Evidence for the Existence of Non-monotonic Dose-response: Does it or Doesn t it?

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1 Evidence for the Existence of Non-monotonic Dose-response: Does it or Doesn t it? Scott M. Belcher, PhD University of Cincinnati Department of Pharmacology and Cell Biophysics

2 Evidence for the Existence of Non-monotonic Dose-response: Does it or Doesn t it? Answer: Yes Every day we accept : The U-Shaped Dose Response Curve for Essential Nutrients Toxic Effects at Low and High Concentrations

3 Modern Toxicology: The Dose Makes the Poison Quintal Responses: e.g. alive vs. dead; tumor vs. tumor sigmoidal C/R curve Atropa belladonna Amanita phalloides Atropa belladonna

4 Paracelsus: a Clinical Pharmacologist who presumed non-monotonic responses Alle Dinge sind Gift und nichts ist ohne Gift, allein die Dosis macht es, dass ein Ding kein Gift ist. ( ) All things are poison and nothing is without poison, only the dose permits something not to be poison. Philippus von Hohenheim (Paracelsus) Atropa belladonna

5 Alle Dinge sind Gift und nichts ist ohne Gift, allein die Dosis macht es, dass ein Ding kein Gift ist. All things are poison and nothing is without poison, only the dose permits something not to be poison. Philippus von Hohenheim (Paracelsus) Improved contraction (decreased arrhythmia) EC50 ( ) Cardiac Gycosides: e.g. digoxin Na +,K + ATPase inhibitors used to treat heart failure

6 Basic Principles Pharmacology/Toxicology: Pharmacodynamics: biochemical and physiological effects of drugs (EDCs) and mechanisms of actions What the agent does to the body Pharmacokinetics: process of drug (EDC) absorption, distribution, metabolism, elimination What the body does to the compound

7 Receptor Occupancy Theory of Drug (EDC) Action Eq. 1: D + R DR Effect Eq. 2: EC 50 = k -1 /k 1 = [D][R]/[DR] Eq. 3: E = E max [D]/EC 50 + [D] Eq. 2b: K d = k -1 /k 1 = [D][R]/[DR] Eq. 3b: B = B max [D]/K d + [D] EFFECT BINDING

8 Concentration Response Curves log transformed dose makes the sigmoidal C/R curve

9 Principals of Pharmacology: Receptor Occupancy Theory - Critical Assumptions

10 Nuclear Hormone Receptors: Mechanisms of Hormonal, Drug, and EDC Actions & Receptor Theory Assumptions Broken One Receptor Multiple Responses to Endogenous Hormone Ligand binding induces a conformation that allows a specific HR/co-regulator interaction Ligand and HRE are allosteric modulators that impact receptor interactions with specific co-activator proteins Ligand bound HR can activate, repress or have no effect on expression of different hormone responsive genes depending on: 1) the nature of specific HRE 2) cell specific expression of co-regulators

11 Nuclear Hormone Receptors: One Receptor Different Responses to Different Ligand Allosteric Modulation by Different ligands Different ligands (e.g. EDC) alter conformation to change co-regulatory interactions

12 Principals of Pharmacology: Receptor Occupancy Theory - Critical Assumptions Assumptions are not valid in relation to mechanisms of hormone actions at Nuclear Receptors?

13 Steroid Hormones Signal Through Nuclear Hormone Receptors and Intracellular Signaling Pathways: Rapid Estrogen Signaling Rapid Signaling Mechanism in Cerebellar Neurons Wong et al., 2003 J. Neurosci.; Belcher et al Endocrinology

14 E2 & BPA Rapidly Activate ERK Signaling via ERb in Developing and Mature Cerebellar Neurons ERK Phosphorylation (Fold Induction) A perk1 perk2 ERK1 ERK2 BPA (M) 10 V B # ** *** ** ** *** *** ERK1 ERK2 Jakab et al., 2001 JCN; Wong et al., 2001 J. Neurosci Methods; Wong et al., 2003 J. Neurosci.; Belcher et al. & Zsarnovszky et al., 2005 Endocrinology Vehicle Bisphenol A (M) 10' -4 10

15 E2 & BPA Rapidly Activate ERK Signaling in Developing Cerebellum of Rats E2 - PND 8 BPA - PND 10 Rapid E2 and BPA signaling involves a high affinity stimulatory and a lower affinity inhibitory binding sites EC50 = 8 pm EC50 = 0.4 nm Zsarnovszky et al., 2005 Endocrinology

16 Concentration-Response Analysis of E2 and BPA Effects on Contractility in Female Rat Myocytes Belcher et al., 2012 Endocrinology

17 Non-Monotonic Dose Responses Do They Exist? YES not limited to EDCs Key Points, Considerations and Comments: Pharmacologically relevant non-monotonic concentration/response relationships exist Examples are well accepted for both therapeutic and toxic actions of natural and synthetic compounds Non-monotonic curves do not violate fundamental understanding of receptor mediated actions All complex biological systems do violate the assumptions necessary for receptor occupancy theory to accurately describe the concentration response relationships for many drug, natural and synthetic compounds Many natural or synthetic compounds (i.e. EDCs) are likely nonselective or have variably selectivity for different receptors Not pre-screened for a receptor specific activity

18 Key Points, Considerations and Comments: Why are non-monotonic effect underappreciated Most studies are not concerned with such effects: Toxicity Assessments: The goals of regulatory toxicity studies (e.g. standard multigenerational) do not include establishing dose response relationships Therapeutics: leads are pre-selected, such effects are likely considered as undesirable side-effects and compounds will not pass therapeutic screening Pharmacokinetic properties (ADME) of many compounds may dominate or mask some pharmacodynamic properties Complex physiological feed-back effects and interactions between systems may impact detection Q: How Does Toxicology and RA Include Complex Dose Response Relationships in the Decision Process?