Investigative and Predictive Approaches to Drug-Induced Steroid Hormone Perturbation Jeff Lawrence Comparative Biology and Safety Sciences Nor Cal SOT 4-Nov-21
Understanding Mechanisms Helps Provide a Path Forward Screening: Require higher throughput, high sensitivity assays There may be false positives Mechanistic Assessment: High specificity assays to distinguish between types of events The assay needs to be a different assay These tend to be lower throughput Risk Assessment: After a hazard is identified and diagnosed, the risk needs to be assessed by determining a safe exposure In animals for Margin of safety determination In the clinical to determine safe exposure and monitor safety compound High sensitivity assay hit High specificity assay(s) diagnosed Integrated model with biomarker to assess Margin of Safety (MOS) hit Decision Risk Assessment 2
Outline of Presentation Program endocrine findings In vitro mechanistic studies In vivo metabolomics study Search for a biomarker Conclusions
28-Day Sprague-Dawley Rat Study with Cmpd 1 Endocrine Findings in Males Prostate and seminal vesicle atrophy with weights Prominent pituitary gonadotrophs Adrenal cortical hypertrophy with weight Thyroid follicular hypertrophy weight H&E PN TSH IHC PI Secondary to hepatic enzyme induction PD LH IHC FSH IHC % Change from Gro oup 5-5 -1-15 -2-25 -3-35 Dose (mg/kg/day) 5 L 2 M 4H Prostate SV Incidence of Microscopic Findings and Estimated Margin Dose 5 2 4 ACTH IHC PR IHC Pituitary 1/1 9/1 5/1 Prostate atrophy /1 2/1 4/1 Seminal vesicle atrophy 1/1 2/1 4/1 Estimated Margin 4 16 25
Cmpd 1 Inhibits Only hcg-induced Testosterone Production ion (pg/ml) Testos sterone product 4 basal hcg 3 2 1.1.1.1 1 1 1 1 Concentration ( M) Method An enriched primary rat Leydig cells were isolated from rat testes by differential centrifugation. Cells were isolated, plated, and incubation with hormones/test articles for 2 hours. Media was assayed for testosterone with an ELISA and cellular ATP measured to assess viability Cmpd 1 only inhibits the stimulated portion of testosterone production Doesn t look like a biosynthetic inhibitor Site of action appears to involve signaling
LH Induced Testosterone Production Signaling Pathway in Leydig Cells LH (or hcg) LHR Forskolin adenylyl cyclase ATP Steroid Steroid transcription SF-1 Steroid camp Synthesis Synthesis + Synthetic activity ERK1 Prolonged stimulation mrna Protein PKA Acute regulation cholesterol l Mit P StAR tochondrial membrane CYP11a1 What aspect of signaling is p g g affected?
Understanding The Mode of Action Will Help Identify The Best Screening System LH (or hcg) 3 LHR Forskolin + adenylyl cyclase 5 camp SF-1 transcription 1 Steroid Synthesis mrna 6 Steroid Synthesis Protein Steroid Synthetic activity 12 Tes stosterone Productio on (pg/ml) 25 2 15 1 5 Tes stosterone Productio on (pg/ml) 4 3 2 1 camp production (pmol/1 6 cells/ 3 min) 8 6 4 2 * * mrna (fold cont trol) 5 4 3 2 1 CYP11a1 CYP17 STAR Te estosterone Production (pg/ml) 1 8 6 4 2 Cmpd 1 Prochloraz AMG 277 AMG 277 + hcg Cmpd 1 Cmpd 1 + Forskolin Cmpd 1 Cmpd 1 +hcg * = below the lower limit of quantitiati Cmpd 1 inhibits hcg and forskolin stimulated testosterone production Cmpd 1 does not inhibit hcg stimulated camp production Cmpd 1 does not inhibit forskolin induced StAR or CYP11a1 mrna CYP17 limited or no induction Cmpd 1 does not inhibit basal testosterone production not a biosynthetic pathway inhibitor Cmpd 1 Cmpd 1 + Forskolin.1.1.1 1 1 1 1 Concentration ( M)
LH Induced Testosterone Production Signaling Pathway in Leydig Cells LH (or hcg) LHR Forskolin adenylyl cyclase ATP Steroid Steroid transcription SF-1 Steroid camp Synthesis Synthesis + Synthetic activity ERK1 Prolonged stimulation mrna Protein PKA Acute regulation Is the PKA-StAR pathway effected by Cmpd 1? 22-OH cholesterol can pass into mitochondria and enter steroid pathway independently of StAR cholesterol l Mit P StAR tochondrial membrane CYP11a1
Effect of Cmpd 1 on 22-OH Cholesterol Stimulated Testosterone Synthesis In Vitro Testo osterone Produc ction (pg/ml) 14 12 1 + Compound 1 + Prochloraz No inhibition of 22-OH cholesterol l stimulated testosterone by Cmpd 1 Strong inhibition of basal, 22-OH 8 cholesterol stimulated, and hcg stimulated testosterone by 6 prochloraz 4 Data suggests Cmpd 1 is not a biosynthetic inhibitor, but acts at 2 cholesterol transport + hcg + hcg + 22-OH holesterol + 22 2OH-Cholesterol + Ch
In Vitro Chemical Survey Effects on hcg-stimulated Testosterone Production in Rat Leydig Cells Viability (ATP) Testosterone Production Cytotoxicity Increased T production Compound 4 Decreased T production to basal levels Compound 5 Decreased T production below basal levels Compound 6 No data Compound 7 Compound 8 Compound 9 Compound 1 Subtle chemical changes can Compound 11 affect in vitro testosterone Compound 12 production markedly Compound 13 No data Still room for good molecules Compound 1 derivative in this series No data Compound 2 derivative How do you sift Compound 14 through them? No data Compound 15 Compound 1 derivative Compound 1 metabolite Only difference is change Compound 3 derivative Compound 2 derivative from F to Me Compound 2 derivative Compound 2 derivative Potential compound 2 metabolite Compound 1 Compound 2 Compound 3 Concentration (μm) Concentration (μm)
Metabolomics Study Demonstrating In Vivo Relevance Group Study Design Number of Animals Day 2 Day 5 Day 14 Day 28 Subgroup 1 2 3 4 Dose Level (mg/kg) Treatment 1 6 6 6 6 1 a 2 6 6 6 -- 2 b 3 6 6 6 -- Cmpd1 1 4 6 6 6 6 Cmpd1 1 5 6 6 6 6 Cmpd1 4 6 6 6 6 -- Prochloraz 75 a 1 (Cmpd1): 2% Captisol, 1% HPMC, 1% Pluronic F68 in RO water, ph 21 2.1 b 2 (prochloraz): corn oil Terminal Procedures Scheduled Necropsy Approximately 6 hours post-dose on days 2, 5, 14, and 28 Blood collection in EDTA tubes and plasma collected Organ weights, tissue collection for histopathology and freezing Analytical Procedures Hormone analysis using a multiplex luminex-based analysis platform Mass Spectrometry based Metabolomics Broad based biochemical profiling High sensitivity quantititative hormone profiling Testicular StAR protein semi-quantification via Western blot
Metabolomics Study Results Histologic Observations Day Organ/ change 2 5 14 28 Prostate/ Decreased secretion Pituitary/ Increased basophilic pituicytes No effect No effect No effect No effect No effect No effect (4) (1, 4) Adrenal/ Vacuolar hypertrophy (4) (1, 4) (1, 1, 4) 1 mpk dose of Cmpd 1 was only tested through 14 days Prochloraz affected prostate t as early as 5 days treatmentt t (1, 4)
Metabolomics Study Plasma Testosterone Pla asma Testosteron e (ng/ml) Plasma a Testosterone (ng g/ml) 16 14 12 1 8 6 4 2 16 14 12 1 8 6 4 2 1 2 Day D22 Day D55 16 A B C D E F g g g g Day D14 14 Day D28 28 16 A B C D E F Pla asma Testosteron ne (ng/ml) Plasma a Testosterone (ng g/ml) 14 12 1 8 6 4 2 14 A B C D E F A- vehicle B- corn oil C- 4 mpk Cmpd 1 D- 1 mpk Cmpd 1 E- 1 mpk Cmpd 1 F- 75 mpk Prochloraz 12 Note variability in 1 vehicle 8 6 testosterone 4 levels 2 A C D At 4 mg/kg/day (C), Cmpd 1 appears to tighten up the testosterone levels Decreased influence of stress? Cmpd 1 did not affect LH hormone levels Cmpd 1 did not affect ACTH levels
45-2-6-4-8-Metabolomics Study Supervized Multivariate Overview of Treatment Cmpd 1 Cmpd 1 Effect16pal componen 8nt 5Prochloraz Q2 (cumulative) =.35 35 Prochloraz ogonal princip al component1q2 (cumulative)=.8724-22orth gonal principaorthog-5p p p 6-4-2Predictive principal component 4principal component-6predictive 2 Weak metabolic effects of Cmpd 1 Strong metabolic effects of prochloraz 1 mpk 1 mpk 4 mpk 75 mpk Score plots of OPLS on log ratios normalized to vehicle control
Metabolomics Study Supervized Multivariate Overview of Treatment Effect Hormone Method Analytes Only Loadin Loadi ngs ngs p1 p1 OPLS OPLS Compound Compound F 3,4-Dihydroxyphenylalanine (DO (DOPA).15 3,4-Dihydroxyphenylacetic acid 3-Methoxytyrosine t Noradrenaline (Norepinephrine).1.5 -.5 -.1 3,4-Dihydroxyphenylglycol Androstenedione 4-Hydroxy-3-methoxyphenylglycol Testosterone 18-Hydroxycorticosterone (DOP Normetanephrine Corticosterone 18-Hydroxycorticosterone (minor:11-deoxycortisol) Histamine Calcitriol Dopamine Serotonin (5-HT) Progesterone Metanephrine Homovanillic acid (HVA) 21-Hydroxyprogesterone (11- Deoxycorticosterone) Adrenaline (Epinephrine) -.15 -.1 -.5.5.1.15 Loadings p1 OPLS Prochloraz Decrease upon Prochloraz Increase upon Prochloraz 1 Increas se upon Cmpd 1 Decreas se upon Cmpd Cmpd 1 Decreases below horizontal line Increases above horizontal line Prochloraz Decreases left of vertical line Increases right of vertical line OPLS loadings SUS plot of log ratios normalized to vehicle control
Metabolomics Study Supervized Multivariate Overview of Treatment Effect of Steroids Only Decrease upon Prochloraz Increase upon Prochloraz Cmpd 1 Loadings p1 OPLS Comp pound F.6.4.2 -.2 -.4 -.6 -.8 Corticosterone 18-Hydroxycorticosterone 18-Hydroxycorticosterone (minor: 11-Deoxycortisol) 21-Hydroxyprogesterone (11-Deoxycorticosterone) Androstenedione Testosterone Progesterone Increase e upon Cmpd 1 Decrease e upon Cmpd 1 All gonadal steroids are decreased by Cmpd 1 Prochloraz Decreased Testosterone Androstenedione Increased Progesterone 21-hydroxyprogesterone 11-Deoxycorticosterstone -.15 -.1 -.5.5.1 Loadings p1 OPLS Prochloraz Consistent with CYP17 inhibition OPLS loadings SUS plot of log ratios normalized to vehicle control
Metabolomics Study Summary Few biochemical changes observed in metabolomics analysis for Cmpd 1 strong effects observed with prochloraz Prochloraz decreased male gonadal steroids distal to CYP17 and increased gonadal steroids proximal to CYP17 consistent with its know mechanisms of testosterone synthesis inhibition (CYP17) Cmpd 1 decreased all gonadal steroids consistent with in vitro testosterone synthesis results cholesterol transport
Biomarker Assessment StAR expression 22-OH cholesterol expt implicates role of transport in defect Herbicide Round-Up affects steroid synthesis by decreasing StAR quantity What does Cmpd 1 do to StAR levels? Can StAR quantity be a biomarker useful in a 4-day study? Eliminate need for 28 day studies to detect changes?
Biomarker Assessment StAR Protein Expression s (% control) StAR Protein in Teste 35 3 25 2 15 1 5 Day 4 Average +/- SD Individual points 1 1 4 Cmpd1 mg/kg/day Rat were treated with Cmpd 1 at 1, 1, and 4 mpk for 4 days At necropsy, testes were harvested and frozen at -7 C A portion of the frozen testes was homogenized and the StAR protein semi-quantified by Western blot StAR content was increased compared to vehicle treated animals at day 4 This suggests that cholesterol transport inhibition may lead to increased StAR protein levels through a feedback loop StAR content may be a useful biomarker in 4 day studies to assess the testosterone disruption effects that manifest in prostate weight changes at 28 days
Mechanistic Summary Cmpd 1 can inhibit testosterone production Only hormone stimulated testosterone production inhibited Does not affect intramitochondrial biosynthetic targets Affects all steroid intermediates equally Is not a transcriptional mechanism Is not a camp production inhibitor mechanism Is not a hormone receptor inhibitor mechanism Is not a LH hormone deficiency mechanism Is not a androgen nuclear receptor antagonist (data not shown) Does not inhibit PKA (data not shown) Defect involves cholesterol transport Does not decrease StAR (cholesterol transporter) quantity Increase observed
What can we conclude? Program compounds affected steroid biosynthesis Cmpd 1 decreased all gonadal steroids in vivo, consistent with in vitro evidence of testosterone synthesis inhibition at cholesterol transport Metabolomics quantitative hormone profiling can distinguish types of testosterone synthesis inhibitors in the in vivo setting Value of in vitro Leydig cell assay: Good positive predictive value (3/3) Negative predictive value less reliable (1/2 for molecules showing no effect up to 3 μm) Edge of sensitivity for distinguishing compounds like Cmpd 2 (1-3 µm) Cytotoxicity limitations Knowing the mechanism can help identify potential predictive biomarkers Cmpd 1 treatment increased StAR protein levels at early time points (Day 4) compared to organ weight or histopathological changes (Day 28) This can be a useful biomarker to assess testosterone synthesis disruption for this mechanism in screening studies
Acknowledgements Michelle Horner Victor Cee Jon Werner Ra Shun Conner Chris DiPalma Yang Xu Rocio Hernandez Han Xu Patrick Cosgrove Min Wong Rong Hu Paul Acton Ihsan Senal