EXAM 4 - "Late Stage" Drug Development Flashcards
Why is early stage ADMET screening important during “late stage” drug development?
We have to think about these factors to make sure we want to proceed with the compound as a drug.
* or design the drug based off what we find during ADMET screening
What are the different screens during early stage ADMET screening?
Absorption
* water solubility: LogP
* intestinal absorption: Caco-2 cell monolayer
Metabolism
* Stability to CYP45o metabolism
* in vivo metabolic stability
* major metabolites: active/inactive?
Toxicity
* general cytotoxicity studies: ATP measurement, MTS, enzyme release
Side-effects
* hERG potassium channel inhibition: arrhythmias
* drug-drug interactions (usually metabolism related)
List the different avenues of drug absorption.
- Passive uptake (paracellular, transcellular)
- active uptake (carrier-mediated)
Explain why P-gp is screened during drug development.
P-gp mediates efflux
* if the compound is a P-gp substrate –> it gets effluxed out of circulation
* We will have to design the drug around the fact that it is going to get effluxed by P-gp
How can we test to see if the drug is a P-gp substrate?
You can incubate the drug with a P-gp inhibitor substrate.
* if the drug is a P-gp substrate –> more drug will enter the basolateral chamber.
Explain what Caco-2 cells are.
Colon cancer cell line that contains all of the uptake and efflux mechanisms of drug absorption.
Explain the process of screening for intestinal absorption using Caco-2 cells.
- Caco-2 cells are grown as a single layer
- 2-chamber model –> (apical - small intestine) chamber inside another chamber (basolateral - blood)
- Drug is added to the apical chamber
- Amount of drug that is isolated in basolateral chamber gives idea of drug permeability
The liver is the major organ responsible for metabolism. What is the primary method to screen for metabolism of potential drugs?
The primary method is through use of microsomes - subcellular fraction of digested liver
* Consists of ER
* Phase 1 (CYP450 and FMO) enzymes only
* easier to use
* contain pooled fractions that have multiple microsomes from multiple people
What is the secondary method used to screen for drug metabolism?
Secondary method (used after microsomes) is use of hepatocytes - the majority of cells in the liver
* isolated from animal and used as intact cell
* contain entire range of metabolic enzymes (phase 1 and 2)
* harder to grow and use
Describe the method of testing drug metabolism using microsomes and hepatocytes.
- Add chemicals into 96-well plate.
- Add hepatocytes or microsomes.
- Incubate plate.
- Extract plate.
- LC-MS analysis.
- Quantification of parent compound disappearance
What does screening for CYP450 inhibition tell you about the drug?
- Determines drug’s ability to inhibit/induce CYP450.
- Drug’s effect on metabolism of other drugs (drug-drug interaction)
- Drug’s effect on digestion of food (drug-food interaction)
How can we test to see if the potential drug inhibits/induces CYP450?
- Add chemicals and hepatocytes to a chamber
- add P450 substrate
- quantify production of metabolites.
- each P450 is tested to see which enzyme the drug inhibits/induces
(If the drug is inhibiting CYP450 –> there will be slower production of metabolite)
(If the drug is inducing CYP450 –> ther will be faster production of metabolite)
B) 30 mins, 15 mins
* inhibitor –> more time needed
* inducer –> less time needed
Describe how toxicity of a drug is quantified at ealy stages.
Hepatocytes - used as model system
* measured as hepatocyte viability in the presence of the drug
* if we give the drug to hepatocytes –> will they survive?
FIbroblasts - used as model of ‘normal’ cells
* connective tissue that is easy to grow
* if fibroblasts are able to grow –> not toxic
Explain what enzyme release tells us about cytotoxicity.
When cells get damaged –> loss of membrane integrity –> enzymes released from cell
* Kits can be purchased to quantify amount of enzyme activity.
* increased enzymatic activity = increased cell death
Side-effects: Explain effect of hERG channel inhibition.
hERG potassium channel is the major protein responsible for conducting K+ iond out of the heart muscle cells.
* When the hERG channel gets inhibited –> leads to arrythmias or death
* if a compound is a hERG inhibitor, it can be designed to not inhibit it
Can a compound be a good drug if it is a hERG channel inhibitor?
Yes. There are major drugs that are hERG inhibitors.
* fluoroquinolones - antibacterial
* terfenadine - antihistamine
Why do we test compound genotoxicity?
It tells us if the potential drug is mutagenic.
* single test sufficient for phase 1
* multiple tests required to progress to phase 2
How do we test for compound genotoxicity?
Cheap, fast, and reliable
* Tube 1: suspected mutagen is incubated with liver extract in culture of His- salmonella
* Tube 2: control (only liver extract) incubated in culture of His- salmonella
* If there is growth of His+ salmonella –> compound is a mutagen
* If there is no growth of His+ salmonella –> compund is not a mutagen
What preclinical studies are needed in order for the FDA to approve for progression to clinical trials?
- proof of prinicple in most appropriate animal model
- acute and chronic toxicity studies
- safety pharmacology (respiratory, CVS, CNS)
- ADME
Name the two in vivo toxicity assays.
Acute toxicity and chronic toxicity
Explain the purpose for conducting in vivo toxicity assays.
Study the relationship between the drug exposure and animal models and provide reliable evidence to explain/predict potential toxicities for later clinical trials.
Describe acute toxicity assays.
Method: one large dose or several large doses given over short period of time (2 weeks)
* dose is outside therapeutic window
Why?: to determine obvious side effects and/or tissues affected at toxic level
Describe chronic toxicity assays.
Method: lower dose levels tested over longer period of time (>2 weeks, length of clinical trial or longer)
* dose given is same as regular therapeutic dose
Why?: to determine if toxicities develop over long period of time after exposure to drug
What organism is used to test toxicity assays?
For both acute and chronic toxicity assays, animals are sacrificed and tissues are studied for damage.
Identify the two different types of species that must be used during toxicity testing.
Must test at least two different species: one rodent and one non-rodent
* rodent - rat or mice
* non-rodent - rabbit, dog, or nonhuman primate
What route of administration has to be tested during toxicity assays?
Drug must be through at least two different routes of administration.
* one must be actual route of thearaputic administration
* unless route of administration is IV –> then only IV needs to be tested
Define LD50.
Dose required to kill 50% of animal group.
Define ED50.
Dose required to have a therapeutic effect on 50% of animals/participants.
Define therapeutic index.
LD50/ED50
What is the typical comparison/experiment done in clinical trials?
New drug vs. Placebo/current treatment on the market
Less common:
* use of already marketed drug for new indication
* change in dose, significantly higher
How many phases are there in a clinical trial?
4 phases
What is the objective of phase 1 trials?
To provide inital pharamcologic and pharamcokinetic data and to determine the maximum tolerable dosage (MTD)
* given to 10-20 healthy volunteers
* also studies food-drug interactions
Define MTD.
Maximum tolerable dose = max dose associated with acceptable levels of toxicity.
Describe what occurs during phase 2 trials.
Objectives:
* to establish if the drug is effective in the targeted patient base
* determine short term safety of drug
* identify dosing regimen
* one location - one clinic
2 subphases: 2a and 2b
Explain the difference between phase 2a and 2b.
2a - limited number of patients to identify any therapeutic valie and obvious side effects (>10 patients)
2b - larger population (10-100)
* double-blind placebo-controlled studies
* placebo patients will still get treated (bc they can’t just go untreated if they’re in the placebo pool)
* new drug is compared to the standard care/typical drug given
Describe what occurs during phase 3 trials.
Objective: to determine clinical effects over a much broader population at various/diverse centers nationally and internationally.
* identify rare, but important side effects based on population differences
* placebo-controlled double-blind studies
* measure effects over long period of time (~3 years)
* patient base - broad and matched (gender and race)
Describe what occurs at phase 4 trials.
Objective: After the drug gets approved to enter the market, continued monitoring of safety and effectiveness.
* AKA post marketing surveillance trials.
Describe characteristics of a patent.
Gives company the exclusive right to produce and sell the drug.
* lasts for 20 years after filing for patent
* usually the patented drug is only on the market for 10-15 years because companies file for patent before drug is ready for the market.
Describe characteristics of a patent.
Gives company the exclusive right to produce and sell the drug.
* lasts for 20 years after filing for patent
* usually the patented drug is only on the market for 10-15 years because companies file for patent before drug is ready for the market.
Describe the two patentable categories.
Composition of Matter - patent to protect the structure of the drug
* specific 3D structure
* strongest patent - hard to get
Method of Use - repurpose an already patented drug
* weaker patent - hard to enforce
* ex: patented as dye –> but want to newly patent as anti-inflammatory drug
What are the 4 main criteria for patentability?
- Novelty - drug must be new compared to what is on the market prior to filing date.
- Inventive step - drug must not be obvious to a person “skilled in the relevant art” of drug development, prior to filing date.
- Enablement - application must clearly describe how the drug can be used by a person “skilled in the art”
- Support - the claims made (drug efficacy) must be justified by the descriptions in the patent.
What happens after a drug goes off patent?
Generic drugs can be made and distributed
Describe the characteristics of a generic drug.
Identical to the name brand “in all ways that matter”
* bioequivalent - works the same
* same API (active pharmaceutical ingredient)
* specific similarities are a moving target - dosage form, route of administration, performance, intended use
* costs much less bc generic manufacturer has not put in the time and money to develop the drug
* differences allowed: color, size, shape, additives, binder, filling agents
What are the two strategies for generic drug before name brand goes off patent?
- Lawsuit challenging brand name drug’s patent
- Brand name company marketing generic drugs
