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• Posicor, anti-hypertensive withdrawn from market because inhibited the metabolism of over 30 drugs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