Dose-Response Concept

Assumptions in Deriving the Dose-Response Relationship The response is due to the chemical administered There is a molecular site(s) with which the chemical interacts to produce the response The response is a function of the [ ] of the compound at the site of action The [ ] of the compound at the site of action is related to the dose of the compound

Assumptions Continued There exist both a quantifiable method of measuring and a precise means of expressing the effect of the compound A chemical that produces cancer through effects on DNA, liver damage through inhibition of a specific enzyme, and CNS effects through ion channel blockage will have three distinct dose-response relationships, one for each endpoint

Molecular Targets of Chemical Compounds Receptors Ion Channel Receptors Carrier Proteins G-Protein Coupled Receptors Tyrosine-Kinase Receptors Ah Receptors Steroid Hormone Receptors

Receptors Binding of a chemical to a receptor Can initiate a cellular response similar to, or identical to, an endogenous chemical – This is termed an agonistic action and the chemical is termed an agonist for the endogenous substance

Example of an Agonist Binding to Receptor Buspirone – attaches to the serotonin IA receptor and activates it, mimicking serotonin action on the receptor, which results in the antianxiety action of clinical significance

Receptors Binding of a chemical near the binding site for an endogenous chemical can facilitate the binding of the endogenous chemical – this is also an agonistic action

Example of an Agonist Binding Near the Receptor Benzodiazepines bind to a site near the GABA-binding site and facilitate the action of GABA. This action allows flow of chloride ions into the neuron, hyperpolarizing the neuron and inhibiting neuronal function. Benzodiazepines are used as sedative and anti-anxiety agents

Receptors Binding to the receptor site normally occupied by an endogenous chemical blocks access of the endogenous chemical to the binding site but does not initiate a normal physiological response – this is an antagonistic action and the chemical is termed an antagonist for the receptor site.

Example of an Antagonist Binding to a Receptor Fluoxetine competes with serotonin for the reuptake protein, blocking access of serotonin to the receptor and prolonging serotonin’s presence in the synaptic cleft. This allows more serotonin stimulation of postsynaptic receptors, leading to down regulation in the number of serotonin receptors and relief of clinical depression

Molecular Targets of Chemical Compounds - Continued Enzymes Lipids Nucleic Acids

Subcellular Organelle Targets Cell Membrane Mitochondria Endoplasmic Reticulum Ribosomes Promotor Regions on DNA

The Dose-Response Graph Classic Example Normal Distribution Popcorn Example

The Normal Distribution

Cumulative Frequency Distribution

Change to a Dose-Response Curve

Classic Dose-Response Curve on Log – Log Coordinates

Probit Scale stat.stanford.edu/~naras/jsm/NormalDe nsity/NormalDensity.html stat.stanford.edu/~naras/jsm/NormalDe nsity/NormalDensity.html 68% of the observations fall within 1 standard deviation of the mean, 95% of the observations fall within 2 standard deviations of the mean, 99.7% of the observations fall within 3 standard deviations of the mean.

Classic Dose-Response Curve on Log – Log Coordinates

Non-Normal Distributions

Types of Exposure to Chemicals Exposure may be classified as Acute Exposure – This usually refers to a single exposure to a chemical. If repeated exposures are given they are given within a 24-hr period The chemical is usually given by injection or by dermal application but would also include oral administration Acute exposure by inhalation refers to continuous exposure for less than 24 hours, usually for 4 hours

Acute Exposure - Continued Mouse and rat are the species most commonly used for testing Both sexes are used Food is withheld the night before testing The number of animals that reach a prescribed endpoint at each dose are tabulated 10 animals per dose 5 dose levels

Acute Exposure - Continued If larger animals are used the dose is increased in the same animal until the prescribed endpoint is reached Endpoints could be Lethal dose (death) Toxic dose (ex. Liver injury) Effective dose (ex. Relief from itching)

Subchronic Testing 90 days is the most common test duration but 30 days to 90 days can be used Usually oral administration of the chemical via food; also implant Used to further characterize the specific organs affected by test compound after repeated administration of the chemical

Subchronic Exposure At least 3 doses A high dose that produces toxicity but death in less than 10% of the animals A low dose that does not produce apparent toxic effects during an acute exposure An intermediate dose

For Drugs Under Development Acute and Subchronic studies must be completed before company can file an IND (Investigate New Drug) application with the FDA (Food and Drug Administration). If the application is approved then Clinical Trials can begin. Chronic tests can begin at the same time.

Chronic Exposure Exposure to a chemical for a period longer than 3 months, usually 6 months to 2 years in rodents Drug Testing – 6 months Food Additives with potential lifetime human exposure – 2 years required

Chronic Exposure - Continued Designed to assess cumulative toxicity of chemicals including consideration of carcinogenic potential Mice – 18 months to 2 years Rats – 2 to 2.5 years Start with 60 animals/sex/dose to end up with 30 animals to survive study

Chronic Exposure - Continued Highest administered dose = “Estimated Maximum Tolerable Dose (MTD) derived from subchronic study The National Toxicology Program defines the MTD as “a dose that suppresses body weight slightly (i.e. 10%) in a 90 day study Also use ½ MTD, ¼ MTD, and a control group

What Can Be Learned From A Dose-Response Curve? LD50 – Median Lethal Dose, quantity of the chemical that is estimated to be fatal to 50% of the organisms LD50 values are the standard for comparison of acute toxicity between chemical compounds and between species TD50 – Median Toxic Dose ED50 – Median Effective Dose LC50 – Median Lethal Concentration

What Can Be Learned From A Dose-Response Curve? LD50, TD50, and ED50 values vary by: Species Gender Genetic strain Age Route of administration Environmental conditions Nutritional status

What Can Be Learned From A Dose-Response Curve? NOAEL Value – No Observed Adverse Effect Level, The highest dose of a chemical that, in a given toxicity test, causes no observable effect in test animals The NOEL for the most sensitive test species and the most sensitive indicator of toxicity is usually employed for regulatory purposes

What Can Be Learned From A Dose-Response Curve? LOAEL Value – Lowest Observed Adverse Effect Level, The lowest dose of a chemical that, in a given toxicity test, does cause an observable effect in test animals

Route of Administration

Comparison of LD50 Values

Why LD50 Values Alone Are Not Very Informative

Dose-Response Graph For A Noncancer Causing Chemical

Relationship Between ED50, TD50 and LD50

How Safe Is A Drug? Therapeutic Index = LD50 / ED50 Margin of Safety = LD1 / ED99

Therapeutic Index Margin of Safety

Potency and Efficacy Potency – Is given by the position of the dose-response curve along the x- axis; farther to the left = more potent Efficacy – Is given by the peak of the dose-response curve; the higher the peak the greater the maximum effect or efficacy

Potency and Efficacy

Carcinogenic Chemical Dose- Response Graph

Tumors Graph

Tumors

Ames Test For Mutagenicity Assumption – Any substance that is mutagenic for the bacteria used in the test may also turn out to be a carcinogen. Benefits of Test – Easy to conduct, low cost Drawbacks – Test gives some false negatives and some false positives

Ames Test - Continued Test strain of bacterium used is a strain of Salmonella typhimurium that carries a mutant gene making it unable to synthesize the amino acid histidine from ingredients in its culture A “back mutation” to this gene will allow the hisidine requiring strain of bacteria to grow on histidine deficient media.

Ames Test - Continued The test involves placing the histidine- requiring strain of bacteria on a culture plate along with the test chemical. If the bacteria grow on the histidine- deficient culture medium, a mutation has occurred Therefore, the test chemical is mutagenic and possibly carcinogenic

Effects of More Than One Chemical Additive Effect:the combined effect of the two chemicals is equal to the sum of the effects of each agent given alone. This is the most commonly observed effect when two chemicals are given together. (2 + 2 = 4)

Effects of More Than One Chemical – Continued Synergistic Effect:occurs when the combined effects of two chemicals are much greater than the sum of the effects of each agent given alone. CCl4 and ethanol are hepatotoxic alone but when given together produce much more liver injury than the mathematical sum of their individual effects. (2 + 2 = 20). Smoking and asbestos exposure is another example. Cocaine use with alcohol use is a third example.

Cocaine + Alcohol Metabolism of ethanol and cocaine together produces a metabolite called cocaethylene. This metabolite of cocaine and ethanol has pharmacological properties similar to those of cocaine but with a longer duration in the blood plasma (three to five times as long).

Effects of More Than One Chemical – Continued Potentiation:occurs when one compound does not have a toxic effect on a certain organ or system but when added to another chemical makes that chemical much more toxic. CCl4 is hepatotoxic, isopropanol is not hepatotoxic, when given together the effect of CCl4 is more than expected. (0 + 2 = 10)

Effects of More Than One Chemical – Continued Antagonism:occurs when two chemicals administered together interfere with each other’s action. Antagonistic interactions are very often desirable in toxicology and are the basis of many antidotes. (2 + (-2) = 0).

Types of Antagonism Functional antagonism occurs when two chemicals counterbalance each other by producing opposite effects on the same physiological function. Chemical antagonism is a chemical reaction between two compounds that produces a less toxic product. Example = a chelator and a metal.

Types of Antagonism- Continued Dispositional antagonism occurs when the disposition of a chemical is altered so that the concentration and/or duration of the chemical at the target organ are diminished. Ex. Metabolism is increased – Excretion is increased, therefore half-life is decreased Receptor antagonism occurs when two chemicals that bind to the same receptor produce less of an effect when given together than the addition of their separate parts. Receptor antagonists are often termed blockers.

Types of Antagonism- Continued Receptor antagonism occurs when two chemicals that bind to the same receptor produce less of an effect when given together than the addition of their separate parts. Receptor antagonists are often termed blockers.