Flashcards in Lecture 19: Safer Drugs Deck (16):
Dioxin and DDT both bind to the aryl hydrocarbon receptor to induce CYPs and induce ROScarcinogenic.
Ames test (1974)—reverse mutation assay
1. An auxotrophic strain of Salmonella bacteria is used that has a single-nucleotide mutation in a histidine biosynthesis gene (also, their DNA repair mechanisms have been knocked out, so that they are more sensitive to mutations)—this Salmonella strain will not grow without histidine supplement.
2. If a compound is mutagenic and reverses the single-nucleotide mutation in the histidine biosynthesis gene (His-) back to its wild-type form (His+) by reversing the mutation, then the Salmonella will grow without the histidine supplement (reverse mutation).
3. Often, a liver preparation that includes microsomes is added to bioactivate compounds.
Disadvantages of this assay: Does not take into account what is happening to the compound in the whole-body (metabolic differences, metabolic gene regulation not taken into account).
More Modern Approaches to Toxicology: Toxicogenomics
Toxicogenomics combines toxicology with genomics or other high-throughput molecular profiling technologies, such as transcriptomics, proteomics, and metabolomics.
Toxicogenomics endeavors to elucidate molecular mechanisms involved in the expression of toxicity, and to derive genomic signatures of toxicity that predict toxic action of chemicals.
DNA or RNA sequencing methods and bioinformatics to sequence, assembly, and analyze the function and structure of genomes
profiling of RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in a cell
Microarray—cDNA microarray to measure levels of mRNA transcripts
methods that allow for the identification and quantifications of large numbers of proteins in biological systems.
proteins are isolated from biological samples and then turned into peptides with proteases (e.g. trypsin)
peptides are analyzed by mass spec—fragmented peptides are analyzed by their mass for peptide sequencing and matched up with genomic databases for protein identification
In vivo Testing
ADME/PK: test materials with respect to absorption, distribution, metabolism, and excretion (ADME); duration: hours to days; animals required: 2 species
Safety Pharmacology: To investigate any toxicity—use single dose with 2 species and test cardiovascular, respiratory, CNS effects
-Acute Toxicity: determine Maximum Tolerated Dose (MTD) and No Observable Effect Level (NOAEL)—14 days after single dose in 2 species—look at mortality, clinical pathology, necropsy, weight change, clinical observations
-Sub Acute Toxicity: 14-28 days of repeated dosing in 2 species—all of the above parameters and toxicokinetics, immunotoxicity
-Subchronic/Chronic Toxicity—30 days to 2 years in 2 species
-Carcinogenicity—testing tumorigenic potential in animals; duration: 12 months+ in mouse or rat—test for tumor development, pathology
-Other tests: Genotoxicity, reproductive toxicity
On- and Off-Target Toxicities
On-Target Toxicity: Toxic effect due to the drug interacting with the intended therapeutic target
Off-Target Toxicity: Toxic effect due to the drug interacting with an unintended biological target or unintended tissue
Idiosyncratic drug reactions: drug reactions that occur rarely and unpredictably
Idiopathic drug reactions: cause unknown
On-Target Toxicities: Rosiglitazone
-Rosiglitazone (or Avandia) was marketed by GlaxoSmithKline
-It was prescribed as an insulin sensitizer for diabetics by binding to PPAR-gamma receptors in adipocytes
-Rosiglitazone causes on-target edema which can put people at risk for congestive heart failure—has been proposed to occur through reduced renal excretion of sodium and increase in sodium and water retention
On-Target Toxicities: Non-Steroidal Anti-Inflammatory Drugs
Non-Steroidal Anti-Inflammatory Drugs—Aspirin, Ibuprofen, Diclofenac, Naproxen, acetaminophen
-Used for pain relief, inflammation, fever reduction and swelling, rheumatoid arthritis, osteoarthritis, headache, migraine, post-operative pain
-Acetylsalicilic acid (active ingredient of aspirin) is produced by plants including willow bark and spiraea
-Hippocrates (460-377 BC) left historical records of how he used powder from willow barks to alleviate headaches, pains, and fevers
On-Target Toxicities: NSAIDs
NSAIDs all inhibit cyclooxygenases type 1 and 2 (COX1 and COX2) and reduce pro-inflammatory eicosanoids such as prostaglandins, prostacyclins, and thromboxanes
- Nonselective COX1/COX2 inhibitors increase risk of gastrointestinal bleeding, because prostaglandins help to maintain the gut mucosal protective barrier (due to COX1 inhibition)
-Rofecoxib (Vioxx) produced by Merck
-COX2-selective inhibitor with very little gastrointestinal side-effects
-Prescribed to over 20 million people as a pain reliever for arthritis
-was found to be responsible for increased risk of heart attack and stroke
-Studies have shown that COX2 is responsible for synthesis of prostacyclins in the vasculature which are cardioprotective
-COX2-selective inhibition reduces the cardioprotective prostacyclin and causes cardiac events
On-Target Toxicities: Cerivastatin
-Statins operate by inhibiting the enzyme involved in cholesterol biosynthesis hydroxymethylglutaryl coenzyme A (HMGCoA) reductase
-HMGCoA reductase also regulates post-translational modification of several muscle proteins through a process called geranyl geranylation
On-Target Toxicities: Benadryl
Diphenhydramine (Benadryl) is an antihistamine that antagonizes the H1 histamine receptor to reduce unpleasant symptoms of histamine release in allergic conditions.
Diphenhydramine also crosses the blood brain barrier where H1 histamine receptor antagonism causes sleepiness.
Modern antihistamines do not cross the blood brain barrier
-Terfenadine (Seldane): a non-sedating antihistamine formerly used for treatment of allergic conditions by Sanofi Aventis
- Terfenadine is a prodrug that is metabolized to fexofenadine by cytochrome P450 CYP3A4.
- People with liver disease or those who consumed grapefruit, erythromycin, or ketoconazole with Seldane led to cardiac arrhythmia—through inhibiting CYP3A4 and increasing the retention of terfenadine
-The therapeutic target of terfenadine bioactivated to fexofenadine is the histamine receptor
-Terfenadine inhibits an off-target cardiac potassium channel (hERG)
Inhibition of hERG led to fatal cardiac arrhythmias
Seldane has been replaced by other brain-impenetrant antihistamines (e.g. claritin)