The

incidence

of

arthropathy

adverse

events

in

efalizumab-treated

patients

is

low

and

similar

to

placebo

and

does

not

increase

with

long-term

treatment

pooled

analysis

of

data

from

Phase

III

clinical

trials

of

efalizumab

A

large-scale

pooled

analysis

of

safety

data

from

five

Phase

III

clinical

trials

-LRB-

including

open-label

extensions

of

two

of

these

studies

-RRB-

and

two

Phase

III

open-label

clinical

trials

of

efalizumab

was

conducted

to

explore

whether

arthropathy

adverse

events

-LRB-

AEs

-RRB-

were

associated

with

efalizumab

treatment

in

patients

with

moderate-to-severe

chronic

plaque

psoriasis

Data

from

patients

who

received

subcutaneous

injections

of

efalizumab

or

placebo

were

stratified

for

analysis

into

phases

according

to

the

nature

and

duration

of

treatment

These

included

the

first

treatment

phase

-LRB-

data

from

patients

who

received

either

efalizumab

mg/kg

once

weekly

or

placebo

in

the

five

placebo-controlled

studies

-RRB-

the

extended

treatment

phase

-LRB-

data

from

seven

trials

for

all

efalizumab-treated

patients

-RRB-

and

the

long-term

treatment

phase

-LRB-

data

from

efalizumab-treated

patients

who

received

treatment

for

up

to

months

in

two

long-term

trials

-RRB-

Descriptive

statistics

were

performed

and

the

incidence

of

arthropathy

AEs

per

patient-year

was

calculated

using

confidence

intervals

-LRB-

CIs

-RRB-

During

the

first

treatment

phase

a

similar

proportion

of

patients

had

an

arthropathy

AE

in

the

efalizumab

group

-LRB-

patients

-RRB-

compared

with

the

placebo

group

-LRB-

patients

-RRB-

the

incidence

of

arthropathy

AEs

per

patient-year

was

in

the

efalizumab

group

-LRB-

CI

-RRB-

and

in

the

placebo

group

-LRB-

CI

-RRB-

Analysis

of

first

treatment

phase

data

from

one

study

-LRB-

n

-RRB-

showed

that

the

incidence

of

psoriatic

arthropathy

per

patient-year

was

lower

in

efalizumab-treated

patients

-LRB-

CI

-RRB-

than

in

those

given

placebo

-LRB-

CI

-RRB-

During

the

extended

treatment

phase

the

incidence

of

arthropathy

remained

low

-LRB-

CI

-RRB-

Data

from

two

long-term

studies

showed

that

there

was

no

increase

in

the

incidence

of

arthropathy

AEs

over

time

in

patients

treated

with

efalizumab

for

up

to

months

Patients

who

had

an

arthropathy

AE

during

treatment

with

efalizumab

appeared

to

be

more

likely

to

have

a

history

of

arthropathy

prior

to

treatment

Efalizumab

does

not

appear

to

increase

the

risk

of

arthropathy

AEs

compared

with

placebo

Introduction

Psoriasis

is

a

chronic

immune-mediated

inflammatory

skin

disorder

that

is

currently

incurable

Consequently

the

majority

of

people

with

psoriasis

require

long-term

treatment

to

maintain

disease

control

Traditional

immunosuppressive

systemic

treatments

such

as

acitretin

methotrexate

cyclosporine

hydroxyurea

and

thioguanine

may

be

effective

in

controlling

psoriasis

in

some

patients

but

significant

toxicity

and

the

need

to

closely

monitor

patients

limit

the

viability

of

these

treatments

for

long-term

continuous

use

-LSB-

-RSB-

Recently

developed

systemic

therapies

that

selectively

target

specific

pathways

in

the

inflammatory

cascade

of

psoriasis

generally

have

a

much

improved

safety

profile

compared

with

traditional

therapies

-LSB-

-RSB-

Efalizumab

-LRB-

Raptiva

-RRB-

is

a

recombinant

humanized

monoclonal

antibody

that

has

been

approved

for

the

treatment

of

moderate-to-severe

chronic

plaque

psoriasis

It

interferes

with

the

pathogenesis

of

psoriasis

via

multiple

mechanisms

including

inhibition

of

T-lymphocyte

trafficking

and

T-lymphocyte

activation

and

reactivation

-LSB-

-RSB-

The

safety

and

efficacy

profile

of

efalizumab

has

been

established

in

numerous

clinical

trials

in

which

more

than

patients

were

enrolled

and

treatment

was

assessed

for

up

to

years

-LSB-

-RSB-

Although

psoriasis

can

be

associated

with

the

co-morbidity

of

psoriatic

arthritis

a

minority

of

patients

with

psoriasis

-LRB-

-RRB-

will

develop

this

joint

disease

-LSB-

-RSB-

Nevertheless

psoriatic

arthritis

constitutes

a

major

consideration

in

patients

who

are

receiving

long-term

treatment

for

their

psoriasis

A

Nordic

study

of

more

than

patients

with

psoriasis

showed

that

patients

with

arthritis

exhibited

greater

impairment

of

psoriasis-related

quality

of

life

-LRB-

QoL

-RRB-

longer

disease

duration

and

greater

self-reported

disease

severity

compared

with

patients

who

had

psoriasis

but

no

co-morbid

arthritis

-LSB-

-RSB-

A

low

incidence

of

arthropathy

adverse

events

-LRB-

AEs

any

form

of

joint

disease

-RRB-

associated

with

efalizumab

treatment

has

been

reported

in

both

clinical

studies

and

routine

clinical

practice

-LSB-

-RSB-

However

anecdotal

reports

of

arthropathy

in

routine

clinical

practice

have

expressed

concern

that

efalizumab

may

be

associated

with

exacerbation

of

arthropathy

-LSB-

-RSB-

To

address

this

concern

we

conducted

a

large-scale

pooled

analysis

of

safety

data

from

five

Phase

III

clinical

trials

-LRB-

including

open-label

extensions

of

two

of

these

studies

-RRB-

and

two

Phase

III

open-label

clinical

trials

of

efalizumab

to

explore

whether

arthropathy

AEs

were

associated

with

efalizumab

treatment

in

patients

with

psoriasis

Methods

The

primary

objective

of

this

pooled

safety

analysis

was

to

assess

the

incidence

of

arthropathy

AEs

in

patients

who

had

received

either

efalizumab

or

placebo

Safety

data

were

pooled

from

five

randomized

double-blind

placebo-controlled

clinical

trials

-LRB-

including

data

from

two

open-label

extension

studies

of

two

of

these

trials

-RRB-

and

two

open-label

clinical

trials

of

efalizumab

-LSB-

-RSB-

Patients

included

in

these

Phase

III

studies

were

aged

years

and

had

moderate-to-severe

chronic

plaque

psoriasis

a

psoriasis

area

and

severity

index

-LRB-

PASI

-RRB-

score

of

at

screening

and

plaque

psoriasis

covering

of

body

surface

area

All

patients

were

candidates

for

either

systemic

anti-psoriatic

therapy

or

had

received

systemic

anti-psoriatic

therapy

Patients

included

in

these

trials

received

subcutaneous

injections

with

efalizumab

mg/kg

once

weekly

or

mg/kg

once-every-other

week

or

placebo

Details

of

individual

study

methodologies

are

described

in

other

publications

-LSB-

-RSB-

Arthropathy

AEs

were

defined

according

to

the

Coding

Symbols

for

Thesaurus

of

Adverse

Reaction

Terms

-LRB-

COSTART

-RRB-

-LSB-

-RSB-

preferred

terms

arthritis

and

arthrosis

or

the

Medical

Dictionary

for

Regulatory

Activities

-LRB-

MedDRA

-RRB-

preferred

terms

arthritis

not

otherwise

specified

-LRB-

NOS

-RRB-

psoriatic

arthropathy

arthropathy

NOS

monoarthritis

polyarthritis

and

osteoarthritis

NOS

Treatment

groups

analyzed

Due

to

the

variety

of

study

designs

five

analyses

were

considered

first-treatment

phase

first

exposure

phase

extended

treatment

phase

re-treatment

phase

and

long-term

treatment

-LRB-

see

Table

-RRB-

Table

of

the

Phase

III

data

from

five

placebo-controlled

clinical

trials

-LRB-

including

data

from

two

open-label

extension

studies

of

two

of

these

trials

-RRB-

and

two

open-label

clinical

trials

of

efalizumab

included

in

the

pooled

safety

analysisPublication

-LRB-

protocol

number

-RRB-

Study

designNumber

of

patients

in

each

analysisFirst

treatment

-LRB-

weeks

-RRB-

Efalizumab

sc

mg/kg

qw

or

mg/kg

qowFirst

exposure

Extended

treatment

-LRB-

weeks

-RRB-

Long-term

treatmenta

-LRB-

months

-RRB-

Re-treatmentPlaceboEfalizumab

mg/kgEfalizumab

mg/kgLeonardi

-LSB-

-RSB-

-LRB-

-RRB-

Randomized

double-blind

parallel-group

-LSB-

-RSB-

-LRB-

-RRB-

Randomized

double-blind

parallel-group

placebo-controlled

Gordon

-LSB-

-RSB-

-LRB-

-RRB-

Randomized

double-blind

parallel-group

placebo-controlled

Papp

-LSB-

-RSB-

-LRB-

-RRB-

Randomized

double-blind

parallel-group

placebo-controlled

Sterry

-LSB-

-RSB-

-LRB-

-RRB-

Randomized

double-blind

parallel-group

-LSB-

-RSB-

-LRB-

-RRB-

Open-label

-LSB-

-RSB-

-LRB-

-RRB-

Open-label

Menter

-LSB-

-RSB-

-LRB-

-RRB-

Open-label

extensionc

of

study

-LSB-

-RSB-

Menter

-LSB-

-RSB-

-LRB-

-RRB-

Open-label

extensionc

of

study

g

-LSB-

-RSB-

Pooled

analysis

once-every-other

week

qw

once

weekly

sc

subcutaneousaNumber

of

patients

at

startbPatients

received

combined

therapy

with

fluocinolone

acetate

-LRB-

n

-RRB-

or

petrolatum

-LRB-

n

-RRB-

for

weeks

for

months

the

dose

of

efalizumab

could

be

escalated

to

mg/kg

per

week

for

up

to

weeks

if

clinically

indicatedcSome

patients

are

included

in

the

analyses

more

than

once

because

patients

in

the

open-label

extension

studies

are

also

included

in

analyses

of

the

parent

studiesdIncluded

patients

who

received

either

efalizumab

or

placebo

in

the

parent

study

-LSB-

-RSB-

eNot

applicable

because

data

are

analyzed

and

reported

separately

for

the

study

by

Gottlieb

et

al.

-LSB-

-RSB-

and

the

study

published

by

Papp

-LSB-

-RSB-

and

Menter

-LSB-

-RSB-

It

is

worth

noting

that

most

of

the

studies

included

in

this

pooled

analysis

were

designed

and

conducted

before

efalizumab

had

received

regulatory

approval

and

before

it

was

known

that

doses

of

more

than

mg/kg

once

weekly

-LRB-

the

approved

dose

-RRB-

did

not

confer

additional

treatment

benefit

-LRB-

EMEA

Raptiva

Summary

of

Product

Characteristics

FDA

US

FDA

Prescribing

Information

for

Raptiva

-RRB-

For

this

reason

only

the

efalizumab

mg/kg

once-weekly

dose

data

are

reported

for

the

first

treatment

phase

of

the

analysis

Due

to

the

wide

variety

of

study

designs

included

in

the

pooled

analysis

data

for

patients

receiving

any

dose

of

efalizumab

are

combined

for

all

other

treatment

phases

analyzed

The

first

treatment

phase

analysis

included

data

from

patients

in

the

five

placebo-controlled

studies

who

received

either

efalizumab

mg/kg

once

weekly

or

placebo

This

analysis

allows

a

comparison

between

the

efalizumab

and

placebo

treatment

groups

The

first

exposure

phase

included

data

from

all

studies

in

patients

who

had

their

first

exposure

to

any

dose

of

efalizumab

and

thus

did

not

include

placebo

data

This

analysis

was

conducted

to

include

the

maximum

number

of

patients

who

received

efalizumab

for

their

first

weeks

of

treatment

-LRB-

i.e.

it

included

those

patients

who

first

received

efalizumab

treatment

after

crossing

over

from

a

placebo

group

as

well

as

the

patients

who

first

received

efalizumab

during

weeks

-RRB-

The

extended

treatment

phase

analysis

included

data

in

patients

given

any

dose

of

efalizumab

who

had

already

received

efalizumab

during

the

first

treatment

phase

The

long-term

treatment

phase

analysis

included

all

patients

who

received

continuous

long-term

treatment

-LRB-

up

to

months

-RRB-

with

any

dose

of

efalizumab

Data

were

analyzed

in

segments

to

assess

change

in

the

incidence

of

arthropathy

AEs

over

time

This

analysis

included

data

from

two

long-term

studies

-LSB-

-RSB-

which

were

analyzed

separately

due

to

differences

in

study

design

The

re-treatment

phase

analysis

included

all

patients

who

re-started

treatment

with

efalizumab

following

a

treatment-free

observation

period

Statistical

analyses

Descriptive

statistics

were

used

to

explore

the

association

between

efalizumab

and

the

occurrence

of

arthropathy

AEs

Results

are

expressed

as

point-estimates

of

the

incidence

rates

-LRB-

ratio

of

the

number

of

patients

with

an

arthropathy

AE

to

the

total

number

of

patient-years

at

risk

of

an

arthropathy

AE

-RRB-

with

their

confidence

intervals

-LRB-

CIs

-RRB-

Descriptive

comparisons

are

provided

no

formal

statistical

tests

were

performed

Analyses

were

also

conducted

to

explore

the

relationship

between

onset

of

arthropathy

AEs

during

efalizumab

treatment

and

a

previous

history

of

arthropathy

-LRB-

reported

as

a

narrative

by

patients

at

the

baseline

visit

-RRB-

and

the

incidence

of

arthropathy

AEs

and

clinical

response

to

efalizumab

treatment

-LSB-

measured

at

weeks

using

the

Physician

Global

Assessment

-LRB-

PGA

-RRB-

and

PASI

scales

-RSB-

Differences

in

patient

and

psoriasis

characteristics

at

baseline

were

also

compared

between

patients

who

had

arthropathy

AEs

and

those

who

did

not

An

additional

analysis

of

data

from

patients

included

in

the

first

treatment

phase

of

the

study

by

Sterry

et

al.

-LSB-

-RSB-

-LRB-

Table

-RRB-

was

conducted

to

assess

the

incidence

of

psoriatic

arthropathy

in

these

patients

This

was

the

only

study

to

define

arthropathy

AEs

according

to

MedDRA

other

studies

used

the

COSTART

which

did

not

include

psoriatic

arthropathy

specifically

as

a

preferred

term

Baseline

demographics

and

psoriasis

characteristics

and

the

proportion

of

patients

with

a

previous

history

of

arthropathy

-LRB-

as

reported

by

patients

at

the

baseline

visit

-RRB-

were

tabulated

by

presence/absence

of

an

arthropathy

event

Results

The

number

of

patients

included

in

each

of

the

pooled

safety

analyses

from

each

of

the

seven

trials

and

two

open-label

extensions

is

summarized

in

Table

Up

to

patients

received

at

least

one

dose

of

efalizumab

A

total

of

patients

were

included

in

the

first

treatment

phase

analysis

of

whom

the

majority

-LRB-

patients

-RRB-

received

efalizumab

mg/kg

per

week

patients

-LRB-

-RRB-

received

placebo

Efalizumab

mg/kg

per

week

regimen

was

given

to

patients

-LRB-

-RRB-

in

two

of

the

five

studies

-LSB-

-RSB-

consequently

these

patients

were

not

included

in

the

first

treatment

analysis

Patient

demographics

and

baseline

psoriasis

characteristics

were

similar

between

treatment

groups

in

the

first

treatment

phase

-LRB-

Table

-RRB-

Table

demographic

and

disease

characteristics

for

patients

in

the

placebo-controlled

first

treatment

phaseCharacteristicsPlacebo

-LRB-

n

-RRB-

Efalizumab

mg/kg

per

week

-LRB-

n

-RRB-

Efalizumab

mg/kg

per

week

-LRB-

n

-RRB-

Mean

age

-LRB-

years

-RRB-

mean

-LRB-

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Weight

-LRB-

kg

-RRB-

mean

-LRB-

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mean

BMIa

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Race

n

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Duration

of

psoriasis

mean

number

of

years

-LRB-

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

of

arthritis

n

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

BMI

body

mass

indexaDue

to

missing

height

data

BMI

was

calculated

for

patients

in

the

placebo

group

patients

in

the

efalizumab

mg

kg

per

week

group

and

patients

in

the

efalizumab

mg/kg

per

week

group

First

treatment

phase

-LRB-

weeks

-RRB-

During

the

first

weeks

of

treatment

a

similar

proportion

of

patients

had

an

arthropathy

AE

in

the

efalizumab

mg/kg

group

-LRB-

-RRB-

and

the

placebo

group

-LRB-

Fig.

-RRB-

Correspondingly

the

incidence

of

arthropathy

AEs

per

patient-year

was

in

the

efalizumab

mg/kg

group

-LRB-

CI

-RRB-

and

in

the

placebo

group

-LRB-

CI

Fig.

-RRB-

The

majority

of

the

arthropathy

AEs

was

mild-to-moderate

in

intensity

in

both

the

efalizumab

-LRB-

events

CI

-RRB-

and

placebo

groups

-LRB-

events

CI

-RRB-

Fig.

Proportion

of

patients

who

had

arthropathy

adverse

events

-LRB-

AEs

-RRB-

during

each

phase

of

the

safety

analysis

and

b

incidence

of

arthropathy

AEs

per

patient-year

for

each

phase

The

additional

analysis

of

data

from

the

study

by

Sterry

et

al.

-LSB-

-RSB-

demonstrated

that

the

incidence

of

psoriatic

arthropathy

per

patient-year

was

lower

in

the

group

treated

with

efalizumab

mg/kg

per

week

-LRB-

CI

-RRB-

than

in

the

placebo

group

-LRB-

CI

-RRB-

the

proportion

of

patients

with

psoriatic

arthropathy

was

-LRB-

patients

-RRB-

in

the

efalizumab

group

and

-LRB-

patients

-RRB-

in

the

placebo

group

First

exposure

phase

In

total

efalizumab-treated

patients

were

included

in

this

analysis

A

small

proportion

of

patients

had

an

arthropathy

AE

-LRB-

Fig.

-RRB-

and

the

incidence

of

arthropathy

AEs

per

patient-year

was

also

low

-LRB-

CI

Fig.

-RRB-

The

incidence

of

arthropathy

AEs

in

this

group

of

patients

was

similar

to

that

in

the

placebo

group

in

the

first

treatment

phase

as

indicated

by

the

overlap

in

CIs

Extended

treatment

phase

-LRB-

weeks

-RRB-

In

total

patients

were

included

in

the

extended

treatment

phase

analysis

During

this

phase

a

low

proportion

of

patients

had

an

arthropathy

AE

-LRB-

Fig.

-RRB-

and

the

incidence

of

arthropathy

AEs

per

patient-year

was

also

low

-LRB-

CI

Fig.

-RRB-

Overlap

in

the

CIs

indicates

that

the

incidence

of

arthropathy

AEs

in

this

group

of

patients

was

also

similar

to

that

in

the

placebo

group

in

the

first

treatment

phase

Long-term

treatment

phase

The

results

of

two

long-term

studies

were

analyzed

separately

to

assess

the

incidence

of

arthropathy

AEs

in

patients

treated

with

efalizumab

In

both

of

these

studies

-LRB-

Fig.

-RRB-

there

was

no

overall

increase

in

the

incidence

of

arthropathy

AEs

over

time

Furthermore

the

incidence

of

arthropathy

remained

similar

to

that

of

the

placebo

group

in

the

first

treatment

phase

and

stable

between

periods

Fig.

of

arthropathy

AEs

in

long-term

studies

of

patients

treated

with

efalizumab

a

for

up

to

months

and

compared

indirectly

with

pooled

placebo

data

from

the

first

treatment

-LRB-

FT

-RRB-

phase

-LSB-

-RSB-

and

b

for

up

to

months

and

compared

with

the

study

's

placebo

group

during

month

-LSB-

-RSB-

Following

the

first

double-blind

placebo-controlled

phase

of

this

study

patients

in

the

placebo

group

who

continued

were

switched

to

open-label

treatment

with

efalizumab

Consequently

the

month

and

results

included

patients

who

had

received

placebo

during

the

initial

months

of

the

study

In

total

patients

were

included

in

the

analysis

of

the

study

by

Gottlieb

et

al.

-LSB-

-RSB-

These

patients

received

continuous

treatment

with

efalizumab

mg/kg

once

weekly

for

weeks

-LRB-

fluocinolone

acetate

or

petrolatum

was

co-administered

during

weeks

-RRB-

followed

by

continuous

maintenance

treatment

with

efalizumab

mg/kg

once

weekly

for

up

to

months

in

patients

who

had

a

improvement

in

PASI

score

For

months

the

dose

of

efalizumab

could

be

escalated

to

mg/kg

per

week

for

up

to

weeks

if

clinically

indicated

then

maintained

at

mg/kg

per

week

During

the

entire

study

period

there

was

little

variation

in

the

incidence

of

arthropathy

AEs

-LRB-

range

Fig.

-RRB-

Reasons

for

discontinuation

were

diverse

and

were

representative

of

the

overall

population

refer

to

Gottlieb

et

al.

-LSB-

-RSB-

for

details

of

discontinuations

For

the

other

long-term

study

the

analysis

included

data

from

efalizumab-treated

patients

in

the

placebo-controlled

first

treatment

phase

of

the

study

-LSB-

-RSB-

and

data

from

patients

who

entered

the

open-label

extension

phase

and

received

efalizumab

treatment

-LSB-

-RSB-

of

the

patients

included

in

the

open-label

extension

had

switched

from

placebo

to

efalizumab

after

completing

the

first

treatment

phase

Patients

who

entered

the

open-label

extension

phase

continued

to

receive

or

initiated

treatment

with

efalizumab

mg/kg

once

weekly

for

up

to

months

continuously

As

in

the

long-term

study

by

Gottlieb

et

al.

-LSB-

-RSB-

there

was

little

variation

in

the

incidence

of

arthropathy

AEs

during

the

entire

study

period

-LRB-

range

Fig.

-RRB-

Re-treatment

phase

In

total

efalizumab-treated

patients

were

included

in

the

re-treatment

phase

of

the

analysis

In

this

phase

a

lower

proportion

of

patients

had

an

arthropathy

AE

-LRB-

Fig.

-RRB-

compared

with

the

first

treatment

phase

and

the

incidence

of

arthropathy

AEs

per

patient-year

was

also

lower

-LRB-

CI

Fig.

-RRB-

The

incidence

of

arthropathy

AEs

in

this

group

of

patients

was

lower

than

in

the

placebo

group

in

the

first

treatment

phase

Baseline

characteristics

and

previous

history

of

arthropathy

There

were

no

differences

in

baseline

demographics

or

disease

characteristics

between

the

patients

who

had

arthropathy

AEs

and

those

who

did

not

Patients

who

experienced

an

arthropathy

AE

during

treatment

with

efalizumab

appeared

to

be

more

likely

to

have

a

history

of

arthropathy

prior

to

treatment

Of

the

patients

who

never

developed

an

arthropathy

AE

during

efalizumab

treatment

reported

a

previous

history

of

arthropathy

compared

with

in

patients

who

did

have

an

arthropathy

AE

During

the

first

treatment

phase

-LRB-

n

-RRB-

and

-LRB-

n

-RRB-

of

patients

who

developed

an

arthropathy

AE

had

a

history

of

arthropathy

prior

to

receiving

placebo

or

efalizumab

mg/kg

once

weekly

respectively

Arthropathy

AEs

and

clinical

response

to

efalizumab

Arthropathy

AEs

appeared

to

be

less

likely

to

occur

in

patients

who

had

a

good

clinical

response

to

treatment

-LRB-

improvement

in

PASI

score

of

patients

had

events

-RRB-

than

in

patients

who

had

a

partial

response

-LRB-

improvement

in

PASI

score

of

patients

had

events

Fig.

-RRB-

and

non-responders

-LRB-

improvement

in

PASI

score

of

patients

had

events

-RRB-

The

corresponding

incidences

of

arthropathy

AEs

per

patient-year

were

in

patients

with

a

good

clinical

response

-LRB-

CI

-RRB-

in

patients

with

a

partial

clinical

response

-LRB-

CI

-RRB-

and

in

patients

who

did

not

respond

-LRB-

CI

-RRB-

Fig.

Proportion

of

patients

with

an

arthropathy

AE

by

response

category

on

the

psoriasis

area

and

severity

index

-LRB-

PASI

-RRB-

and

physician

global

assessment

-LRB-

PGA

-RRB-

scales

and

b

incidence

of

arthropathy

AEs

per

patient-year

by

response

category

on

the

PASI

and

PGA

scales

When

assessed

using

the

PGA

scale

arthropathy

AEs

also

appeared

to

be

less

likely

to

occur

in

patients

who

had

better

clinical

responses

to

treatment

with

efalizumab

-LRB-

Fig.

-RRB-

During

the

extended

treatment

phase

the

incidence

of

arthropathy

AEs

per

patient-year

was

in

patients

with

responses

categorized

as

cleared

excellent

or

good

on

the

PGA

scale

-LRB-

CI

-RRB-

and

in

patients

with

responses

categorized

as

fair

slight

unchanged

or

worse

on

the

PGA

scale

-LRB-

CI

-RRB-

Discussion

The

placebo-controlled

results

of

this

large-scale

pooled

analysis

of

arthropathy

data

from

seven

clinical

trials

show

that

efalizumab

does

not

appear

to

increase

the

risk

of

developing

arthropathy

AEs

compared

with

placebo

during

the

first

weeks

of

treatment

In

addition

for

patients

treated

with

efalizumab

the

incidence

of

arthropathy

AEs

did

not

appear

to

increase

over

time

The

proportion

of

patients

who

had

an

arthropathy

AE

within

any

treatment

period

was

low

-LRB-

-RRB-

through

all

treatment

phases

-LRB-

first

treatment

first

exposure

extended

treatment

re-treatment

long-term

treatment

-RRB-

Joint

disease

has

also

been

reported

as

a

side-effect

of

other

approved

biological

treatments

for

psoriasis

namely

infliximab

-LRB-

EMEA

public

statement

on

infliximab

-RRB-

-LSB-

-RSB-

alefacept

-LRB-

Biogen

safety

presentation

on

Alefacept

to

the

FDA

-RRB-

-LSB-

-RSB-

and

etanercept

-LRB-

EMEA

Scientific

discussion

for

the

approval

of

Enbrel

-RRB-

Indeed

placebo-controlled

studies

of

infliximab

and

alefacept

indicate

that

in

patients

with

moderate-to-severe

psoriasis

the

incidence

of

arthralgia

-LRB-

joint

pain

-RRB-

is

and

respectively

-LRB-

Biogen

safety

presentation

on

Alefacept

to

the

FDA

-RRB-

-LSB-

-RSB-

Psoriatic

arthritis

has

been

reported

as

serious

treatment-related

AE

in

three

placebo-controlled

studies

of

etanercept

in

the

treatment

of

chronic

plaque

psoriasis

-LRB-

incidence

data

have

not

been

published

-RRB-

-LRB-

EMEA

Scientific

discussion

for

the

approval

of

Enbrel

-RRB-

The

incidence

of

arthropathy

AEs

in

the

current

analysis

of

efalizumab

appears

to

be

similar

to

that

for

arthralgia

in

studies

of

infliximab

and

alefacept

Moreover

the

term

arthropathy

used

in

the

current

study

encompasses

a

variety

of

joint

diseases

not

just

a

single

joint

condition

such

as

arthralgia

or

psoriatic

arthritis

and

therefore

has

greater

potential

to

include

more

patients

However

this

between-study

comparison

is

indirect

and

thus

should

be

treated

with

caution

Moreover

no

arthropathy

event

-LRB-

defined

by

any

of

the

MedDRA

or

COSTART

preferred

terms

-RRB-

was

excluded

from

the

analysis

Also

data

from

first

treatment

phase

of

the

study

by

Sterry

et

al.

-LSB-

-RSB-

indicate

that

the

proportion

of

patients

with

psoriatic

arthropathy

specifically

was

low

-LRB-

-RRB-

in

patients

treated

with

efalizumab

mg/kg

per

week

in

fact

lower

than

in

the

placebo

group

-LRB-

-RRB-

It

should

be

noted

however

that

psoriatic

arthropathy

events

were

not

confirmed

by

a

rheumatologist

this

is

a

potential

limitation

of

the

study

However

the

umbrella

term

arthropathy

was

designed

to

capture

all

joint

diseases

including

psoriatic

arthropathy

Also

the

incidence

of

psoriatic

arthropathy

in

the

study

by

Sterry

et

al.

was

in

line

with

the

incidence

of

arthropathy

in

the

overall

pooled

analysis

To

put

the

results

of

this

pooled

analysis

which

by

its

very

nature

included

select

patient

populations

-LRB-

determined

by

the

inclusion/exclusion

criteria

and

study

designs

-RRB-

in

the

context

of

routine

clinical

practice

post-marketing

surveillance

data

were

assessed

During

post-marketing

surveillance

of

efalizumab

which

accounts

for

approximately

patient-years

to

date

serious

arthropathies

requiring

hospitalization

were

reported

with

a

frequency

of

about

per

patient-years

in

patients

receiving

efalizumab

It

should

be

noted

however

that

underreporting

of

AEs

in

routine

clinical

practice

setting

may

lead

to

an

underestimate

of

the

true

incidence

of

arthropathy

For

both

the

first

treatment

and

first

exposure

phases

of

the

current

analyses

the

proportions

of

patients

reporting

an

arthropathy

AE

appeared

to

be

lower

in

the

efalizumab

groups

than

in

the

placebo

group

in

the

first

treatment

phase

Correspondingly

the

incidences

of

AEs

per

patient-year

in

these

treatment

phases

were

also

lower

in

the

efalizumab

groups

than

that

observed

in

the

placebo

group

in

the

first

treatment

phase

However

the

proportion

of

AEs

that

were

moderate

or

severe

was

greater

in

the

efalizumab

groups

than

in

the

placebo

groups

too

few

patients

had

events

to

draw

any

meaningful

conclusions

During

the

extension

phase

-LRB-

weeks

-RRB-

the

incidence

of

arthropathy

AEs

in

efalizumab-treated

patients

remained

similar

to

the

placebo

group

in

the

first

treatment

phase

Previous

history

of

arthropathy

and

poor

clinical

response

may

potentially

indicate

a

risk

for

occurrence

of

new

arthropathy

AEs

during

treatment

Indeed

arthropathy

AEs

were

most

frequent

in

patients

who

did

not

respond

to

therapy

with

efalizumab

or

in

patients

with

a

history

of

arthropathy

Importantly

the

data

from

the

two

long-term

studies

of

efalizumab

indicate

that

the

incidence

of

arthropathy

AEs

remains

stable

and

low

for

up

to

years

of

continuous

treatment

These

results

coupled

with

efficacy

data

showing

that

the

clinical

improvements

of

the

skin

after

months

of

efalizumab

therapy

are

maintained

throughout

months

of

continuous

dosing

-LSB-

-RSB-

support

the

suitability

of

efalizumab

for

the

chronic

continuous

treatment

of

patients

with

psoriasis

Reasons

for

patients

discontinuations

in

the

study

by

Gottlieb

et

al.

-LSB-

-RSB-

were

diverse

and

representative

of

the

overall

population

included

in

this

analysis

and

have

been

described

previously

When

considering

the

long-term

analysis

of

the

study

-LRB-

Fig.

-RRB-

it

should

be

noted

that

the

number

of

patients

who

remained

in

the

study

decreased

over

time

This

discontinuation

rate

is

not

unexpected

for

a

study

that

is

years

in

duration

but

by

month

there

is

a

small

number

of

patients

on

which

to

base

comparisons

with

the

first

treatment

phase

Another

factor

that

may

confound

between-phase

analysis

comparisons

was

the

possible

use

of

concomitant

medications

for

psoriasis

after

the

first

treatment

phase

in

the

study

by

Gottlieb

et

al.

-LSB-

-RSB-

which

permitted

the

use

of

topical

corticosteroids

and

ultraviolet

B

phototherapy

Accordingly

it

should

be

noted

that

comparisons

of

the

results

between

any

of

the

treatment

phases

of

this

analysis

are

observational

-LRB-

i.e.

not

direct

-RRB-

but

do

confirm

the

results

of

the

long-term

treatment

phase

and

the

placebo-controlled

first

treatment

phase

studies

suggesting

that

the

risk

of

joint

disease

is

not

increased

with

continued

efalizumab

treatment

and

that

the

incidence

of

arthropathy

is

low

and

similar

to

placebo

Further

investigation

is

needed

to

confirm

the

results

of

this

preliminary

analysis

of

arthropathy

events

during

long-term

treatment

with

efalizumab

In

patients

who

re-started

treatment

for

a

further

weeks

following

an

intervention-free

period

the

proportion

of

patients

who

had

an

arthropathy

AE

was

lower

than

during

the

first

treatment

phase

the

same

was

true

for

the

incidence

of

arthropathy

AEs

per

patient-year

in

re-treated

patients

Although

this

scenario

is

likely

to

occur

infrequently

in

clinical

practice

these

data

show

that

if

a

patient

needs

to

stop

-LRB-

e.g.

during

pregnancy

-RRB-

and

then

restart

treatment

there

appears

to

be

no

increased

risk

of

arthropathy

AEs

Although

arthritis

in

patients

with

psoriasis

has

a

significant

impact

on

QoL

-LSB-

-RSB-

it

can

in

most

cases

be

managed

effectively

-LSB-

-RSB-

In

the

small

minority

of

patients

who

develop

arthropathy

during

treatment

the

symptoms

can

be

managed

successfully

with

non-steroidal

anti-inflammatory

drugs

-LSB-

-RSB-

In

conclusion

the

results

of

this

pooled

analysis

show

that

efalizumab

does

not

appear

to

increase

the

risk

of

developing

arthropathy

AEs

compared

with

placebo

Long-term

studies

of

efalizumab

indicate

that

the

incidence

of

arthropathy

AEs

remains

stable

and

low

for

up

to

years

of

continuous

treatment

O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O B O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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arthropathy arthritis efalizumab psoriasis


Caloric restriction counteracts age-dependent changes in prolyl-4-hydroxylase domain -LRB- PHD -RRB- 3 expression Caloric restriction remains the most reproducible measure known to extend life span or diminish age-associated changes . Previously , we have described an elevated expression of the prolyl-4-hydroxylase domain -LRB- PHD -RRB- 3 with increasing age in mouse and human heart . PHDs modulate the cellular response towards hypoxia by regulating the stability of the α-subunit of the transcriptional activator hypoxia inducible factor -LRB- HIF -RRB- . In the present study we demonstrate that elevated PHD3 , but not PHD1 or PHD2 , expression is not restricted to the heart but does also occur in rat skeletal muscle and liver . Elevated expression of PHD3 is counteracted by a decrease in caloric intake -LRB- 40 % caloric restriction applied for 6 months -RRB- in all three tissues . Age-associated changes in PHD3 expression inversely correlated with the expression of the HIF-target gene macrophage migration inhibitory factor -LRB- MIF -RRB- , which has been previously described to be involved in cellular HIF-mediated anti-ageing effects . These data give insight into the molecular consequences of caloric restriction , which influences hypoxia-mediated gene expression via PHD3 . Introduction One of the hallmarks of ageing is the decline of physiological functions . This is accompanied with progressive organ failure and an increased incidence of age-related diseases . Caloric restriction remains the most reproducible measure known to extend the maximum lifespan and to retard a variety of age-associated changes in several species -LRB- Speakman and Hambly 2007 -RRB- . Recent evidence suggests that caloric restriction , besides preventing accumulation of energy-related metabolites like reactive oxygen species or advanced glycation end products , triggers a regulatory response -LRB- Koubova and Guarente 2003 -RRB- . As a result of the limited energy intake , a modulated expression of genes involved in various regulatory integrative functions has been discovered . In a recent study we described age-related changes in the expression of the prolyl-4-hydroxylase domain -LRB- PHD -RRB- 3 and as a consequence in the expression of the hypoxia-inducible factor -LRB- HIF -RRB- -1 α transcription factor -LRB- Katschinski 2006 ; Rohrbach et al. 2005 -RRB- . PHD3 and HIF belong to the cellular oxygen sensing system and regulate therefore the adaptation of cells towards a decreased oxygen supply via hypoxia-inducible gene expression . This involves genes associated with angiogenesis , pH control , glucose metabolism , oxygen transport etc. -LRB- Wenger et al. 2005 -RRB- . In this regard , it should be noted that some ageing-associated diseases such as ischemic diseases , atherosclerosis and cancer are associated with an altered oxygen supply . Moreover , the tolerance to ischemia and hypoxia is reduced in elderly -LRB- Abete et al. 1999 ; Bosch-Marce et al. 2007 ; Mariani et al. 2000 ; Paolucci et al. 2003 -RRB- . HIF is a heterodimer comprising the oxygen labile α-subunit and the oxygen-independently expressed β-subunit , which is also called ARNT . The expression of HIF-1α is regulated by three described PHDs , i.e. PHD1 , PHD2 and PHD3 , at the protein level -LRB- Ivan et al. 2001 ; Jaakkola et al. 2001 -RRB- . PHD-dependent hydroxylation is directly depending on the oxygen availability -LRB- Bruick and McKnight 2001 ; Epstein et al. 2001 -RRB- . Hydroxylation of HIF-1α allows binding of the von Hippel Lindau tumor suppressor protein , which triggers rapid ubiquitination and degradation -LRB- Maxwell et al. 1999 -RRB- . An age-dependent decline of HIF-1α protein levels and HIF-target gene expression has been described in different organs like rat cerebral cortex , mouse heart , carotid body and smooth muscle cells isolated from rabbit aorta -LRB- Di Giulio et al. 2005 ; Rivard et al. 2000 ; Rohrbach et al. 2005 -RRB- . The age-related expression pattern of PHDs in other organs besides heart is not known . Caloric restriction has been consistently described to challenge biological ageing -LRB- Rohrbach et al. 2006b -RRB- . Regarding the HIF system , it is interesting to note that caloric restriction counteracts age-related changes in the angiogenic response and can protect cardiomyocytes from hypoxic death in rodent models of ischemic diseases -LRB- Ahmet et al. 2005 ; Facchetti et al. 2007 -RRB- . To gain more insight into the impact of nutrient availability on the adaptive transcriptional response towards hypoxia , we quantified the expression of the HIF-1α regulating PHD1 -- 3 in heart , liver and skeletal muscle in young and old rats undergoing a well-defined protocol of caloric restriction . Materials and methods Animals and diet protocol , 40 % caloric restriction for 6 months Male young -LRB- 4 months -RRB- and senescent -LRB- 22 months -RRB- Sprague-Dawley rats were obtained from Charles River -LRB- Germany -RRB- , caged individually with a light/dark cycle of 12 hrs and with tap water ad libitum . Food -LRB- AltrominR 1244 ; 2550 cal/g -RRB- was offered ad libitum . Prior to the application of the specific diet protocols , daily food intake of the normal standard diet was monitored for 14 days and averaged for each rat individually . Thereafter , rats at the age of 6 months -LRB- young rats -RRB- or at the age of 24 months -LRB- old animals -RRB- were randomly assigned to one of the following diets for the next six months : rats on `` control diet '' received their individual prediet average of AltrominR 1244 -LRB- 2,550 cal/g -RRB- , but not more , in order to avoid any degree of diet-induced obesity . Rats subjected to caloric restriction received also their prediet average , but of a calorically reduced , fibre-rich diet -LRB- AltrominR 1344/1500 ; 1,550 cal/g -RRB- . Thus , in young rats on control diet -LRB- n = 6 -RRB- , the daily energy intake during six months was 64.8 ± 2.2 kcal and in young rats on -40 % caloric restriction -LRB- n = 6 -RRB- the daily intake was 37.8 ± 1.9 kcal . In old rats the daily energy intake amounted to 57.9 ± 1.8 kcal with control diet -LRB- n = 3 -RRB- and to 36.6 ± 0.1 kcal with 40 % caloric restriction -LRB- n = 3 -RRB- . All animals were fasted for 12 hours before killing . RNA extraction , RT-reaction and quantification by PCR The RNA was isolated from left ventricle , liver and skeletal muscle -LRB- M. gastrocnemicus -RRB- tissue as described before -LRB- Rohrbach et al. 2007 -RRB- . Integrity and quality of the RNA was confirmed by agarose gel electrophoresis and the concentration determined by measuring UV-absorption . Real time PCR Reverse transcription -LRB- RT -RRB- of RNA samples was carried out for 30 min at 42 °C . Real-time PCR and data analysis were performed using the Mx3000P Multiplex Quantitative PCR System -LRB- Stratagene -RRB- . DNA amplification was performed as follows : initial denaturation at 95 °C for 10 min , 40 cycles of amplification -LRB- denaturation at 95 °C for 30 s , annealing at 60 °C for 60 s , and extension at 72 °C for 60 s -RRB- , followed by a denaturation at 95 °C for 60 s and a melting curve over the range from 55 °C up to 95 °C . Fluorescence data were collected at the end of the annealing stage of amplification . We performed Real-time PCR of PHD1 , PHD2 , PHD3 , MIF-1 and 18S rRNA -LRB- 18S rRNA Control kit , Yakima Yellow ® - Eclipse ® Dark Quencher , Eurogentec -RRB- in samples derived from rat left ventricles , skeletal muscle and liver . The following primers were used : PHD1 sense CGTGAGGCATGTTGACAATC , PHD1 antisense AACACCTTTCTGTCCCGATG ; PHD2 sense TACAGGATAAACGGCCGAAC , PHD2 antisense GGCTTGAGTTCAACCCTCAC ; PHD3 sense GGCCGCTGTATCACCTGTAT , PHD3 antisense TTCTGCCCTTTCTTCAGCAT ; MIF sense CAGAACCGCAACTACAGCAA , MIF antisense GAACAGCGGTGCAGGTAAGT . Each assay was performed in duplicate and validation of PCR-runs was assessed by evaluation of the melting curve . All data of mRNA are given as relative units of 18S rRNA concentrations . Western blot Liver tissue was rapidly homogenized in a buffer containing 50 mmol/l Tris · HCl , 1 % SDS , 1 mmol/l sodium-orthovanadate , 5 mmol/l EGTA , 1 mmol/l PMSF , 1 μg/ml aprotinin , and 1 μg/ml leupeptin . Proteins were quantified using the bicinchoninic acid protein assay -LRB- Pierce -RRB- . Protein -LRB- 50 μg -RRB- in 2 × Laemmli SDS sample buffer were boiled for 5 min and after centrifugation loaded onto a SDS-PAGE gel . After electrophoresis , proteins were transferred to a nitrocellulose membrane at 100 V for 90 min . The filters were blocked with 0.01 % Tween , 2 % nonfat milk , and then incubated with antibodies directed against MIF -LRB- Abcam -RRB- and GAPDH -LRB- Abcam -RRB- . Statistical analysis For statistical comparison of age groups or the influence of caloric restriction , a non-paired t-test was used . A P-value < 0.05 was considered to represent a significant difference . Results Prevention of age-dependent increase of PHD3 in heart , liver and skeletal muscle Age-dependent changes of PHD1-3 expression were investigated in three different organs obtained from young -LRB- 12 months old -RRB- and old -LRB- 30 months old -RRB- rats . PHD1 and PHD2 mRNA expression did not differ significantly between the two age groups neither in heart , liver nor in skeletal muscle -LRB- Figs. 1 and 2 -RRB- . However , in all three different organs investigated , there was a significant increase in PHD3 mRNA expression in the tissues obtained from the old rats compared to the young animals -LRB- Fig. 3 -RRB- . Fig. 1Effect of age and caloric restriction on PHD1 mRNA expression . Tissue samples -LRB- heart , liver and skeletal muscle -LRB- M. gastrocnemicus -RRB- -RRB- were obtained from young -LRB- 6 months old -RRB- and old -LRB- 24 months old -RRB- rats with or without a 40 % caloric restriction applied for 6 months . Subsequently , RNA was extracted and PHD1 mRNA quantitated by real time PCRFig . 2Effect of age and caloric restriction on PHD2 mRNA expression . Tissue samples -LRB- heart , liver and skeletal muscle -LRB- M. gastrocnemicus -RRB- -RRB- were obtained from young -LRB- 6 months old -RRB- and old -LRB- 24 months old -RRB- rats with or without a 40 % caloric restriction applied for 6 months . Subsequently , RNA was extracted and PHD2 mRNA quantitated by real time PCRFig . 3Effect of age and caloric restriction on PHD3 mRNA expression . Tissue samples -LRB- heart , liver and skeletal muscle -LRB- M. gastrocnemicus -RRB- -RRB- were obtained from young -LRB- 6 months old -RRB- and old -LRB- 24 months old -RRB- rats with or without a 40 % caloric restriction applied for 6 months . Subsequently , RNA was extracted and PHD3 mRNA quantitated by real time PCR Young and old rats were subsequently challenged by caloric restriction . Daily caloric intake was reduced to 40 % in young and old animals for 6 months . Control animals received their prediet average caloric intake . Using this regimen , we previously have observed that thioredoxin reductase 2 is significantly reduced in ageing skeletal and cardiac muscle and renormalized after caloric restriction -LRB- Rohrbach et al. 2006a -RRB- . Whereas no significant effect of caloric restriction was observed on the expression of PHD1 or PHD2 -LRB- Figs. 1 and 2 -RRB- , the reduced food intake significantly counteracted the age-dependent increase in PHD3 expression in heart , liver and skeletal muscle -LRB- Fig. 3 -RRB- . PHD3 levels in old rats after caloric restriction was comparable to the PHD3 expression levels in young animals . Caloric restriction affects the expression of the macrophage migration inhibitory factor Numerous HIF-target genes , including erythropoietin , vascular endothelial growth factor , carbonic anhydrase IX etc. , have been identified -LRB- Wenger et al. 2005 -RRB- . Recently , the expression of the macrophage migration inhibitory factor -LRB- MIF -RRB- has been demonstrated to be inducible by hypoxia . In addition , functional HIF binding sites in the promoter have been identified . In a recent study evidence was provided that HIF-1 plays a critical role in delaying the onset of senescence in rodent cells via transcriptional activation of MIF and thereby inhibition of the p53-mediated pathway -LRB- Welford et al. 2006 -RRB- . To gain insight into the functional consequences of age and caloric restriction-mediated changes on the HIF-induced signal transduction pathways , we analyzed the mRNA expression of MIF as a function of age and caloric restriction . In line with the suggestion that HIF-dependent gene expression is hampered with increasing age , MIF RNA and protein expression was significantly lower in the heart , liver and skeletal muscle obtained from the old rats compared to the expression found in the young rats -LRB- Figs. 4 , 5 -RRB- . Restriction of food intake reversed the age-dependent decline in MIF expression -LRB- Fig. 4 -RRB- . MIF levels after caloric restriction were comparable to the expression levels in young rats in all three tissues investigated . Fig. 4Effect of age and caloric restriction on MIF mRNA expression . Tissue samples -LRB- heart , liver and skeletal muscle -LRB- M. gastrocnemicus -RRB- -RRB- were obtained from young -LRB- 6 months old -RRB- and old -LRB- 24 months old -RRB- rats with or without a 40 % caloric restriction applied for 6 months . Subsequently , RNA was extracted and MIF mRNA quantitated by real time PCRFig . 5Effect of age and caloric restriction on MIF protein levels . Liver tissue samples were obtained from young -LRB- 6 months old -RRB- and old -LRB- 24 months old -RRB- rats with or without a 40 % caloric restriction applied for 6 months . Subsequently protein was extracted and MIF and GAPDH protein levels were investigated by immunoblot analysis Discussion Caloric restriction has been described to extend life in a variety of different mammalian and non-mammalian species -LRB- Masoro 2000 -RRB- . In addition , restriction of food intake delays the progression of several age-associated diseases . Identification of age-dependently expressed genes is one of the most direct approaches in ageing research . In a previous study , we identified PHD3 as one age-dependently expressed gene in human and mouse heart -LRB- Rohrbach et al. 2005 -RRB- . The data presented here demonstrate that PHD3 expression is additionally increased in rat heart , liver and skeletal muscle with increasing age . These findings indicate that age-dependent changes in PHD3 expression are tissue - and species-independent . Furthermore our data presented here demonstrate that PHD3 expression is decreased by caloric restriction in old rats , whereas no change in the expression of PHD1 or PHD2 was observed . Previous findings indicate that each PHD-isoform displays its own tissue and cell-line-specific expression pattern -LRB- Appelhoff et al. 2004 -RRB- . In addition , the susceptibility of the PHD isoform expression by change in the oxygen availability differs greatly . Whereas PHD3 is highly inducible by hypoxia , there is just a slight hypoxia-mediated induction described in case of PHD2 . In case of PHD1 no hypoxia-inducible expression has been observed -LRB- Appelhoff et al. 2004 ; Marxsen et al. 2004 -RRB- . Including the age-dependent and caloric restriction-induced changes in PHD3 expression , PHD3 seems to be the most flexible PHD isoform regarding stimuli-induced change in expression . In an unbiased screen using white adipose tissue it was found that energy restriction lowers the expression of genes linked to angiogenesis indicating that hypoxia-inducible gene expression is affected by caloric restriction -LRB- Higami et al. 2006 -RRB- . Similarly , the age-dependent and caloric restriction-induced expression of PHD3 described here seems to have consequences . Quantification of the HIF-target gene MIF demonstrates a decrease of MIF expression in heart , liver and skeletal muscle in the old animals , which was reversed by caloric restriction . In line with our observation , in previously published microarray experiments increased levels of MIF RNA were found in liver lysates from young adult mice treated with caloric restriction or with a methionine-deficient diet as well as in young mice of the long-lived Snell dwarf stock -LRB- Dozmorov et al. 2002 ; Miller et al. 2005 ; Miller et al. 2002 -RRB- . Since MIF affects cellular senescence -LRB- Welford et al. 2006 -RRB- , changes in PHD3 expression with increasing age seem to be one important feature of the molecular senescence phenotype . This conclusion is in line with a recent report demonstrating increased HIF-1α hydroxylation in aged versus young primary murine fibroblasts -LRB- Chang et al. 2007 -RRB- . Together with a previously published study demonstrating that under hypoxic conditions senescent cells failed to induce HIF-1α compared to presenescent cells our data indicate that the age-dependent changes in the oxygen sensing system may indeed have impact for the ageing process -LRB- Coppe et al. 2006 -RRB- . Up to now a reduced protein expression of HIF-1α with increasing age has been demonstrated in smooth muscle cells isolated from rabbit aorta , rat cerebral cortex , mouse lung , liver , kidney , heart and in the carotid body -LRB- Di Giulio et al. 2005 ; Frenkel-Denkberg et al. 1999 ; Rivard et al. 2000 ; Rohrbach et al. 2005 -RRB- . A decreased HIF-1α expression was also demonstrated in tissue lysates from ischemic limbs comparing 20 months old mice with 2 months old mice -LRB- Bosch-Marce et al. 2007 -RRB- . Hwang et al described a diminished HIF-1α stabilization in old rats in response to hypoxia , although in nomoxic breathing animals there was an age-related increase in basal levels of HIF binding to DNA . Finally , mouse and human fibroblasts isolated from different age groups express less HIF-1α as a matter age -LRB- Chang et al. 2007 -RRB- . However , it also should be noted that Kang et al demonstrated an increased HIF activity and HIF-1α expression in old rats and conflicting to the presented results a decrease in HIF activity as a result of caloric restriction in liver lysates -LRB- Kang et al. 2005 -RRB- . Comparing the chow protocols , these rats were fed a soybean protein diet , which may have resulted in the described increase in redox metabolites and HIF-1α protein expression . The degree and management of caloric restriction is not described -LRB- Hwang et al. 2007 -RRB- . Taken collectively , several papers indicate a diminished HIF-response with increasing age with some exceptions , which may be tissue or diet-specific . Whereas ageing thus is mostly associated with a decreased hypoxia-inducible gene expression , in endometrial cancer cells mutation of PHD2 has been associated with increased expression of HIF-1α and immortality -LRB- Kato et al. 2006 -RRB- . Downregulation of HIF-1α by overexpression of functional PHD2 altered the cancer cell phenotype and led to the occurrence of senescence markers -LRB- Kato et al. 2006 -RRB- . The impact of the HIF-system for cell fate decisions additionally was described by Welford et al demonstrating in a well defined nontransformed/nonimmortalized knock out cell model that HIF-1 delays the onset of senescence via transcriptional activation of MIF and inhibition of p53 dependent pathways -LRB- Welford et al. 2006 -RRB- . Alltogether these data demonstrate the necessity of tight regulation of the HIF system to prevent unrestricted cell proliferation or early cellular senescence .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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