Sart Exam 3 - Drug Toxicity Testing & Clinical Trials

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Title: Baby Elephant
Artist: Dave White

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Pharmacology

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study of the effect of drugs on the function of living systems

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Toxicology

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study of the effect of poisons on the function of living systems

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Chemical agents that cause toxicity

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1. drugs
2. insecticides/herbicides
3. plant toxins
4. animal toxins
5. chemical weapons
6. radioactive elements

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Paracelsus

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-'grandfather of toxicology'
-"all things are poison and nothing is w/out poison, only the dose permits something not to be poisonous"
-the dose makes the poison!

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Adverse Drug Reactions (ADRs)

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-noxious or unintended responses occurring at therapeutic doses (bad reaction to normal dose)
~5% of all acute hospital admissions
-Type A vs. Type B

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Type A ADRs

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effects:
-related to known pharmacology; undesirable
-common, dose-related
-predictable
examples:
-hemorrhage w/anticoagulants
-respiratory depression w/opioids
-sedation w/anxiolytic & older antihistamine
drugs

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Type B ADRs

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effects:
-unrelated to known pharmacology
-rare
-unpredictable
-often idiosyncratic
examples:
-anaphylaxis w/penicillin
-allergic liver damage by halothane
-bone marrow suppression by chloramphenicol
-individual allergy/genetic basis

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Pharmacokinetics

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-the effects of the body of poison (related to ADME)
-with this info, it's possible to predict concentration
of toxin that reaches site of injury & resulting
damage

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ADME - Absorption

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ingestion: mercury & dioxin in fish
pesticides in produce
salmonella (dairy), botulinum (meat) toxins

inhalation: asbestos, nerve gases

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ADME - Distribution

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via blood stream

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ADME - Metabolism

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-Phase 1 by cytochrome p450 (oxidation, reduction,
hydrolysis - make molecules more polar)
-Phase 2 conjugation to allow excretion in urine &
bile
-Detoxification
-Toxification

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Detoxification

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compound rendered less toxic

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Toxification

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relatively inert compound converted into toxin

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ADME - Excretion

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Toxins not excreted may be stored in:
-bone (eg. lead)
-fat (eg. DDE a metabolite of pesticide DDT
dichlorodiphenyl trichloroenthane)
-the toxin may be released slowly into body

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Molecular Mechanisms of Toxicology

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1. Allergic responses
2. Receptor, ion channel, & enzyme-mediated toxicity
3. Biochemical pathways
4. Organ-directed toxicity --> liver & kidney
5. Mutagenesis & carcinogenesis
6. Teratogens

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Molecular Mechanisms of Toxicology: Allergic Responses

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-common form of ADR, w/different time course to pharmacological effects
-4 basic clinical syndromes - types I, II, III, IV
-TYPE I hypersensitivity reactions
-can trigger anaphylactic shock!
-TYPE II antibody-mediated cytotoxic hypersensitivity
-Type III immune complex-mediated hypersensitivity
-involve haematological reactions (those
pertaining to bl. cells & bl. forming organs)
-Type IV delayed-type hypersensitivity

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Molecular Mechanisms of Toxicology: Receptor, ion channel & enzyme-mediated toxicity

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-4 major superfamilies' of receptors
-enzymes: metabolic & catabolic pathways
-carriers: uptake/transport systems
-others: proteins involved in vesicle release
-Animal toxins block ion-conduction
-a-bungarotoxin on nicotinic acetylcholine
receptor (nAChR)
-bind irreversibly & competitively to nicotinic
acetylcholine receptor found at neuromuscular
junction - paralysis, resp. failure, death
-Voltage-gated K+ channels block by dendrotoxins
-prolong duration of action potentials &
increase acetylcholine release at neuro-
muscular junction - muscle hyperexcitability,
& convulsive symptoms

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4 Major Superfamilies' of Receptors

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1. ligand-gated ion channels
-ionotropic receptors
-voltage-gated ion channels
2. GPCRs - G protein coupled receptors
(metabotropic receptors)
3. enzyme-linked receptors (tyrosine kinase activity)
4. nuclear receptors (regulate gene transcription)

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Molecular Mechanisms of Toxicology: Biochemical Pathways

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-cyanide inhibits mitochondrial cytochrome c oxidase to prevent cellular respiration
-carbon monoxide: displaces oxygen from hemoglobin causing hypoxia

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Molecular Mechanisms of Toxicology: organ-directed toxicity

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Hepatic: -necrosis --> acetaminophen poisoning
-hepatic inflammation (hepatitis):
halothane covalently binds to liver
proteins to trigger autoimmune reaction
-chronic liver damage (cirrhosis)
-long-term ethanol abuse causes cellular
toxicity & inflammation
Nephrotoxicity:
-changes in glomerular filtration rate
(GFR) --> largely due to bl. flow altering drugs
(NSAIDs)
-allergic nephritis --> reaction to NSAIDs &
antibiotics
-chronic nephritis --> long-term NSAID &
acetaminophen use

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Molecular Mechanisms of Toxicology: Mutagenesis & Carcinogenesis

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-mutagens cause changes to cell DNA that are
passed on when cell divides - if this produces a
neoplastic cell the agent is termed a carcinogen
-2 major classes of gene involved in carcinogenesis
-proto-oncogenes
-tumor-suppressor genes

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Proto-oncogenes

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promote cell cycle progression
-eg. constitutive activity of growth factor tyrosine-
kinase receptors can cause neoplastic
transformation

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Tumor-suppressor genes

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inhibit cell cycle progression
-mutations in tumor suppression gene product
p53 (prevalent in smokers)
-p53 = cellular policeman

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Molecular Mechanisms of Toxicology: Teratogens

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-Teratogenesis: creation of birth defects during fetal
development
-Teratogens: substances that induce birth defects

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Pre-Clinical Drug Testing

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-in vitro cytogenetic evaluation of chromosome
damage in response to drug
-carcinogenicity --> chronic drug dosing - look for
tumors
-repro (teratogenicity) testing:
preg. females from one rodent species & one
non-rodent species does
w/drug at diff. organogenesis stages - look for
birth defects
-preliminary toxicity testing
-therapeutic index

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Preliminary Toxicity Testing

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-max. non-toxic dose (given for 28 days to 2 species)
-animals examined post-mortem & tissue damage
accessed
-lethal dose LD50 - dose of drug which kills 50% of
treated animals w/in specified time
-NOAEL
-LOAEL

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NOAEL

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No Observed Adverse Effects Level
-highest concentration that does not have toxic
response
-determine NOAEL & convert to 'Human Equivalent
Dose"
-adjust for anticipated exposure in humans
-adjust for inter-species difference in affinity &
potency
-apply >10 fold safety factor

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LOAEL

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Lowest Observed Adverse Effects Level
-lowest concentration that produces a toxic response

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Therapeutic Index

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ratio of dose of drug the produces unwanted (toxic) effect to that producing a wanted (therapeutic) effect

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Why do we need Toxicity Testing?

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-Elixir Sulfanilamide disaster of 1937 - most
consequential mass poisonings of 20th century
-sulfanilamide diluted in diethylene glycol to give a
red Elixir Sulfanilamide --> over 100 patients died
-under existing drug regulations, toxicity testing was
not required
-in reaction: US Congress passed 1938 Federal Food,
Drug, & Cosmetics Act - required proof of safety
before releasing new drugs

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Clinical Trials

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1. NIH
2. study participant recruitment:
3. ethical norms of clinical trials
4. protection of human subjects
5. 9 keys points
6. vulnerable populations

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Clinical Trials: NIH definiton

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prospective biomedical or behavioral research study of human subjects that is designed to answer specific questions about biomedical or behavioral interventions
-drugs, treatments, devices, & new ways of using
the three

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Clinical Trials: Study Participant Recruitment

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1. identify eligible participants
2. explain study
3. provide informed consent
4. reassess eligibility
5. assign to groups (treatment vs. placebo)
6. participants should be told:
-may have side effects (adverse effects)
-time commitment
-benefits & risks
-may withdraw at any time
-enrollment 100% voluntary

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Clinical Trials: Ethical Norms

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sound study designs take in account
-randomization of sharing of risks
-proper use of placebo
-processes to monitor safety of rx/tx
-competent investigators
-informed consent
-equitable selection of participants
-compensation for study related injuries

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Clinical Trials: Protection of Human Subjects

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-rely on integrity of investigator but outside groups
also have oversight
-participants' rights protected by Institutional Review
Boards (IRBs)
-IRB = "any board, committee, or other group
formally designated by an institution to review,
approve the initiation of, and conduct periodic
review or biomedical research involving human
subjects"

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Clinical Trials: Key Points

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1. voluntary informed consent
2. experiment must be for good of society & results
not obtainable by other means
3. experiment based upon prior animal studies
4. physical & mental suffering & injury avoided
5. no expectation of death/disabling injury occurring
6. risk vs. benefit
7. protect subjects against injury, disability, death
8. only scientifically qualified persons involved
9. subject can terminate his/her involvement

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Why/Why Not Participate In Study?

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-Participate: give back to society, exhausted all other
treatments, health care services, payment &
incentives, support
-Not participate: mistrust of studies, don't want to
be 'guinea pig', do not meet criteria, cannot give up
time for study visits, barriers (language, distance)

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Stages of Drug Development: Phase I

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-tests new biomedical intervention in small group of people (20-80) for first time to evaluate safety
-dose range
-side effects in humans

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Stages of Drug Development: Phase 2

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-studies biomedical or behavioral intervention in larger group of people (several hundred) to determine efficacy and further evaluate safety

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Stages of Drug Development: Phase 3

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-investigates the efficacy of the biomedical or behavioral intervention in large groups of humans (100's to 1,000's)
-comparing intervention to other standard or
experimental interventions
-monitor adverse effects
-collect info that will allow intervention to be used
safely

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Stages of Drug Development: Phase 4

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-studies are conducted after intervention has been
marketed
-designed to monitor effectiveness of approves
intervention in the general population and to
collect information about any adverse effects
associated with widespread use

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TGN1412 Disaster

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highlighted need for accurate toxicity testing:
-TGN1412 is monoclonal antibody (MAB) designed to
bind CD28 protein to activate leukocytes
-TGN1412 could fight leukemia by triggering cytokine
release
-animal studies of TGN1412 indicated no toxicity
-6 volunteers were given 1:500 dilutions of doses
used in animal studies at 30 min. intervals according
to agreed protocols
-w/in minutes of 6th volunteer receiving dose,
serious side effects occurred: severe headache,
fever & pain --> brief coma, kidney failure, head
swelling

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Potential Flaws in TGN1412 Study

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-lack of biological knowledge (of how CD28 works)
-use of healthy volunteers w/intact immune
response could trigger a 'cytokine storm'
-TGN1412 works differently between species
-dose regime too short (given too frequently)
-testing should have been staggered over several
days
-blister test: expose small amount of skin to drug to
check adverse reaction prior to whole body
exposure

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Summary: Treatment & Prevention of Toxicity

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1. preclinical toxicity testing vital part of drug
development
2. new compounds must be assessed in particular
for mutagenic, carcinogenic & teratogenic potential
3. preliminary toxicity testing typically uses LD50,
NOAEL, & LOAEL values
4. LD50 experiments are not perfect
5 Prevention of toxicity is based on knowledge of
molecular mechanisms of toxin action