Epatotossicità da farmaci antiretrovirali: ruolo della

farmacocinetica edella patologia epatica sottostante

Raffaele BrunoUniversità degli studi di Pavia

Liver injury in HIV-infected patients

Opportunistic diseases

Alcohol abuse/IVDU

HCV treatment

Immunereconstitution

Hepatitis viruses

HIV treatmentNRTIs, NNRTIs, PIs; OI Rx

Preexisting diseases

Fatty Liver Disease

Liver toxicity is the most common severe adverse event in the HAART era

Coinfection with hepatitis B or C associated with 4.15(95% CI 2.26–7.60) hazard ratio of Grade 4 liver toxicity

Reisler RB, et al. J Acquir Immune Defic Syndr 2003;34:379–86

Inci

denc

e/

100

pers

ons

How is hepatotoxicity defined?

Grade 3 toxicity

Grade 4 toxicity

Normal

Grade 1 or 2 toxicity

ALT or AST level (number of times ULN)

1

5

10

0

ULN →

“Severe hepatotoxicity”

Hepatotoxicity spectrum

Fulminant Hepatitis - Hepatic Failure

Reversible Clinical Drug-induced Liver Injury

Asymptomatic>5x ULN ALT/AST

Asymptomatic≤5x ULN ALT/AST

Background noise:HCV/HBV/HIV alcohol,

infections,medication

Liver Disease and Antiretroviral Therapy

In case series of patients with HIV infection, the prevalence of liver enzyme abnormalities is:

– 40%-70% for elevations in ALT,– 20%-40% for elevated alkaline phosphatase, – 0%-10% for increased bilirubin.

- Patterns of liver injury

Bruno et. Al. Am J Gastroenterol 2002

++ ++ + +Gamma glutamyl transpeptidase (GGT)

++ ++ + +Alkaline phosphatase

+ + ++ ++Aspartate aminotransferase (AST)

+ + ++ ++Alanine aminotransferase (ALT)

HepatocellularMixedCholestatic

Patterns of Hepatic Injury

Liver Disease and Antiretroviral Therapy

Lactic acidosis, steatosis? Impaired mitochondrial polymerase gamma

NNRTIs

Hepatocellular injury, steatosis

? Inhibition of retinoic binding proteinPIs

Eosinophilic hepatic injury? Immune-mediated hypersensitivity

Unconjugated hyperbilirubinemia

UDP-glucuronyl transferase competition

Lactic acidosis, steatosisImpaired mitochondrial polymerase gamma function

NRTIs

ManifestationsMechanismDrugClass

Probable Mechanisms of Antiretroviral-Induced Hepatotoxicity

Liver Disease and Antiretroviral Therapy

NRTI-related hepatotoxicitySyndrome of mitochondrial toxicity– Mitochondria have their own DNA (mtDNA) that encodes 13%

of mitochondrial proteins– mtDNA is replicated by DNA polymerase γ

• Differs from nuclear DNA

NRTIs inhibit DNA polymerase γ– Mitochondrial dysfunction– Cellular toxicity

High-risk drugs– Stavudine, didanosine, zalcitabine

Low-risk drugs– Abacavir, zidovudine, lamivudine, tenofovir

0.01 0.02 0.04

3.69 11 13

0.0010.010.1

110

100

Abacav

irZidovu

dineLam

ivudine

Stavidine

Didanosin

eZalc

itabine

FIAU

NRTIs: Inhibition of DNA polymerase γ

Martin JL, et al. Antimicrob Agents Chemother 1994;38:2743-9

Rel

ativ

e P

oten

cy o

f Inh

ibiti

on*

A: HepG2 Liver Cells

Birkus G, et al. Antimicrob Agents Chemother. 2002;46:716-723

1000

NRTI concentration (µmol/L)0.1 1 10 100

0

20

40

60

80

100

120

140

Rel

ativ

e m

tDN

A c

onte

nt (%

)

Tenofovir3TC

Abacavir

ZDV d4T

ddI ddC

0.1 1 10 100 10000

20

40

60

80

100

120

140

Tenofovir3TC

Abacavir

ZDV

d4T

NRTI concentration (µmol/L)

ddI ddC A B

B: Skeletal Muscle Cells

Effect of NRTIs on mtDNA Content in Liver and Muscle Cells

Mitochondria

Energy power-houses Have their own DNAMitochondrial DNA is replicated by a separate enzyme to nuclear DNA

Mitochondrion

Cell

mtDNA

Polymerase γ

mtDNA

Polymerase γ NRTI

function

dysfunction

mitochondrion

mtDNA mtDNA encoded protein

nDNA encoded protein

Inhibition of Mitochondrial DNA Synthesis

Compound IC50 (µ M) IC50 (µ M) mt DNA

virus3TC >200 0.7

ABC >100 5.3ZDV >100 0.01d4T 10 0.9ddC 0.002 0.5ddI not determined 3.9

Kakuda et al Clin Ther 2000

Progenitor cell showing heteroplasmy of mitochondria

Normal MitochondriaDysfunctionalMitochondria

At cell division, each daughter cell gets the same nDNA, but varying mtDNA

80% mutantDISEASE

60% mutantNORMAL

40% mutantNORMAL

100%NORMAL

Threshold70% mutant

Segregation of mitochondria during cell division

Neuromuscular:polyneuropathy ddC, ddI, d4Tmyopathy ZDV, ddIcardiomyopathy ZDV, ddC, ddI

Hepatic:steatosis, lactic acidosis ddI,d4T,ddC

gastro-intestinal:pancreatitis ddI, d4T

hematological:pancytopenias ZDV

nephrological:prox. renal tubular dysfunction adefovir

metaboliclipodystrophy d4T

Mitochondrial dysfunction due to NRTIs

Energy production in mitochondriaglucose

pyruvate + NADH

FFA triglycerides

Acetyl CoATCAcycleNADH

CO2 + H2O

NAD+

ketone bodiesNAD+

Resp chain

cytosol

mitochondrion

Lactic acidosisglucose

pyruvate + NADH lactate + NAD+

FFA triglycerides

Acetyl CoATCAcycleNADH

CO2 + H2O

NAD+

ketone bodiesNAD+

Resp chain

cytosol

mitochondrion

From Hyperlactatemia to Acidosis

Source: Medscape

NRTI-based liver toxicity:Clinical presentation

Non-specific symptoms– Abdominal pain, vomiting, anorexia, pain (right

upper quadrant), weight loss

Hepatomegaly

Mixed cholestatic/hepatocellular pattern of liver enzymes

Evidence of extrahepatic mitochondrial toxicity– Amylase/lipase, CPK, lactate, metabolic acidosis,

loss of bicarbonate

Falco et al. Clin Infect Dis 2002

HCV and NNRTI-associated hepatotoxicity

Sulkowski MS, et al. Hepatology 2002;35:182-9

Risk of mitochondrial toxicity: NRTI + RBV in HIV/HCV-coinfected patients

US FDA Reporting System of HIV/HCV patients treated with IFN/RBV

31 cases (58 adverse events) suggestive of mitochondrial toxicity– Pancreatitis and/or

increased lipase (n=21)– Lactic acidosis (n=20)– Elevated LFTs (n=8)– Hepatic steatosis (n=6)– Elevated creatinine,

neuropathy, multiorgan failure (n=1 each)

Fleischer R, et al. Clin Infect Dis 2004;38:e79-e80

DidanosineDidanosine

Didanosine +Didanosine +stavudinestavudine

StavudineStavudine

AbacavirAbacavir

LamivudineLamivudine

ZidovudineZidovudine0.01 0.1 1.0 10 1000.01 0.1 1.0 10 100

Odds Ratio (95% CI)Odds Ratio (95% CI)

RBV +RBV +

Mitochondrial ToxicityMitochondrial ToxicityRiskRisk

12.412.4

8.08.0

3.33.3

1.11.1

0.20.2

0.060.06

ROS

MITOCYPs

Fat deposits

Lipid peroxidation

Interactions between Mito and CYPs 450

Mitochondrial disfunction

ROS

TNF- α

Depletion of antioxidans

FAT

LipidPeroxidation

Vicious cycles involving ROS and Mitochondria

1

23

4

ROS

From ROS to steatohepatitis fibrosis

Lipid peroxidation productsCytokines

(IL-2, IFN-γ ,TNF-α, TGF-β, PDGF)

Kupffer cellKupffer cell

ActivationActivation

FIBROSISFIBROSIS

Cell killing

ApoptosiApoptosiss

Hepatocytes

TGFTGF--ββ

Hepatic stellate cellsHepatic stellate cells

Some ARV Pharmacogenetic Variants Abacavir HSR (HLA-B*5701)white > blackEfavirenz PK (CYP2B6) black > whiteNevirapine LFTs (ABCB1) black > whiteAtazanavir jaundice (UGT) white > blackAtazanavir PK (PXR?) white > blackNelfinavir (CYP2C19) Asian > black, white

To date, every genetic variant that affects HIV disease progression, ARV pharmacokinetics, efficacy, or toxicity differs considerably in frequency depending on geographic region of ancestry.From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Drug Phenotype GeneAbacavir Hypersensitivity HLA-B*5701Lancet 2002:359,727 & 1121; PNAS 2004:101,4180

Indinavir Jaundice UGT-1A1Atazanavir PNAS 2001:98,12671; ICAAC 2002; JID 2005;192,1381

NRTI Lipoatrophy TNF-α promoter, HFEAIDS 2002:16,2013; AIDS 2003:17,121; JID 2008:197, 858

Nevirapine Hypersensitivity HLA-DRB1*0101, -Cw8, -B3505Hepatotoxicity ABCB1Pharmacokinetics CYP2B6

AIDS 2005:19,97; Pharmacogenet Genom 2005;15,1; Clin Inf Dis 2006;43,779 & 783; AIDS 2006:20,1621; AIDS2007;21,264; Pharmacogenet Genom 2009;19,139

Associations Between Allelic Variants and HIV Treatment Response

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Drug Phenotype GeneEfavirenz CNS effects, Pharmacokinetics CYP2B6

Viral response, Resistance ABCB1, CYP2A6Lancet 2002;359:30; AIDS 2004;18:2391; Pharmacogenet Genom 2005;15:1; JID 2005;192:1931; CID 2006;42:401; CROI 2008; JID 2009;199:872

Nelfinavir Viral response, Pharmacokinetics CYP2C19JID 2005;192:1931

PIs Dyslipidemia APOC, RETNJID 2005;191:1419; PLoS Med 2006;3:e52; AIDS 2008;22:1561.

NRTIs Peripheral neuropathy Mitochondrial AIDS 2005;19:1341, AIDS 2006;20:1503 haplogroup T,

HFEHAART CD4 recovery Proliferation & JID 2006;194:1098 apoptosis

Genetic Associations (continued) Slide 28

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Drug Phenotype GeneTenofovir Renal toxicity MRP2 (ABCC2)JID 2006;94:1481

Others…

?

?

?

?

?

?

?

Genetic Associations (continued) Slide 29

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Approved Studies with ACTG Human DNA from US (A5128)

Project PapersNWCS 213 – efavirenz, nelfinavir PK & response ◄ 2NWCS 214 – efavirenz CNS toxicity and PK ◄ 2NWCS 215 – T cell turnover rates ◄ 1NWCS 224 – metabolic complications 1NWCS 233 – CD4 increase on ART ◄ 1NWCS 237 – metabolic complications *NWCS 238 – peripheral neuropathy ◄ 3NWCS 239 – efavirenz resistance after Rx interruption ◄ 1NWCS 243 – HLA and viral evolution *NWCS 248 – efavirenz & lopinavir PK 1NWCS 253 – efavirenz response ◄NWCS 254 – efavirenz response ◄NWCS 256 – peripheral neuropathy ◄NWCS 259 – metabolic complications NWCS 262 – innate immunity and responses to ARTNWCS 266 – treatment responses to ARTNWCS 268 – efavirenz PK ◄NWCS 273 – peripheral neuropathy ◄NWCS 275 – pharmacogenomics of HIV therapy ◄NWCS 279 – immune controlNWCS 280 – lipoatrophy ◄NWCS 291 – tenofovir renal toxicity ◄

DNA banked on >10,000 individuals

Slide 30

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Abacavir hypersensitivity

&HLA-B*5701

Slide 31

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Nolan et al J HIV Ther 2003;8:36

INDIA 5-20%

JAPAN 0%

CHINA 0%(NB 2.5% N.E. provinces)

UK ~8%

MIDDLE EAST 1-2% (NB 5-7% Ashkenazi Jews)

AUSTRALIA ~8%

US Caucasian

~8%US Asian

~1%

US African-

American ~2.5%

W. EUROPE 5-7%

THAILAND 4-10%*

*THAILAND B*57 carriage:Urban Bangkok 3.6%

Thai Dai Lue (NE Thai) ~11%Southern Thai Muslim 3%

MEDITERRANEAN 1-2%

S. AMERICAN Caucasian

5-7%

US Hispanic

~2%

Subsaharan AFRICA

<1%

Slide 32FrequencyFrequency of of HLAHLA--B*5701B*5701

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

• 6-Week Observation Period

ABC-naïve subjects ABC-containing regimen

HSR monitoring according toStandard of Care

plus HLA-B*5701 screening2

(screening arm)

ABC-containing regimen HSR monitoring according to

Standard of Care1

(control arm)

Randomise (1:1) HLA-B*5701positive

subjects excluded

HLA-B*5701 negative subjects

continued

1. retrospective high resolution typing2. prospective high resolution typing Mallal et al; NEJM 2008.

“PREDICT” Double-Blind Study Design

HSRNo HSR

Pos Neg

Immunologically Confirmed HSR1

HLA-B*5701

23 025 794

Pos PV

48%

Neg PV

100%

Sens 100%Spec 97%

Clinically Suspected HSR1

HLA-B*5701

Pos Neg

30 3619 762

Pos PV Neg PV

62% 96%

Sens 46%Spec 98%

1 Control Arm Data Only Mallal et al; NEJM 2008

Performance Characteristics ofHLA-B*5701 Screening for HSR

DHHS Panel Guidelines (12/07)(http://aidsinfo.nih.gov)

“The Panel recommends HLA-B*5701 testing prior to initiating abacavir therapy... HLA-B*5701-positive patients

should not be prescribed abacavir…, positive status should be recorded as an abacavir allergy in the patient’s medical record….”

From DH Haas, MD, at San Francisco, CA: April 20, 2009, IAS–USA.

Why TDM in HIV therapy?

Adapted from Acosta EP, et al AIDS Res Human Retro 2002

ART and the liver: Conclusions

Liver-related morbidity and mortality are assuming an increasing importance in HIV care

Each class of antiretroviral agents may be associated with hepatic toxicity

Viral hepatitis co-infection increases the risk of drug-induced liver injury; all co-infected patients must be monitored carefully

Data suggest that treatment of HIV slows progression of fibrosis and reduces liver-related mortality