Metabolism-driven in vitro/in vivo disconnect of an oral ERɑ VHL-PROTAC.

Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.

Identification and design of a novel series of MGAT2 inhibitors.

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to naїve, p<0.01) in plasma triacylglycerol (TAG) concentration.

Identification, optimisation and in vivo evaluation of oxadiazole DGAT-1 inhibitors for the treatment of obesity and diabetes.

A novel series of DGAT-1 inhibitors was discovered from an oxadiazole amide high throughput screening (HTS) hit. Optimisation of potency and ligand lipophilicity efficiency (LLE) resulted in a carboxylic acid containing clinical candidate 53 (AZD3988), which demonstrated excellent DGAT-1 potency (0.6 nM), good pharmacokinetics and pre-clinical in vivo efficacy that could be rationalised through a PK/PD relationship.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

PI3Kα/δ inhibition promotes anti-tumor immunity through direct enhancement of effector CD8+ T-cell activity.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.

Modeling and design of challenge tests: Inflammatory and metabolic biomarker study examples.

Given the complexity of pharmacological challenge experiments, it is perhaps not surprising that design and analysis, and in turn interpretation and communication of results from a quantitative point of view, is often suboptimal. Here we report an inventory of common designs sampled from anti-inflammatory, respiratory and metabolic disease drug discovery studies, all of which are based on animal models of disease involving pharmacological and/or patho/physiological interaction challenges. The corresponding data are modeled and analyzed quantitatively, the merits of the respective approach discussed and inferences made with respect to future design improvements. Although our analysis is limited to these disease model examples, the challenge approach is generally applicable to the vast majority of pharmacological intervention studies. In the present five Case Studies results from pharmacodynamic effect models from different therapeutic areas were explored and analyzed according to five typical designs. Plasma exposures of test compounds were assayed by either liquid chromatography/mass spectrometry or ligand binding assays. To describe how drug intervention can regulate diverse processes, turnover models of test compound-challenger interaction, transduction processes, and biophase time courses were applied for biomarker response in eosinophil count, IL6 response, paw-swelling, TNFα response and glucose turnover in vivo. Case Study 1 shows results from intratracheal administration of Sephadex, which is a glucocorticoid-sensitive model of airway inflammation in rats. Eosinophils in bronchoalveolar fluid were obtained at different time points via destructive sampling and then regressed by the mixed-effects modeling. A biophase function of the Sephadex time course was inferred from the modeled eosinophil time courses. In Case Study 2, a mouse model showed that the time course of cytokine-induced IL1ß challenge was altered with or without drug intervention. Anakinra reversed the IL1ß induced cytokine IL6 response in a dose-dependent manner. This Case Study contained time courses of test compound (drug), challenger (IL1ß) and cytokine response (IL6), which resulted in high parameter precision. Case Study 3 illustrates collagen-induced arthritis progression in the rat. Swelling scores (based on severity of hind paw swelling) were used to describe arthritis progression after the challenge and the inhibitory effect of two doses of an orally administered test compound. In Case Study 4, a cynomolgus monkey model for lipopolysaccharide LPS-induced TNFα synthesis and/or release was investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test compounds. Case Study 5 contains data from an oral glucose tolerance test in rats, where the challenger is the same as the pharmacodynamic response biomarker (glucose). It is therefore convenient to model the extra input of glucose simultaneously with baseline data and during intervention of a glucose-lowering compound at different dose levels. Typically time-series analyses of challenger- and biomarker-time data are necessary if an accurate and precise estimate of the pharmacodynamic properties of a test compound is sought. Erosion of data, resulting in the single-point assessment of drug action after a challenge test, should generally be avoided. This is particularly relevant for situations where one expects time-curve shifts, tolerance/rebound, impact of disease, or hormetic concentration-response relationships to occur.

Optimization of brain penetrant 11ß-hydroxysteroid dehydrogenase type I inhibitors and in vivo testing in diet-induced obese mice.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has been widely considered by the pharmaceutical industry as a target to treat metabolic syndrome in type II diabetics. We hypothesized that central nervous system (CNS) penetration might be required to see efficacy. Starting from a previously reported pyrimidine compound, we removed hydrogen-bond donors to yield 3, which had modest CNS penetration. More significant progress was achieved by changing the core to give 40, which combines good potency and CNS penetration. Compound 40 was dosed to diet-induced obese (DIO) mice and gave excellent target engagement in the liver and high free exposures of drug, both peripherally and in the CNS. However, no body weight reduction or effects on glucose or insulin were observed in this model. Similar data were obtained with a structurally diverse thiazole compound 51. This work casts doubt on the hypothesis that localized tissue modulation of 11ß-HSD1 activity alleviates metabolic syndrome.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors still improve metabolic phenotype in male 11ß-HSD1 knockout mice suggesting off-target mechanisms.

The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11ß-HSD1 inhibitor (compound C) inhibited liver 11ß-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)-fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11ß-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11ß-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting "off-target" mechanisms. Based on the patent literature, we synthesized another 11ß-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11ß-HSD1 inhibitors occurs via 11ß-HSD1-independent pathways, and only limited efficacy is achievable from selective 11ß-HSD1 inhibition. These data challenge the concept that inhibition of 11ß-HSD1 is likely to produce a "step-change" treatment for diabetes and/or obesity.

Free-Wilson and structural approaches to co-optimizing human and rodent isoform potency for 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been a target of intensive research efforts across the pharmaceutical industry, due to its potential for the treatment of type II diabetes and other elements of the metabolic syndrome. To demonstrate the value of 11ß-HSD1 in preclinical models, we required inhibitors with good potency against both human and rodent isoforms. Herein, we describe our efforts to understand how to co-optimize human and murine potency within the (5-hydroxy-2-adamantyl)-pyrimidine-5-carboxamide series. Two approaches are described-a data-driven (Free-Wilson) analysis and a structure-based design approach. The conclusions from these approaches were used to inform an efficient campaign to design compounds with consistently good human/murine potency within a logD(7.4) range of 1-3. Compounds 20 and 26 demonstrated good rodent PK, which allowed us to demonstrate a PK/PD relationship in rat and mouse. We then evaluated 26 against glycemic and body weight end points in murine disease models, where it demonstrated glucose and body weight efficacy at 300 mg/kg/day but only body weight efficacy at 50 mg/kg/day, despite providing >90% target engagement in the liver.

(1R,2R)-N-(1-cyanocyclopropyl)-2-(6-methoxy-1,3,4,5-tetrahydropyrido[4,3-b]indole-2-carbonyl)cyclohexanecarboxamide (AZD4996): a potent and highly selective cathepsin K inhibitor for the treatment of osteoarthritis.

Directed screening of nitrile compounds revealed 3 as a highly potent cathepsin K inhibitor but with cathepsin S activity and very poor stability to microsomes. Synthesis of compounds with reduced molecular complexity, such as 7, revealed key SAR and demonstrated that baseline physical properties and in vitro stability were in fact excellent for this series. The tricycle carboline P3 unit was discovered by hypothesis-based design using existing structural information. Optimization using small substituents, knowledge from matched molecular pairs, and control of lipophilicity yielded compounds very close to the desired profile, of which 34 (AZD4996) was selected on the basis of pharmacokinetic profile.