BIOCHEMICAL MECHANISMS OF DRUG TOXICITIES

Lance R. Pohl, Pharm.D., Ph.D.

Chief, Section of Molecular and Cellular Toxicology Laboratory of Molecular Immunology

pohll@nih.gov

1/4/2007

U.S. Department of Health and Human

Services

National Institutes of Health

National Heart, Lung, and Blood Institute

TOXICITIES PRODUCED BY DRUGS

Anaphylaxis

Hemolytic anemia

Granulocytopenia

Thrombocytopenia

Aplastic anemia

Vasculitis

Hepatic

Skin

Renal

Pulmonary

Cardiac

Neurological

Lupus

LEADING CAUSES OF DEATH IN USA IN 1994

Heart disease Cancer Stroke Adverse drug reactions Pulmonary disease Accidents Pneumonia Diabetes

Lazarou et al., JAMA, 279, 1208 (1998)

743,460 529,904 150,108 106,000 101,077 90,523 75,719 53,894

TYPE A ADRs

• Accounts for 80% of ADRs• Dose-dependent and often predictable • Often due to excessive expression of

known pharmacologic effects• Often caused by drug-drug interactions• Often reproduced in animals

Role of transporters in drug interactions, C.J. Endres, et al., European Journal of Pharmaceutical Sciences, 27, 501 (2006)

EXAMPLES OF TYPE A ADRS

• Drowsiness from the use of antihistamines

• Exacerbated therapeutic responses of warfarin and phenytoin-low therapeutic indices

• Posicor, anti-hypertensive withdrawn from market because inhibited the metabolism of over 30 drugs

• Acetaminophen• Fialuridine

FIALURIDINE-INDUCED MITOCHONDRIAL INJURY IN PATIENTS

• FIAU is a uridine analog developed for hepatitis B treatment

• Administration to 15 patients resulted in 7 developing severe mitochondrial liver damage with 5 dying and 2 receiving liver transplants

• Toxicity was not predicted from rodent studies• Toxicity of FIAU is apparently due to FIAU-TP which

inhibits mitochondrial DNA polymerase-γ and DNA synthesis

• Humans and not rodents have human nucleoside transporter 1 (hENT1) in the mitochondrial membrane

E.W. Lee, et al., J.Biol.Chem., 281, 16700 (2006)

TYPE B ADRs

• Accounts for 20% of ADRs• Rare, unpredictable, and highly host-

dependent• Mechanisms usually unknown, but in

some cases they have been attributed to: Allergic reactions Pseudoallergic reactions Deficiency in cytoprotective factors Polymorphisms

• Rarely reproduced in animals

HAPTEN HYPOTHESIS AND DRUG-INDUCED ALLERGIC REACTIONS

+ Drug orMetabolite

B and T C ell R esponsesT oxicity

FORMATION OF PROTEIN ADDUCTS WITHOUT DRUG METABOLISM

R CH2

Penicillins

R CH2 CONH CH

CO

NH

R1 CONH

O

R1 CONH CHS

HNR2

COOH

CO

NH

N

S

R2

COOH

CONH

ON

S CH3

CH3

COOH

HN

S

COOH

CH3

CH3

Cephalosporins

Penicillamine

HOOC C

NH2

H

C

CH3

SH

CH3 HOOC C

NH2

H

C

CH3

S

CH3

Cys

S

Protein

Protein

Lys

Protein

Lys

Captopril

N

COOH

C

O

CH

CH3

CH2 SH

N

COOH

C

O

CH

CH3

CH2 S S Cys Protein

ROLE OF IMMUNE SYSTEM IN INITIATING DRUG-INDUCED ALLERGIC REACTIONS

Drug Protein Conjugate Formed in a Cell

Secreted

Injured Cell

Immature Dendritic Cell

Ag Processing

Ag Presentation byMHC Class 1 and 2

CD8+

CD4+ CD4+CD8+

HSPs, INF-,TNF-, IL-1

Migrate to Lymph Nodes Immunization of T Cells

Migrate to Lymph NodesTolerization of T Cells

IL-10, PGE2,TGF-

Regulatory T CellsMigration to Periphery Block Drug Allergy

Migration to Periphery Drug Allergy

B Cell

IgG, IgE,IgA

PATHWAYS OF IMMUNOPATHOLOGY

DRUG-INDUCED LIVER DISEASE IS A MAJOR HEALTH PROBLEM

It is a major cause of acute liver failure and a major safety reason for:

• Stopping preclinical development of drugs

• Terminating clinical trials of drugs • Withdrawing drugs postmarketing

F. Ballet, J. Hepatol., 26 (Suppl. 2), 26 (1997)

LIVER DISEASE IS CAUSED BY MANY COMMONLY USED CLASSES OF DRUGS

• Antihypertensives• Lipid-lowering

agents• NSAIDs• Antimicrobials• Antidiabetic agents

• Anticonvulsants• Psychotropic

agents• Inhalation

anesthetics• Herbal medicines

DRUGS WITHDRAWN / NOT APPROVED DUE TO LIVER DISEASE

Iproniazid Ibufenac (Europe)Ticrynafen Benoxaprofen Perhexilene (France)Dilevalol (Portugal and Ireland)Bromfenac Troglitazone Nefazodone (Serzone)Ximelagatran (Exanta)

1956197519801982198519901998200020032004

Initiation Progression Secondary Injury Repair

COMPLEXITY OF DRUG-INDUCED LIVER DISEASE

Reactive Metabolites

Protein Adducts

ROS, RNS

Injury

Innate Immune System Activation

Dendritic Cells

Kupffer Cells

NK and NKT Cells, Monocytes, PMNs, and Eosinophils

Adaptive Immune System Activation

Effector Abs and T Cells

Death

Release of Protein Adducts

Release of Adjuvants

PATHWAY OF HALOTHANE-INDUCED ALLERGIC HEPATITIS

P 4 5 0

H e p a to c yte

H u m o ra l a n d C e llu la r Im m u n e R e s p o n s e s

H a lo th a n e T riflu o ro a c e ty l C h lo rid e

T o x ic ity

C

F

C

F

H

C l

B rF C

F

C

F

F C l

OC F

F

C

O F

HALOTHANE HEPATITIS PATIENTS’ SERUM ANTIBODIES (% REACTIVITY)

Antigen TFA-Protein Native-Protein

PDI 10 5

PDI isoform 55 25

Carboxylesterase 13 5

Calreticulin 5 3

ERP72 30 25

GRP94 65 28

CYP2E1 45

OTHER FLUORINATED INHALATION ANESTHETICS FORM IDENTICAL

PROTEIN ADDUCTS

H a lo th a n e

C

F

C

F

H

C l

B rF C

F

O

F

HCC

H

C lF

F

F

C

F

O

F

HCC

H

FF

F

F

Is o flu ra n e D e s flu ra n e

H e p a to c y te

C F

F

C

O F

P 4 5 0

CYP AUTOANTIBODIES ASSOCIATED WITH OTHER DRUGS CAUSING ALLERGIC HEPATITIS

Drug

Tienilic acid

Dihydralazine

Ethanol

Antigen

CYP2C9

CYP1A2

CYP2E1, CYP3A4, CYP2E1-hydroxyethyl radical

T CELL REACTIVITY ASSOCTIATED WITH DRUGS CAUSING ALLERGIC HEPATITIS

CotrimoxazoleErythromycinKetoconazoleAmpicillinAllopurinolIbuprofenCaptopril-MethyldopaEnalapril

Maria and Victorino, Gut, 41, 534 (1997)

Chlorpromazine

Amineptine

Dothiepine

Phenytoin

Carbamazepine

Tamoxifen

Glibenclamide

Lovastatin

Propylthiouracil

MECHANISM OF ACETAMINOPHEN (APAP) HEPATOTOXICITY

SERUM CYTOKINE LEVELS AFTER APAP TREATMENT OF MICE

Time (hr)

4 8 12 240

3000

6000

9000

12000

15000ALT

IU/L

4 8 12 240

100

200

300IL-4

pg/m

L

4 8 12 240

100

200

300IL-10

pg/m

L

4 8 12 240

100

200

300

400

500IL-13

pg/m

L

IL-6

4 8 12 240

40

80

120

160

200

pg/

mL

IL-10 DEFICIENCY INCREASES APAP-INDUCED LIVER INJURY AND DEATH

Time (hours)

0 10 20 30 40 50

Su

rviv

al

(%)

0

20

40

60

80

100

Wild Type IL-10 KO

APAP-LIVER NECROSIS IN IL-10-/- MOUSE

IL-10 REGULATES iNOS L

iver

iNO

S m

RN

A L

evel

(Arb

itra

ry U

nit

)

0.00

0.25

0.50

0.75

*

Wild-Type Knockout

Wild-Type Knockout

IL-10/iNOS KO MICE ARE NO MORE SUSCEPTIBLE TO APAP THAN WT

MICE

A B C D0

1000

2000

3000

4000

5000

*A= Wild Type APAPB= IL-10 KO APAPC= iNOS/IL-10 KO APAPD= iNOS KO APAP

AL

T (

IU/L

)

• Diclofenac: G. P. Aithal et al., Gastroenterology, 118(4), 1077, Part 1, Supplement. 2. April 2000

• Ethanol: J. Grove et al., Gut 46, 540-545 (2000)

IL-10 PROMOTER POLYMORPHISMS IN DRUG-INDUCED LIVER DISEASE

OTHER HEPATOPROTECTIVE FACTORS UNCOVERED WITH KNOCK OUT MICE

• IL-6: liver regeneration, prevents apoptosis, induces HSPs

• COX-2: prostaglandin products induce HSPs and prevent inflammation

• IL-4 and IL-13: mechanisms not yet defined• Nrf2: regulates induction of GSH synthesis,

UGTs, GSTs, NADPH quinone reductase, heme oxygenase, catalase

• CCR2: down regulates INF-γ and TNF-α

Y. Masubuchi K. Chan, et. al., PNAS, 98, 4611 (2001); C.M. Hogaboam, et. al., Am. J. Pathol., 156, 1245 (2000).

NIH FUNDS NETWORK TO STUDY DRUG-INDUCED LIVER INJURY (DILIN)

• Sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) in 2004

• The DILIN centers are located at U. of North Carolina, Duke, U. of Michigan, U. of Connecticut, and U. of California in SF

• The goal is to establish a registry of patients who have experienced severe drug-induced liver disease and collect biological samples for biochemical, serological, and genetic testing by investigators throughout the country

DRUG-INDUCED CUTANEOUS ADRS

• Exanthems-95% are erythematous or maculo-papular in nature and are usually self-limiting when the drug is withdrawn, Steven-Johnson syndrome and toxic epidermal necrolysis are life-threatening

• Exanthems from β-lactam antibiotics are often mediated by drug-specific IgE antibodies while many others by CD4+ and CD8+ T cellsN. Yawalkar, Toxicology, 209, 131(2005)

T CELL REACTIVITY TO DRUGS CAUSING CUTANEOUS ADRS

• Lidocaine• Sulfonamides -Lactam antibiotics • Phenytoin• Carbamazepine

Lebrec et al., Cell Biology and Toxicology, 15, 57 (1999)

MACULO-PAPULAR EXANTHEM AND TOXIC EPIDERMAL NECROLYSIS

MECHANISM OF T-CELL MEDIATED CUTANEOUS ADRS

S. Roychowdhury and. C.K. Svensson, The

AAPS Journal, 7, E834 (2005)

MECHANISMS OF DRUG-INDUCED IMMUNE-MEDIATED BLOOD DYSCRASIAS

D rug

Anti-D rugAb

B ind and activatecom plem ent

B ind to Fc receptoro f m acrophages

C ell lys is

Im m uneC om plex

Lysis by com plem entand /or m acrophages

HAPT EN M EC HANISM

IM M UNE C O M PLEX M EC HANISM

AUT O ANT IB O D Y M EC HANISM

AutoAbLysis by com plem entand /or m acrophages

DRUG-INDUCED IMMUNE-MEDIATED HEMOLYTIC ANEMIA

Hapten Mechanism

Immune Complex Mechanism

Autoantibody Mechanism

Ampicillin Diclofenac Cephalosporins

Carbenicillin Isoniazid Diclofenac

Penicillin Tetracycline Levodopa

Methicillin Quinidine Methyldopa

Cephalosporin Thiopental Procainamide

Tetracycline Chlorpromazine Nomifensine

Tolbutamide Nomifensine TolmentinT. Deloughery, Immunol. Allergy Clin. of North Am. 18, 829 (1998)

Lists drugs causing aplastic anemia, agranulocytosis, and thrombocytopenia

IgE-MEDIATED ANAPHYLACTIC DRUG REACTIONS

Alcuronium

Cephalosporins

Penicillins

Protamine

Streptokinase

Park et al., Chem. Res. Toxicol., 11, 969 (1998);

B.Y.H. Thong and Y. Chan, Ann. Allergy Asthma Immunol., 92, 619 (2004).

Sulfamethoxazole

Suxamethonium

Thiopentone

Trimethoprine

Tubocurarine

MECHANISM OF DRUG-INDUCED ANAPHYLAXIS

IgE-secretingplasm a cell

M ast cell or basophil

Drug

1. Airway sm ooth m uscle contractionleading to bronchospasm2. Increase perm eability of b lood vesselsand m ucous gland secretion3. Inflam m ation (eosinophils andneutrophils)4. Respiratory, gastrointestinal, cutaneous,and cardiovascular system s can be involved

Histam ine, leukotrienes, and cytokines

ASSOCIATION OF GENETIC VARIANTS OF HLA-B AND HSP70 WITH HYPERSENTIVITY

TO ABACAVIR

• Abacavir is a nucleoside reverse transcriptase inhibitor used to treat HIV type 1

• Approximately 5% of patients who receive abacavir develop a treatment-limiting hypersensitivity reaction, characterized by fever, rash, gastrointestinal symptoms (nausea, vomiting, diarrhea or pain) and lethargy or malaise

• Median time to onset is 11 days, but symptoms occur within hours when patients are rechallenged

• Susceptibility is associated with specific variants of HLA-B and HSP70 and CD8+ T cells that are activated in presence of abacavir to produce TNF

A.M. Martin, et. al., PNAS, 101, 4180 (2004)

PSEUDOALLERGIC (ANAPHYLACTOID) REACTIONS WITH NSAIDS

• Most cases occur with aspirin, although other NSAIDs can be involved except for COX-2 inhibitors

• Reactions include asthma, rhinitis, urticaria and/or anaphylactic shock.

• Not mediated by IgE antibodies• COX-1 inhibitors may divert arachidonic

acid metabolism to 5-lipoxygenase pathways and formation of proinflam-matory cysteine-leukotrienes

PSEUDOALLERGIC (ANAPHYLACTOID) REACTIONS WITH NSAIDS

• Leukotrienes cause bronchial smooth muscle contraction, mucous secretion, vasodilation, and cellular infiltration

• Cysteine-leukotriene receptor 2 poly-morphisms associated with aspirin intolerance

J.S. Park et al., Pharmacolgenics and Genomics, 15, 483 (2005)

ANAPHYLACTOID REACTIONS WITH IODINATED RADIOCONTRAST AGENTS

• Reactions can be similar to aspirin and include asthma, rhinitis, urticaria, gastrointestinal symptoms, and/or anaphylactic shock consisting of hypotension, pulmonary edema, respiratory arrest, cardiac arrest, and convulsions

• Mechanisms?

J. Szebeni, Toxicology 216, 106 (2005)

MECHANISMS OF REACTIONS TO RADIOCONTRAST REAGENTS

ENZYME POLYMORPHISMS AND TYPE B ADVERSE DRUG REACTIONS

• Sensitivity to warfarin and phenytoin due to metabolic deficiency caused by CYP2C9*3Rettie et al., Epilepsy Research, 35, 253 (1999); Brandolese et al., Clin. Pharmacol. Ther., 70, 391 (2001)

• Irinotecan side effects of serious and potentially fatal diarrhea and neutropenia are associated with metabolic deficiency caused by UGT1A1*28 and other allelic forms of UGT1A

Han et al., J. Clin. Oncol., 24, 2237 -2244 (2006)

THIOPURINE-S-METHYLTRANSFERASE POLYMORPHISMS AND TYPE B ADR

• Thiopurine drugs 6-mercaptopurine and aza-thioprine are used to treat acute lymphoblastic leukemia in children, inflammatory bowel disease, autoimmune disease, and organ transplant recipients

• Polymorphisms of TPMT that lead to low enzyme activity result in life-threatening pancytopenia

L. Wang and R. Weinshilboum, Ongene, 25, 1629-1638 (2006)

THIOPURINE METABOLISM RESULTS IN THERAPEUTIC ACTIVITY AND TOXICITY

DRUG-INDUCED-LONG QT SYNDROME

• Results in prolonged ventricle repolarization that can lead to polymorphic ventricular tachycardia (torsade de pontes), and ultimately ventricular fibrillation and death

• Rare, unpredictable, and a major reason for withdrawal or restriction of many drugs

• Antiarrhythmic drugs such as quinidine, procainamide, sotalol and others

• Other classes of drugs cause this syndrome including astemizole (antihistamine), terfenadine (antihistamine), mibefradil (anti-hypertensive), and grepafloxacin (antibiotic)

• Linked to genetic variants of drug-metabolizing enzymes• Linked to cardiac K ion channel subclinical mutations

P.J. Kannankeril and D.M Roden, Current Opinion in Cardiology, 22, 39 (2007)

Summary

• Drug-drug interactions are a major cause of Type A ADRs and are in many cases predictable

• Type B ADRs may be caused by rare allelic forms of enzymes, receptors, ion channels, transporters, transcription factors, etc

• Many Type B ADRs also appear to be mediated by specific Abs and T cells that are induced by protein adducts of reactive metabolites

• It is likely that numerous factors protect most people from getting Type B ADRs and rare polymorphisms of one or more of these factors determine individual susceptibility

• Toxicogenomics and proteomics should provide new information about other important regulatory factors that normally protect cells from TYPE B ADRs

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REFERENCES

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REFERENCES

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