Lecture 05-08 - Quantitative DMPK Flashcards
What is absorption?
The transfer of a drug from its site of administration into the blood stream for circulation
What is bioavailability?
The extent to which a drug can overcome the barriers to absorption (including first pass metabolism) and enter the systemic circulation
Mention the 4 steps that happen when a solid drug is given orally
In general
1) Disintegration
2) Dissolve
3) Permeates through gut wall
4) Transports to the liver or lymphatic circulation
Why can absorption delay the desired therapeutic effect?
The drug needs to reach site of action before effect can be observed
Mention the regions and dosages forms for the site of administration: Parenteral
- Region: Intravenous, Intramuscular, Intraperitoneal, Subcutaneous
- Dosage form: Solution-emulsion, solution-suspension
Mention the regions and dosages forms for the site of administration: GI Tract
- Region: Intestine, rectum
- Dosage form: Solution, suspension, capsule, tablet, suppository, enema
Mention the regions and dosages forms for the site of administration: Buccal cavity
- Region: Mouth
- Dosage form: Lozenge, solution, powder, aerosol
Mention the dosage form the site of administration: Skin
Solution, cream, emulsion, lotion
Mention the dosage form the site of administration: Lung
Inhaler, aerosol
Mention the dosage form the site of administration: Vaginal
Pessary, cream
Mention the dosage form the site of administration: Eye and ear
Drops, cream, insert
What is the F for IV administration?
100%
What are the factors that affect oral bioavailability?
Dissolution, absorption, and first pass metabolism
What is dissolution?
The process whereby the drug moves from solid state into solution
Disintegration into smaller granules aids dissolution
What are the consequences of poor solubility?
- Increased risk,cost and time to development
- Incomplete oral absorption
- Enabling tecnologies may be required
- Bridgin gap between formulation adds complexity to clinical programme
What is bioequivalence?
Two drugs with identical active ingredients or two different dosage forms of the same drug that possess similar bioavailability and produce the same effect at the site of physiological activity, are said to be bioequivalent
What are the key requirements for a rapid dissolution?
- Aqueous solubility (LogWS) across a pH range
- Salt form for ionisable drugs
- Appropriate particle size
- Crystal morphology
What are the advantages in terms of PK profile for controlled release formulations?
- Increased duration of action
- Can be sustained release where prolonged release is intended, pulse release, delayed release
- Lower peak-trough range (maintains drug levels within the therapeutic window to avoid potentially hazardous peaks)
Where must the drug be absorbed for controlled release?
Delivered orally and absorbed through the intestine / May also include gels, implants, devices (contraceptive implant) and transdermal patches
What are the advatages of enteric coating?
- Prevents contact between compound and the acidic environment
- Increases bioavailability of acid labile compounds
- Protects the stomach from potentially harmful drugs
- Can be used to delay release
What are some of the physicochemical drug properties that may significantly influence the rate and or extent
- Drug lipophilicity (Log P)
- Drug ionization (pKa)
- Molecular weight
Mention a disadvantage of ionised drugs
Poor partition through the cell membrane
Mention an advantage of lipophilic drugs
Readily partinions into blood, therefore absorption rate is normally quick
Mention different absorption mechanisms and the direction (from apical to basal or from basal to apical)
- Transcellular diffusion (passive) - Apical to basal
- Active Efflux - Basal to apical
- Tight junction - Between cells from basal to apical
- Carrier-mediated transport - apical to basal
- Paracellular permeation - Between cells from apical to basal
- Endocytosis - apical to basal
Describe what happens in paracellular permeation
Lipid membrane act like filters permitting passage of very small dissolved drug particles / Drug passes through the gap between cells
What type of compounds are usually transported through paracellular permeation
Low MW and hydrophilic compounds
Mention disadvantages of paracellular permeation
Lower surface area than transcellular route (can still achieve a reasonable absorption). Dogs have larger tight junctions so they may show a higher absorption (Implications for preclinical studies).
Describe what happens in transcellular permeation (through the cells)
Passage is through diffusion
Why is the majority of successful oral drugs target transcellular permeation
- Large absorptive surface area available
- Many intracellular targets need membrane permeability to reach pharmacological target (kinases), so compounds already optimised for this route
What types of drugs are favoured for transcellular absorption?
Hydrophobic drugs
What physicochemical properties determine membrane permeation?
- Area of absorptive surface
- Lipophilicity (Log P)
- pKa
- Hydrogen bonding
- Molecular size
Describe carrier mediated absorption system
It acts like a transporter pump.
* Facilitated transport: Does not require energy
* Active transport: Requires energy - Efflux transporter/symporter and antiporter that indirectly require ATP
Mention examples of solute carrier family
- PepT1/SLC15A1
- MCT1/SLC16A2
- OATP1A2/SLC21A3
- OATP2B1/SLC21A9
Disadvantages for carrier mediated system of absorption
- Saturable at high drug concentrations
- Drug-Drug Interactions could involve competition for transporters
Mention one example of drugs absorbed by carrier mediated transporters
Acyclovir (PepT1)
What is the p-glycoprotein recognized by?
An 170kDa ATP binding cassette drug efflux pump on the apical surface of cells at many sites around the body (GI tract, liver, blood-brain barrier, testes). It recognizes xenobiotics. It provides protection against xenobiotic susbtances including drugs. Pumps drugs back into the lumen.
Was previously called multidrug resistance 1
Describe the process of absorption through receptor mediated endocytosis
- May be receptor mediated (Recognition)
- Drug is enclosed into a vesicle
- Vesicle moves through the cell and then delivered to the other side
- Very specific process
Is the less common route for drug absorption
What is first pass metabolism?
Drugs metabolised in the gut wall and in the liver before entering systemic circulation
What enzyme contributes to metabolism in the gut, liver and intestine
Cytochrome P450
Its major isoform is CYP3A
What types of compounds are more likely to be substrates for Pgp and CYP3A4
Basic compounds
What can slow down permeation across the gut wall membrane and make drugs more susceptible to efflux by PGP?
Lipophilicity and MW
A weak acid where will it be ionised?
In the stomach or in the blood?
Unionised in the stomach (high pH) and ionised in the blood (pH 7.4)
Does a drug need to be ionised or unionised in the stomach in order to be absorbed in there?
Unionised
Can ionised molecules partition membranes?
No
Can ionised drugs go back from the blood into the stomach?
No
Mention the 3 models to study absorption in vitro
- Caco-2 cells
- Recombinant PGP expression
- PGP ATPase activity
Mention the models to study absorption in situ
- Ussing chamber
- Isolated perfused rat gut
Mention models to study absorption in vivo
- Portal vein sampling radiolabelled compound
- PGP-ko mice
What do the oral drugs require for absorption?
Movement across the intestinal epithelial barrier
Mention methods to assess permeability
- Artificial membranes: PAMPA (parallel artificial membrane permeability assay)
- Epithelial cell monolayers (intestine-like cell lines): Madin Darby Canine Kidney (MDCK) and Caco-2 (human colon carcinoma) cell line
- In vitro affinity against efflux transporters: cell-free ATPase assays and cell based assays with recombinant transporters
What is the purpose of PAMPA assay and describe it
Estimates passive, transcellular permeability
Avoids the complexities of active transport, allowing test compounds to be ranked based on a simple permeability property alone
There are NO CELLS but instead artificial semi-permeable lipidic membrane on 96well plates to mimic the cell membrane
Evaluate permeability over a large pH range (valuable for an early understanding how new oral compounds might be absorbed across the entire gastrointestinal tract)
What is a high apparrent permeability in an assay?
Higher than 10x10^-6
What physicochemical properties affect the PAMPA assay?
- Lipophilicity (LogP/LogD) plays a major role in passive diffusion
- Adequate lipophilicity required for a compound to travel across the phospholipid membrane
- Molecular descriptors of a drug can influence overall passive permeability
- Polar surface area, molecular volume/flexibility, hydrogen bonding
Describe the assay with Caco-2 cells
Originated from a human colonic adenocarcinoma
Spontaneously differentiates to mimic small intestine
Cultured as monolayers (polarised epithelium) on plastic filters (14-21 days)
Determine Papp (cm/sec) from fitting time course or assume linearity
Why is it necessary to create a standard curve to determine Caco-2 absorption
Variability in cell lines, expression levels of transporters. Allows to establish permeability guidelines
What can be determined with Caco-2 cells?
- Measure permeability in both A-B and B-A directions
- Determine mechanisms: Paracellular passive/ Transcellular passive/ Active accumulation: Amino acids, dipeptide, glucose, OATPs / Efflux: PGP, MRP2 but low BCRP
- Determine affinity for active processes
- Metabolism?: CYP3A4 and UGT «< liver / Esterase activity, some CYP1A & Glutathione S-transferase
What does a high flux from apical to basolateral side mean?
High oral absorption
What can you determine by the Caco-2 cell assay?
If there is efflux to the blood, uptake from blood to the gut or passive movement.
- Determine or identify mechanism of efflux
What happens if a drug is in the presence of a PGP inhibitor like verapamil?
Blocks PGP mediated efflux. The efflux ratio will decrease in the presence of verapamil.
How many days take MDCK cells to grow vs how many days take Caco-2 cells to grow?
3 days vs 21 days
Why would you perform an assay with MDCK cells?
To assess the impact of transporters on permeability
Can be genetically altered to express only one transporter
Name one example of in situ models of drug absorption
IPRG – isolated perfused rat gut
How is the IPRG (isolated perfused rat gut) assay performed?
- Intestinal segments of the anesthetised animals are cannulated and perfused by a solution of the drug
- Calculate the amount of disappearance from perusate
What is the purpose of IPRG (isolated perfused rat gut) assay?
- Estimates in vivo permeability Peff rather than Papp (in vitro apparent permeability)
- Though done in rats, there is a very good correlation between rat permeability and human permeability
- Method commonly used for the BCS assessment of drugs
Mention an example of an in vivo model for drug absorption
Human intestinal perfusion
What is the purpose of the human intestinal perfusion assay?
- Measures drug absorption in human directly
- Closest to the normal situation
How is the human intestinal perfusion assay performed?
A tube is inserted into the jejunum and two tubes are inflated which isolates a region of the the jejunum for drug absorption studies and the absorption rate is calculated by the disappearance rate of the drug from the section of the gut
What are some of the disadvantages of doing the human intestinal perfusion assay?
More complex data handling
Need to de-convolute contribution from absorption and first pass metabolism
What is Distribution?
- Relationship between amount of drug in the body and blood/plasma concentration
- The amount of drug in the body at any one time is important to know
- The reversible movement of a drug from one compartment in the body to another
What are the physiochemical properties that affect distribution?
Describe each
- Perfusion (driving by blood flow rate)
How quickly the drug is delivered to the tissue. Drugs distributes rapidly into highly perfused organs like liver, intestine and spleen. - Membrane permeability
Once it has been delivered, how quickly does it get in?. The single layered endothelial cells on the wall of the very thin capillaries favours permeation of small lipophilic molecules - Transporter mediated distribution
Not all well perfused organs have high tissue distribution. Organs may have tightly packed endothelial cells and transport and high levels of transporter proteins to aid distribution into them. E.g the brain (BBB)
What other factors may influence distribution?
Aside from perfusion, membrane permeability and transporter mediated dis
- Tissue partitioning
Lipophilic drugs distribute readily to fat rich organs like the adipose, while hydrophilic drugs distribute into water rich organs like the muscles. - Protein binding
Drugs bind reversibly to plasma proteins. Only unbound fraction in the blood leaves circulation and distributes into the tissues - Binding to tissue components (specific or non specific binding)
Driven by high affinity of drug to tissue components e.g tetracyline distribution to bones. Lipophilic bases have high affinity for RBCs. Basic drug may be trapped in the lysosomes e.g azithromycin leading to high distribution
What are physicochemical properties of drugs that may affect its distribution?
- Log P (lipophilicity)
- pKa (ionisation)
- Molecular weight
- Nature of compound (acidic, basic or neutral)
Drug distribution into the tissues can be (space) limited and (space ) limited
Perfusion rate limited and Permeability rate limited
What does it mean that drug distribution can be perfusion rate limited?
Tissue membranes tend not to be substantial barriers to the rapid movement of lipophilic drugs
In this case, the time taken for a drug to distribute into a tissue will mainly depend on the perfusion rate of that tissue
Under these circumstances, distribution is said to be perfusion rate-limited
Perfusion Rate Limitations
At equilibrium the amount of drug in the tissue is dependent on what? What property can characterize the time taken for this?
The drug’s extent of distribution
Distribution half-life
Highly ionised drugs exhibit ___________ distribution
Limited
At equilibrium, concentrations of unbound drug are the same in - and -
Blood and tissue
What is the tissue with the best perfusion rate? what is the tissue with the worst perfusion rate?
What is the Brain Blood Barrier?
It is essentially the CNS vascular endothelium
Tight endothelial junctions
Lack of endothelial gaps or channels
Which types of drugs exhibit limited permeability distribution in the BBB?
Hydrophobic or hydrophilic?
Hydrophilic
If perfusion rate is identical, what is what determines which drug distributes faster?
Physochemical properties. pKa, cLogP, etc.
Why are in situ brain perfusion methods used?
To measure the rate of brain penetration indicated by the the permeability surface area product (PS) while extent of brain penetration can be estimated by determining steady state brain to plasma ratio (logBB)
How is brain:plasma ration calculated for clinically-used drugs
The brain:plasma ratio of clinically-used drugs can be (semi-quantitatively) predicted from a simple calculated parameter, PSA
It is safe to assume that lowe PSA, better brain distribution or pharmacological activity?
No, this is not always the case
What is the volume of dristribution?
- The volume in the body into which a drug appears to be distributed into in order to reflect a particular plasma drug concentration
- A proportionality constant relating the blood/plasma concentration to the amount of drug in the body
- Amount of drug in body at time (t) = Volume of distribution at time (t) x blood/plasma concentration at time (t)
The rate and extent of distribution of a drug on what it depends on?
- The extent of distribution of a drug into a particular tissue depends on its affinity for that tissue relative to blood (affinity depends on lipophilicity, pKa, protein binding and intracellular binding)
- The rate of distribution of a drug depends on its blood perfusion and membrane permeability
The extent of distribution tends to be greater for acids or for bases?
It tends to be greater for bases and neutrals:
* Acids
Can be highly bound to plasma proteins
Low tissue affinity
* Bases
Have high tissue affinity
* Neutrals
Have affinity for both tissue and plasma proteins which is driven by lipophilicity
Changes in lipophilicity tend to impact tissue and plasma binding to the
Is plasma protein binding species-specific?
Yes
Why is plasma protein binding a reversible process?
(Drugs binding to plasma proteins)
Because they are formed by non-covalent interactions (H-bonds, van der waals forces)
Why drugs binding to plasma protein may cause DDIs?
Drug-drug interactions
Different drugs may compete for the same protein
What is the major blood plasma protein?
Describe some of its characteristics
Serum albumin (35-50 g/L)
* Contains lipophilic amino acid residues
Trend for increased binding to albumin with increasing lipophilicity
* Rich in lysine (basic aa)
Acidic drugs tend to be more highly bound due to charge-charge interaction with lys
Which, bases or acids interact with alpha1-acid glycoprotein (0.4-1.0 g/L) or β-globulin?
Bases
What three factors does the amount of drug bound to protein depends on ?
- Drug concentration
- Affinity of drug for the binding site on protein
- Concentration of the protein
What happens to plasma protein binding with an increase in lipophilicity?
An increase in plasma protein binding
Which, acids or bases are shifted to higher protein plasma binding?
Acids are generally shifted to higher PPB for their lipophilicity compared to neutrals, bases and zwitterions
How is volume in a steady state related to free fraction in plasma?
What is the volume of plasma water, extracellular fluid, total body water?
Plasma water aprox 3 litres
Extracellular fluid aprox 12 litres
Total body water aprox 40 litres
What is a large amount of Vd that indicates storage in drug tisues and what is the lower limit that indicates that the drug is restricted to plasma and interstitial fluid
Vd > 40 litres: storage of drug in tissues
Vd < 10 litres: drug is largely restricted to plasma and interstitial fluid
What is one of the functions of efflux transporters for drugs?
Membranes can act as barriers between tissue
Efflux transporters can “protect” tissues from exposure (eg limit CNS exposure)
Mention the effect of pgp in drug transport for CNS
Pgp limits penetration of some drugs to the CNS
How may you assay efflux limited-CNS penetration
Transfected cell lines can be used to determine if a compound could have efflux-limited CNS penetration
MDCK-MDR1
MDCK-BCRP
What is drug elimination?
The irreversible removal of drugs from the body. It may be a combination of metabolism and excretion
Describe metabolism and excretion
- Metabolism: the enzymatic conversion of a drug to a different form
- Excretion: the removal of the drug from the body
Why is drug elimination important?
Controls the amount of drugs leaving the body, hence Important for designing dosage regimen
What is drug clearance?
Drug clearance (CL) is used to “quantify” the process of elimination (may be expressed as total body clearance or organ specific clearance e.g. hepatic clearance or renal clearance)
Mention routes of elimination
- Metabolism
- Renal excretion
- Biliary excretion
- Other minor routes
In terms of PK, is metabolism a route for clearance and why?
Once a compound has undergone metabolism, it is no longer the original compound and, in PK terms, it has been eliminated
Metabolism is therefore a route of clearance
What happens to metabolites of a drug in terms of PK? How does this affect elimination?
Metabolites may undergo distribution, excretion and sometimes further metabolism in the same way as the parent drug
* As long as the metabolite is not converted back to the original compound, this does not affect elimination of the parent drug
What is the biological function of metabolism? What could happen to drugs during metabolism?
Metabolism has evolved to deal with environmental toxins
Reactions increase water solubility, reactivity and size
Promotes excretion and reduces binding affinity for biological targets
May lead to active metabolites (prodrugs)
May lead to side effects from active metabolites
Where does metabolism happen?
Most metabolism occurs in the liver
Some enzyme degradation occurs in gut wall
What are the function of enzymes in metabolism?
Enzymes with broad selectivity act to make drugs more water soluble (and therefore more readily excreted)
What are some of the enzymes involved in metabolism?
Cytochrome P450
Alcohol dehydrogenase (ethanol)
Xanthine oxidase (6-mercaptopurine)
Esterases
How many steps are involved in metabolism and what happens in each?
Phases
Two step process
Phase I = oxidation, reduction, hydrolysis
Phase II = conjugation
What is the function of Phase I metabolism?
Functionalisation reaction (adding or exposing functional groups by oxidation, reduction or hydrolysis)
Mention examples of foods that can increase CYP expression
- CYP2B6 induced by phenytoin
- CYP1A2 induced by broccoli
Mention examples of drugs/foods that can inhibit CYP isoforms
CYP2C8 inhibited by gemfibrozil
CYP3A4, 5 & 7 are inhibited by grapefruit juice
What can happen when metabolites of a drug are more active than the drug itself?
- Side effects (eg paracetamol N-acetyl-β-benzoquinone imine)
- Prodrugs (eg azathioprine => 6-mercaptopurine)
What is the function of Phase II metabolism?
- Addition of polar/charged groups to reactive centres
―OH (glucuronyl, methyl, sulphate)
―NH2 (glucuronyl, acetyl)
―COOH (glucuronyl, glycine) - Most common conjugation is catalysed by UDP-glucuronyl transferases (UGT)
Addition of glucuronic acid - Makes drugs less reactive, and more readily excreted by kidney
Mention examples of drugs affected by Phase I metabolism
Phase I – principally oxidation, reduction or hydrolysis
* Propranolol oxidation
* Debrisoquine oxidation
* Nitrazepam reduction
Mention examples of drugs affected by Phase II metabolism
Phase II – glucuronidation
Chloramphenicol conjugation
Mention examples of drugs affected by Phase I and Phase II metabolism
Codeine (weak analgesic) to Morphine-6-glucuronide (Strong analgesic)
CYP2D6 -> UGT2B7 -> UGT2B7
Describe CYP450S
An extensive family (~60 in humans) of haem-containing monooxygenases
Originally defined by high absorbance at 450 nm due to the reduced carbon monoxide-complexed haem component of the cytochromes
Where are CYP450s enzymes concentrated in the body and mention the most common isoforms
Although the majority of CYP450 enzyme activities are concentrated in the liver, the extrahepatic enzyme activities also contribute to patho/physiological processes
* CYP3A isoform accounts for 82% of all CYP450 in the SI
* CYP3A isoform accounts for 40% of all CYP450 in the liver
Isoform CYP3A4/5 accounts for the highest portion (36%) of drug metabolism
What are the effects of genetic variation of CYP450s?
Likely underlies a significant proportion of the individual variation to drug administration
Species orthologue often do not mediate metabolism of the same substrates
What are the requirements of CYP450 catalysis?
Catalytic mechanism requires O2 and NADPH
Oxidises drugs by cyclic reduction/oxidation of haem iron
Mention most common CYP involved in clearance mechanisms
CYP1A1 and CYP2D6
Mention most common UGT involved in clearance mechanisms?
UGT2B7 and UGT1A1
Why is it biliary clearance a complex process?
Active transport of drug from hepatocyte into the bile duct
Dependent upon transporter specificity, MW, ionization state
MW influence can also be species dependent
Extrapolation across species is difficult
What happens to a drug that has been secreted via the bile in the small intestine?
Drug which has been excreted via the bile reaches the small intestine and is available for reabsorption (enterohepatic recirculation)
* Enterohepatic recirculation of excreted drug is not a clearance method
* Biliary excretion of a metabolite which is subsequently converted back to parent and reabsorbed is not a clearance mechanism
Mention examples of human biliary transporters?
Mention its location too
- BSEP/ABCB11
Bile salt export protein - Cannalicular - BCRP/ABCG2
Breast cancer resistance protein - PGP/ABCB1
P-glycoprotein - Cannalicular - MRP2/ABCC2 - Cannalicular
Multi-drug resistant protein 2 - MATE1/SLC47A
Multidrug and toxin extrusion 1
Mention some examples of minor routes of transport
- Volatile drugs may be exhaled via the lungs (e.g. alcohol)
Some drugs are metabolized in the lungs - Compounds can be excreted in milk
Minor route for the mother but can result in exposure of the baby - Transfer across the GI tract from blood to lumen does occur
Excretion from reverse absorption - Compounds can be excreted in sweat and other body fluids
Mention examples of in vitro renal models
Advatages and disadvantages if any
- Transfected cells lines
Parental cell lines (MDCK, HEK) transfected with:
Uptake transporters – OCTs, OATs
Efflux transporters – BCRP, MRPs - Primary cell cultures
Primary proximal tubule cells - Kidney slices
Requires organ donation - Isolated perfused kidney
Intact organ structure
Expensive, short term viability < 2h
Mention the usefullness of using microsomes and how to use them in vitro
- Prepared from cellular smooth endoplasmic reticulum by differential centrifugation
- Microsomes contain all the membrane bound proteins of the cell
Cytochrome P450
UDP-glucuronyltransferases - Cofactors (eg NADPH/UDPGA) needed to initiate metabolism
- Lots of species readily available, including human
- Easy to use and store at -80 C
Mention the usefullness of using hepatocytes and how to use them in vitro
- Hepatocytes are isolated liver cells, prepared by liver digestion and centrifugation
- Contain all metabolising enzymes of the liver (microsomal and cytosolic)
- Often prepared fresh, but cryopreserved hepatocytes are a validated and valuable resource
- Used for assessing metabolic stability and metabolite identification
Ideal for assessing species differences in metabolism
How would you determine intrinsic clearance?
Substrate disappearance measured at set [E] and [S]
Plot of the substrate disappearance vs. time will be exponential decline
Natural log transformation will be a straight line
Slope of line is rate of metabolism (min-1) and can be converted to the disappearance half-life
Substrate as Drug
Can data in vitro obtained from hepatocyte or microsomal essays be scaled to in vivo? How?
Yes:
What is IVIVE?
In vitro – in vivo extrapolation
What are some of the IVIVE assumptions?
- Major clearance pathway must be metabolic
- The appropriate in vitro system must be selected
- Human and animal metabolism must be similar
- Metabolism must occur under a first order process
- No inhibition by substrate of product
- Disappearance of substrate must be detectable
Can use metabolite appearance
Mention an example of a “well-stirred” model? And what is it useful?
The “well stirred” model views the liver as a well-stirred compartment with concentration of drug in the liver in equilibrium with that in the emergent blood
- Useful for IVIVE
What are drug-drug interactions?
- A large number of pharmaceutical drugs have metabolism as their major clearance route
- Drugs compete for the same metabolic (or transport) pathway
- When inhibitors and substrates are co-administered there is a potential for drug-drug interactions (DDI)
- Some have been shown to be potent inhibitors of metabolizing enzymes or transporters
- Polypharmacy is commonplace, particularly in the elderly
How can CYP450 mediated interactions be classified?
- Inhibition
-Reversible
Competitive
Non-competitive
-Irreversible
Reactive metabolic intermediate
Inactivation (time dependent inhibition TDI) - Induction
Describe Terfenadine metabolism
What is it for, metabolism enzymes involved, MOA
Anti-allergy antihistamine prodrug
* Almost complete first pass metabolism in man
CYP3A4
Not normally measurable in plasma
* Kv11.1 potassium channel blocker
* Also known as human ether-a-gogo (hERG)
Cardiotoxicity
- Fexofenadine
Metabolite responsible for efficacy in man
No Kv11.1 activity
No cardiotoxicity
What is Ketoconazole for and what do we need to consider in regards of metabolism
Antifungal agent
Also a potent inhibitor of CYP3A4
IC50 < 1 uM
Antifungal dose is high
400 mg, twice daily
Circulating concentrations exceed the IC50 for CYP3A4
Consequently, oral administration results in low CYP3A4 activity
What happens if you co-administer terfenadine and ketoconazole
Since ketoconazole is an inhibitor of CYP3A4, terfenadine will have
* High circulating levels
* Potential to prolong Qt interval in ECG
* Abnormal heart rhythm
* Small number of patients developed Torsade de Pointes
* Led to the withdrawal of terfenadine from the market in 1998
What enzymes can be inhibited by grapefruit juice and what are the effects in DDIs?
Grapefruit juice known to inhibit CYP3A4 and CYP1A2
* Half-life estimated to be 12 h, but may last from 4-24 h
* Effects more pronounced with regular consumption prior to ingestion of drug
* May be a cumulative increase in [drug] with continued grapefruit juice intake
What questions do you need to ask before launching a drug about DDIs?
Does the compound inhibit or induce the clearance of other drugs?
How do other drugs influence the disposition of the new drug?
Mention examples of assays that could be used to test in vitro DDIs?
- CYP450 inhibition
HTS with over-expressed enzyme - Transporter inhibition
Generation of in vitro IC50 using recombinant cell lines expressing specific transporters - CYP450 induction
HTS with genetically engineered cell lines that express PXR reporter gene
Other lower throughput assays using cultured hepatocytes available
How would you estimate in an in vitro assay if there is likely to result in a DDI?
In vitro studies using recombinant human CYP450s and other human tissues
* Measure effect of the compound on the rate of metabolism of substrate and calculate an IC50 value
* The lower the IC50 value, the more likely to result in a DDI
