Stephanie Dodd

The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR–ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR–ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment.

The Development of Self-Emulsifying Oil-in-Water Emulsion Adjuvant and an Evaluation of the Impact of Droplet Size on Performance

Microfluidization is an established technique for preparing emulsion adjuvant formulations for use in vaccines. Although this technique reproducibly yields high-quality stable emulsions, it is complex, expensive, and requires proprietary equipment. For this study, we developed a novel and simple low shear process to prepare stable reproducible emulsions without the use of any proprietary equipment. We found this process can produce a wide range of differently sized emulsions based on the modification of ratios of oil and surfactants. Using this process, we prepared a novel 20-nm-sized emulsion that was stable, reproducible, and showed adjuvant effects. During evaluation of this emulsion, we studied a range of emulsions with the same composition all sized below 200; 20, 90, and 160 nm in vivo and established a correlation between adjuvant size and immune responses. Our studies indicate that 160-nm-sized emulsions generate the strongest immune responses.

Co-crystal formation based on structural matching.

A co-crystal is defined as a single crystalline structure composed of two or more components with no proton transfer which are solid at room temperature. Our group has come up with the following rationale selection of co-formers for initial co-crystal screening: 1) selection of co-formers with the highest potential for hydrogen bonding with the API and 2) selection of co-formers with diversity of secondary structural characteristics. We demonstrate the feasibility of this technique with a Novartis drug candidate A. In the first tier, 20 co-formers were screened and two hits were identified. By examining the two co-formers, which worked from the first round, a second round of screening was undertaken with more focused chemical matter. Nineteen co-crystal formers closely related to the two hits in the first screen were screened in the second tier. From this screen five hits were identified. All the hits were compared for their physical and chemical stability and dissolution profile. Based on the comparison 4-aminobenzoic co-crystal was chosen for in-vivo comparison with the free form. The co-crystal had 12 times higher exposure than the free form thus overcoming the solubility limited exposure.

COUNTERION QUANTIFICATION FOR DISCOVERY CHEMISTRY AND PRE-FORMULATION SALT SCREENING

A fast method was developed to determine the amount of anions and cations present in discovery samples using a reagent free ion chromatography system. The system separates and quantifies counterions present in novel discovery compounds from an accurately weighed sample of 1 mg. The advantage of using these methods in the discovery phase and early development to accurately characterize salt identity and stoichiometry has been demonstrated. Likewise, a further advantage is the ability to accurately quantify undesirable counterions that may be present due to process related manipulation of material and to quantify salt equivalents for “mixed” salt samples. The instrumentation described also has the ability to utilize a reagent-free automated eluent generation approach which in addition to saving time, eliminates the variability that can occur with manually prepared eluents. Several examples have been collated to highlight the usefulness of this approach within discovery.

Discovery and Evaluation of Clinical Candidate IDH305, a Brain Penetrant Mutant IDH1 Inhibitor

Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Orally Bioavailable and Brain Penetrant Mutant IDH1 Inhibitors

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

Abstract A144: Inhibition of 2-HG production in IDH mutant xenograft models.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) oxidize isocitrateto α-ketoglutarate (α-KG), a cofactor needed for the function of over 100 enzymes1. Heterozygous mutations of IDH1/2 occur in a variety of tumor types including glioma, acute myeloid leukemia (AML), cholangiosarcoma, chondrosarcoma and melanoma2. Mutant IDH is crippled for wild-type function but gains the ability to convert α-KG to 2-hydroxyglutarate (2-HG), a believed “oncometabolite” that may alter cell biology through, in part, changes in global histone and DNA methylation 1,3,4. However, the dearth of in vivo models dependent upon mutant IDH has made understanding the biological relevance of IDH mutations and 2-HG production in cancer challenging. To better understand the role that IDH mutations play in oncogenesis, and the potential effects of inhibition of these mutations, we introduced a heterozygous point mutation of IDH1 (R132H) into the endogenous locus of the HCT116 colon carcinoma cell line. When compared with the parental IDH1 wild-type HCT116 model, HCT116 IDH1R132H/+ xenograft tumors produce significantly higher levels of 2-HG. In addition, we have identified a patient-derived melanoma model, HMEX2838, which harbors an endogenous IDH1R132C/+ mutation and expresses high levels of 2-HG. To better explore the biology of mutant IDH inhibition, we developed a mutant-selective IDH inhibitor with favorable in vivo properties. Using this compound we demonstrate that 2-HG production is strongly inhibited in both the engineered HCT116 IDH1R132H/+ xenograft model and the endogenously mutant HMEX2838 patient-derived xenograft model. The data we have generated suggests that this inhibition of 2-HG directly correlates with the area under the curve (AUC) of free-drug above the HCT116 IDH1R132H/+ cellular IC50, as determined in vitro. Using these models and our IDH inhibitors we hope to gain a better understanding of how IDH mutations contribute to cancer and how inhibition of the mutant enzyme could benefit patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A144. Citation Format: Kelly L. Slocum, Julia Downall, Ty Gould, Mohammad Zafari, Stephanie Dodd, Brant Firestone, Ray Pagliarini, Julian Levell. Inhibition of 2-HG production in IDH mutant xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A144.