Dose-Response Concept

1. Dose-Response Concept

2. 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

3. 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

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

5. 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

6. 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

7. 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

8. 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

9. 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.

10. 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

11. Molecular Targets of Chemical Compounds - Continued • Enzymes • Lipids • Nucleic Acids

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

13. The Dose-Response Graph • Classic Example • Normal Distribution • Popcorn Example

14. The Normal Distribution

15. Cumulative Frequency Distribution

16. Change to a Dose-Response Curve

17. Classic Dose-Response Curve on Log – Log Coordinates

18. Probit Scale • http://www-stat.stanford.edu/~naras/jsm/NormalDensity/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.

19. Classic Dose-Response Curve on Log – Log Coordinates

20. Non-Normal Distributions

21. 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

22. 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

23. 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)

24. 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

25. 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

26. 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.

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. Route of Administration

35. Comparison of LD50 Values

36. Why LD50 Values Alone Are Not Very Informative

37. Dose-Response Graph For A Noncancer Causing Chemical

38. Relationship Between ED50, TD50 and LD50

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

40. Therapeutic Index Margin of Safety

41. 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

42. Potency and Efficacy

43. Carcinogenic Chemical Dose-Response Graph

44. Tumors Graph

45. Tumors

46. 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

47. 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.

48. 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

49. 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)

50. 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.