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

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

Modern Toxicology: “The Dose Makes the Poison”

Quintal Responses: e.g. alive vs. dead; tumor vs. tumor “sigmoidal C/R curve”

Atropa belladonna

Atropa belladonna

Amanita phalloides

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

(1493 -1541)

“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)

(1493 -1541)

Cardiac Gycosides: e.g. digoxin

Na+,K+ ATPase inhibitors used to treat heart failure

Improved contraction

(decreased arrhythmia)

EC50

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”

Basic Principles

BINDING

Receptor Occupancy Theory of Drug (EDC) Action

Eq. 1: D + R ↔ DR → Effect

Eq. 2: EC50= k-1/k1 = [D][R]/[DR] Eq. 2b: Kd = k-1/k1 = [D][R]/[DR]

Eq. 3: E = Emax [D]/EC50 + [D] Eq. 3b: B = Bmax [D]/Kd + [D]

EFFECT

Concentration Response Curves

log transformed dose makes the “sigmoidal C/R curve”

Principals of Pharmacology: “Receptor Occupancy Theory”- Critical Assumptions

Nuclear Hormone Receptors: Mechanisms of Hormonal, Drug, and EDC Actions & Receptor Theory Assumptions Broken

One Receptor – Multiple Responses to Endogenous Hormone

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

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

Different ligands (e.g. EDC) alter conformation to change co-regulatory interactions

Nuclear Hormone Receptors:

One Receptor – Different Responses to Different Ligand Allosteric Modulation by Different ligands

Principals of Pharmacology: “Receptor Occupancy Theory”- Critical Assumptions

Assumptions are not valid in relation to mechanisms of hormone actions at Nuclear Receptors

?

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. 2005 Endocrinology

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1

2

3

4

5

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ERK1ERK2

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BPA (M) 10’

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A

B

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pERK2

ERK1

ERK2

1 2 3 4 5 6

BPA (M) 10’

V10’ 10–12 10-10 10-8 10-6 10-4

pERK1

pERK2

ERK1

ERK2

1 2 3 4 5 6

A

B

E2 & BPA Rapidly Activate ERK Signaling via ERb in Developing and Mature Cerebellar Neurons

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

EC50 = 8 pM

EC50 = 0.4 nM

Zsarnovszky et al., 2005 Endocrinology

E2 & BPA Rapidly Activate ERK Signaling in Developing Cerebellum of Rats

Rapid E2 and BPA signaling involves a high affinity

stimulatory and a lower affinity inhibitory binding sites E2 - PND 8 BPA - PND 10

Concentration-Response Analysis of E2 and BPA Effects on Contractility in Female Rat Myocytes

Belcher et al., 2012 Endocrinology

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 non-selective or have variably selectivity for different receptors

• Not pre-screened for a receptor specific activity

“Non-Monotonic Dose Responses” Do They Exist?

YES – not limited to EDCs

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?