Factors affecting the bioavailability

Question 1

Models to predict or measure 3. Permeability

Answer 1

• Computational
• Physicochemical
• Biological
– In vitro (in the lab) o Cell culture
o Excised tissues
– In situ studies
– In vivo studies
o Animal o Human

Question 2

Physicochemical approaches to predict permeability

Answer 2

• Partition coefficient
– Shake-flask method 
• Log P or log D
– Computational 
• clogP
• mlogP
– HPLC (coat column with octanol) – measure how much of the drug is absorbed onto
the column (indication of logP)
– Immobilised artificial membranes (IAM)

Question 3

Caco-2 cells: cancer colon 2 cells

Answer 3

cell line that is immortal
• Placed on top of a semi-permeable membrane
• This helps us predict how well a drug crosses the epithelium in the GI tract (small intestine)
• We can then measure how much of the drug has moved from the reservoir through those cells and the membrane and into the bottom
• The cells are alive so we need nutrients and buffers in our solution

Question 4

Transport experiment

Answer 4

At regular time intervals we take samples from the receptor chamber to see how much of the drug has moved down
• Over time there is an increase in the concentration of drug
• This means the drug crosses the cell membrane at a certain time interval
PAPP CALCULATION
Graph is not linear
• For our drug to be highly soluble we want the absorption to be above 90%

Question 5

This model can show absorption mechanisms as well

Answer 5

• If we have a linear line, this indicates a passive diffusion process – only way that the drug gets from the donor to the receptor part is by passive diffusion
• If we get a sigmoidal curve like above, then that means active transport is taking place trying to move the drug
• Once we get up to ~15 we don’t get anymore active transport because of saturation of transporter
• By looking at the graph we can get an idea about the mode of transport involved in getting our drug across the epithelium

Question 6

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Answer 6

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Question 7

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Answer 7

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Question 8

Effect of calcium chelation

Answer 8

Effect of calcium chelation on permeation of furosemide through Caco2 epithelia
• Calcium ions are involved in opening tight junctions in between cells – calcium ions keep the cells together
• If we add something like EGTA, a chelating agent for free ions, we open up the tight junctions in between
• We can then see if that has an effect on the drug concentration absorbed
• If we look at just furosemide without any EGTA, we get the max conc. of 130ng/cm2
• Once EGTA gets added, we open the tight junctions and we get almost double the amount of furosemide going through
• This shows that furosemide also moves paracellularly – inbetween cells

Question 9

Advantages of Caco-2 model

Answer 9

• Can give mechanisms of drug absorption
• Non-animal
• Can use small amounts of drug
• Rapid screening of many potential new drugs
• Potential toxicity can also be seen – toxic drugs will kill the Caco2 cells
• But:
• paracellular route too tight for intestine and no mucus present in the model

Question 10

Other techniques that can be used

Answer 10

• Live tissue is present in the middle • Effort needed to keep the intestinal epithelium alive
• Can test for concentrations across the intestinal epithelium
• Read up on this in Aultons

Question 11

Permeability experiment

Answer 11

• We pump drug solution into the intestine of the animal
• We measure the concentration of drug that goes in and the concentration that comes out
• We can then assume that any drug that is lost is absorbed in the intestine of the rat
Bile infused via cannulated bile duct
• We need to keep the section of gut alive

Question 12

Assessment of permeability in humans

Answer 12

We can isolate 10cm sections in the jejenum
• Depending on where the balloons are inflated we can check how much of the drug is being absorbed there
• Called a Loc-I-Gut
• This method is basically used to check where in the gut the drug is being absorbed

Question 13

Pre-systemic metabolism: oral administration

Answer 13

• Gut wall
– Brush border membranes
– Gut wall homogenate – if we blend up the gut wall and add our drug to it we can see if any of the enzymes present will degrade the drug
• Liver
– Liver microsomes - phase I
– Isolated hepatocytes – phase I and phase II – Liver slices - phase I and phase II
• Perfusion models involving intestine and liver

Question 14

brush border

Answer 14

One of the models that we can use
• A brush border (striated border or brush border membrane) is the microvilli-covered surface of simple cuboidal and simple columnar epithelium found in different parts of the body.

Question 15

Liver microsomes

Answer 15

as vesicles of the hepatocyte endoplasmic reticulum,
contain membrane phase I enzymes namely CYPs, flavine-containing monooxygenases (FMO), esterases, amidases, and epoxide hydrolases, and also the phase II enzymes such as UGTs.
• This is how you would isolate the liver microsomes

Question 16

Physiologically based pharmacokinetic (PBPK) modelling

Answer 16

Describes conc. profile of drug in various tissues over time on basis of:
• Physicochemical properties of drug
• Site and means of administration
• Physiological processes to which drug is subjected
Parameters from in vitro experiments used in in silico models to predict in vivo data • E.g. GastroPlus, Symcyp, PK-SIM

Question 17

So we use computer modeling to process the data

Answer 17

Physiological based pharmacokinetic modeling and simulation (PBPK) is a computer modeling approach that incorporates blood flow and tissue composition of organs to define the pharmacokinetics (PK) of drugs.
• Simply put, PBPK is a tool to assess factors responsible for patient variability that impacts the PK of drugs.

Question 18

Possible SAQ

Answer 18

From the information given below put each drug in the appropriate BCS class. State whether the drug is likely to have good oral bioavailability and, if not, what can be done to improve its oral bioavailability

Question 19

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Answer 19

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Question 20

BCS Class Boundaries

Answer 20

Solubility:
• A drug substance is considered highly soluble when the highest dose strength is soluble in 250 mL or less of water over a pH range of 1–7.5 at 37 °C
Permeability:
• A drug substance is considered highly permeable when the extent of absorption in humans is greater than 90% of an administered dose, based on mass-balance or compared with an intravenous reference dose
Dissolution:
• A drug product is considered rapidly dissolving when 85% or more of the labelled amount of drug substance dissolves within 30 min using USP Apparatus 1 or 2 in a volume of 900 mL or less of buffer solutions

Question 21

Effect of food on absorption of furosemide and bumetanide

Answer 21

• Without food we get a much higher concentration than with food for both furosemide and bumetanide
• Onset is about 3 times as long if you’ve eaten food with furosemide
• Onset is about 2.5 times as long if you’ve eaten food with bumetanide
• The area under the curve and bioavailability is not affected nearly as much by food in bumetanide than it is in furosemide
• Probably to do with furosemide being absorbed further down in the small intestine

Question 22

Effect of disease on bioavailability: Chronic Heart Failure

Answer 22

• CHF patients
– Lower Cmax
– Longer Tmax
– CHF leads to a decreased ability to transport drug into urine compared to healthy
subjects
• Likely reasons
– Delayed gastric emptying
– Decreased GI motility
– Decreased renal function
– Bowel wall oedema associated with CHF