# Presentation: 316427

## Identifying susceptible persons with markers

**Paul B. Watkins**
**University of North Carolina**
**Chapel Hill, N.C.**

## SAFE

**Not safe**

**SAFE**

**Concept of idiosyncratic reaction**

## Hypothesis:

- <date/time>

**Hypothesis:**

**Genetic factors largely account for an individual’s susceptibility to an idiosyncratic toxicity**** **

**Therefore, molecular diagnostics could **
**identify patients at risk for toxicity.**

## Science:294,2274,2001

## 
FDA Working Group on Pharmacogenetics and Pharmacogenomics 

Subcommittee of the Advisory Committee for Pharmaceutical Science, 
October 23, 2002. 

- <date/time>

**FDA Working Group on Pharmacogenetics and Pharmacogenomics **
**Subcommittee of the Advisory Committee for Pharmaceutical Science, **
**October 23, 2002. **

**Sympathetic Regulatory Climate**

## Topic No. 3

- <date/time>

**Topic No. 3**
**Scientific and Practical Considerations in the Use of Pharmacogenetic Tests to Determine Drug Dosage and Administration**
*“ ****It sounds like, for TPMT... there is considerable enthusiasm and considerable use of having these genetic tests available...”***
**	****	****Bill Jusko, Acting Chair.**

**http://www.fda.gov/ohrms/dockets/ac/02/transcripts/3898T1.htm**

## 1). Candidate gene/functional polymorphism
		one gene – one hypothesis

- <date/time>

**Three approaches**

**1). Candidate gene/functional polymorphism**
**	****	*****one gene – one hypothesis***

**2). Candidate gene/linkage disequilibrium**
**	*****multiple genes – multiple hypotheses***

**3). Genome-wide linkage disequilibrium**
**	*****every gene – no hypotheses***

## 1). Candidate gene/functional polymorphism
		one gene – one hypothesis

- <date/time>

**Three approaches**

**1). Candidate gene/functional polymorphism**
**	****	*****one gene – one hypothesis***

**2). Candidate gene/linkage disequilibrium**
**	*****multiple genes – multiple hypotheses***

**3). Genome-wide linkage disequilibrium**
**	*****every gene – no hypotheses***

**Use of Tranilast to treat restenosis after percutaneous transluminal coronary revascularisation**
**Double-blind placebo trial of 11,500 patients**
**4% of patients in the trial developed unconjugated hyperbilirubinemia**

**Example of ****Candidate Gene Approach**** **
**2001 GSK PRESTO Trial**

## Gilbert’s Syndrome

**First described in 1901 by Gilbert and Lereboullet **
**Mild, chronic hyperbilirubinemia**
**Absence of liver disease**
**Hepatic glucuronidating activity reduced **
**	****by @ 30%**
**Not life threatening**

## Genetics of Gilbert’s Syndrome

**	****polymorphic (TA)****n**** repeat element in the TATAA box of UDP-glucuronosyltransferase 1 (UGT1A1)UGT1A1**

***Bosma et al,***** *****NEJM,333:1171,1995***** **

## Results

**92% of Tranilast treated patients with hyperbilirubinemia were 7/7 -  none were 6/6  **
**Subjects with 7/7 who received placebo did not have elevations in baseline bilirubin**
**50% of 7/7 treated with Tranilast did not have hyperbilirubinemia**

*Nature Reviews, July 2002*

## Clinical Characteristics of Drug Induce Liver Injury (DILI)

**Many different types of injury including steatosis due to impaired ******** -oxidation, phospholipidosis, cholestatic injury, “chronic occult ” injury (methotrexate).****   **

## Liver problems post marketing (1995-2000)

- <date/time>

**Liver problems post marketing ****(1995-2000)**

**Second Line**
**pemoline**
**tolcapone**
**trovafloxacin**
**felbamate**

**Strong Warning**
**valproic acid**
**ketoconazole**
**nictonic acid**
**acetaminophen**
**chlorzoxazone**
**isoniazid**
**dantrolene**
**rifampin**

**Withdrawals**
**bromfenac**
**troglitazone****	**

## DILI of major concern in drug development is:

** ****Hepatocellular (acute)**
** ****Idiosyncratic****   **

## Acute idiosyncratic hepatocellular injury

- <date/time>

**Acute idiosyncratic hepatocellular injury**

**1). Elevation in serum ALT and AST ****	****(delayed)**

**	**
**2).  Serum bilirubin elevation means ****	****life-threatening injury**

## SAFE

**liver failure**

**SAFE**

**Increased ALT**

**Concept of idiosyncratic hepatocellular injury**

## Zileuton

- <date/time>

**Zileuton**

**     ****NDA           target       population     3 X ULN**
**	**
**   ****~1996    ****	****5-LO****	****       Asthma****	****        3.5%***

**     ****ALT >**

*** *****Single case of hepatocellular jaundice***

## Zileuton Phase # 3 Safety Trial

- <date/time>

**Zileuton Phase # 3 Safety Trial**

**12-month surveillance study of approx. 2500 treated and 500 control patients**
**ALT monitored at months 1-5, 7, 10, 12**** **

## Zileuton Long-Term Surveillance Study

- Unpublished data

## Zileuton Metabolism

- 80%

**5-LO**
**pseudoperoxidase**

- Zileuton

**CYP1A2**

- gut bacteria

- reactive oxygen species (ROS)

- ring hydroxides

**CYP3A (4, 5, 7)**
**CYP2C (8, 9, 18)**

- R(+)/S(-) glucuronide

- sulfoxide

**CYP1A2**

**CYP3A (4, 5, 7)**
**CYP2C (8, 9, 18)**

- A-66193

- sulfoxide

- ring hydroxides

- extracellular transport by MRP2/cMOAT, deficiency in this transporter causes Dubin-Johnson hyperbilirubinemia, which is not consistent with the form of hepatotoxicity observed in Zyflo patients

- unknown subtype:
**UGT1**
**UGT2B4**
**UGT2B7**
**UGT2B10**
**UGT2B15**
**UGT2B17**

- reaction depends on
- cellular glutathione state

## Arachidonic Acid Metabolism

**calmodulins (I, II, III)***

- *	high calcium levels inhibit the effect of glutathione depletion on 5-LO induction
- ¶ 	elevated arachidonic acid has been shown to cause changes in rat hepatocytes consistent with hepatocellular injury

**annexin II, annexin III, calpactin I/annexin II ligand**

- stimulation

- inhibition

**lipocortin I/annexin I/calpactin II**

**cPLA2**

- cellular
- membranes

**5-LO**

- leukotriene A4
- (see “Leukotriene Pathway”)

**12-LO**

- arachidonate hydroxides

**CYP1A, CYP2C**

**CYP-AA/CYP2J2**

**COX 1,**
**COX 2**

**15-LO A.**
**15-LO B**

- 12(S)-hydroperoxy-5,8,10,14-cis-
- eicosatetrænoic acid (12-HPETE)

- 15(S)-hydroperoxy-5,8,11,13-cis-
- eicosatetrænoic acid (15-HPETE)

- epoxyeicosatetrænoic acids, lipoxins (may involve epoxide hydrolase and/or various CYPs)

- prostaglandin H2

- prostaglandin D2

- prostaglandin I2

- thromboxanes

**TXA****2**** synthase/CYP5**

**prostacyclin synthase/CYP8**

**PGH****2**
**reductase**

- inactivation/
- urinary excretion

**prostaglandin 15-OH**
**dehydrogenase**

- cytoplasmic arachidonic acid¶

- regulation

## Leukotrienes & Glutathione State

- glutathione depletion in leukocytes activates 5-LO,
- except when Ca++ is in excess

**G6PDH**, **phosphogluconate decarboxylase** & **malate decarboxylase** are cellular sources of NADPH

- Formation of ROS in activated eosinophils

**glutathione synthase**

**glutathione reductase**

- reduced glutathione

****** ****-glutaminyl-cysteine synthase**

- NADPH

- NADP+

**LTC****4**** synthase** -or-
**microsomal GST**

- leukotriene A4

- arachidonic acid

**5-LO**

**FLAP** is a necessary partner

- ROS product of 5-LO pseudoperoxidase action on Zyflo is most likely detoxified via glutathione
- (see “Zyflo Metabolism”)

- Zyflo is both an inhibitor and an alternate substrate

**LTA****4**** hydrolase**

- leukotriene B4

- mediates inflammation, leukocyte recruitment & activation

**LTB****4**** 12-OH dehydrogenase**
**LTB****4**** ********-hydroxylase/CYP4F2**

**LTB****4**
**receptor**

- inactivation, excretion

**renal dipeptidase**

- leukotriene E4 (-Gly)

- leukotriene D4 (-Glu)

****** ****-glutaminyl transpeptidase/ glutaminyl cleaver**

- leukotriene C4

- mediate allergy, bronchoconstriction & vascular permeability

## 1). Candidate gene/functional polymorphism
		one gene – one hypothesis

- <date/time>

**Three approaches**

**1). Candidate gene/functional polymorphism**
**	****	*****one gene – one hypothesis***

**2). Candidate gene/linkage disequilibrium**
**	*****multiple genes – multiple hypotheses***

**3). Genome-wide linkage disequilibrium**
**	*****every gene – no hypotheses***

## Abbott-Genset Zileuton Genetics Project

**Selected 37 candidate genes**
**Zileuton metabolism & mechanism**
**Hepatic homeostasis**
**200 markers (4 - 6 SNPs / candidate gene)**
**69 cases & 104 controls genotyped**

## Single Nucleotide Polymorphisms (SNPs)

- <date/time>

**Single Nucleotide Polymorphisms (SNPs)**

***Key Concepts:***

**1). >99.9% of DNA sequence is identical in **
**	****all human beings.**

**2). The majority of the <0.1% differences **
**	****involve single nucleotide substitutions **
**	****at fixed locations in the genome.**

## Single Nucleotide Polymorphisms (SNPs)

- <date/time>

**Single Nucleotide Polymorphisms (SNPs)**

***Key concepts continued:***

**3). A principle called “linkage disequilibrium” makes it possible to use SNPs to identify regions of the genome that may contain genes relevant to susceptibility.**
**	**

## Concept behind SNP profile

**candidate gene**

**SNPs**

## Zieluton Project: Lessons Learned

**Strongest evidence for association with:**
**Two genes in zileuton metabolism pathway**
**Two genes in arachidonic acid metabolism pathway**
**Unconfirmable due to lack of additional case samples**
**Utility unclear**

## Drug

- <date/time>

**Drug**

**elimination**

**Reactive **
**Metabolite**

**Necrosis**

**Safe pathways**

**many**
**enzymes**

**many enzymes**

**Apoptosis**

## 1). Candidate gene/functional polymorphism
		one gene – one hypothesis

- <date/time>

**Three approaches**

**1). Candidate gene/functional polymorphism**
**	****	*****one gene – one hypothesis***

**2). Candidate gene/linkage disequilibrium**
**	*****multiple genes – multiple hypotheses***

**3). Genome-wide linkage disequilibrium**
**	*****every gene – no hypotheses***

## The SNP Consortium (TSC) Membership

**Charity**

**THE****WELLCOME****TRUST**

**Academic****Centres**
- COLD SPRING HARBOURLABORATORY
- SANGER CENTRE
- STANFORD HUMAN GENOME CENTRE
- WASHINGTON UNIVERSITYSCHOOL OF MEDICINE
- WHITEHEAD INSTITUTE FORBIOMEDICAL RESEARCH

**Notes:**
The SNP Consortium (a consortium of pharmaceutical companies, technology companies and the Wellcome Trust) is producing an ordered high-density SNP map of the human genome that is being placed in the public domain and will be completed by 2001.

There will be approximately 1 million SNPs in this map.

## Concept behind SNP profile

**candidate gene**

**SNPs**

## Expense  now ~$0.10/SNP

Selection of optimal SNPs (~200,000?)

Still ~ $20,000/patient 

**Selection of optimal SNPs (~200,000?)**
**Still ~ $20,000/patient **

**Current limitations**

**Abacavir**

**Reverse transcriptase inhibitor**
**~4% incidence of hypersensitivity syndrome**
**Candidate gene analysis pointed to TNF******** and HLA-B57 genes**
**Candidate gene SNP profile 30-70% predictive in white men**

## Large patient numbers required 
if ALT elevations rare
		>3 X	trial size
Zileuton		3.5%	>6,000
Troglitazone	1.5%	>15,000 

**if ALT elevations rare**
**	****	****>3 X****	****trial size**
**Zileuton****	****	****3.5%****	****>6,000**
**Troglitazone****	****1.5%****	****>15,000 **

**$3 million for SNP analysis of 150 subjects with **
**current technology**

**Current limitations**

## SAFE

**liver failure**

**SAFE**

**Increased ALT**

- <date/time>

**Adaptation**

**  ****hepatitis**

**INH****	****	****  15%****	****	****<1%**
**diclofenac****	****     5%****	****	****<0.01%**
**troglitazone****	****     2%****	****	****<0.1**

## Treatment with tacrine through ALT elevations

- <date/time>

**Treatment with tacrine through ALT elevations**

*unpublished*

**Common Observation**

**1). ALT elevations often reverse with continuation of therapy**
**2). True liver injury is usually occurring and goes away on drug**

- <date/time>

*unpublished*

**Reversed on treatment****	****Treatment stopped**

## Drug

- <date/time>

**Drug**

**elimination**

**Reactive **
**Metabolite**

**Necrosis/Apoptosis**

**Safe pathways**

**immune**
**mechanisms**

**non-immune**
**mechanisms**

- <date/time>

**Toxicol. Sci.**
**64,269-280, 2001**

- <date/time>

**Adaptation**

**1). Important key to idiosyncrasy**
**2). Not easily studied in clinical trials**
**3). Argument for a national **
**	****	****DNA/tissue bank**