Late Stage Drug Development Flashcards
what do you use to test for intestinal absorption>
a Caco-2 cell monolayer
if a drug has stability against CYP450, less than ____ metabolism to the drug will occur after 1 hour
50%
for in vivo metabolic stability studies, greater than _______ percent of the drug should remain after 1 hour in rodents
20
general cytotoxicity studies include
ATP measurement, MTS, enzyme release
to check for mitochondrial dysfunction:
look at oxygen consumption
hERG potassium channel inhibition can cause
life-threatening arrhythmias
drug-drug interactions are usually _________ related
metabolism
paracellular and transcellular are forms of ____ uptake
passive
carrier mediated uptake is a form of ____ uptakes
active
Efflux is mediated by __________
p-glycoprotein
Caco-2 cells exhibit __________ mechanisms
all forms of uptake and efflux
the small intestine side of a membrane is called the _______ side
apical
the blood side of a membrane is called the __ side
basolateral
how many layer of cells are used in Caco-2 testing
one, a monolayer
hepatocytes are isolated from ____ and used as _______ cells. They contain the entire range of metabolic enzymes. A downside is that they are ____
animal/ intact/ harder to grow and use
microsomes:
subcellular fraction of digested liver
microsomes consist of ________
endoplasmic reticulum
microsomes contain ____ enzymes only
phase I
____ and ______ are phase I enzymes
CYP450/ FMO
microsomes are _____ to use than hepatocytes
easier
metabolism stability: half life should definitely be over _______, and _____ would probably be good to proceed to the next step
10 minutes/ 20 minutes
To do stability tests: start with a 96 well plate. Add ________. Then add ________. Now _________. Then ________ and ______ –> quantification of parent compound disappearance
chemicals/ hepatocytes or microsomes/ incubate/ extract/ analyze
to see if a drug inhibits CYP450, add ___, then ____ and incubate. Then add ____, incubate, and check for ______.
chemicals/ hepatocytes/ P450 substrate/ metabolites
toxicity can be measured as hepatocyte viability in the presence of the drug using _____
MTS/ MTT colorimetric assay
name three cytoplasmic enzymes that can be tested for to analyze the amount of cell death
lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase
_____ percent of the cell’s energy is produced in the mitochondria
90
where is the programmed cell death control center located?
in the mitochondria
what are three main screens for mitochondrial toxicity?
disrupted energy metabolism, increased free radical generation, altered apoptosis
name four screening assays to check for energy metabolism disruption
ATP production (reduction = toxicity), membrane potential, oxygen utilization, mt biogenesis
name a screening assay that checks for free radical production
ROS production
name a screening assay that checks apoptosis induction
PARP activation
name three mechanistic assays that check energy metabolism disruption
OXPHOS Enzyme activity, phospo + acetyl, metabolic protein expression panel
name three mechanistic assays used to check for free radical production
protein nitration panel, protein oxidation panel, antioxidant protein expression panel
name two mechanistic assays use to check for apoptosis induction
cell fractionation and protein translocation panel, apoptosis protein expression panel
normal heart rhythms are governed by
electrical pulses (based on ion transport)
disruption of the electrical pulses that regulate heart rhythms results in
prolonged QT interval (prolonged heart beat) eventually arrythmias and possibly death
the hERG potassium channel is the major protein responsible for conducting K+ ions ____ the heart muscle cells
out of
name two drugs that are hERG inhibitors
fluroquinolones, terfenadine
what test can you use to check for genotoxicity?
Ames test
You need ___________, _____________, ________ and ____ studies to open an IND
proof of principle in most appropriate animal model, acute and chronic toxicity studies, safety pharmacology (respiratory, CVS, CNS), ADME panels
acute toxicity studies:
one large dose or several large does given over less than two weeks
chronic toxicity studies:
should be for the length of the clinical trial
therapeutic index:
LD50/ED50
phase I trials provide ___________ and determine _______
initial pharmacologic and pharmacokinetic data/ maximum tolerable dosage (MTD)
initial dose is usually _____ for phase I trials
1/10 of MELD10
______ ______ volunteers are used in a phase I trial
20-100/ healthy
________ interactions studies are performed in phase I trials
drug-food
how long are drug patents for?
20 years
four main criteria for patentability:
novelty, inventive step, enablement, support (justification for claims of efficacy)
patent claims are written to cover ____
a wide range of compounds in a given class (scaffold)
terfenadine is a prodrug _____ converted to ______– by ________. It is a _______.
antihistamine/ fexofenadine (Allegra)/ CYP 450 3A4/ hERG channel inhibitor
name four hERG channel inhibitors
HIV protease inhibitors, Gleevec (cancer drug), ketoconazonle, grapefruit juice
name two types of hERG channel inducers
barbiturates, HIV reverse transcriptase inhibitors
