1

PHARM 630

Toxicological Aspects of Drug Metabolism/ Factors affecting drug

metabolism

Toxicological Aspects of Drug Metabolism

• We interact with an environment that introduces us to thousands of unique compounds. (~8g/day of food additives alone)

• Humans have responded to these chemicals with phase I and phase II metabolic enzymes that process xenobioticsand encourage their elimination. This was originally termed detoxication after it was assumed that a xenobioticwas transformed into a metabolite less toxic than the parent molecule.

• However, metabolism can generate compounds with:a) Increased activity, b) Toxicity and c) Carcinogenicity

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Metabolic Conversions Leading to Toxic Metabolites

• This is called activation or bioactivation

• Therefore, xenobiotics can be activated by the phase I or phase II metabolic pathways into:a) Active metabolites.b) Reactive metabolites (electrophiles).- These electron deficient molecules can covalently attach to DNA, protein, and lipids.

I) Toxicity

1) Toxicity of Acetaminophen (Tylenol)• Acetaminophen was identifies as a safe drug with

analgesic and antipyretic properties more than 100 years ago.

• It is widely used in many countries as an alternative to aspirin.

• Even with widespread use over many years, reports of poisoning did not appear in the literature until 1966.

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How can Acetaminophen cause toxicity?

a) Metabolism - Major Metabolites.

How can Acetaminophen cause toxicity?

b) Metabolism - Minor Metabolites.

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How can Acetaminophen cause toxicity?

Metabolism can generate a reactive intermediate. However, at therapeutic doses, glutathione in the liver combines with the reactive metabolite to form the glutathione conjugate.

How can Acetaminophen cause toxicity?

OVERDOSE• Following an overdose, glutathione becomes depleted and

the reactive metabolite binds to hepatic proteins.

• After a massive overdose, metabolism by oxidations will increase because the glucuronidation and sulfationpathways become saturated.

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2) Toxicity of Halothane• Widely used anesthetic that occasionally results in severe

hepatitis• About 15% is metabolized by the cytochrome P450• Hepatotoxicity is mainly due to an immune response to

metabolite-protein complex.

II. Carcinogenicity

• Most chemical carcinogens are not active in themselves but require bioactivation to electrophiles (pro-carcinogens).

• Electrophiles can bind covalently to DNA.• This process can lead to mutations and potentially cancer.

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II. Carcinogenicity

1) The Polycyclic Aromatic Hydrocarbon (PAH)• PAH are ubiquitous environmental contaminants formed

from:- Auto emissions.- Cigarette smoke.- BBQ.

It is the electrophilic diol epoxide metabolite that readily reacts with DNA to form covalently bound adducts.

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II. Carcinogenicity

2) Aflatoxin b1Aflatoxin is a secondary metabolite secreted into the environment by the fungus Aspergillus flavus.

II. Carcinogenicity

3) Tamoxifen (antiestrogen)• Inhibits the binding of estradiol to estrogen receptors.• Is the current agent of choice for treating all stages of

breast cancer.

Of concern are the findings that:a) Tamoxifen increases the incidence of human endometrial cancer.b) Tamoxifen causes hepatocellular carcinoma in rats

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Metabolism of Tamoxifen

Induction and inhibition of Drug Metabolizing enzymes

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Induction of Drug Metabolism

- Many drugs and chemicals can induce drug metabolizing enzymes upon repeated exposure/administration.

- Enzyme systems inducible by xenobiotic are cytochromeP450s, glutathione S-transferase, glucuronyl transferaseand epoxide hydrolase.

- Not all cytochrome P450 isoenzymes are inducible.- Clinically important enzyme inducers are carbamazepin,

phenobarbital, phenytoin, rifampicin and griseofulvin.

Synthesis and Degradation of MFO System

Increased synthesis

Reduced degradation

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Mechanisms of induction of multiple forms of Cytochrome P450

Induction of drug metabolism may arise as a consequence of increased Synthesis, decreased degradation, activation of pre-existing componentsOr a combination of these three processes

233

TCDD

Nucleus

Hepatocyte

DRE

cyp1a1 mRNA

cyp1a1 proteinTranscription Translation

Regulation of cyp1a1 by AhR

Cyp1a1 gene

Arnt

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Time - dependent induction of cyp1a1 mRNA by Hg2+

Time (h) 0 1 3 6 12 24

Hg2+

TCDD

TCDD+

Hg2+

Time (h)

0 5 10 15 20 25 30

Fold

of i

nduc

tion

0

25

50

75

100

125

150TCDDTCDD + Hg2+

Hg2+

150

125

100

75

50

25

5 10 15 20 250

Time (h)

Fold

of I

nduc

tion

30

Korashy and El-Kadi. Toxicol Sci, 88:39-51, 2005

mRNA steady-state level

mRNA SS level

Synthesis Degradation

Hg2+ increased SS TCDD-induced cyp1a1 mRNA

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Does Hg2+ increase the synthesis of cyp1a1 mRNA at the transcriptional level?

Cyp1a1

Gapdh

Control

Act D Hg2+

Hg2+

+ Act D

TCDDTCDD + Act

D

DRECyp1a1 mRNA

Transcription

Inhibitor

Korashy and El-Kadi. Toxicol Sci, 88:39-51, 2005

Does Hg2+ increase the synthesis of cyp1a1 mRNA at the transcriptional level?

Control

CHXHg

2+

Hg2+

+ CHX

TCDDTCDD + CHX

Cyp1a1

Gapdh

CYP1A1 mRNA

Cyp1a1 proteinTranslationTranscriptionTranscription

CHX

Korashy and El-Kadi. Toxicol Sci, 88:39-51, 2005

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Cyp1a1 mRNA and protein decay assays

TCDD12 h

cyp1a1 mRNA

24 h

cyp1a1 protein

Transcription inhibitor

Translation inhibitor

½ h

block RNA synthesis

0,1,3,6,12,24 h

incubation

0,1,3,6,12,24 h

incubation

Northern blot

analysis.

½ h

block protein synthesis

Hg2+

Western blot

analysis.

Time (h) 0 1 3 6 12 24

TCDD

TCDD+

Hg2+

Time (h)

0 5 10 15 20 25 30

% o

f cyp

1a1

mR

NA

rem

aini

ng

0.1

1

10

100

1000

t½=3.9±0.4 h

t½=4.3±0.6 h

Actinomycin D Chase Experiment

TCDDTCDD + Hg2+

Effect of Hg2+ on the stability of cyp1a1 mRNA

1000

100

10

1

0.1

% o

f cyp

1a1

mR

NA

rem

aini

ng

5 10 15 20 25

Time (h)

300

P>0.05Korashy and El-Kadi. Toxicol Sci, 88:39-51, 2005

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Time (h) 0 1 3 6 12 24

TCDD

TCDD+

Hg2+

Time (h)

0 5 10 15 20 25 30%

of c

yp1a

1 pr

otei

n re

mai

ning

0.1

1

10

100

1000

t½=4.8±0.4 h*

t½=3.3±0.1 h

Cyloheximide Chase Experiment

TCDD

TCDD + Hg2+

Effect of Hg2+ on the stability of cyp1a1 protein

1000

100

10

1

0.1%

of c

yp1a

1 pr

otei

n re

mai

ning

* p<0.05

5 10 15 20 25

Time (h)

300

Korashy and El-Kadi. Toxicol Sci, 88:39-51, 2005

Induction of extrahepatic Drug Metabolism

-Induction depends not only on the nature of the inducing agent and the extrahepatic tissue but also on the particular drug substrate.

Ex. Induction of phenacetin and benzo[a]pyrene by cigaratesmoke

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Tissue-specific effects of polychlorinated biphenyl (PCB) mixtureon monooxygenase activities in rabbit liver, lung and kidney

Alvares et al., Life Sci 30:747, 1982

Constitutive Cytochrome P450 regulated by endogenous compounds

N.B. the Cytochrome P450 discussed below are not induced by xenobioticsAnd are essential to survival and reproduction of the organism.

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Induction of non-Cytochrome P450 Drug-Metabolizing Enzymes

Inhibition of Drug Metabolism

- Drugs or chemicals inhibit the metabolism of another drug; often competitive in nature.

- Some potent inhibitors of P450 form inactive complexes with the haemoprotein e.g. cimetidine.

- Inhibitors for nonmicrosomal enzyme: disulfiram inhibits alcohol and ALDH, carbidopa inhibits dopa decarboxylase, allopurinol inhibits xanthine oxidase and phenelzineinhibits Monoamine oxidase.

- Clinically important enzyme inhibitors are allopurinol, dicoumarol, isoniazid, clotrimazole, ketoconazole, cimetidine, chloramohenicol and oral contraceptives.

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Inhibition of Drug Metabolism

1. Competitive inhibition2. Destruction of hepatic Cytochrome P4503. Metal ions4. Forming inactive complexes with hepatic

cytochrome P4505. Miscellaneous drugs and xenobiotics

1. Competitive inhibition

Two drugs can compete for the same drug metabolizing enzyme depending on their relative affinities and inhibitory potencies and can result in inhibition of one of the drugs. Ex. Clotrimazole, Saquinavir and Nicardipine.

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2. Destruction of hepatic CytochromeP450

- Many therapeutic drugs and environmental xenobioticshave the ability to destroy Cytochrome P450 in the liver by a variety of mechanisms.

- Xenobiotics containing an olefinic (C=C) or aceylenic(C=C) functions are porphyrinogenic resulting in the formation of green pigments (substrate-heme adduct) in the liver.

- These olefinic and acetylenic compounds are inert and require metabolic activation by Cytochrome P450 therefore classified as “suicide substrates” of the hemeprotein.

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2. Destruction of hepatic Cytochrome P450

- Inhibition of drug metabolism by olefinic and acetylenicdrugs and xenobiotics dependes not only on the chemical nature of the drug it self but also on the prevailing complement of Cytochrome P450 enzymes and their substrate specificities.

- EX. Influence of allylisopropylacetamide (AIA) on the pharmacological activity of hexobarbitone and zoxazolamine.

477.8257.6Zoxazolamine paralysis time (min)

235.637.5Hexobarbitone sleeping time (min)AIAControlDrug

Unseld F and DeMatteis F, Int J Biochem 9:865, 1978.

Drugs and xenobiotics that destroy hepatic Cytochrome P450

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3. Metal ions

Metal ions such as cobalt exert their inhibitory influences on drug metabolism by:

1. Inhibiting the heme biosynthesis (inhibit aminolevulinicacid synthase).

2. Increasing the heme metabolism (increase hemeoxygenase activity).

Biosynthesis of hepatic Cytochrome P450 heme

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Effect of cobalt on hepatic drug metabolism and hemebiosynthesis

4. Forming inactive complexes with hepatic cytochrome P450

- These compounds are substrates of Cytochrome P450 and require metabolism to exert their inhibitory effect.

- The metabolic intermediate binds tightly to the hemeprotein and preventing its further participation in drug metabolism.

- The substrate-hemeprotein complexes exhibit spectral characteristics and normally absorb maximally at 448-456 nm with the reduced (ferrous) form of Cytochrome P450.

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Drugs and xenobiotics inhibiting drug metabolism by complexing with Cytochrome P450

Examples of Drugs inhibiting drug metabolism by complexing with Cytochrome P450. (a) Amphetamine, (b) methylenedioxybenzene.

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Examples of Drugs inhibiting drug metabolism by complexing with Cytochrome P450. Influence of triacetyl-Oleandomycin on hexobarbitone metabolism and sleeping time

5. Miscellaneous drugs and xenobiotics

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Biological Significance of Induction and Inhibition of Drug Metabolism

1. Drug tolerance2. Variability in drug response3. Drug-drug interactions4. Drug and chemical toxicity

Factors Affecting Drug Metabolism

I. Internal Factors:1. Species differences2. Age3. Hormonal control4. Disease states5. Genetic differences

II. External Factors:1. Dietary factors2. Environmental factors

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Internal Factors1. Species differences

A. Hexobarbitone metabolism

B. Caffeine metabolism

Internal Factors1. Species differences

C. Plasma levels of oxyphenbutazone

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Internal Factors1. Species differences

D. Phenol conjugation

Internal Factors2. Age differences

Development of Phase IA. Aromatic and aliphatic hydroxylation reactions e.g. aniline 4-hydroxylationB. N-demethylation reactionsC. Hydroxylation of 4-methylcoumarinD. Aryl hydrocarbon hydroxylase activity

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Internal Factors2. Age differences

Development of Phase IDevelopment of the steroid 16alpha-hydroxylase in male and female rat liver

Internal Factors2. Age differences

Development of Phase IN-demethylation is fully developed in fetal human and primate but O-demethylation arenot presentFetal CYP3A7 disappear at birth and replaced by the adult forms CYP3A4/5

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Internal Factors2. Age differences

Development of Phase II (in Rat)A. Enzymes belong to the late fetal cluster and metabolizes exogenous compoundsB. Enzymes belong to the late neonatal cluster and metabolizes endogenous compoundsN.B. in man little glucuronidation activity is seen until after birth)

Internal Factors3. Hormonal control

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Internal Factors3. Hormonal control

A. Sex differences:Effect of growth hormone on the expression of CYP2C11 and 2C12

Internal Factors3. Hormonal control

B. Adrenal glands:Effect of adrenalectomy and corticosteroid replacement therapy on the hepatic metabolism of ethylmorphine and aniline.

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Internal Factors3. Hormonal control

C. Thyroid gland:Effect of thyroidectomy on hepatic drug metabolism.

Internal Factors3. Hormonal control

D. Pancreas:Effect of streptozotocin-induced diabetes on hepatic drug metabolism.

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Internal Factors3. Hormonal control

D. Pancreas:Effect of streptozotocin and insulin treatment on blood glucose levels and N-deethylation of lignocaine in rat liver.

Internal Factors3. Hormonal control

E. Pregnancy:Effect of pregnancy on the metabolism of coumarin and progesterone in the rat.

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Internal Factors4. Disease states

Disease States that affect drug metabolism:1. Cirrhosis of the liver2. Alcoholic liver disease3. Liver carcinoma4. Infections and inflammation

Internal Factors4. Disease states

1. Cirrhosis of the liver:Cirrhosis appear to depress phase I but has no effect on glucuronidation.

Effect of liver cirrhosis on drug metabolism

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Internal Factors4. Disease states

2. Alcoholic liver disease:-Acute ethanol exposure: decreases phase I (inhibit electron flow from the

reductase) and II metabolism (increase NADH/NAD+, this inhibit the production of UDPGA which require NAD+)

- Chronic ethanol exposure: increase drug metabolism (CYP2E1)

Effect of chronic ethanolexposure on aniline 4-hydroxylase activity and total Cytochrome P450

Internal Factors4. Disease states

3. Liver Carcinoma:

Drug metabolism in hepatoma tissue and surrounding liver disease

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Internal Factors4. Disease states

3. Infection and inflammation:

• Infection and inflammation diminish the rate of cytochromeP450 (P450) mediated hepatic metabolism of drugs in humans and experimental animals.

• Influenza infection impairs theophylline clearance in humans.

• The inhibition of P450 appears to be mediated by cytokines.

Endotoxin Administration to Humans Inhibits Hepatic Cytochrome P450-mediated Drug Metabolism

Shedlofsky et al., J Clin Invest. 94:2209, 1994

Endotoxin Administration to Humans Inhibits Hepatic Cytochrome P450-mediated Drug Metabolism

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Effect of inflammation on the ability of hepatocytes (from control rabbit Hcont or rabbit with inflammatory reaction) to biotransform theophylline

HCONT HINFLA

Theo

phyl

line

met

abol

ites

(μg/

ml)

0.0

0.2

0.4

0.64.0

8.0

12.0

16.0 3 MX 1 MU 1,3 DMU

* *

*

HCONT HINFLA

P45

0 or

MD

A(n

mol

/mg

prot

ein)

0.00.20.40.60.81.01.21.41.6

*

*P450 MDA

*p<0.05 compared with HCONT

El-Kadi et al., Br J Pharmacol, 121:1164, 1997

Amount of 3 MX, 1 MU, and 1,3DMU recovered in a 24-h urinary collection from five volunteers with and without an upper respiratory viral infection after oral intake

of 300 mg of theophylline.

Bleau et al. Drug Met Dispos. 29:1007 , 2001

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II. External Factors:1. Dietary factors

A. Dietary nutrient:Effect of dietary nutrients on drug metabolism: + = increase, - = decrease

II. External Factors:1. Dietary factors

A. Dietary nutrient:Effect of essential fatty acid on a) aniline 4-hydroxylation and b) P450

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II. External Factors:1. Dietary factors

A. Dietary nutrient:Effect of high fat, fasting and diabetes on plasma acetone levels, hepatic anilinee 4-hydroxylation and CYP2E1

II. External Factors:1. Dietary factors

A. Dietary nutrient:Effect of RAR and RXR on Cytochrome P450 levels

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II. External Factors:1. Dietary factors

A. Dietary nutrient:Effect of Vitamin C deficiency on CytochromeP450 activities and levels

II. External Factors:1. Dietary factors

B. Dietary non-nutrient:Effect of dietary non-nutrient on drug metabolism

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II. External Factors:1. Dietary factors

B. Dietary non-nutrient:Effect of smoking on phenacetin metabolism

II. External Factors:Environmental factors

A. Heavy metals:Effect of cadmium on hepatic drug metabolism in male and female rats

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II. External Factors:Environmental factors

A. Heavy metals:Effect of cadmium and testosterone treatment on hepatic drug metabolism in male rats

II. External Factors:Environmental factors

B. Industrial Pollutants:Such as TCDD, solvents, PCB, pesticides

Effect of xylene on hepatic drug metabolism

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II. External Factors:Environmental factors

B. Industrial Pollutants:

Effect of mirex, kepone, 3-methylcolanthrene (3MC) and phenobarbitone (PB) on hepatic drug metabolism