Introduction to Pharmacologyand Drug Metabolism

Luke Lightning, PhD

Outline of topics to be discussed:

Introduction Quantitative aspects of drug-receptor interactions Fundamental mechanisms of drug action Drug dose and clinical response Factors modifying effects of drugs ADME

Text: B.G. Katzung, Basic & Clinical Pharmacology, chapters 1 & 2

Introduction

Pharmacology: study of interactions between chemical compounds and biological systems.

i.e. - how drugs work- where drugs act- how the body processes drugs, etc. (mechanisms of drug action)

The receptor is the cornerstone of pharmacology

Explains how the organism interacts with a drug and initiates a chain of biochemical events that results in observed effects

An agonist is a drug whose interaction with the receptor stimulates a biological response

Purpose of Drug Therapy

To produce the characteristic effect(s) of the drug being used. The drug must achieve adequate concentrations at its site(s) of action.

To achieve the maximal positive effect of the drug while minimizing undesired effects.

No drug will have only one effect (i.e. adverse effects)!

Magnitude of Response Following Drug Therapy

Dependent on various factors:– amount of drug administered (dose)– concentration at site of action

» dependent on rate of absorption and blood flow to the site

– amount of time the drug remains at the site of action» dependent on biotransformation (metabolism) and elimination

Appropriate dose of a drug:– amount of drug needed at a given time that results in the appropriate

concentration at the site of action (where biological effect occurs)

Effect of Drugs on Organs and Tissues

Drugs only modify cellular function – do not create effects

– Pharmacodynamics: Drug Biological Effects– drugs alter the normal biochemical functions of an organ, tissue, or cell

e.g. laxatives increase the activity of the GI tract (i.e. stimulation)

general anesthetics decrease activity of cells in the CNS (i.e. depression)

DRUG RECEPTOR RESPONSE

Drugs, Dose, Receptor, and ResponseDrugs Dose Target (Receptor/Enzyme) Response

Lipitor 10-80 mg HMG-CoA Reductase Decreases LDL

Singulair 10 mg Leukotriene Receptors Prevents Bronchochonstriction

Lexapro 5-20 mg Serotonin Receptors Relieves Anxiety

Nexium 20-40 mg Proton Pump Decreases Gastric Secretion

Plavix 75 mg Purinergic Receptors Anticoagulation

Drug-Receptor Interactions

Receptors largely determine the quantitative relationship between dose or concentration of drug and their pharmacological effects.

Receptors are responsible for selectivity of drug action

– binding to the receptor is dependent on the 3-D characteristics of the drug– size, shape (e.g. stereochemistry), and electrical charge of a drug molecule

– changes in the chemical structure of a drug can affect receptor binding– different types of bonds can be formed between drug and receptor (e.g. H-bond)

» explore these 2 aspects in more detail in Dr. Dave’s section of MCMP 407

Drug-Receptor Interactions (cont.)

Receptors mediate the actions of pharmacologic agonists and antagonists

– Agonists: drugs that bind to a receptor and stimulate a biological response

– Antagonists: » drugs that bind to a receptor but do NOT alter receptor function

(i.e. stimulating a response)» alter the interaction of the receptor with another drug » effect depends completely upon its ability to prevent binding of an agonist to its

receptor and blocking their biological activity» possess affinity, but lack intrinsic activity

Drug-Receptor Interactions

LSD

CNS effects Br

LSD is an agonist at the 5-HT2A receptor

2-Bromo-LSD is anantagonist

LSD

Effect of Drugs on Organs and Tissues (cont.) site of drug action: where the drug acts to initiate the chain of events leading to a

biological effect

– extracellular sites:» some drugs do not need to enter the cell to exert their effects » intracellular reactions (i.e. signaling pathways) are responsible» more on these biochemical pathways later

– intracellular sites:» usually involve a lipid-soluble drug that is able to cross membranes

– sites on the cell surface:» usually involve transmembrane receptors

Concentration-Effect Curves and Receptor Binding of Agonists

Responses to low concentrations of a drug increase proportionally

As the dose increases, the incremental response decreases

Finally, concentrations may be reached at which no further increase in response can be achieved with increasing concentration

akin to Michaelis-Menten kinetics (principles of Km, Vmax)

Concentration-Effect Relationship

0%

25%

50%

75%

100%

0 200 400 600 800 1000

- difficult to accurately extrapolate quantitative information due to the constantly changing slope of the curve

- difficult to compare multiple curves at the low concentrations

EC50 = concentration of drug required to produce half-maximal effect

At lower concentrations:drug effect is changing rapidly

At higher concentrations:drug effect is changing slowly

EC50

Drug Concentration (µM)

Drug Effect

log plot

Concentration-Effect Relationship (cont.)

0%

25%

50%

75%

100%

DrugEffect

1 10 100 1000

Drug Concentration (µM)

Relatively linear portion in the curve about its central point more accurate quantitation

EC50

easier to compare concentration-effect (dose-response) curves graphically

expansion of scale at lower concentrationscompression of scale at higher concentrations

there is no biological significance to this change in graphical presentation

Pharmacological Descriptors of the Receptor

KD:

– describes the interaction between the drug and receptor– drug concentration where drug binding to the receptor is half-maximal– constant for a given drug-receptor system– The lower the KD, the stronger the interaction

Bmax:

– total amount of receptor present in a cell or tissue

Homer Simpson and KD

+ beer

+ champagne

low KD

high affinity

very high KD

very low affinity

Receptor Binding and Drug Concentration

50 % occupancywhen [Drug] = KD

0.01 0.1 1 10 100 10000.00.10.20.30.40.50.60.70.80.91.0

Drug concentration ( )mMR

atio

occ

up

ied

rec

epto

r

50 % occupancywhen [Drug] = KD

Rat

io o

ccu

pie

d r

ecep

tor

0 100 200 3000.00.10.20.30.40.50.60.70.80.91.0

Drug concentration ( )mM

arithmetic scale the drug-receptor binding curve is hyperbolic

log scale the drug-receptor binding curve is sigmoidal

KD is constant for a drug-receptor system

Concept of Affinity affinity: ability of the drug to interact with the receptor

KD is a measure of affinity

affinity is a determinant of potency– lower KD higher affinity more potent

a single drug: different affinities for different receptors

relative affinities among drugs may change from receptor to receptor

Concept of Potency potency: dose of a drug required to produce a particular effect of given intensity

compare drug doses that produce the SAME effect (usually at ED50)

more potent if less drug is required (higher affinity)

higher KD or EC50 less potent

potency may be over-rated– imperfect: our world of D + R DR response

instead determine efficacy

Concept of Efficacy efficacy: the biological response resulting from the drug-receptor interaction

– not all DR same amount of response

a strong agonist has high affinity and high efficacy

maximal efficacy is often limited by toxicity– high doses

efficacy is more important than potency as a drug property

log dose-response curves good for visualinspection

Foye’s: page 90

Homer and Agonists

Agonists

Partial AgonistRemember LMA: conformational change in R response

k1

[D] + [R] [DR] Effect

k-1

what about this step?

full agonist full occupancy maximal effect

some agonists full occupancy less than maximal effect

effects of these agonists are less efficiently coupled to receptor occupancy

= “partial agonists”

Partial Agonist

0

0.2

0.4

0.6

0.8

1 A

gonis

t E

ffect

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 Log Agonist Concentration

Drug

Effect

log [Drug]

0.6

0.4

full agonist A

partial agonist B

partial agonist C

A

B

C

does NOT same maximal effect as a full agonist regardless of the concentration used

Partial Agonist (cont.)

reduced response even at 100% receptor occupancy

may competitively inhibit the response to a full agonist

can have the same affinity for the receptor as full agonists– decreased affinity is not the reason for a less than maximal response

mechanisms complex but probably related to drug binding to inactive form of receptor – receptor can take on two forms (active and inactive)– partial agonist can bind to both forms

Example of Concepts Potency

Efficacy

Agonist

Partial Agonist

log (Dose)

Response

A

B

C

Receptor Antagonism a D-R interaction that inhibits the drug response produced by an agonist

binds to the receptor, but does NOT activate it

4 major types of receptor antagonists:

– competitive: almost all antagonists in clinical use are of this type– irreversible: these covalent modifications of the receptor– mixed: we won’t discuss– noncompetitive: we won’t discuss

exhibit very different concentration-effect and concentration-binding curves

Competitive Antagonist Reversible or equilibrium competitive antagonism:

– antagonist combines with the same binding site on the receptor as the agonist

– can be reversed by increasing the dose of the agonist

– e.g. heroin overdose is treated with competitive antagonist naloxone

Competitive Antagonist (cont.)

0%

25%

50%

75%

100%

Dru

g E

ffect

0 100 200 300 400 500 Drug Concentration

Drug

Effect

[Drug]

AB

C

A : agonist alone

B: (+) competitive antagonist

C: (+) more comp. antagonist

In presence of comp. antag.:

Higher [ agonist ] required to:

- overcome inhibition

- produce effect

Competitive Antagonist (cont.)

- increase [ antagonist ] increase EC50 of the agonist

- potency decreases

- efficacy is unchanged

- magnitude of the shift is

proportional to [antagonist ]

0%

25%

50%

75%

100%

Dru

g E

ffect

-6 -5 -4 -3 -2 Log Drug Concentration

Drug

Effect

log [Drug]

Increasing

[ antagonist ]

EC50

Log Dose-Response Curve in the Presence of a Competitive Antagonist

the shape of the log dose-response curve and the maximal response are not altered by the competitive antagonist

at very high [antagonist], raising the [agonist] should still response

a competitive antagonist has affinity, but lacks significant intrinsic activity (efficacy)

Irreversible Antagonist

an irreversible antagonist will usually bind to the same site as the agonist, but will not be readily displaced

irreversible inhibition is generally caused by a covalent reaction between antagonist and receptor

inhibition persists even after an irreversible antagonist is removed!

Irreversible Antagonist (cont.)

0%

25%

50%

75%

100%

Dru

g E

ffect

-6 -5 -4 -3 -2 Log Drug Concentration

curve is shifted to the right

at high [ irrev. antag. ]:

- max effect decreases- covalent bond is formed

Drug

Effect

log [Drug]

increasing

[ antagonist ]

higher [ agonist ] does not:

- overcome inhibition

- produce max. effect

0

0.2

0.4

0.6

0.8

1

Dru

g E

ffect

0 1 2 3 4 5 6 7 8 9 10Time (hr)

Time-Action Curve

Time to Peak Effect

Addresses two main questions for every drug:

How quickly will the drug act? How long will the drug effect last?

Minimum Effective Concentration

Time to onset Duration of action

Residual Effects

after the primary effects are terminated, it is possible for a drug to exert a residual effect that is unmasked when another dose of the same drug is given– e.g. impaired psychomotor skills following anesthesia

may not be due to the binding at the receptor responsible for the primary effects

can only be observed if another dose or a dose of another drug is given– e.g. cognitive decline (sleep disorders, impaired memory, etc.) with chronic MDMA use

Can last for long periods of time (months, years)

may also occur when another entirely different drug is given and the phenomenon of antagonism or potentiation is manifested– e.g. 2nd drug bind to receptor responsible for primary effects 1st drug released

Residual Effects (cont.)

0

0.2

0.4

0.6

0.8

1

Dru

g E

ffect

0 1 2 3 4 5 6 7 8 9 10Time (hr)

residualeffect

1° drug effectsterminated

impaired neuropsychology(attention, memory, etc.)

women > men

Marijuana Use

Pharmacokinetics (PK) Section

BODILY PROCESSES DRUG

Drug Absorption and Transport

Text: Katzung, Basic & Clinical Pharmacology, chapters 3-4

Foye’s, Principles of Medicinal Chemistry, chapters 7-8

Pharmacodynamics: Drug Biological Effects

Drug

Absorption

EliminationMetabolism

Distribution

Pharmacokinetics

Biological Effect

Pharmacodynamics

Pharmacokinetics and Pharmacodynamics

Katzung: page 36

ADME:

- Absorption

- Distribution

- Metabolism

- Elimination

PK Curve May Not Correlate with PD Curve

Problem:

– PK ≠ PD » average: 6-8 hr activity, 22 hr t1/2» individualized dosing is required

– Prescriptions are increasing– contributed to 3,849 deaths in 2004 (790 in 1999)

» 82% of those deaths listed as accidental

CH3N

CH3

H3C

O*

methadone

Definitions once thought that the biological response to a drug was due to its pharmacologic activity

– it is now apparent that this is NOT the case

Absorption: movement of a drug FROM the site of administration the circulation

Distribution: movement of drug FROM circulation tissues (e.g. plasma receptor)

Metabolism: biotransformation of drugs into metabolites

Elimination: removal of unchanged drug and metabolites from the body

Introduction in order for a drug biological activity, it MUST be present at its target site in the body ADME processes occur simultaneously and determine the time course of [drug] at its

target

in combination with the affinity of the drug for its target site:

– ADME processes serve to regulate the pharmacological activity of a drug ADME processes play an important role in the overall drug effect:

– drugs are rarely administered directly to the site of action (e.g. topical administration)

an understanding of cell membrane

properties and structure is required

Foye’s: page 145

Transport of Drugs: drug transport = movement of a drug molecule across a series of membranes and spaces

most often: drug is given into one body compartment and must move to its site of action in another– requires that the drug be absorbed into the blood and distributed to its site of action

drug action (time of onset and duration) depends on ALL of the rates of ADME processes

elimination can occur by metabolism and/or directly excreted– should occur at a reasonable rate so length of drug effect is appropriate for therapy

the rate of uptake/release by a tissue is a function of:– blood flow to that tissue– affinity (partition coefficient) of tissue for drug

rates of absorption can depend upon the rate of blood perfusion at the site of absorption

Drug Absorption:

Routes of Administration

Drug Absorption for most routes of administration, drugs must cross epithelial membranes in order to reach

the blood

– e.g. GI, oral

– but NOT injection (sc, im, or iv)

therefore, (except for injection) drugs must go through the cells in the membrane

– cannot go between cells by bulk flow

drug absorption is usually limited by:

– the rate the drug can cross cell membranes by drug transport mechanisms:

(diffusion, filtration, ion-pairing, endocytosis, facilitated transport, or active transport)

– perfusion (i.e. circulation at the site of absorption) and concentration gradient

– surface area

Routes of Administration choice will have a profound effect upon the rate and efficiency with which the drug acts

– enteral = drug placed directly in the GI tract (epithelial barriers – stomach)

» oral – swallowing

» rectal – absorption through the rectum

» sublingual – placed under the tongue

– parenteral - BYPASS GI system (endothelial barriers)

» injection - sc, im, iv

– topical - (epithelial barriers - skin)

– inhalation - (epithelial barriers - lung)

remember: no single method of drug administration is ideal for all drugs in all situations

Bulk Flow (cont.)

+

-o

Plasma

+

-

ocapillary endothelium

(loose junctions)

epithelium

(tight junctions)

Environment

GI

Skin

Lung

Absorption Distribution

+

-o

+-

o

ORAL

SC, IM

Enteral Absorption formulation: controls the ability of the active ingredients to dissolve and go into solution

– essential 1st step for absorption

– especially important at gastric pH (very low)

– achieve delayed release into small intestine with pH sensitive coatings – avoid stomach

microbial metabolism:

– proteolytic and hydrolytic enzymes of intestinal microflora may metabolize drugs

– altered rate of absorption OR

– altered biological activity (metabolites)

Enteral Absorption (cont.) FOOD (generally decreases absorption)

– delays gastric emptying

– increases hydrolysis by gastric enzymes

– increases intestinal blood flow and subsequent absorption

– complexes with drugs to retard absorption

» e.g. tetracycline: complexes with Ca2+ in food and milk products

Effect is considerable can reduce absorption of tetracyclines by 80%

Solution: leave a 2 hour gap between eating and taking tetracycline

Advantages:– convenient: can be self-administered, pain-free, easy to take– absorption: takes place along the entire GI tract– cheap: compared to parenteral routes

Disadvantages:– sometimes inefficient: only part of the drug may be absorbed– 1st pass effect: drugs absorbed orally are initially transported to the liver via the

portal vein– irritation to gastric mucosa nausea and vomiting– destruction of drugs by gastric acid and digestive juices– effect too slow for emergencies– unpleasant taste of some drugs– unable to use in an unconscious patient (patient compliance is a problem)

Routes of Administration: Oral

1st Pass Effect

drug is absorbed from the gut and delivered to the liver by the portal circulation

enzymes in the liver metabolize the drug to an inactive species before it reaches the systemic circulation– inactive product = metabolite that does not possess the desired pharmacological activity

the greater the 1st pass effect:– the less the drug will reach the systemic circulation

when administered orally

Routes of Administration: Sublingual

barrier is oral mucosa (epithelial cells) surface area is limited (< 1 m2), but well perfused cell layer is relatively thin absorption is rapid if lipid/water partition coefficient is high

pKa is the major rate limiting factor - saliva pH is 7.0 absorption direct to general circulation - thus bypasses 1st pass metabolism limiting factors: dissolution and transit time in oral cavity

– some drugs are taken as smaller tablets which are held in the mouth or under the tongue

» advantages: rapid absorption, drug stability, avoid 1st pass effect

» disadvantages: incovenient, small doses, unpleasant taste of some drugs

GI Absorption

size of the absorptive surface of the various parts of the GI tract (in m2):

– oral cavity: 0.02– stomach: 0.1-0.2– small intestine 100– large intestine 0.5-l .0– rectum 0.04-0.07

pH in Body Compartments

Blood 7 Mouth 6-7 Colon 8 Cerebral spinal fluid 7 Urine 5-8 Sweat 4-7

pH 1-3

7-8

6-7

5-7

Foye’s: page 144

note: stomach pH is variable

SI and LI pH is near neutral

Other Routes of Administration: Advantages Rectal:

– Bypasses:

» low pH of GI, hydrolytic enzymes in GI, first-pass metabolism

» good for drugs affecting the bowel (laxatives)

– useful for unconscious or vomiting patients or uncooperative patients (children)

Topical:

– generally produces only local effects e.g. dermatology: antibacterial, antifungal, sunscreens, antiviral agents

Lung:

– very highly vascularized and absorption RATE in the lungs is considerably higher than that in the small intestine

Parenteral Administration

barrier is endothelial cells

can bypass epithelial barriers via injection

subcutaneous (sc): bypass epidermis - only barrier is dermis

intramuscular (im): bypass epidermis and dermis – injected into skeletal muscle

– faster absorption than s.c. due to better perfusion and lateral diffusion

transdermal: diffusion through intact skin

intravenous (iv): bypass ALL barriers (membranes) to absorption

– drug injected directly into the blood stream

– produces essentially immediate response

Advantages of Intravenous Administration

absorption phase is bypassed (drug is 100% bioavailable)

almost immediate onset of action

obtain precise plasma levels; excellent compliance; fairly pain free

large quantities can be given

good for drugs with narrow therapeutic index (accurate route of administration)

useful for rapidly metabolized or labile drugs – bypass 1st pass and absorption phase

especially good for drugs which are poorly absorbed by other mechanisms

especially good for very large drug molecules (macromolecules that can’t cross membranes)

Disadvantages of Intravenous Administration

very rapid response potential for overdose (OOPS! factor is high)

non-recoverable – can’t “suck out the poison”

requires skilled administration (costly)

potential for tissue necrosis

potential for embolism – drug or particulate in formulation blocks the flow of blood

potential for microbial or viral contamination in preparation

IV vs Oral Administration Bioavailability (F) Calculation:

– Amount of drug available after oral administrationcompared to:

– Amount of drug available after IV administration (F = 100%)

– Tells you: » amount of first pass metabolism» if there were absorption problems new formulation?» etc.

Time-Action Curve (PK)

Ideal Situation:

PD and PK Time-Action

Curves are Correlated

0

0.2

0.4

0.6

0.8

1

Dru

g E

ffect

0 1 2 3 4 5 6 7 8 9 10Time (hr)

Dru

g P

lasm

a L

evel

s

Cmax

Tmax

AUC T1/2

General Scheme of Drug Metabolism

Metabolism

Lipophilic Hydrophilic

increase eliminationdecrease biological activity

Phase I(oxidative)

Phase II(synthetic)

Parent compound

Metabolites ConjugatedMetabolites

polarityfunctionality ionization

water solubility

Human P450 Isoforms

major drug metabolizing P450s % of drugs metabolized by P450s

Foye’s pages 178-179

Clinical Considerations of CYP450 Metabolism

CYP450 CYP450 + MetaboliteDrug

Elimination

Loss of Drug EffectNo Toxicities

Substrate Oxidation

CYP450 + Drug + electrons Activated CYP450 CYP450 + Metabolite

(capable of oxidations)

bound

molecular

oxygen

substrate

NADPH2

endoplasmic

reticulum

(membrane)P450

cytoplasmic

side

luminal side

P450

Oxidations

OxidationR R

*H

O*H

R

HO

*H

RHO*H

HO

H2O

Dihydrodiol

Arene Oxide(carcinogen)

:Nu

RHO*H

Nu

Aromatic Oxidation

[O]

inactivation vs. bioactivation

bioactivation

cellular toxicities

MDMA and Cytochrome P450 Metabolism

O

O

NCH3

H

CH3

MDMA (“Ecstasy”)

P450 1A2

O

O

NH

H

CH3

P450 2D6

HO

HO

NCH3

H

CH3

MAJOR

MINOR

CYP450s

ISOZYME SUBSTRATES INDUCERS INHIBITORS

CYP1A2(2%)

AcetaminophenTheophylline

BarbecueSmoking

AntibioticsQuinolone

CYP2C fam(20%)

DiazepamPhenytoin

Rifampin Fluoxetine

CYP2D6(25%)

CodeineImipramine

None known QuinidineAntidepressants

CYP3A4(52%)

QuinidineWarfarin

PhenobarbitalPhenytoin

AntifungalsAntibiotics

approximate % of drugs metabolized by this CYP450

P450-catalyzed reactions: Epoxidation - ring (aromatic)

P450

EpoxidationO

Benzo[a]pyrene – polycyclic aromatic hydrocarbon

present in cigarette smoke, smog, charcoal grilled meat

1A

known carcinogen in fish, insects, humans, and other animals epoxide reacts w/ DNA and macromolecules LC50: cricket = 15mg/g (oral)

P450 Inhibited P450

Drug A(Inhibitor)

Drug B(Substrate)

Drug B

Prolonged or Enhanced EffectUndesirable Toxicities

(Drug-Drug Interaction)

slow release of inhibitor

Clinical Considerations of Cytochrome P450 Inhibition

Competitive Inhibition

Drug-Drug Interaction (DDI)

Time-Action Curve – Competitive Inhibitor

0

0.2

0.4

0.6

0.8

1

Dru

g E

ffect

0 1 2 3 4 5 6 7 8 9 10Time (hr)

Dru

g P

lasm

a L

evel

so

r

+ inhibitor

PK and PDare affected

Why are we so interested in DDIs??

FDA: 2006

FDA Draft Guidance – Metabolism and DDIs September 2006

– Study design, data analysis methods– Implications for dosing and labeling– Mostly concerned with effects on CYP450

DDIs can be due to metabolism but also:– Changes in PK, transporters, etc.

Does not establish legally enforceable responsibilities Describe the FDA’s current thinking View only as recommendations, not required

– May be best to be running experiments described to stay ahead of or with the rest of the pack

– “Negative findings from early in vitro and early clinical studies can eliminate the need for later clinical investigations.”

– i.e. potentially fewer protocols!!

Adverse Events Reported to FDA FDA has a website devoted to ADRs:http://www.fda.gov/cder/aers/default.htm

This figure illustrates the patient outcome(s) for reports in AERS since the year 1999 until the end of 2008. Serious outcomes include death, hospitalization, life-threatening, disability,

congenital anomaly and/or other serious outcome.

Factors Modulating Xenobiotic Metabolism (cont.)

DRUG INTERACTIONS (DI’s):

competitive inhibition by other drugs and xenobiotics can decrease metabolism of drugs

especially important with multiple drug treatments

potential DI’s with:

– herbal drugs and illegal drugs relatively unexplored

very important with elderly patients who are

often taking multiple drugs simultaneously

4 or more drugs68%

3 drugs13%

2 drugs12%

1 drug7%

approx. 1000 patients at

VA Medical Center, Wichita, KS

Steps of the Experiment

Test Articles

Combined with tissues of interestand other reaction ingredients

Mixture undergoes vigorous shaking for a period of time

Purification and AnalysisCentrifuged to precipitate protein

Injected onto the LC/MS for analysis

Data Analysis and Next Steps

Go home and let the LC/MS work overnight Process the data

disseminate tothe Project Team

I think we should performthis experiment

next!

No MoreBailouts or DDIs!

Competitive Inhibition of Cytochrome P450s

(B) coordination to the heme iron atom - usually through a nitrogen

(esp. imidazole ring)

(A) lipophilic and H- bonding interactions

Inhibitor BInhibitor A

NN

NN Fe

P450

NN

NN Fe

P450

Contaminants commonly found:

• MDMA structural derivatives: legal, cheaper• caffeine and ephedrine (“herbal ecstasy”): mimic speedy feeling• LSD (very rare)

MDMA and Cytochrome P450 Inhibition

O

O

NCH3

H

CH3

• dextromethorphan (“green triangles”)anti-tussive (cough medicines)raises body tempinhibits sweating

MDMA

MDMA

CH3O

NCH3

NCH3

H

CH3

O

O

Dextromethorphan

P450 2D6

plasma levels of MDMA

Drug-Drug Interaction

P450 2D6-Dextromethorphan

cheaper

drugs

N

N

H

SNMeHN

NCN

Cimetidine (Tagamet)

Drug-Drug Interactions• H2 receptor antagonist (anti-ulcer agent)• general inhibitor of human P450s• inhibits hepatic elimination of many drugs:

warfarin alprazolamacenocoumarol triazolamphenadion theophyllinephenytoin imipraminecarbamazepine caffeinechlormethiazole propanololdiazepam labetalolchlordiazepoxide metoprolollidocaine ethanol

H

• imidazole ring able to coordinate to theheme iron atom of several different P450s

NN

NN Fe

undesirable toxicities

O

N Me2

SNMeHN

N O-

HO

Ranitidine (Zantac)

• H2 receptor antagonist

• replacement of imidazole w/ furan ring: circumvents cimetidine drug interactions

• knowledge of which structural features of a drug were important for P450 inhibition

H

Drug-Drug Interactions

N

N

H

SNMeHN

NCN

Cimetidine (Tagamet)

Hdesign of a safer drug

Mechanism-Based Inhibition (Irreversible)

FDA Draft Guidance

Metabolic activity will not be restored until enzyme is re-synthesized

Pathways of Mechanism-Based Inhibition of CYP450

NN

NN

MBI*

MBI

Fe

Fe

NN

N N

MBI*

Cys

NN

NN

FeN

N

NN

Fe

Mechanism-based Inactivators of CYP450s

Raloxifene (osteoporosis)

Bergamottin (Grapefruit Juice Component)

RU-486 (morning after)

Phencyclidine (street drug)

Ritonavir

“BOOSTER” for

other HIV drugs

Mechanism-based

inactivator of CYP3A4

+ ritonavir

Experimental Design: Mechanism-based Inactivation

• human liver microsomes• MBI (e.g. 8-MOP for

CYP2A6)• initiate rxn

+ NADPH

time time

1˚ rxn 2˚ rxn• CYP450 selective substrate (e.g. coumarin at 2X KD)

• initiate rxn with P450 from 1˚ reaction

≥ 20-fold dilution ANDexcess substrate to displace MBI

(now <<< KD)

product analysis(e.g. 7-OH coumarin)

• HPLC/fluorescence• LC/MS• GC/MS

(0-10 min)

Enzyme-Drug Interaction - Concepts

E

EE

[E + I]

[E + I]E

I

KI

[E-I]

[E + I]E

kinact

time

timeMetabolites

[E-I]

[E + S][E + S] 20X

dilution

S

KD

I

RU486 and CYP2B6 (2008)

31% remaining

KI

kinact

competitiveinhibition

0-25 µM

Esterases > 70 different human esterase genes

– Esterases are present in every tissue and blood

a/b hydrolase-fold family (>15,000 members)– Carboxylesterases (hCE-1, 2, 3) – broad substrate specificities– Acetylcholinesterase (AChE) – specific for acetylcholine– Butyrylcholinesterase (BChE) – broad substrate specificity

Others:– Proteases (Chymotrypsin, Trypsin, etc.)– Albumin– Paraoxonases (hPON-1, 2, 3) – broad substrate specificities

Famous Esters

Esther Rolle“Good Times!!”

O O

OO

n

polyester

OO

O

CH3

O

H3C

O

N

CH3

heroin

OHO

O CH3

O

aspirin

General Esterase Activity

R1 O

O

R2R1 OH

O

R2OH+esterase

acid alcohol

H2O

ester

+

Human Carboxylesterases

Enzymes known to be involved in drug metabolism– Human carboxylesterases-1 and -2 (hCE-1 and hCE-2)

hCE-1liver

hCE-2intestine

microsomescytosol

Twopurified

enzymes

Inhibitors of Esterases: Biological Weapons

P

S

N

H3C

CH3

CH3

CH3H3C

OH3C

O

VX

AChE inhibitor – developed as a pesticide (1952)most deadly nerve agent in existence

3X more deadly than sarin300 g is fatal

F

P

H3C

O

O

CH3

CH3

Sarin

O

P

O

N

CH3

N

H3C

CH3

Tabun

"It's one of those things we wish we could disinvent." - Stanley Goodspeed, on VX nerve agent

Factors Modulating Xenobiotic MetabolismAge and Ontogeny:

decreased:

– absorption (decreased absorptive surfaces, blood flow, and GI motility)

– tissue perfusion

– general metabolism and liver function

– P450 levels in very young and very old

– different P450 are expressed

altered drug distribution:

– increased % body fat

– decreased: serum albumin (plasma protein), muscle mass, total body water

Factors Modulating Xenobiotic Metabolism (cont.)PHARMACOGENETICS: sex differences (generally small in humans)

ethnic differences (P450)

– isoniazid - slow vs. fast acetylators

species differences (P450)

– MAJOR problem: drug testing in animals and extrapolation to humans

individual genetic variability (relative amounts of P450s and Phase II enzymes)

organ-specific differences (P450, bioactivation)

individualized drug therapy is the goal

DRUG Phase II Reactions

Metabolite

P450

FMO

ADH

esterases

amidases

Glucuronosyl TransferasesSulfotransferasesGlutathione Transferases

Amino Acid TransferasesAcetyltransferasesMethyltransferases

Phase I

Reactions

elimination

elimination

elimination

Drug Elimination Pharmacological activity of drug can be reduced by:

– metabolism

– plasma protein binding

– redistribution to other compartments (i.e. fat)

Elimination:

– required to remove the chemical from the body and terminate biological activity

» especially if drug is minimally metabolized

– necessary to prevent accumulation of xenobiotics in the body

Major Routes of Drug Elimination: are highly dependent on metabolism:

– KIDNEYS (renal)» represent approx. 1% of of total body weight, » but receive 25% of cardiac output» blood flow rate is approx. 8X more that exercising muscle

– Liver– Intestines– Lungs– Sweat, Saliva, Milk – not really significant

same physiological mechanisms govern drug elimination as absorption– i.e. cell membranes are the barriers.

Methadone Problems:

– PK ≠ PD (average: 6-8 hr activity, 22 hr t1/2)» F = 36-100%, t1/2 = 5-130 hr» individualized dosing is required

– Lots of interindividual variability– Long t1/2 and high tissue distribution

– DDIs

– Prescriptions are increasing

CH3N

CH3

H3C

O*

Methadone Metabolism

CH3N

CH3

H3C

O

EDDP(inactive,

renally excreted)

MethadoneCmax ~ 0.6 µM

NCH3

H3CCYP2B6: S > RCYP3A4: S = R

CYP2C19: R >> S

Several DDIs possible

The End