Endocrine Targets in Drug Discovery: Focus on 11β-Hydroxysteroid Dehydrogenase PDF

Prof Scott Webster Centre for Cardiovascular Science The Queen’s Medical Research Institute University of Edinburgh Endocrine Physiology & Pharmacology October 2024 ▪ Overview of cortisol action and the HPA axis ▪ Overview of the drug discovery process ▪ 11b-Hydroxysteroid de...

Prof Scott Webster Centre for Cardiovascular Science The Queen’s Medical Research Institute University of Edinburgh Endocrine Physiology & Pharmacology October 2024 ▪ Overview of cortisol action and the HPA axis ▪ Overview of the drug discovery process ▪ 11b-Hydroxysteroid dehydrogenase (11b-HSD1) as a therapeutic target – Metabolic disease – Inflammatory disease – CNS disease – Cardiovascular disease ▪ Overview of drug discovery programmes – Big pharma – University of Edinburgh  Abiraterone Finasteride Letrozole Ketoconazole Source: Yin et al (2013) Nat Rev Urol 11: 32.  Discovery Development Preclinical validation Clinical validation Pharmacology Disease Pharmacology Efficacy & in animals Target Compound in humans pathway safety in selection design PK/PD, Safety, research PK/PD, Safety patients Efficacy Phase 1 Phases 2 & 3 Confidence in Target Confidence in Compound Pathway modulation Drug Exposure (Patho)physiological regulation Target Engagement Disease modification Functional Target Modulation Confidence in Target-Disease Linkage Proof of Concept  Discovery Development Preclinical validation Clinical validation Pharmacology Disease Pharmacology Efficacy & in animals Target Compound in humans pathway safety in selection design PK/PD, Safety, research PK/PD, Safety patients Efficacy Phase 1 Phases 2 & 3 Confidence in Target Confidence in Compound Pathway modulation Drug Exposure (Patho)physiological regulation Target Engagement Disease modification Functional Target Modulation Confidence in Target-Disease Linkage Proof of Concept Main aspects to target selection/identification: ▪ Is the target biologically relevant to the disease? ▪ Will modulation of the target be efficacious in disease? ▪ Will the medicine be safe (are there any downsides to modulating the target?)? ▪ Is the target accessible to a drug molecule? Is it druggable? ▪ Can modulation of the target be measured in an animal model which closely mimics human disease? ▪ Mobilise fuel - Glucose - Fatty acids - Proteins ▪ Increase blood pressure ▪ Euphoria ▪ Inhibit - Inflammation - Immune responses - Wound healing - Digestion - Growth/bone formation - Reproduction - Detailed learning and memory  Hypothalamus CRH + - Anterior pituitary - ACTH Adrenal gland Adrenal androgens Agonist at glucocorticoid receptor (GR) Cortisol and mineralocorticoid receptor (MR) ▪ High blood pressure ▪ Diabetes ▪ High blood cholesterol ▪ Central obesity ▪ Stretch marks ▪ Bruising ▪ Osteoporosis ▪ Muscle weakness ▪ Infertility ▪ Depression ▪ Memory loss ▪ Hypertension ▪ Type 2 diabetes ▪ Dyslipidaemia ▪ Coronary heart disease ▪ Looks like Cushing’s syndrome but… blood cortisol is normal  Hypothalamus CRH + - Anterior pituitary 11b-HSD2 protects high - affinity MR receptors in ACTH kidney from glucocorticoid activation → sodium retention and raised BP Adrenal gland Adrenal androgens 11b-HSD1 Cortisol Cortisone 11b-HSD2 active inactive  11β-HSD1 11β-HSD2 cortisone Human (R=OH) cortisol 11-DHC Rodent (R=H) corticosterone (inactive) (active) ▪ 11b-HSD1 acts as a reductase in vivo ▪ Reversibility observed in vitro ▪ Low sequence homology with 11b-HSD2 Compulsory ordered mechanism: cortisol NADP+ NADPH cortisone catalysis ▪ NADPH-dependent short chain reductase ▪ Active site variability between species  THE and THF excreted in urine The ratio of [THE]/([aTHF]+[bTHF]) can be used to determine peripheral 11b-HSD1 activity  Increased adipose 11b-HSD1 Tissue-specific dysregulation of activity in human obesity in vivo 11b-HSD1 in Zucker obese rats Ref: Livingstone et al. (2000) Endocrinology Hypothesis: Excess tissue 11b-HSD1, by increasing intracellular cortisol levels, will cause ‘Cushing’s disease’ of specific tissues. In fat this might produce the Metabolic Syndrome. ▪ Transgenic aP2/11b-HSD1 mice created (Masuzaki et al (2001) Science) ▪ 2.5-fold increased 11b-HSD1 activity in adipose – no change in circulating CORT Male Tg Female Tg Male non-Tg Female non-Tg How do we further validate 11b-HSD1 as target for the treatment of metabolic disease? Knockout the gene in mice and analyse the phenotype (most top selling drugs validated by mouse KO) Hypothesis: Deficiency of 11b-HSD1, by reducing local intracellular cortisol levels, will prevent the Metabolic Syndrome and its complications. ▪ Mice have normal weights, growth, reproduction and lifespan ▪ Mice have normal plasma electrolytes and blood pressure 11b-HSD1-/- mice eat more, but weigh less  Discovery Development Preclinical validation Clinical validation Pharmacology Disease Pharmacology Efficacy & in animals Target Compound in humans pathway safety in selection design PK/PD, Safety, research PK/PD, Safety patients Efficacy Phase 1 Phases 2 & 3 Confidence in Target Confidence in Compound Pathway modulation Drug Exposure (Patho)physiological regulation Target Engagement Disease modification Functional Target Modulation Confidence in Target-Disease Linkage Proof of Concept  Discovery Development Preclinical validation Clinical validation Pharmacology Disease Pharmacology Efficacy & in animals Target Compound in humans pathway safety in selection design PK/PD, Safety, research PK/PD, Safety patients Efficacy Phase 1 Phases 2 & 3 Confidence in Target Confidence in Compound Pathway modulation Drug Exposure (Patho)physiological regulation Target Engagement Disease modification Functional Target Modulation Confidence in Target-Disease Linkage Proof of Concept  TOXIC Maximum Tolerated Safety Concentration (MTC) Margin Elimination phase (t½) Minimum Effective Concentration (MEC) INEFFECTIVE If we can increase the potency and exposure of a drug then we can administer a smaller dose → less chance of toxicity Phase 1 (0.5 – 1 year) ▪ 20 -100 healthy volunteers ▪ Safety & dosage Phase 2 (1-2 years) ▪ 100-500 patients with disease Increasing cost ▪ Evaluate efficacy ▪ Look for side effects Phase 3 (2-4 years) ▪ 1000-5000 patients with disease ▪ Confirm effectiveness ▪ Monitor side-effects with long term use ▪ Most pharmaceutical companies have pursued drug discovery programmes - >200 new molecular entity patents ▪ Most pharma companies focused on metabolic disease ▪ Many companies with drugs in development - Merck (MK-0916, MK-0736) - Amgen/Biovitrum (AMG221) - Incyte (INCB-13739, INCB-20817) - Pfizer/Wyeth (PF-915275, HSD-016) - Bristol-Myers Squibb (BMS-770767, BMS-016336) Cardiometabolic - Boehringer Ingleheim/Vitae (BI-135585) - Eli Lilly (LY-2523199) - Roche (RO-7234, RO-4929) - AstraZeneca (AZD4017, AZD6925) - AbbVie (ABT-384) CNS - University of Edinburgh/Actinogen (UE2343/Xanamem)  ▪ The 11b-HSD1 active site is Q177 relatively hydrophobic – Y177 (mouse) implications for inhibitor design ▪ There is generally high sequence homology across species, but there A226 are significant changes in active site E226 residues involved in substrate (mouse) binding ▪ Significant differences in potency across species for many drugs – Active Site Residue Human Monkey Pig Dog Rat Mouse implications for study of efficacy in 124 T T A T T T rodents 126 L L M M L L 177 Y Y N S Q Q 186 V V V V I I 218 L L L L F L 226 A A A A E E 227 V V V V T I 280 Y Y S S S S  H-bond Acceptor (interactions with S170 and Y183) H-bond Acceptor (interaction with entrance of binding pocket) lipophilic H-bond Acceptor/Donors (interactions with NADPH and Steroid substrate interior of binding pocket) HAcc or HDon HAcc or HDon Non Steroidal lipophilic HAcc Pharmacophore group lipophilic group PF-915275 ▪ Possible to develop a 3-dimensional pharmacophore from X-ray data  AMG221 Entrance to the binding pocket is Interior of binding highly flexible pocket is fairly rigid Y177 Y183 S170 Y280 NADPH small hydrophobic residues Overlay of multiple 11β-HSD1 inhibitor structures Y280 UE1976 hHSD1: IC50(cell) = 39nM Y177 O N N HN S ▪ Binding of UE1976 in an averaged pharmacophore ▪ V-shaped pharmacophore for typical 11β-HSD1 inhibitor ▪ Structure of INCB-13739 has not been disclosed ▪ Programme has not progressed beyond Phase 2  IC50(nM) PD (mouse) hHSD1 mHSD1 hHSD2 mHSD2 1h 4h 8 98 >3000 >4000 59% 17% ▪ decreases body wt, food intake and fad pad wt in DIO mice ▪ lowers fasting glucose and improves insulin resistance in murine models ▪ lowers serum lipids in murine models of metabolic syndrome and atherosclerosis Compound 544 ▪ decreases aortic lesions in apoE deficient mice IC50(hHSD1) = 2nM PD(mouse): 1h=99%, 4h=98% PK(mouse): Cl= 273ml/min/kg, F=21% MK-0916 IC50(hHSD1) = 5nM PD(mouse): 4h=91% PK(mouse): Cl= 11ml/min/kg, t½ = 1.5h, F=100% MK-0736 IC50(hHSD1) = 3nM PD(mouse): 16h=63% Sulfone blocks metabolism of parent compound  ▪ Phase 2 study in T2DM and hypertension (2008) ▪ Dose: 0.5-6mg/day ▪ No improvement in fasting plasma glucose ▪ HB1Ac -0.3% MK-0916 ▪ LDL +10.4% at 6mg/day ▪ Dose-dependent decrease in BP ▪ Adrenal androgens +20-30% Ref: Feig et al (2011) Diabetes Obes Metab ▪ Phase 2 study in hypertension (2008) MK-0736 ▪ No reduction in BP ▪ LDL -12.3% ▪ HDL -6.3% ▪ Body weight -1.4kg ▪ Adrenal androgens +30-50% Ref: Shah et al (2011) J Am Soc Hyper MK-0736 and MK-0916 no longer in clinical development Biovitrum Lead in vivo epimerisation IC50=52nM IC50=12nM IC50=770nM -OH active metabolite formed in AMG221: vivo discontinued after Phase II IC50=189nM IC50=13nM ▪ excellent PK profile in rodents and dog ▪ clear CYP450 and off-target profile ▪ lowers blood glucose in DIO mice Human Phase I Data IC50=126nM IC50=4.5nM Ref: Veniant et al (2010) J. Med. Chem. 53, 4481-4487. Gibbs et al (2010) J. Clin. Pharm.  Hippocampal volume and memory correlates with cortisol levels Ref: Lupien et al (1998) Nat Neurosci 1: 69 hsd11b1 mRNA in rat brain hsd11b1 mRNA in human brain hippocampus cerebellum ▪ THFs/THE ratio is a measure of peripheral 11b-HSD1 activity Ref: MacLullich et al (2012) Neurobiol Aging CSF cortisol concentrations by APOE genotype Transgenic mice treated with glucocorticoids show in 64 patients with AD (open circles) and 34 increased levels of Ab in brain regions nondemented older control subjects (filled circles) Ref: Peskind et al. (2001) Neurology 56:1094-1098 Ref: Green et al (2006) J. Neurosci 26: 9047 Ref: Yau et al (2001) Proc Natl Acad Sci. ADMET: Absorption, Distribution, Metabolism, Excretion & Toxicity PK: Pharmacokinetics Heavily metabolised in liver microsomes IC50 = 0.7nM UE2316 A potent, rodent active compound ▪ Improvement in passive avoidance test demonstrates a positive impact on cognition in AD mice Passive Avoidance Test W ild - ty p e Tg2576 d a r k c o m p a r tm e n t (s ) 300 L a te n c y to e n te r ** Experimental Details 200 V e h ic le Mouse strain: Tg2576 U E2316 Age: 15 month old males Dose: 10mg/kg/day by s.c. minipump for 28 100 days 0 2-way ANOVA effect of UE2316, *p