Synthesis and biological evaluation of novel PET tracers [18F]AG120 & [18F]AG135 for imaging mutant isocitrate dehydrogenase 1 expression.

Mutations in isocitrate dehydrogenase 1 (IDH1) are commonly found in various human malignancies. Inhibitors of several mutant IDH1 enzymes have entered clinical trials as target therapeutic drugs for the treatment of patients with IDH1 mutations. Herein, we report the synthesis and evaluation of two 18F-labeled tracers, [18F]AG120 and [18F]AG135 for imaging expression of mutated IDH1 in positron emission tomography (PET). [18F]AG120 and [18F]AG135 were synthesized in decay-corrected radiochemical yield of 1 % and 3 %, respectively, high molar activity (52-66 MBq/nmol and 216-339 MBq/nmol, respectively) and high radiochemical purity (>99%). Both tracers showed good in vitro stability, selective uptake into mutated IDH1-expressing cells and good pharmacokinetic profiles with low uptake in most organs/tissues. Furthermore, [18F]AG120 micro-PET/CT imaging displayed significantly greater uptake in IDH1-mutant than in wild-type tumors, Relatively, uptake of [18F]AG135 was observed neither in IDH1-mutant tumor xenografts nor in wild-type tumors. This study suggests that [18F]AG120 is a promising radiotracer for PET imaging of IDH1 mutation, However, further optimization and investigation are necessary for [18F]AG135 due to the limited uptake in mutated IDH1-expressing tumors.

Differentiation syndrome during ivosidenib treatment with immunohistochemistry showing isocitrate dehydrogenase R132H mutation.

We report a case of differentiation syndrome in a patient receiving the IDH1 inhibitor ivosidenib, with skin biopsy showing isocitrate dehydrogenase (IDH) R132H-mutated leukemia cutis. A 72-year-old man with IDH1-mutated acute myeloid leukemia (AML), status-post allogeneic cell transplantation, on ivosidenib for 6 months, was admitted for culture-negative neutropenic fever, pink and purpuric plaques and patches on the legs, abdomen and back, edema, hypotension, and shortness of breath. Skin biopsy revealed an infiltrate of atypical, immature, myeloperoxidase-positive mononuclear cells compatible with leukemia cutis or Sweet syndrome. Although dermal edema and interstitial neutrophilic infiltrate with karyorrhexis characteristic of Sweet syndrome were not seen, the atypical cells lacked expression of CD117 and CD34, which were expressed in the original leukemia. Additional immunohistochemical staining of suspected blasts was strongly positive for IDH1 R132H, suggesting a diagnosis of leukemia cutis. As the immunophenotype of blasts in skin infiltrates can significantly differ from the immunophenotype seen in blood and bone marrow, this case shows that mutation-specific antibodies such as anti-IDH1 R132H may be useful to help distinguish malignant from non-malignant infiltrates in the skin. Furthermore, differentiation syndrome may show histopathologic features of leukemia cutis on skin biopsy.

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors.

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79 ±â€¯6% yield (n = 9), and 18F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ±â€¯1.6% (n = 5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with 18F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.

Discovery and structure-activity-relationship study of novel imidazole cyclopropyl amine analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors.

The discovery and optimization of imidazole cyclopropyl amime analogues as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, moderate liver microsome stability and PK properties.

Discovery and structure-activity-relationship study of novel conformationally restricted indane analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors.

The discovery and optimization of various of indane amides as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, favorable PK properties and great selectivity against IDH1wt and IDH2R140Q.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

The first-in-human phase I study of a brain-penetrant mutant IDH1 inhibitor DS-1001 in patients with recurrent or progressive IDH1-mutant gliomas.

BACKGROUND: Approximately 70% of lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) mutations, resulting in the accumulation of oncometabolite D-2-hydroxyglutarate (D-2-HG); this leads to epigenetic dysregulation, oncogenesis, and subsequent clonal expansion. DS-1001 is an oral brain-penetrant mutant IDH1 selective inhibitor. This first-in-human study investigated the safety, pharmacokinetics, pharmacodynamics, and efficacy of DS-1001. METHODS: This was a multicenter, open-label, dose-escalation, phase I study of DS-1001 for recurrent/progressive IDH1-mutant (R132) glioma (N = 47) (NCT03030066). DS-1001 was administered orally at 125-1400 mg twice daily. Dose-escalation used a modified continual reassessment method. RESULTS: The maximum tolerated dose was not reached. Eight patients were continuing treatment at the data cutoff. Most adverse events (AEs) were grade 1-2. Twenty patients (42.6%) experienced at least 1 grade 3 AE. No grade 4 or 5 AEs or serious drug-related AEs were reported. Common AEs (>20%) were skin hyperpigmentation, diarrhea, pruritus, alopecia, arthralgia, nausea, headache, rash, and dry skin. The objective response rates were 17.1% for enhancing tumors and 33.3% for non-enhancing tumors. Median progression-free survival was 10.4 months (95% confidence interval [CI], 6.1 to 17.7 months) and not reached (95% CI, 24.1 to not reached) for the enhancing and non-enhancing glioma cohorts, respectively. Seven on-treatment brain tumor samples showed a significantly lower amount of D-2-HG compared with pre-study archived samples. CONCLUSIONS: DS-1001 was well tolerated with a favorable brain distribution. Recurrent/progressive IDH1-mutant glioma patients responded to treatment. A study of DS-1001 in patients with chemotherapy- and radiotherapy-naïve IDH1-mutated WHO grade 2 glioma is ongoing (NCT04458272).

FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress.

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

The Landscape of the Anti-Kinase Activity of the IDH1 Inhibitors.

Isocitrate dehydrogenases constitute a class of enzymes that are crucial for cellular metabolism. The overexpression or mutation of isocitrate dehydrogenases are often found in leukemias, glioblastomas, lung cancers, and ductal pancreatic cancer among others. Mutation R132H, which changes the functionality of an enzyme to produce mutagenic 2-hydroxyglutarate instead of a normal product, is particularly important in this field. A series of inhibitors were described for these enzymes of which ivosidenib was the first to be approved for treating leukemia and bile duct cancers in 2018. Here, we investigated the polypharmacological landscape of the activity for known sulfamoyl derivatives that are inhibitors, which are selective towards IDH1 R132H. These compounds appeared to be effective inhibitors of several non-receptor kinases at a similar level as imatinib and axitinib. The antiproliferative activity of these compounds against a panel of cancer cells was tested and is explained based on the relative expression levels of the investigated proteins. The multitargeted activity of these compounds makes them valuable agents against a wide range of cancers, regardless of the status of IDH1.

Non-Invasive Measurement of Drug and 2-HG Signals Using 19F and 1H MR Spectroscopy in Brain Tumors Treated with the Mutant IDH1 Inhibitor BAY1436032.

BACKGROUND: BAY1436032 is a fluorine-containing inhibitor of the R132X-mutant isocitrate dehydrogenase (mIDH1). It inhibits the mIDH1-mediated production of 2-hydroxyglutarate (2-HG) in glioma cells. We investigated brain penetration of BAY1436032 and its effects using 1H/19F-Magnetic Resonance Spectroscopy (MRS). METHODS: 19F-Nuclear Magnetic Resonance (NMR) Spectroscopy was conducted on serum samples from patients treated with BAY1436032 (NCT02746081 trial) in order to analyze 19F spectroscopic signal patterns and concentration-time dynamics of protein-bound inhibitor to facilitate their identification in vivo MRS experiments. Hereafter, 30 mice were implanted with three glioma cell lines (LNT-229, LNT-229 IDH1-R132H, GL261). Mice bearing the IDH-mutated glioma cells received 5 days of treatment with BAY1436032 between baseline and follow-up 1H/19F-MRS scan. All other animals underwent a single scan after BAY1436032 administration. Mouse brains were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: Evaluation of 1H-MRS data showed a decrease in 2-HG/total creatinine (tCr) ratios from the baseline to post-treatment scans in the mIDH1 murine model. Whole brain concentration of BAY1436032, as determined by 19F-MRS, was similar to total brain tissue concentration determined by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS), with a signal loss due to protein binding. Intratumoral drug concentration, as determined by LC-MS/MS, was not statistically different in models with or without R132X-mutant IDH1 expression. CONCLUSIONS: Non-invasive monitoring of mIDH1 inhibition by BAY1436032 in mIDH1 gliomas is feasible.