EXAM 4 - "Late Stage" Drug Development

Question 1

Why is early stage ADMET screening important during “late stage” drug development?

Answer 1

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

Question 2

What are the different screens during early stage ADMET screening?

Answer 2

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)

Question 3

List the different avenues of drug absorption.

Answer 3

• Passive uptake (paracellular, transcellular)
• active uptake (carrier-mediated)

Question 4

Explain why P-gp is screened during drug development.

Answer 4

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

Question 5

How can we test to see if the drug is a P-gp substrate?

Answer 5

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.

Question 6

Explain what Caco-2 cells are.

Answer 6

Colon cancer cell line that contains all of the uptake and efflux mechanisms of drug absorption.

Question 7

Explain the process of screening for intestinal absorption using Caco-2 cells.

Answer 7

• 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

Question 8

The liver is the major organ responsible for metabolism. What is the primary method to screen for metabolism of potential drugs?

Answer 8

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

Question 9

What is the secondary method used to screen for drug metabolism?

Answer 9

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

Question 10

Describe the method of testing drug metabolism using microsomes and hepatocytes.

Answer 10

1. Add chemicals into 96-well plate.
2. Add hepatocytes or microsomes.
3. Incubate plate.
4. Extract plate.
5. LC-MS analysis.
6. Quantification of parent compound disappearance

Question 11

What does screening for CYP450 inhibition tell you about the drug?

Answer 11

• 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)

Question 12

How can we test to see if the potential drug inhibits/induces CYP450?

Answer 12

• 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)

Question 13

Answer 13

B) 30 mins, 15 mins
* inhibitor –> more time needed
* inducer –> less time needed

Question 14

Describe how toxicity of a drug is quantified at ealy stages.

Answer 14

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

Question 15

Explain what enzyme release tells us about cytotoxicity.

Answer 15

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

Question 16

Side-effects: Explain effect of hERG channel inhibition.

Answer 16

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

Question 17

Can a compound be a good drug if it is a hERG channel inhibitor?

Answer 17

Yes. There are major drugs that are hERG inhibitors.
* fluoroquinolones - antibacterial
* terfenadine - antihistamine

Question 18

Why do we test compound genotoxicity?

Answer 18

It tells us if the potential drug is mutagenic.
* single test sufficient for phase 1
* multiple tests required to progress to phase 2

Question 19

How do we test for compound genotoxicity?

Answer 19

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

Question 20

What preclinical studies are needed in order for the FDA to approve for progression to clinical trials?

Answer 20

• proof of prinicple in most appropriate animal model
• acute and chronic toxicity studies
• safety pharmacology (respiratory, CVS, CNS)
• ADME

Question 21

Name the two in vivo toxicity assays.

Answer 21

Acute toxicity and chronic toxicity

Question 22

Explain the purpose for conducting in vivo toxicity assays.

Answer 22

Study the relationship between the drug exposure and animal models and provide reliable evidence to explain/predict potential toxicities for later clinical trials.

Question 23

Describe acute toxicity assays.

Answer 23

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

Question 24

Describe chronic toxicity assays.

Answer 24

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

Question 25

What organism is used to test toxicity assays?

Answer 25

For both acute and chronic toxicity assays, animals are sacrificed and tissues are studied for damage.

Question 26

Identify the two different types of species that must be used during toxicity testing.

Answer 26

Must test at least two different species: one rodent and one non-rodent * rodent - rat or mice * non-rodent - rabbit, dog, or nonhuman primate

Question 27

What route of administration has to be tested during toxicity assays?

Answer 27

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

Question 28

Define LD50.

Answer 28

Dose required to kill 50% of animal group.

Question 29

Define ED50.

Answer 29

Dose required to have a therapeutic effect on 50% of animals/participants.

Question 30

Define therapeutic index.

Answer 30

LD50/ED50

Question 31

What is the typical comparison/experiment done in clinical trials?

Answer 31

New drug vs. Placebo/current treatment on the market Less common: * use of already marketed drug for new indication * change in dose, significantly higher

Question 32

How many phases are there in a clinical trial?

Answer 32

4 phases

Question 33

What is the objective of phase 1 trials?

Answer 33

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

Question 34

Define MTD.

Answer 34

Maximum tolerable dose = max dose associated with acceptable levels of toxicity.

Question 35

Describe what occurs during phase 2 trials.

Answer 35

*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

Question 36

Explain the difference between phase 2a and 2b.

Answer 36

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

Question 37

Describe what occurs during phase 3 trials.

Answer 37

*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)

Question 38

Describe what occurs at phase 4 trials.

Answer 38

*Objective:* After the drug gets approved to enter the market, continued monitoring of safety and effectiveness. * AKA post marketing surveillance trials.

Question 39

Describe characteristics of a patent.

Answer 39

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.

Question 40

Describe characteristics of a patent.

Answer 40

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.

Question 41

Describe the two patentable categories.

Answer 41

**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

Question 42

What are the 4 main criteria for patentability?

Answer 42

1. **Novelty** - drug must be new compared to what is on the market prior to filing date. 2. **Inventive step** - drug must not be obvious to a person "skilled in the relevant art" of drug development, prior to filing date. 3. **Enablement** - application must clearly describe how the drug can be used by a person "skilled in the art" 4. **Support** - the claims made (drug efficacy) must be justified by the descriptions in the patent.

Question 43

What happens after a drug goes off patent?

Answer 43

Generic drugs can be made and distributed

Question 44

Describe the characteristics of a generic drug.

Answer 44

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

Question 45

What are the two strategies for generic drug before name brand goes off patent?

Answer 45

* Lawsuit challenging brand name drug's patent * Brand name company marketing generic drugs