75435479 Drug Metabolism

8/3/2019 75435479 Drug Metabolism

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Drug Metabolism and Protein binding

By

Hamid saeedPhD., Mphil., B.Pharm

Usman Akhtar (B.Sc English PU)

4th prof.MLOVELY CR


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Drug Bio-transformation reactions

Biotransformation enzymes play an important role for the inactivation and

subsequent elimination of drugs.

E.g Theophylline, phenytoin and acetaminophen direct relation between

drug metabolism and elimination half life.

In most cases the metabolite become more polar than lipid soluble.

Some drugs are pro-drugs and are bio-transformed into active metabolite

e.g Prontosil which is reduced to anti-bacterial agent sulfanilamide.

LEVODPA decarboxylated in brain into L-dopamine


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The major site of drug metabolism is the liver where the microsomal

enzyme systems of hepatocytes play an important role. Other sites are the

Kidney,

Lung,

Intestinal mucosa,

Plasma and

Nervous tissue.


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The number of microsomal enzymes is influenced by factors such as

Drugs

hormones,

Age and sex

stress,

Temperature,

Nutritional status and

Pathological state.

This is what is referred to as enzyme induction and could result in increase

in enzyme activity or reduced activity.


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Reactions involved:

Oxidation

Reduction

Hydrolysis

Conjugation

Enzymes responsible for oxidation and reduction of drugs

(Xenobiotics) are monoxygenase enzymes knownas Mixed function

Oxidases.


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Hepatic parenchymal cells contain the MFOs in association with the

endoplasmic reticulum

MFOs are structural enzymes that constitute and electron transport system that

requires reduced

NADPH (NADPH2)

Molecular oxygen

Cytochrome P-450

NADPH-cytochrome P-450 reductase

Phospholipid

Phospholipid is invloved in the binding of the drug to the cytochrome P-450

Many lipid soluble drugs bind to cytochrome P-450, resulting in oxidation (or

reduction) of the drug


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Common Bio-tranformation reactions

Phase I Reactions Phase II Reactions

Oxidation Glucuronide conjugation

Armoatic Hydroxylation Ether glucuronide

Side chain hydroxylation Ester glucuronide

N-O- and S-dealkylation Amide glucuronide

N-hydroxylation Peptide Conjugation

Reduction Glycine Conjugation

Azo-reduction Mehytlation

Nitro-reduction N-methylation

Alcohal Dehydrogenase O-methylation

Hydrolysis Acetylation

Ester hydrolysis Sulfate conjugation

Amide hydrolysis Mercapturic acid synthesis


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Bio-transformation reactions and Phamarcologicalactivity of the medicine

REACTION EXAMPLE

Active drug to Inactivemetabolite

Amphetamine Deamination Phenylacetone

Phenobarbital Hydroxylation Hydroxy-phenobarbital

Active drug to Active metabolite

Codeine Demethylation Morphine

Procainamide Acetylation N-acetylprocainamide

Phenylbutazone Hydroxylation Oxyphenbutazone

Inactive drug to Activemetabolite

Hetacillin Hydrolysis Ampicillin

Sulfasalazine Azoreduction Sulfapyridine+5-aminosalycylic acid

Active drug to ReactiveIntermediate

Acetaminophen Hydroxylation RM (Hepatic necrosis)

Benzopyrene Hydroxylation RM (Carcinogenic)


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Importance of Drug Metabolism

Metabolism => Termination of Drug Action

Bioinactivation -and/or-

Detoxification -and/or-

Elimination -and/or-

Metabolism => Bioactivation

Active Metabolites

Prodrugs

Toxification


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Metabolism => Termination of Drug Action

Bioinactivation


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Prodrugs

Active metabolite


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Metabolism => Drug interactions


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Metabolism => stereochemical implications


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Phase I Reaction (Non- synthetic phase)

Enzymic modification of a substance by oxidation, reduction, hydrolysis,

hydration, dehydrochlorination, or other reactions catalyzed by enzymes of

the cytosol, of the endoplasmic reticulum (microsomal enzymes) or of other

cell organelles. Usually Phase 1 reactions occur first that resulted in the introduction and

exposure of a functional group on the drug molecule.

For example: oxygen is introduced into the phenyl group on phenylbutazone

by aromatic hydroxylation to form oxyphenbutazone a more polar

metabolite.


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1) Oxidation reaction.

Reaction results in proton enriched products. There are two types.

A)Microsomal oxidation reactions take place mainly in the liver.

The following reactions are grouped under microsomal reactions

(a) Oxidation of alkyl chains. Alkyl compounds or alkyl side chains of aromatic

compounds

compounds with carbonyl, aldehyde, carboxyl or amino groups undergo

oxidation

e.g.

i. ethanol is broken down to acetaldehyde then to acetic acid

ii. amines undergo deamination eg.5HT to 5- hydroxyl indoleacetic acid


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b).oxidation of aromatic ring

eg acetanilide oxidation to acetaminophen.

c) Oxidative dealkylation.

This is either on an oxygen (O-dealkylation) or on a nitrogen (N-dealkylation)

O-dealkyalation eg codeinemorphine

or phenacetinacetaminophen

N-deakylation- eg mephobarbital to phenobarbital

d)N-oxidation eg aniline oxidation to nitrobenzene e) Sulfoxidation

The thioethers are oxidized to their corresponding sulfoxides derivatives eg

chlorpromazine is oxidized to chlorpromazine sulfoxide.


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B)Non microsomal oxidation reactions.

These are oxidation reactions catalyzed by enzyme in mitochondria,

cytoplasmic plasma and other organelles.

2) Reduction reactions:-

Conversion of aldehydes to primary alcohols. eg chloral hydrate reduction to

trichloethanol, cyclic ketone reduction to alcohol.

e.g progesterone - pregnandiol

Conversion of prontosil to sulfanilamide


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3). Hydrolytic Reactions.

These reactions involves the break down of ester, linkages (c-o-c)

eg esters of choline, amide bonds, hydrazide and glycosides.

The ester bonds in atropine are broken to give tropine and tropic acid.

Cocaine is hydrolysed to benzoic acid and ecgonine methyl ester.

Procaine to p-amino acid (PABA) and diethyl amino ethanol.

Acetylcholine to acetic acid and choline.


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PHASE II (SYNTHETIC REACTIONS)

It is usually the last step in detoxification reactions and almost always results in

loss of biological activity of a compound. It may be preceded by one or more of

phase one reaction.

The synthetic or conjugations involves chemical combination of a compound

with a molecule provided by the body.

The conjugating agent is usually a carbohydrate, amino acids or compounds derived

from them.

For conjugation to take place, a compound should have an appropriate group or

centre eg COOH, -OH, -NH, or SH.

A compound having non, can acquire it from the non synthetic reaction.

Conjugated metabolites are in variably less lipid soluble than their parent

compound.


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Phase-2 Metabolism Description

Phase 2 = "Conjugation" Reactions

Acts on parent drug or

Acts on phase 1 metabolite.

Links to endogenous, polar, ionizable cpd.

Purpose: enhance excretion.

Reaction types include:

Glucuronidation

Sulfate formation


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Conjugation reactions include

a) Glucuronide Conjugation

This is the most frequently occurring conjugation.

This is the conjugation of glucuronide by UDP- glucuronic acid in

hepatocytes. D- glucuronic acid is derived from D- glucose in which the

terminal primary alcoholic group is oxidized to carboxyl.

The immediate donor of glucuronic acid for conjugation reaction is UDP-

glucuronic acid which arises from breakdown to glycogen.


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glycogen ------------> a glucose phosphate

a glucose - Iphosphate + UTPUDPglucose + phophosphate

UDP-glucose -> UDPglucuronic acid OR UDPGA (active high energy

conjugating agent)

The glucuronide are easily secreted in urine and bile because they are

highly soluble.

They are broken down in the intestine by bacteria and may result in

enterohepatic circulation of the drug.


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Other conjugating agents are

Acetyl Co-A

3-phosphoadenosine-5-phosphosulphate (PAPS)

S-adenosylmethionine (SAM)

At very high drug concentration, Glucoronidation reaction follow non-linear

kinetics (saturation)

While glycine, sulphate and glutathione conjugation demonstrate non-linear

kinetics at therapeutic doses

Glucoronidation and sulphate conjugation aremost common phase-II reactions

that result in water-soluble metabolites rapidly excreted in bile (high molecular

weight) or urine


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Acetylation and Mercapturic acid synthesis are conjugation reactions often

implicated in the toxicity of the drug

Acetylation

Acetylated drug is less polar than the parent drug

Drugs such as, sulfanilamide, sulfadiazine and sulfoxazole produces

metabolites that are less water soluble, therefore in sufficient concentrations

participate in kidney tubules causing damage and crystaluria

Moreover, less polar metabolite will be reabsorbed in the renal tubules and

have longer elimination half-life


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Mercapturic acid conjugation

Glutathione (GSH) main compound that protect cells against reactive

electrophilic compounds

GSH reacts enzymatically or non-enzymatically via gluathione-S-tranferasewith reactive electrophilic oxygen intermediates

These reactive electrophilic intermediates react with nucleophilic

macromolecules such as proteins in cells causing cell injury and cellular

necrosis

GSH detoxify reactive oxygen intermediates

The resulting GSH conjugates are precusors for a group of drug conjugates

known asMercapturic acid derivatives


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ConjugationReaction

Conjugation agentHigh energyintermediate

Functional groupscombined with

Glucoronidation Glucoronic acid UDPGA -OH, -COOH, -NH2, SH

Sulphation Sulphate PAPS -OH.NH2

Amino acid

conjugation Glycine

Co-enzyme A

thioesters -COOH

Acetylation Acetyl CoA Acetyl CoA -OH, -NH2

MethylationCH3 from S-

adenosylmethionine

S-

adenosylmethionine-OH, -NH2

Glutathione

(mercapturic acid

conjugation)

GlutathionArene oxides,

epoxides

Ary halides, epoxides,

arene oxides


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Kidney

Main excretory organ

Maintain salt and water

balance

Endocrine function

Secretion of renin for BP

control

Secretion of erythropoietin

stimulate red blood cell

production


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Some facts

0.5% of total body weight

20-25% of cardiac output

Basic unit is nephron

1 1.5 milliion nephrons

Water is mainly re-absorbed in longer loop of henle in medulla

Renal blood flow (RBF) 1.2min/L or 1700L/day

Renal Plasma Flow (RPF) RBF volume of red cells present

RPF = RBF (RBF x Hct) , where Hct is the hematocrit (fraction of blood

cells in the blood)


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RPF = RBF(1-Hct)

Glomerular filteration rate (GFR) = 125ml/min

About 180L of fluid is filtered per day

While urine volume is 1 1.5 L


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Quantitative aspects of urine formation

Per 24 hours

Substance Filtered Reabsorbed Secreted Excreted %Reabsorbed

Sodium ion 26,000 25,850 150 99.4

Chloride ion 18,000 17,850 150 99.2

Bicarbonate ion 4,900 4,900 0 100

Urea (mM) 870 460 410 53

Glucose (mM) 800 800 0 100

Water (mL) 180,000 179,000 1,000 99.4

Hydrgoen ion variable Variable

Potassium ion 900 900 100 100 100


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Process of drug elimination/excretion

Glomerular fiilteration

Active tubular secretion

Tubular reabsorption


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Glomerular filteration

Unidirectional processoccurs for most small molecules (MW < 500)

Ionized and non-ionized drugs

Proteinbound drugs are large moleculesnot get filtered at glomerulous

Main driving force hydrostatic pressure within the glomerulous capillaires

Blood receive 25% of the total cardiac output

GFR is measured by using a drug that is eliminated by filteration only (drug is neither

absorbed nor secreted)

Examples of such drugs are: Inulin and creatinin

GFR of a drug relates to free or non-protein bound drug

GFR of a drug increases as the free bound drug in the plasma increases


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Active tubular or renal secretion

Requires energy inputdrug is transported against the conc. Gradient

System can be saturated

Drugs with similar structure may compete for the same carrier

Two active renal secretion systems have been identified

Weak acids

Weak bases

For example: Probenecid will compete with penciline for the same carrier

Its rate depends upon renal plasma flow

Commonly used drugs to measure active renal secretion are

P-amino-hippuric acid (PAH)

Iodopyracet (Diodrast)


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Protein binding effects half-life of drugs solely excreted by GF

While protein binding has very little effect on the elimination half-life of drugs

excreted mostly by active secretion

Drugs such as Penicillins are extensively protein bound but their elimination

half-life are short because of rapid elimination by active secretion.

Tubular Reabsorption

Occurs after the drug is filtered through glomerulous and can be active or passive

Drugs - completely reabsorbed have Zero clearance value

Drugs that are partially reabsorbed have clearance values less than the GFR of 15130

mL/min


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Reabsorption of a drug that is weak acid or weak base is influnced by pH of the fluid in the renal

tubule (urine pH)

pKa of the drug

Generally, un-dissociated drugs are more lipid soluble and have greater membrane permeability

pKa of the drug is contant while urine pH can vary from 4.5 to 8 depending upon diet,

pathophysiology and drug intake

Vegetable diets or diets rich in carbohydrates will result in higher urinary pH

Diets rich in protein will result in lower urinary pH

Drugs such as, ascorbic acid and antacids such as, sodium bicarbonate by acidfy or alkalinize

urine, respectively

Most important changes in urinary pH is caused by fluids administered IV

pH = pKa + log [ionized]

[non-ionized]

HendersonHesselbalch equation


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Protein binding

Drugs interact with many molecules

Plasma

Tissue proteins OR

Melanin

DNA

To form macro-molecule complex

This formation of drug-protein complex is calledDrug-protein binding

- can be reversible or irreversible

Irreversible protein binding

result from chemical activation of a drug

Attaches strongly to the protein

Via covalent chemical bonding


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Example:

High doses of acet-aminophen

Form reactive metabolites

That interact with liver proteins

Most drugs bound by reversibly with the proteins

Have weaker chemical bonding (hydrogen or wander-waals forces)

Normally amino acids of the proteins have hydroxyl, carboxyl or other sites available

Drug binding to macro-molecules

Albumin

A1-gylcoprotein

Lipoprotein

Immunoglobulins (igG)

Erytherocytes (RBC)


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Albumin:

Sythesized in the liver

MW ~ 65,00069,000

Major component of plasma protein

Responsible for irreversible protein binding

Elimination half-life is 1718 days

Conc. is normally maintained at low level i-e., 3.55.5 %

Responsible for maintaining osmotic pressure, transport protein for exogenous and

endogenous substances

Endogenous substancesinclude, free fatty acids, bilirubin, various hormones (e.g

cortisone), trytophan and other compounds


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Weak acidic drugs are highly bound to albumin such as

Salicylates

Phenylbutazone

PencillinA1- acidic glycoproteins

-A globulin having MW ~ 44,000 d.

- plasma conc. is low ~ 0.4 1%

- binds mainly basic drugs

-Imipramine

- Propanalol


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Globulins (, , ):

Responsible for the transport of certain endogenous substances e.g, corticosteroids

Have low capacity but high affinity for endogenous substances

Lipoproteins

Macro-molecule complexes of lipids and proteins

VLDLvery low density, LDL low density, HDLhigh density, lipoproteins

Transport plasma lipids

Binding occurs if albumin sites are saturated


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Erythrocytes: (RBCs)

Bind both endogenous and exogenous compounds

RBCs constitute 45% of the total volume of the blood

Drugs such as pehnobarbitol and amobarbitol have RBC/plasma water ratio of 4:2,

means they preferentially bind to RBCs

Protein bound drugs are large molecules and therefore, have restricted distribution

Usually protein bound drugs are in-active


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Factors affecting protein bound drugs

The drug itself

Physicochemical properties

Total concentration

The protein

Quantity of protein

Quality of protein synthesized

- Affinity between the drug and the protein

- Includes magnitude of association constant

- Drug interactions

- Competition of drugs for protein binding sites

- Alteration of protein by drugs e.g. aspirin acetylates lysine residues of albumin

- Pathophysiological condition of the patient

- E.g, drug protein binding me be reduced in uremic patients and patients with hepatic disease


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Plasma drug concentration:

Total drug conc in the plasma

Protein bound + unbound (free)

Kinetics of Protein binding:

reversible drug-protein binding with one single binding site can be defined bylaw of

mass action

Protein + drug Drug-protein-complex

P + D (PD)

Ka = (PD)

(P) (D)

Ka is an association constantprotein-drug binding is dependent upon Ka


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Binding behavior of drugs

r = moles of drug bound

total moles of protein

Moles of durg bound (PD), total moles of protein (P) + (PD)

r = (PD)

(PD) + (P)

We know from previous equation that (PD) = Ka (P) (D)

So r becomes

r = Ka (P) (D)

Ka (P) (D) + (P)

r = Ka (D)

1 + Ka (D)


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Clinical significance of protein-bound drugs Most drugs bind reversibly to proteins

Fraction of drug bound changes with plasma drug conc. and dose of the drug

Plasma protein conc. is controlled by different variables

Protein synthesis

Protein catabolism

Distribution of albumin

Excessive elimination of plasma protein i-e., albumin

Number of diseases, age, and trauma affect plasma protein conc. E.g liver disease results in

decrease plasma protein conc. due to reduced protein synthesis

Severe burns results in increase plasma albumin into extracellular fluid

Highly protein bound drug is displaced from the binding site by a second drug or agent results

in sharp increase in the free drug conc. of the former

E.g increase in free warfarin when co-administered with phenylbutazone


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Albumin A1-glycoprotein Lipoprotein

Decreasing Age Fetal conc. Hyperthyroidism

Becterial pneumonia Nephrotic syndrome Injury

Burns Oral contraceptives Liver disease

Cirrhosis of liver Trauma

GI disease

Malignant neoplasms

Malnutrition

Nephrotic syndrome

Pancreatitis

Renal failure

Trauma

Increasing Benign tumor Age Diabetes

Exrecise Crohns disease Hypothyroidism

Hypothyroidism Injury Liver disease

Neurological disease Myocardial infarction Nephrotic syndrome

Psychosis Rehumatoid arthritis

Physiological and pathological conditions altering protein binding

75435479 Drug Metabolism

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