CHEM 07490 SEU Treatment and Prevention of Asthma Essay

DISCUSSION 4:

Consider the cost/benefit analysis of genetic tests of important pharmacogenetic variants prior to placing a patient on a new medication. Those placed in charge of patient treatment as clinicians (who are primarily tasked with patient health) or as health care system administrators (who are tasked with keeping the system financially solvent) often have competing mandates. How would you try to craft a general policy for pharmacogenetic testing?

DRUG: LISINOPRIL

Assignment 8 – Semester-Long Research Project: second draft. You’ll submit your second draft of your semester-long research project. Your draft should reflect an understanding of the material covered in weeks 5-10 and how it all relates to your drug of interest. At a minimum, your draft should include new information on the each of these topics:

1. ADME properties of your drugFormulations & routes of administrationDistribution patterns in the bodyGeneral PK parameters (Vd, t1/2, Clearance rate, etc.)Metabolism & excretion of drugs, including key interactions with CYPs/transporters/etc.Important drug-drug PK interactions?Likely & confirmed biological targets of the medication and the mechanism(s) of actionEfficacy? Potency? Selectivity?Important drug-drug PD interactions?

Your drafts will be peer reviewed by two classmates (both reviews are due a week after the draft submission deadline). See the assignment rubric on Canvas for grading criteria. This rubric is also what your peer reviewers will use.

Assignment 9 – Peer evaluations of research project drafts. You will be assigned to read and provide useful feedback to two other students’ research project second drafts. The peer review process is designed to provide constructive feedback, and should be given and taken with honesty, charity, and grace. Ideally, when preparing your peer reviews, you will recognize things you could improve in the next update of your own draft! Giving useful feedback to others will also be a part of your course grade, so be thoughtful and generous. See the assignment rubric on Canvas for grading criteria.

Assignment 10 – Research project: pharmacogenetic/pharmacogenomics sources. Find pharmacogenetic/pharmacogenomics information for your target medication to add to your draft report. For this assignment you will merely need to submit a list of properly cited sources that provide key information on these topics, including genetic interactions that may be critical to the risks and benefits of using the drug in certain populations. This will help you prepare for the submission of your final paper in Week 15. See the assignment rubric on Canvas for grading criteria.

Pharmacokinetics: Drug
Metabolism
General Aspects of Pharmacology
CHEM 07490 & 07590
1
Absorption
Drug elimination is the
combined effects of drug
metabolism (converting the
drug into a metabolite) and
excretion of metabolites and
unchanged drug.
2
How that Cp curve gets its shape…
Plasma drug concentration curve
At the peak of this curve (Cmax):
drug absorption = drug elimination
Drug in the central compartment (i.e.,
absorbed into the bloodstream) is
eliminated by metabolism & excretion.
Cmax
1st order elimination kinetics!
The moment any drug enters the body,
metabolism & excretion start…
½ Cmax
Metabolism tends to facilitate excretion,
but it is not always necessary.
t1/2
Tmax
Tmax + t1/2
Drug elimination often follows 1st-order
kinetics, which means you can model it
with a simple half-life (t1/2)
3
Drug metabolism
• Hepatic metabolism – especially 1st-pass metabolism
• Phase I – Cytochrome P450 (CYP450 or just CYP) enzymes & other
oxygenases, esterases, & amidases
• Phase II – transferases  conjugation
• Other major drug metabolism organs:
• Gut
• Kidneys
• Lungs (for inhaled drugs)
• Major metabolic enzymes found largely in the brain:
• MAO (Monoamine oxidase)
• COMT (Catechol-O-methyltransferase)
4
DRUG
PHASE-I
PHASE-II
EXCRETION
5
Metabolic enzymes
Xenobiotic = substance that is
foreign to the body
Xenobiotics include drugs,
environmental toxins, and dietary
components
6
Phase I metabolism: P450s
• Cytochrome P450 (CYPs) are
hemoproteins—they contain
a heme cofactor
• Found in all domains of life
(and some viruses!)
• CYPs account for ~3/4 of total drug metabolism
• Found primarily in endoplasmic reticulum membranes
Fe2+ (ferrous) +
Porphyrin ring
• Some expression in mitochondrial inner membranes and cell surface
• Highest expression in liver, but different CYPs are found all throughout the
body
7
CYP450s
8
Phase I metabolism examples
• CYPs typically oxidize a substrate
• This most commonly involves the insertion of an OH group
• General reaction: O2 + NADPH + H+ + RH → NADP+ + H2O + ROH
Oxidation of Δ⁹-tetrahydrocannabinol to 11-Hydroxy-Δ9-tetrahydrocannabinol
9
CYP450 oxidations
10
Phase I metabolism examples
• Sometimes Phase I enzymes will hydrolyze a drug
• This also occurs in the acid environment of the stomach
• Example: R-COO-R’ + H2O ⇒ R-COOH + R’-OH
• Example: R-CO-NH-R’ + H2O ⇒ R-COOH + R’-NH2
• This can be accomplished by P450s; more typically performed by systemic esterases
& amidases
Deacetylation by arylesterase enzymes, highly expressed in red blood cells
11
Phase I metabolism examples
• Reductase enzymes will reduce a substrate
• This will often reduce things like nitro compounds (R-NO2) into amines (R-NH2),
which may react more readily with Phase II enzymes
• Together, Phase I metabolism reactions:
1) modify the drug chemical structure to facilitate detoxification and
elimination &
2) reduce the concentration of the drug molecule
• Remember: these reactions can activate or inactivate the biological
effects of a drug
• Many metabolites are still active, or gain new activity!
12
Prodrugs
• Sometimes Phase I metabolism creates the (more) active form of a
medication! Prodrugs typically have little to no activity until converted into
an active metabolite.
• Familiar example: aspirin ↓
Active drug with
primary biological
activity
Pro-drug with
little biological
activity
Acetylsalicylic acid
Readily absorbed by GI tract
Salicylic acid
COX-1 inhibitor
13
Phase I metabolism: why?
What’s the point of oxidizing random molecules?
1. It’s an important component of synthesizing & regulating
important biomolecules
Many steroid hormones are synthesized by or broken down by
CYPs
2. Oxidizing a molecule typically increases its water solubility
Why would that be important?
3. Added functional groups can be reaction sites for further
metabolism (Phase II)
14
Phase II metabolism
• Phase II reactions feature conjugation
• Conjugation reactions are typically performed by enzymes known as
transferases
• Transferases can react with a variety of electrophiles or nucleophiles
found within hydrophobic compounds, sometimes (but not always!)
added through phase I reactions
•
•
•
•
-OH (hydroxyl)
-COOH (carboxyl)
-SH (sulfhydryl)
-NH2 (amine)
15
Phase II metabolism
• A variety of groups can be conjugated to
electrophiles or nucleophiles found within
hydrophobic compounds
Glucuronic acid
• Glucuronidation (Glucuronidases, UDPglucuronosyltransferases)
• Adds glucuronic acid
• Glutathione (Glutathione S-transferases)
• Sulfation (Sulfotransferase):
ROH + PAPS  RS(=O)2OH + PAP
Glutathione
• PAPS = 3′-phosphoadenosine-5’phosphosulfate
• Acetylation (N-acetyltransferase: transfers acetyl
from acetyl coA)
Sulfate
16
Phase II metabolism
• Glucouronidation is the most common Phase II reaction
• Occurs primarily in liver, but enzymes (UDPglucuronyltransferases) found widely
• Dramatically increases water solubility
• Glycosidic bond
• Some hormones are glucuronidated
for transport purposes
D-glucose
Glucuronic acid
UDP = Uridine 5′-diphospho-glucuronic acid
addition @ O, N, S, or carboxyl
17
18
Phase II metabolism
• Usually gives metabolites
that are:
• More polar
• Pharmacologically inactive
• Non-toxic
• However, some sulfated &
acetylated metabolites can
be toxic
• Some glucuronidated
compounds are still active!
19
Overall conjugation scheme
X = O, N, S
Phenol, alcohol, or carboxylic acid
Amine (aliphatic or aromatic)
Thiol
20
Metabolism example:
Phenytoin, an anticonvulsant
(Dilantin™, Phenytek™)
21
Metabolic enzymes
Figure 6–3. The fraction of clinically used drugs
metabolized by the major phase 1 and phase 2
enzymes. The relative size of each pie section
represents the estimated percentage of drugs
metabolized by the major phase 1 (A) and phase 2
(B) enzymes, based on studies in the literature. In
some cases, more than a single enzyme is
responsible for metabolism of a single drug.
CYP, cytochrome P450;
DPYD, dihydropyrimidine dehydrogenase;
GST, glutathione-S-transferase;
NAT, Nacetyltransferase;
SULT, sulfotransferase;
TPMT, thiopurine methyltransferase;
UGT, UDP-glucuronosyltransferase.
22
Predicting drug metabolism
• In silico: predictive models of metabolism
• Typically use machine learning tools to break down molecular characteristics and
correlate to known outcomes
23
In vitro testing for
drug metabolism
• Microsomes are preparations
of endoplasmic reticulum
• Liver has highest CYP450
expression
• ER is site of highest CYP450
expression  requires
ultracentrifugation to pellet!
25
Testing for drug metabolism
• In vitro: liver microsomal incubations
Strategy: 1,2,3-triazole is a bioisosteric
replacement for amide linker
26
Testing for drug metabolism
drug
• In vitro: CYP inhibition assays
antifungal
antihypertensive
HIV antiviral
27
Testing for drug metabolism
PG648 (10mg/kg) Plasma Levels
• In vivo: animal drug
exposure
IV
How would you calculate
bioavailability from these data?
[PG648] (ng/mL)
10000
PO
1000
100
10
1
0
1
2
3
4
Time after dosing (hours)
Mean concentration profiles for (±)-PG648 in mouse plasma following i.v. () or p.o. ( )
administration of 10 mg/kg. Data are presented as means ± SEM.
28
Testing for drug metabolism
• In patients: clinical trials
• As personalized medicine
Roche Amplichip™
CYP2D6 & CYP2C19 variants
29
Toxicity & metabolism
• Metabolism can affect toxicity
• Acetaminophen (Paracetamol, Tylenol™) is typically directly
glucuronidated
• Its CYP metabolite NAPQI is quite toxic
CYP
• Healthy livers will readily inactivate NAPQI with glutathione
• In unhealthy livers, or in cases of overdose, NAPQI overwhelms the
glutathione reserves, builds up & damages the cell
30
31
Toxication
Neurotoxic,
cytochrome c oxidase
inhibitor
Kidney toxicity
32
Drug-metabolic enzyme interactions
• Enzymes clearly affect drugs…But drugs can affect metabolic
enzymes, too!
• Substrates: drugs that are metabolized by an enzyme
• Two substrates can compete against each other
• Blockers/Inhibitors: drugs that inhibit enzyme activity—they bind to
the active site of the enzyme but don’t get metabolized
• Inducers: drugs that increase the expression of a metabolic enzyme
• CYP inducer  more CYP protein  more CYP activity!
33
CYP inducers
34
CYP inducers
35
CYP inhibitors & inducers
36
St. John’s Wort:
Notorious
CYP3A4 inducer
37
Predict the effects…
A patient takes a daily statin to help treat hypercholesterolemia. It is a
substrate for CYP3A4. What might happen if the patient takes their
medication with grapefruit juice (a CYP3A4 inhibitor)?
Pharmacology in Drug Discovery
and Development (2nd Edition),
Academic Press, 2017,
10.1016/B978-0-12-8037522.00016-8.
38
https://www.psychiatrist.com/jcp/psychopharmacology/fruitjuice-organic-anion-transporting-polypeptides/
https://www.sciencedirect.com/science/article/pii/S18180876
16301878
39
Pharmacokinetics: Drug
Excretion & Clearance
General Aspects of Pharmacology
CHEM 07490 & 07590
1
Absorption
2
How that Cp curve gets its shape…
Plasma drug concentration curve
At the peak of this curve (Cmax):
drug absorption = drug elimination
Drug in the central compartment (i.e.,
absorbed into the bloodstream) is
eliminated by metabolism & excretion.
Cmax
1st order elimination kinetics!
The moment any drug enters the body,
metabolism & excretion start…
½ Cmax
Metabolism tends to facilitate excretion,
but it is not always necessary.
t1/2
Tmax
Tmax + t1/2
Drug elimination often follows 1st-order
kinetics, which means you can model it
with a simple half-life (t1/2)
3
Elimination half-life
Cmax
Cp = plasma concentration of drug
𝐶𝑝 = 𝐶𝑚𝑎𝑥 𝑒 −𝑘𝑒𝑡
½ Cmax
t1/2
@ t½ 𝐶𝑝 =
1
𝐶
2 𝑚𝑎𝑥
Tmax
0.5 = 𝑒 −𝑘𝑒 𝑡½
Tmax + t1/2
ln 0.5 = −0.693 = −𝑘𝑒 𝑡½
0.693
𝑡½ =
𝑘𝑒
ke = elimination rate
constant
Units?
4
Elimination half-life
Cp = plasma concentration of drug
𝑪𝒑 = 𝑪𝒎𝒂𝒙 𝒆−𝒌𝒆 𝒕
1
2
@ t½ 𝐶𝑝 = 𝐶𝑚𝑎𝑥
0.5 = 𝑒 −𝑘𝑒 𝑡½
ln 0.5 = −0.693 = −𝑘𝑒 𝑡½
𝟎. 𝟔𝟗𝟑
𝒕½ =
𝒌𝒆
Note that half-life is completely independent of [drug]!
5
Elimination kinetics
Plasma drug concentration curve
Zero-order kinetics!
Cmax
½ Cmax
t1/2
1st order kinetics!
Tmax
Tmax + t1/2
6
So why
st
1 –
or 0-order kinetics?
• In order for a liver enzyme to metabolize the drug, they have to bump into each
other! In order for the kidneys to excrete the drug, it has to pass through the
filtration system. Etc.
• 1st order rate constant: probabilistic measure of drug-enzyme interaction,
dependent on drug concentration
k
𝑑[𝐵]
A→B
= 𝑘[𝐴]
𝑑𝑡
• Zero-order rate constant: not dependent on drug concentration because
enzymes/systems are saturated
k
A→B
𝑑[𝐵]
𝑑𝑡
=𝑘
7
8
Elimination kinetics
Can you calculate a half-life for zero-order kinetics?
Zero-order kinetics!
Cmax
½ Cmax
t1/2
1st order kinetics!
Tmax
Tmax + t1/2
9
Dose vs. AUC
AUC = total
drug exposure
AUC is directly proportional to
the drug dose if drug
elimination follows 1st-order
kinetic processes
t1/2
[drug]*time
Cmax
i.e., if dose doubles, AUC
doubles…
½ Cmax
t1/2
…as long as that higher dose
didn’t alter the elimination
kinetics
∞
Tmax
Tmax + t1/2

Area under the Curve

10
Drug excretion
• Main routes of excretion:
• Kidney  urine
• Liver  into GI tract, via bile
• Minor routes
• Lungs  expiration (negligible,
except for inhaled anesthetics)
• Sweat
• Tears
• Breast milk
11
Renal excretion
• Water soluble drugs and metabolites are typically secreted via the kidneys
• Kidney anatomy basics:
To bladder
12
Renal excretion

Renal Excretion

13
Renal excretion
• Drugs are transferred from the
plasma into the urine via:
• Glomerular filtration: Unbound drug
molecules of 1 mM [EtOH]
• BAC of .08 g/dL ≈ 17 mM
Blood alcohol concentration (BAC) after the rapid
consumption of different amounts of alcohol by eight adult
fasting male subjects.* (Adapted from Wilkinson et
al., Journal of Pharmacokinetics and
Biopharmaceutics 5(3):207-224, 1977.)
https://pubs.niaaa.nih.gov/publications/aa35.htm
26
Clearance
Example:
• Drug A is cleared from plasma at 4.0 mL/min/kg
• For 60 kg person: CL = 240 mL/min
• Overall removal of drug depends upon [drug] in plasma
• Drug B is cleared from plasma at 1.0 mL/min/kg
• For 60 kg person: CL = 60 mL/min
Why would drugs differ in clearance rate?
27
Clearance
(3.5 L)
(14 L)
• CL and VD combine to determine the overall half-life!
• CL rates near ~120 mL/min are near the baseline glomerular
filtration rate (GFR) for healthy individuals
(42 L)
28
Textbook example
• Plasma clearance for antibiotic cephalexin =
4.3 mL/min/kg, 90% of drug excreted unchanged in the urine
• 70 kg man: plasma cephalexin clearance = 301 mL/min
• Renal clearance = 90% of this elimination, thus the kidney is able to
excrete cephalexin at ~270 mL/min of plasma every minute
• If Cp = 32 µg/mL, how much drug will be eliminated in next minute?
29
Now you should understand just about everything about this curve…
Plasma drug concentration curve
AUC = total
drug exposure
Cmax
½ Cmax
Tmax
Tmax + t1/2
30
When & how long will a therapeutic effect be present?
• PK affects onset & duration of
therapeutic effect (TE)
• MEC = minimum effect
concentration
31
But we don’t always take a single dose
Doses taken at a regular rate will result in a steady-state drug
concentration
32
But we don’t always take a single dose…
• Doses taken at a regular rate will result in a
steady-state drug concentration as long as you
remain in 1st-order elimination kinetics
• Red line = pattern of drug accumulation during
repeated administration of a drug at intervals
equal to its elimination half-time when drug
absorption is 10 times as rapid as elimination.
• Blue line = pattern during administration of
equivalent dosage by continuous intravenous
infusion.
ഥ 𝒔𝒔
𝑫𝒐𝒔𝒊𝒏𝒈 𝒓𝒂𝒕𝒆 = 𝑪𝑳 × 𝑪
𝐶ҧ ss = average steady-state plasma concentration
33
But we don’t always take a single dose…
ഥ𝒔𝒔
𝒅𝒐𝒔𝒊𝒏𝒈 𝒓𝒂𝒕𝒆 𝑪𝑳 × 𝑪
𝑴𝑫 =
=
𝑭
𝑭
𝐶ҧ ss = desired steady-state plasma concentration
MD = maintenance dose
F = bioavailability
34
Rearranging for
st
1 -order
ഥ𝒔𝒔
𝑪𝑳 × 𝑪
𝑴𝑫 =
𝑭
kinetics…
𝑪𝑳𝒔𝒚𝒔𝒕𝒆𝒎𝒊𝒄
𝑫𝒐𝒔𝒆
=
= 𝑽𝒅 × 𝒌𝒆
𝑨𝑼𝑪
ഥ𝒔𝒔 𝑽𝒅 × 𝒍𝒏(𝟐) × 𝑪
ഥ𝒔𝒔
𝑽𝒅 × 𝒌𝒆 × 𝑪
𝑴𝑫 =
=
𝑭
𝑭 × 𝒕½
35
Simulations of multiple doses
a | A sufficient dose (400 mg ibuprofen) hits the analgesic window (therapeutic range) after the first and second dose.
Low doses, given repeatedly, allow for putative recovery phases (such as in the endothelium of the vasculature) as the
plasma concentration falls below substantial COX2 inhibition. This might be associated with a loss of analgesic activity,
which can be compensated for by increasing the dose to 800 mg ibuprofen. However, prolonged effect time might be
associated with more ADRs (when the concentrations exceed the therapeutic range) and the disappearance of the
recovery phases.
Pharmacokinetics data for ibuprofen simulation were derived with t50 = 2 h. Pharmacodynamic data for therapeutic
range (ibuprofen) were taken from Laska et al.
Abbreviations: ADRs, adverse drug reactions; COX, cyclo-oxygenase.
Nature Reviews Rheumatology 6, 589-598 (October 2010)
36
Simulations of multiple doses
b | Slow accumulation of a long half-life drug. When a maintenance dose of 30 mg etoricoxib is given once daily (blue
line), it takes 2–3 days to reach the therapeutic window. However, using a loading dose of 90 mg etoricoxib at day one
will ensure immediate onset of action and speed up the time to reach steady state conditions (black line).
Data for etoricoxib were adopted from Dallob et al.
Abbreviations: ADRs, adverse drug reactions; COX, cyclo-oxygenase.
Nature Reviews Rheumatology 6, 589-598 (October 2010)
37
Simulations of multiple doses
c |Plasma concentrations of etoricoxib were simulated for a case when high maintenance doses of etoricoxib (120 mg)
are used. The simulation indicates fast onset of action, the development of critically high concentrations and the lack of
recovery phases. Simulation was performed using a two compartment model with 1storder input and 1st order
elimination rate (WinNonlin® Vers. 3.3, Pharsight Corp., USA).
Data for etoricoxib were adopted from Dallob et al.21
Abbreviations: ADRs, adverse drug reactions; COX, cyclo-oxygenase.
Nature Reviews Rheumatology 6, 589-598 (October 2010)
38
Examples—putting all of ADME together
• Physician’s Desk Reference: http://www.pdr.net/
• Prozac (fluoxetine HCl):
• http://www.pdr.net/drug-summary/prozac?druglabelid=3205&id=2826
• http://www.medsafe.govt.nz/profs/datasheet/f/fluoxetineafforalsoln.pdf
• Antidepressant, SSRI
• Absorption: (Single 40mg dose) Cmax=15-55ng/mL, Tmax=6-8 hrs.
• Distribution: Plasma protein binding (94.5%); crosses the placenta; found in
breast milk.
• Metabolism: Liver (extensive) via CYP2D6; demethylation into norfluoxetine
(active metabolite).
• Elimination: Kidney; t1/2=1-3 days (acute administration), 4-6 days (chronic
administration)
• VD: 30-40 L/kg
• F%: 80-95%
39
Fluoxetine  Norfluoxetine
• Norfluoxetine has a similar pharmacodynamic profile
• That means that much of the therapeutic benefit of Prozac comes from the
active metabolite…
• Elimination: Kidney; t1/2= 4-16 days
• Therapeutic range for parent + metabolite (120-300 ng/mL) at steady
state
40
Fluoxetine dosing
ഥ𝒔𝒔 𝑽𝒅 × 𝒍𝒏(𝟐) × 𝑪
ഥ𝒔𝒔
𝑽𝒅 × 𝒌𝒆 × 𝑪
𝑴𝑫 =
=
𝑭
𝑭 × 𝒕½
• t1/2= 4 days (96 h)
• VD = 30 L/kg
• F = 0.9
• 𝐶ҧ ss = 200 ng/ml = 200 μg/L = 0.2 mg/L
𝟑𝟎
𝑴𝑫 =
𝒎𝒈
𝑳
× 𝟎. 𝟔𝟗𝟑 × 𝟎. 𝟐
𝒎𝒈
𝑳
𝒌𝒈
= 𝟎. 𝟎𝟒𝟖
𝟎. 𝟗𝟎 × 𝟗𝟔 𝒉
𝒌𝒈 × 𝒉
for 60 kg patient: 2.88 mg/h = 69 mg/day
Imagine how this would differ if a patient needed different values…
41
Radiolabeled Absorption, Distribution, Metabolism
and Excretion Studies of Drug Development: Why,
When and How?
Drug Discovery – focus on ADME
Early discovery, and preclincial development of drugs uses various methods:
In silico = computer-based screening
In vitro = testing outside of living organisms
In vivo = testing inside of living organisms
Typically in vivo studies yield critical information unavailable via other means:
• Test species naturally create, and are exposed to, major metabolites
• Excretion routes can be identified
• Determine absorption rates, VD, clearance rates, bioavailability
In silico PK
In silico PK studies will
predict:
• Compound
physiochemical
properties
• Solubility
• Likely PK parameters
• etc.
http://www.swissadme.ch/ 
Bupropion SMILES: Clc1cccc(c1)C(=O)C(NC(C)(C)C)C
In vitro PK
In vitro studies may use:
• Animal or human hepatocellular and
subcellular fractions
•
•
•
•
S9: Supernatant of centrifuged organ cells;
contains microsomes and cytosol
Mitochondria: A concentration of
mitochondria organelles
Microsomal fractions: Endoplasmic
reticulum spun into vesicles; P450 enzyme in
the supernatant
Cytosolic fractions: Breaking cells by
ultracentrifugation to form a supernatant
and insoluble pellet
• Recombinant enzymes
In vivo PK
In vivo studies use a variety of animal models:
Rodent (usually rats or mice)
Non-rodent (usually dogs, monkeys, or pigs)
https://health.arlingtonva.us/environmental-health/rats-mice/
http://www.yourpurebredpuppy.com/reviews/beagles.html
http://dmd.aspetjournals.org/content/32/4/398
Current limitations
The techniqes described in the previous slides:
Are limited by analytical techniques
Are limited by animal metabolism
Can reliably predict human pharmacokinetics (PK) of ~80% of compounds
Many times this is insufficient
Radiolabeled in vivo ADME studies provide key PK data that address
these issues
Objectives of Radiolabeled ADME Studies
Primary Objectives:
1. Determine extent of drug absorption
2. Characterize the distribution of the compound into tissues and organs
3. Identify circulatory and excretory metabolites
4. Explore whether metabolites contribute to the pharmacological/toxicological
effects
5. Determine clearance mechanisms & routes of elimination (biliary, fecal, renal)
6. Determine exposure levels of the parent compound and key metabolites
7. Help validate the animal species used for toxicological testing
Objectives of Radiolabeled ADME Studies
Can more sensitively & reliably detect molecular entities
Radiolabeled ADME studies are required by regulatory authorities for the
registration of a new drug
These studies are typically conducted in 2 preclinical species and often in
humans
The results are used for long-term safety assessment of the drug
Objectives of Radiolabeled ADME Studies
Characterizing excretion routes critical for clinicians who may prescribe
medications that cannot be excreted well (e.g., renal excretion in patient
with kidney disease)
Determining the amount of unchanged drug and the quantity and identity
of major circulating metabolites to fully characterize metabolism
Low levels of parent drug + high plasma radioactivity indicates presence of
metabolite(s) with a long half-life that can accumulate over multiple
dosings, altering pharmacological and toxicological profile of the drug
Objectives of Radiolabeled ADME Studies
Results from human studies are used to design drug-drug interaction
(DDI) studies
Bioequivalence studies
“If two products are said to be
bioequivalent it means that they
would be expected to be, for all
intents and purposes, the same”
Demonstrated bioequivalence can reduce/
eliminate need for some clinical trials.
http://www.slideshare.net/muliksudip/bioavailability-and-bioequivalence
Choice of Radioisotope and Their Position in the Drug Molecule
Since hydrogen and carbon are in all drug molecules, the two most common
radioisotopes used for radiolabeling drugs are:
Radioisotope
Pharmaceutical Companies
Half-Life (years)
Carbon-14
12/15 = 80%
5730 ± 40
Hydrogen-3 (Tritium)
2/15 = 13.3%
12.32
*1/15 = 6.7% use both
antineutrino
electron
http://www.physics.utah.edu/~jui/5110/y2009m03d09/KATRIN.htm;.htm

Choice of Radioisotope and Their Position in the Drug Molecule
Important parameters to consider when picking a radioisotope label:
Position in the drug molecule
Radiochemical purity
Specific activity
14C
3H
• Carbon atoms are
present in all drug
molecules and located in
stable positions
• Fast and easy synthesis
• Radioisotope has a
relatively long half-life
• Preferred if 14C synthesis
is extremely difficult or
impossible
• Used in the majority
(80%) of radiolabeled
ADME studies
• High specific activity
• Biological instability
• Largely limited to protein
and ligand-binding studies
http://www.radiolabeling.org/theory/synthesis.htm
Mass Balance Studies:
Characterization of Absorption and Routes of Excretion
What are mass balance studies?
•
Investigation of the ADME of a drug
following a single dosing.
What are they used for?
•
•
•
•
•
Characterizing excretion patterns
Identifying mechanisms of clearance
Defining the rate of elimination
Estimating the extent of absorption 
Determining the differences in
absorption, excretion, and metabolism
between normal and poor
metabolizers (slide 20)
Dose Administration and Sample Collection
Mice:
• PO or IV, depending on clinical route of
administration
• Terminal blood samples are collected from
multiple mice for each time point
• Radiolabeled studies in mice is conducted to
support carcinogenicity assessment
Dose Administration and Sample Collection
Rats:
• PO and IV arms are administered to both
male and female rats
• Excreta is collected for mass balance
• Bile is collected from bile duct cannulated
(BDC) animals
• Terminal plasma samples collected from 2
animals/sex/time point
Dose Administration and Sample Collection
Rabbits:
• PO or IV administration, depending on clinical
route of administration
• Serial plasma sample collection from 2-4
rabbits with PK analysis for total radioactivity,
[parent drug], and metabolic profile
• Studies in female rabbits used in embryo fetal
development evaluation
Dose Administration and Sample Collection
Dog/Monkey:
• Dogs are preferred; monkeys are used
when use of dog is not justified
• Oral dose administered to both sexes
• Serial blood points are collected at several
time points for PK analysis
• Samples from time points are used for
metabolite profiling
Dose Administration and Sample Collection
Humans:
• Radiolabeled studies in humans are gold standard for
understanding metabolism of drug candidate.
• Same route of administration as clinical use.
• Sample collection is chosen to fully characterize routes
of elimination, metabolic profile, and to identify
circulating and excreting metabolites.
Dose Administration and Sample Collection
•
15/15 surveyed pharmaceutical companies used 14C-labeled
• ~70% of these companies only conducted single radiolabeled dose
tests
• ~30% conducted multiple radiolabeled doses
• Radiolabeled doses can be given after non-labeled doses
(tigecycline binds to bone)
Subject confinement is between 7 to 10 days until ~90% of administered
radioactivity is excreted OR excreta contain less than 1% of the
administered dose for two consecutive samples
Blood sample collection follow the same patterns as animals
•
Additional human sampling can be used to identify metablism outliers
•
•
Characterization of Absorption and Routes of Excretion
Tmax= 12 h
Subjects: six healthy male
adults, 18-40 years old
BMI between 19-27
normal clinical blood results
free of significant disease
normal physical exam
non-smoker
Given a single dose of [14C]
desloratadine
Distribution / Tissue Targeting
Analyses performed in vivo to track the distribution of radiolabeled compound
throughout the body and analyze target binding.
Method
Operation
Applications
QWBA/WBAL
creates an image based on density in
tissue areas or parent, metabolite,
and degradant drug-related
compounds
used in tumor therapy; determining
developmental toxicity to a fetus or
passing of metabolites across placenta
or through milk
PET
noninvasive highly sensitive
radioactivity imaging technique;
preclinical and clinical application
used in cancer medication imagery;
used in brain imagery due to
noninvasive operation
22
QWBA/WBAL
Quantitative whole body autoradiology/whole
body autoluminography (QWBA/WBAL) is a
technique used to quantify the amount of
radioactivity, or specifically, the amount of a
specific radiolabel, distributed in the various
tissues of a body
QWBA/WBAL studies are conducted on preclinical
species before the administration of a drug
candidate to a human, to ensure safety and
determine the maximum radioactive dose that
can be safely administered to humans.
Drug regulatory agencies require tissue
distribution studies in rats used to predict
tissue exposure to radioactivity in humans
Image of radiolabeled drug distribution over a 72
hour period – darkness is representative of drug
concentration intensity
Positron Emission Tomography (PET)
PET used for screening NMEs and their
distribution, target occupancy, and
pharmacological effects in preclinical
and clinical studies
11C
and 18F are used because of their short halflives
18F-labeled
2-fluoro-2-deoxy-D-glucose (FDG)
detects unusual increased glucose
metabolism in tumor cells
PET used to understand influence of Pglycoprotein (P-gp) transporter on the
penetration of drugs on the brain
Expressed in human blood-brain barrier
Limits its substrates transport into the brain
PET Scans: indicates regions of higher concentration of tracer molecule in red, the
tracer here is attached to glucose so regions with a brain tumor are more
reproductively active, consuming more glucose
Metabolism/Clearance Mechanisms
Studying metabolism can reveal:
Excreted drug unchanged or changed to metabolites
Enzymes involved in metabolism
Routes of elimination
Understanding clearance patterns can help to understand DDI
Drugs reacting with other drugs as well as enzymes
Quantification of metabolites
% dose for metabolite = (% dose excreted in a given sample) x (% radioactivity for a
metabolite)
Metabolism/Clearance Mechanisms
Routes of elimination and clearance mechanism
1) Compounds Extensively Metabolized by Multiple Metabolic Pathways:
If no, or very little, radioactive dose is recovered unchanged, parent drug was extensively
metabolized
Less chance of DDI due to many clearance mechanisms
2) Compounds Extensively Metabolized Predominantly by a Single
Metabolic Pathway
Drugs that are extensively metabolized by a single pathway are susceptible to DDI
3) Metabolically Stable Compounds
eliminated primarily as an unchanged drug
Little to no DDI
Characterization of Unusual and Unexpected Metabolites
Unusual or unexpected metabolites
can lead to toxicity
Are caused by unexpected reactions
with drug
e.g., N-dealkylation, oxidation,
aromatization, and sulfation in
torcetrapib 
Radiolabeled compounds could help
identify unexpected metabolites
Radiolabeled ADME Studies used to compare metabolite exposure for compound C
Microdosing / Microtracer Studies
Introduced to address clinical trial failures due to PK or ADME properties
Humans given doses >[P] so Δ[L] is
effectively negligible (see previous slide)
25
What does θ look like over time?
Imaginary Protein (P) & Ligand (L) interaction: @ t0, [L] = 1200, [P] = 80, [PL] = 0 (completely arbitrary units)
B in d in g r e a c t io n
1250