A/S; Boehringer Ingelheim