# Presentation: 394453

## 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**

## 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)

**Notes:**
TRANSPORTER PROTEINS PLAY IMPORTANT ROLE IN THE ABSORPTION, DISTRIBUTION, AND ELIMINATION OF A VARIETY OF DRUGS.
THIS REVIEW SUGGESTS DRUG-DRUG INTERACTIONS MAY OCCURS AT THE TRANSPORTER LEVEL

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**Posicor, anti-hypertensive withdrawn from market because inhibited the metabolism of over 30 drugs**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

**Notes:**
Drowsiness and benadryl;

## 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)

**Notes:**
FIAU CAUSED SEVERE MULTISYSTEM TOXICITY INVOLVING THE LIVER, PANCREATITIS, NEUROPATHY, OR MYOPATHY.
ONCE IN MITOCHONDRIA FIAU IS METABOLIZED BY THYMIDINE KINASE 2, A MITOCHONDRIAL SPECIFIC ENZYME, TO THE MONOPHOSPHATE THAT IS THE RATE-LIMITING STEP IN THE FORMATION OF THE TRIPHOSPHATE, A POTENT INHIBITOR OF MITOCHONDRIAL DNA POLYMERASE-GAMMA
RODENTS HAVE THE TRANSPORTER ONLY IN THE PM WHILE HUMANS HAVE IT IN PM AND M MEMBRANES.  RODENT hENT1 DOES NOT HAVE THE PROPER TARGETING SEQUENCE TO BE INCORPORATED INTO THE MIT0CHONDRIAL MEMBRANE.
hENT1 PLAYS IMPORTANT ROLE IN MODULATING ACTIVITY OF OTHER NUCLEOSIDE DRUGS INCLUDING DRUGS FOR TREATMENT OF CANCER (5-FLUOROURACIL) AND VIRAL DISEASE (RIBAVIRIN) 

## 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

**Notes:**
MENTION SYMPTOMS INDICATING ALLERGIC REACTION

## FORMATION OF PROTEIN ADDUCTS WITHOUT DRUG METABOLISM

**Notes:**
Penicillamine used for chelation therapy of heavy metals (and Wilson’s disease) and rheumatoid arthritis; causes cutaneous diseases and lupus erythematosus;

Captopril is a angiotensinogen II converting enzyme inhibitor; it causes cough is 10-20 % patients and rarely liver, skin, and anaphylaxis

## ROLE OF IMMUNE SYSTEM IN INITIATING DRUG-INDUCED ALLERGIC REACTIONS

## PATHWAYS OF IMMUNOPATHOLOGY

**PATHWAYS OF IMMUNOPATHOLOGY**

**Notes:**
Cytotoxic T cells can kill by granules containing perforin (pore-forming, similar structurally and functionally to C9) and granzymes (collection of serine esterases), causing apoptosis via fas ligand and TNF-alpha on surface, or via release of cytokines such as INF-gamma which activates macrophages.
ADCC can be mediated by macs, NKs, and PMNs which all have Fc receptors 

## 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)**

**Notes:**
DILD and Idiopathic causes are major causes of acute liver failure, not hepatitis A and B.  It has been estimated that 2000 deaths per year due to acute liver failure and they account for at least 5% of of liver transplantations.

## 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)****	**

**1956**
**1975****	**
**1980**
**1982**
**1985**
**1990**
**1998**
**2000**
**2003**
**2004 **

**Notes:**
Serzone (nefazodone)-anti-depression drug made by BMS.
Exanta oral thrombin anticoagulant inhibitor – Astra Zeneca, voted against approval for long-term prevention of stroke and other venous thromboembolitic conditions resulting from atrial fibrillation; incidence was 1/2300. Approved in some European Countries

Perhexilene is cardiac vasodilator used to treat angina.

## COMPLEXITY OF DRUG-INDUCED LIVER DISEASE

**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

## 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 |

**Notes:**
Mention that sulfonamides antibodies, autoantibodies against PDI and GRP78 and one patient had antibodies against hapten; anti-sulfamethoxazole sera reacted with all proteins that autoantibodies were directed against....Allaistaire Cribb

## OTHER FLUORINATED INHALATION ANESTHETICS FORM IDENTICAL PROTEIN ADDUCTS

## 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

**Cotrimoxazole****	**
**Erythromycin**
**Ketoconazole**
**Ampicillin**
**Allopurinol**
**Ibuprofen**
**Captopril**
******-Methyldopa**
**Enalapril**
**Maria and Victorino, Gut, 41, 534 (1997)**

**Chlorpromazine**
**Amineptine**
**Dothiepine**
**Phenytoin**
**Carbamazepine**
**Tamoxifen**
**Glibenclamide**
**Lovastatin**
**Propylthiouracil**

**Notes:**
56% positive reactivity with prostaglandin inhibitor present in lymphocyte proliferation assay reaction mixture

## MECHANISM OF ACETAMINOPHEN (APAP) HEPATOTOXICITY

## SERUM CYTOKINE LEVELS AFTER  APAP TREATMENT OF MICE

**Notes:**
IL-4 and IL-13 inhibit ischemic-reperfusion liver injury via activating STAT6, which results in decrease in TNF-alpha  (IL-13 also decrease MIP-2, macrophage inflammatory protein 2, chemokine) and neutrophil infiltration.  IL-10 not prevent ischemic-reperfusion liver injury in mice.  IL-13 protection due in part to protecting endothelial cells from damage in this model via IL-13 KO study, as measured by a decrease in hyyaluronic acid in serum.  IL-13 also shown to protect hepatocytes in vitro against hydrogen peroxide-induced damage.

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

**Notes:**
Found that TNF-alpha and IL-1 alpha levels went up significantly in IL-10 KO mice after APAP, but not in WT mice treated with APAP

Known that IL-10 regulates iNOS and NO reported to possibly have a role in APAP toxicity

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

## IL-10 REGULATES iNOS 

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

**Notes:**
150 mg/kg

## IL-10 PROMOTER POLYMORPHISMS IN DRUG-INDUCED LIVER DISEASE

- 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).

**Notes:**
UGT-UDP-glucuronosyl transferase; CCR2, C-C chemokine receptor 2; mention INF-gamma KO mice less susceptible; NK-Tcells involved

NRF2 and antioxidant responsive element (ARE).

## 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 cells**
- 	N. Yawalkar, Toxicology, 209, 131(2005)

**Notes:**
EXANTHEMS-DEFINED AS ERUPTIONS ON THE SKIN
ACCOUNT FOR 2%-3% OF HOSPITALIZEDS
MENTION THAT VIRTUALLY ALL KNOWN CUTANEOUS DISEASES ARE ALSO INDUCED BY DRUGS
MACULA-COLORED SPOT NOT ELEVATED
PAPULAR-CROPS OF SMALL URTICARIAL PAPULAE AND WHEALS

## 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

**Notes:**
Mentin that can have similar reactions with other tissues

## 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 | Tolmentin |
| T. 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

**Notes:**
Mention that similar reactions could cause skin urticaria and other forms of dermititis. Release of histamine, leukotrienes LTC4, D4, and E4, and inflammatory cytokines leading to eosinphil and neutrophil infiltration.

Mention skin reactions at last because good transition to T cell-induced dermatitis

## 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)**

**Notes:**
HLA-B*5701 and HSP70-Hom variant. Some patients also have respiratory symptoms

## 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**

**Notes:**
Stated in one paper that PGE2 inhibits LT synthesis.  Also Leucotrienes can cause increase in bronchial smooth muscle contriction, vasodilation, mucous secretion, and cellular infiltration.  It is also know (reference I have not used yet) that Aspirin worsens chronic indiopathic urticaria which is associated with autoantibodies against the alpha chain of the high affinitiy IgE receptor present on mast cells and serum levels of mast cell trypase, i.e., activated mast cells.

## 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)

**Notes:**
Mention lesser degrees of Symptoms.  Nephrotoxicity is also associated with these drugs
Susceptible patients maybe pretreated 1 hour before treatment with prednisone and diphenhydramine.

## MECHANISMS OF REACTIONS TO RADIOCONTRAST REAGENTS

**Notes:**
FIGURE REPRESENTS MAST CELL OR BASOPHIL
Anaphylatoxin receptors C3aR and C5aR
C-complement activation

## ENZYME POLYMORPHISMS AND TYPE B ADVERSE DRUG REACTIONS

**Sensitivity to warfarin and phenytoin due to metabolic deficiency caused by CYP2C9*3**
**	**Rettie *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)** **

**Notes:**
IRINOTECAN COMMONALY PRESCRIBED FOR COLORECTAL AND LUNG CANCER
It IS A PRODRUG CONVERTED TO AN ACTIVE METABOLITE BY A CARBOXYLESTERASE WHICH IS 100-1000 TIME MORE CYTOTOXIC THAN PARENT DRUG, WHICH IS CONVERTED IN AN INACTIVE METABOLITE BY UGTS.

## 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)

**Notes:**
NAT2 polymorphism can lead to hydralazine and procainamide-induced immune complex disease. 
Procainamide is an antiarrhythmic drug.

Thiopurine S-methyltransferase  polymorphism can lead to bone marrow suppression (myelosuppression) of thiopurine containing drugs used for acute lymphoblastic leukaemia in children, inflammatory bowel disease, and organ transplant patients.

## THIOPURINE METABOLISM RESULTS IN THERAPEUTIC ACTIVITY AND TOXICITY

**Notes:**
XO-XANTHINE OXIDASE
AO-ALDEHYDE OXIDASE
TPMT-THIOPURINE S-METHYLTRANSFERASE
HPRT-HYPOXANTHINE GUANINE PHOSPHORIBOXYLTRANSFERASE
6-MP MAINLY ELIMINATED BY XO AND AO EXEMPT IN BONE MARROW WHERE TPMT
METABOLIC ACTIVATION OF 6-MERCAPTOPURINE TO 6-THIOGUANINE NUCLEOTIDES

## 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)

**Notes:**
DRUG-INDUCED PROLONGED QT SYNDROME MORE COMMONLY SEEN WITH ANTIARRHYTHMIC DRUGS

##    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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