Efficacy of Low-Dose FK506 in the Treatment of Myasthenia gravis ...

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Efficacy of Low-Dose FK506 in the Treatment of Myasthenia gravis ...

Original Paper

Eur Neurol 2005;53:146–150

DOI: 10.1159/000085833

Received: December 9, 2004

Accepted: March 22, 2005

Published online: May 17, 2005

Efficacy of Low-Dose FK506 in the

Treatment of Myasthenia gravis

A Randomized Pilot Study

Yuriko Nagane Kimiaki Utsugisawa Daiji Obara Ryushi Kondoh

Yasuo Terayama

Department of Neurology, Iwate Medical University, Morioka , Japan

Key Words

FK506 Interleukin-2 Myasthenia gravis

Peripheral blood mononuclear cells

Abstract

To determine the efficacy of low-dose FK506 in the treatment

of myasthenia gravis (MG), untreated de novo patients

were randomly selected to receive treatment with

(n = 18) or without (n = 16) FK506, and were evaluated

for 1 year after treatment with limitation of daily dose of

prednisolone. Low-dose FK506 reduced the duration of

early-phase therapy in hospital (p ! 0.05) and the need

for combined therapy with plasmapheresis and highdose

intravenous methylprednisolone or high-dose intravenous

methylprednisolone alone (p ! 0.05). It also

reduced the daily dose of prednisolone (p ! 0.05) required

to maintain minimal manifestations of MGFA

postintervention status. None of the patients exhibited

significant side effects up to 1 year after treatment. These

findings suggest that low-dose FK506 is safe and efficacious

for the treatment of de novo MG patients.

Copyright © 2005 S. Karger AG, Basel

Introduction

Myasthenia gravis (MG) is an autoimmune disease

generally mediated by antibodies against the acetylcholine

receptor (AChR) of skeletal muscle (AChR Ab) [1] .

Production of these antibodies in B cells depends upon

AChR-specific T cells [2] . Although both Th1 and Th2

cytokines generally play important roles in the pathogenesis

of MG [3] , T-cell proliferation and activation are apparently

induced by interleukin-2 (IL-2), particularly in

severe cases of MG [4] . Treatment with the IL-2 fusion

toxin DAB 389 IL-2, which is toxic to cells with high-affinity

IL-2 receptors, prevents experimental autoimmune

MG [5] . These findings suggest that the IL-2-mediated

immunoresponses in MG arise from functional abnormalities

of T cells. The immunosuppressant FK506 binds

to a cytoplasmic protein (FK binding protein) and inhibits

calcineurin [6] . The principal biological effects of calcineurin

inhibition include a decrease in antigen-stimulated

IL-2 production in T cells and a decrease in IL-2

receptor expression on T cells [6, 7] . FK506 has been

proven safe and effective in preventing organ rejection in

clinical organ transplantation [8] . Furthermore, in noncontrolled

studies, it has been observed that even a low

dose of FK506 can be used as an effective supplementary

treatment for MG and produces no significant side effects

[9–11] . In the present study, we examined the efficacy of

low-dose FK506 for treatment of de novo MG patients

in a randomized prospective clinical trial.

Fax +41 61 306 12 34

E-Mail karger@karger.ch

www.karger.com

© 2005 S. Karger AG, Basel

0014–3022/05/0533–0146$22.00/0

Accessible online at:

www.karger.com/ene

Kimiaki Utsugisawa

Department of Neurology, Iwate Medical University

19-1 Uchimaru

Morioka 020-8505 (Japan)

Tel. +81 196 51 5111, Fax +81 196 54 9860, E-Mail kutsugi@iwate-med.ac.jp


Methods

Subjects and Randomization

Thirty-six de novo MG patients between the ages of 21 and 82

years (mean age 55.4 8 16.4 years; 10 males and 26 females) were

consecutively enrolled into the study, and were randomly and reciprocally

selected to receive treatment with or without FK506.

However, the present study design was unblinded and nonplacebo

controlled. Informed consent was obtained from all subjects. FK506

(Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan) was administered

orally at a daily dose of 3 mg, which is approximately 20% of

the dose given initially to organ transplant patients [8] . The diagnosis

of MG was based on widely accepted criteria [12] . Factors for

exclusion from the present study were previous immunosuppressant

therapy, elevated levels of serum liver enzymes, renal insufficiency,

pregnancy and age younger than 20 years.

Study Design and the Primary Endpoints

Early-Phase Therapy

The patients were admitted to our hospital for early-phase therapy,

which was started with FK506 and/or prednisolone (PSL) administration.

For patients older than 50 years, thymectomy was not

strongly recommended if there were no signs of thymoma. In patients

who received thymectomy, no pharmacological intervention

was performed before thymectomy, and administration of FK506

and/or PSL was started within 2 days after thymectomy. Normal

quality of life corresponding to the category ‘Minimal Manifestations’

of Myasthenia Gravis Foundation of America (MGFA)

Postintervention Status (MM) [13] was achieved in both groups of

patients by administering the following treatment as required.

Treatment in the early-phase therapy during hospital stay consisted

of oral PSL not exceeding a daily dose of 20 mg, and combined

therapy with plasmapheresis (immunoadsorption method [14] ; IA)

and high-dose intravenous methylprednisolone (HMP) (IA + HMP)

[14] . HMP was performed by serial intravenous injection of 1.0 g

methylprednisolone/100 ml saline immediately after IA and on the

morning of the following 2 days [14] . Anticholinesterase medication

was prohibited in the early-phase therapy. PSL dose was tapered

and adjusted to the minimal dose to maintain MM.

The endpoint of the early-phase therapy was defined as the time

at which clinical symptoms remained MM with PSL not exceeding

10 mg per day. The efficacy of FK506 was evaluated by comparison

of the frequency of IA + HMP and the duration of early-phase

therapy between the two groups of patients treated with or without

FK506 (see table 2 ).

Follow-Up Phase Therapy

After discharge from the early-phase therapy in hospital, the

patients were followed up and treated to maintain MM for 1 year

after the beginning of treatment. In the follow-up phase therapy,

IA + HMP, HMP alone, oral PSL (not exceeding a daily dose of

20 mg) or pyridostigmine bromide (60 or 120 mg per day) was administered

as needed to maintain MM.

The endpoint of the follow-up phase therapy was defined as 1

year after the beginning of treatment. Efficacy of FK506 was evaluated

by comparison of the number of treatments with IA + HMP

or HMP alone during the follow-up phase and the dose of oral PSL

per day required at the endpoint between the two groups of patients

treated with or without FK506 (see table 2 ).

Table 1. Profile of patients treated with and without FK506

Type of therapy

with FK506

(n = 18)

without FK506

(n = 16)

Age, years 56.6817.0 54.2816.1

Gender, m/f 5/13 4/12

Time since onset, months 11.587.7 10.5813.5

AChR Ab-positive cases, % 72 (13/18) 50 (8/16)

Thymectomy, % 39 (7/18) 38 (6/16)

Thymic histology, %

Hyperplasia

Thymoma

Remnant

0

29 (2/7)

71 (5/7)

0

50 (3/6)

50 (3/6)

Classification a , %

I

II

III

IV

V

22.2

38.9

22.2

11.1

5.6

12.5

37.5

25.0

18.8

6.2

Severity score b 8.584.6 9.386.5

No significant difference was observed.

a

MGFA Clinical Classification [13].

b

MGFA Quantitative MG score [13].

The protocol for this study was approved by the ethics committee

of our institute.

Clinical Parameters

Clinical classification was performed according to MGFA [13]

( table 1 ). Clinical severity was determined by patients and participating

physicians (Y.N., D.O. and R.K.) (not blinded to the treatment

type) according to MGFA quantitative MG score [13] ( table

1–3 ). Each patient was assessed by the same physician throughout

the course of the present study. The ratios (posttreatment to

pretreatment) of clinical severity score were calculated. Severity

score and severity ratio were estimated for individual patients before

and at 1 month, 6 months and 1 year after the beginning of

treatment ( table 3 ).

Laboratory Parameters

Blood sampling and examination of IL-2 production by peripheral

blood mononuclear cells (PBMs) were performed as described

elsewhere [15] . In brief, PBMs from the patients were cultured in

standard RPMI medium supplemented with 1% fetal bovine serum

and 20 g/ml of phytohemagglutinin for 48 h. Then, the IL-2 concentration

(pg/ml) of the supernatant was measured by solid-phase

enzyme-linked immunoassay (ELISA) [15] . Serum AChR Ab titer

was estimated by the immunoprecipitation method using 125 I-alpha-bungarotoxin.

The ratios (posttreatment to pretreatment) of

PBM IL-2 production and AChR Ab titer (for patients with elevated

AChR Ab titer) were calculated. PBM IL-2 production, PBM

IL-2 production ratio and AChR Ab ratio were estimated for individual

patients before and at 1 month, 6 months and 1 year after

the beginning of treatment ( table 3 ).

Effects of FK506 on MG Eur Neurol 2005;53:146–150 147


Table 2. Comparison of the items for estimation at the endpoints (early phase and follow-up phase) in patients

treated with and without FK506

Type of treatment

with FK506

(n = 18)

without FK506

(n = 16)

Early-phase therapy

Total early-therapy period, weeks 3.781.2* 5.882.9

Severity score a /severity ratio after total early therapy 1.781.3/0.1980.13 2.282.0/0.2380.23

Number of IA + HMP treatments 1.381.5* 3.182.3

Dose of PSL at the endpoint, mg 5.184.1 6.783.0

Follow-up phase therapy (up to 1 year)

Number of IA + HMP treatments 0.280.5* 1.181.6

Number of HMP treatments 0.480.9* 1.882.7

Dose of PSL at the endpoint, mg 4.684.1* 8.182.6

* p < 0.05 (Mann-Whitney U test) compared with patients treated without FK506.

a

MGFA Quantitative MG score [13].

Table 3. Comparison of clinical parameters after beginning of therapy in patients treated with and without FK506

Type of treatment

with FK506 (n = 18) without FK506 (n = 16)

Before treatment

Severity score a 8.584.6 9.386.5

IL-2 production, pg/ml 1,036.28647.1 1,167.98808.1

One month after beginning of treatment

Severity score a /severity ratio 2.981.5***/0.3880.19*** 3.983.8**/0.5580.47*

IL-2 production, pg/ml/IL-2 production ratio 530.88193.7***/0.6680.30** 874.68566.3*/0.8480.48

Anti-AChR antibody ratio 0.9780.30 b (n = 13) 0.6680.29* (n = 8)

Plasma FK506, ng/ml 6.681.2

Six months after beginning of treatment

Severity score a /severity ratio 1.981.6***/0.2280.17*** 2.383.1*/0.2180.23*

IL-2 production, pg/ml/IL-2 production ratio 495.88215.2** ,b /0.6280.38** ,b 890.68635.4*/0.9080.48

Anti-AChR antibody ratio 0.6580.28** (n = 13) 0.3880.26** (n = 8)

Plasma FK506, ng/ml 6.781.4

One year after beginning of treatment

Severity score a /severity ratio 1.981.6***/0.2480.18*** 1.981.6*/0.2380.13*

IL-2 production, pg/ml/IL-2 production ratio 528.88250.3** , b /0.6480.32** ,b 925.08622.6*/0.9880.50

Anti-AChR antibody ratio 0.5980.28** (n = 13) 0.4380.29** (n = 8)

Plasma FK506, ng/ml 6.681.4

* p < 0.05; ** p < 0.01; *** p < 0.001 (Wilcoxon signed-rank test) compared with values before treatment.

a

MGFA Quantitative MG score [13].

b

p < 0.05 (Mann-Whitney U test) compared with patients treated without FK506.

148

Eur Neurol 2005;53:146–150

Nagane/Utsugisawa/Obara/Kondoh/

Terayama


Statistics

Differences in the evaluation items of the efficacy of FK506

between the two groups were estimated with the Mann-Whitney U

test. Changes in clinical and laboratory parameters in each patient

group at 1 month, 6 months and 1 year into the treatment (compared

with before treatment) were estimated with Wilcoxon signedrank

test. The significance level was set at p = 0.05.

Side Effects

A side effect questionnaire was completed by the patient at each

visit. Blood test results were monitored by the nonblinded doctor.

Results

Two patients selected to receive treatment without

FK506 were excluded from the study because daily dose

of PSL exceeded 20 mg to achieve MM in the early-phase

therapy. The final analysis was performed for 18 patients

treated with FK506 and 16 patients treated without

FK506. There was no difference in background, including

the frequency of thymectomized patients between the

two groups ( table 1 ).

The Primary Endpoints

At the endpoint of the early-phase therapy ( table 2 )

and at 6 months and 1 year into the treatment ( table 3 ),

MM was achieved in both groups of patients, and the

mean severity score and the mean severity ratio were significantly

decreased in both groups of patients, compared

with before treatment, with no difference between the two

groups ( tables 2 and 3 ).

The number of treatments with IA + HMP was significantly

lower in the patients treated with FK506 than

in those treated without FK506 during early-phase therapy

(p ! 0.05; table 2 ). The period of early-phase therapy

was significantly shorter in the group treated with FK506

(p ! 0.05; table 2 ). During the follow-up phase therapy,

the number of treatments with IA + HMP and the number

of treatments with HMP alone were significantly lower

for the patients treated with FK506 than for those treated

without FK506 (p ! 0.05 and p ! 0.05, respectively;

table 2 ). At 1 year into the treatment, the PSL dose required

was significantly lower for the patients treated

with FK506 than for those treated without FK506 (p !

0.05; table 2 ).

Laboratory Parameters

At 6 months and 1 year into the treatment, PBM IL-2

production and PBM IL-2 production ratio were more

markedly decreased in the patients treated with FK506

than in those treated without FK506 (p ! 0.05; table 3 ).

Although the mean AChR Ab ratio was significantly

decreased in the patients treated without FK506 at 1

month, 6 months and 1 year into the treatment ( table 3 ),

it was not decreased in the patients treated with FK506

up to 6 months into the treatment ( table 3 ). The mean

AChR Ab ratio was significantly lower in the patients

treated without FK506 than in those treated with FK506

at 1 month (p ! 0.05; table 3 ), but there was no difference

between the two groups at 6 months or 1 year ( table 3 ).

Side Effects

FK506 was well tolerated over the 1-year treatment

period. The mean concentration of FK506 did not change

throughout the study in the patients treated with FK506

( table 3 ). None of the patients exhibited significant side

effects up to 1 year into the treatment. Only 1 patient who

had been suffering from hypertension exhibited increased

levels of serum creatinine (1.4–1.5 mg/dl; normal range

0.4–1.1 mg/dl).

Discussion

In the present study, we demonstrated that using lowdose

FK506 to treat de novo MG patients reduces the

number of treatments with IA + HMP or HMP alone,

reduces duration of the early-phase therapy period in hospital,

and reduces the dose of PSL at 1 year into the treatment.

None of the patients exhibited significant side effects

up to 1 year into the treatment, which is consistent

with previous reports [9–11] , probably because of the

low-dose administration (daily dose of 3 mg) of FK506

[8] . Furthermore, among the 18 patients treated with

FK506, 4 patients (22%) maintained MM sufficiently

with FK506 alone during the follow-up phase (severity

score: 6.2 8 3.3 before treatment; 1.3 8 1.1 at 1 year).

This indicates that low-dose FK506 is safe and efficacious

for treatment of de novo MG patients. Among the 4 patients

who maintained MM sufficiently with FK506

alone, 3 exhibited elevated PBM IL-2 production

( 1 1,250 pg/ml [15] ) before treatment, generalized symptoms

of MG and elevated serum AChR Ab titer. This appears

consistent with previous reports that MG patients

with elevated PBM IL-2 production exhibited immediate

effects after FK506, with clinical characteristics including

generalized symptoms of MG and serum AChR-Ab

positivity [11, 15] .

The therapeutic protocol in the present study would

be somewhat different from an orthodox method that attaches

more importance to oral steroid therapy. However,

Effects of FK506 on MG Eur Neurol 2005;53:146–150 149


given that IA + HMP can be safely applied not only to

overcome acute deterioration but also to reduce the dose

of oral PSL and aim at remission in MG patients [14] ,

that the number of treatments is simple for evaluation,

and that low-dose PSL is the preferred treatment to avoid

side effects, the present methods can be considered for

treatment or clinical trial of MG.

The frequency of thymectomy was relatively low in the

patients with and without FK506 (39 and 38%, respectively;

table 1 ), possibly due to their relatively higher age

(56.6 8 17.0 and 54.2 8 16.1 years, respectively; table 1 ).

Although no significant difference in the effects of lowdose

FK506 was observed between thymectomized

(n = 7) and nonthymectomized (n = 11) patients at up to

1 year (not shown), longer-term observation is needed to

investigate the effects of low-dose FK506 on the need for

thymectomy.

In the patients treated with FK506, significant amelioration

of symptoms was observed at 1 month into the

treatment with FK506. However, serum AChR Ab titer

was unchanged at 1 month and then gradually decreased

at 6 months and 1 year ( table 3 ). In our previous study,

despite amelioration of symptoms, serum AChR Ab titer

was not affected in the first month after FK506 treatment

of MG [11] . The mechanisms of the early effect of FK506

regardless of AChR Ab titer levels remain to be elucidated.

The most recent report showed that the early effect of

FK506 probably results from its pharmacological actions

on the ryanodine receptor because of the early effects of

FK506 on MG patients with autoantibodies against the

C-terminus of ryanodine receptor [16] ; however, this issue

could not be addressed in the present study.

Although the present study is the first randomized trial

of the effects of FK506 on de novo MG patients, there

are limitations due to its unblinded design and the relatively

small number of patients. Larger-scale, multicenter,

double-blind and placebo-controlled clinical trials to confirm

the efficacy of FK506 in the treatment of MG are

currently in progress in Japan.

In conclusion, early administration of low-dose FK506

for the treatment of MG reduces the number of treatments

with IA + HMP or HMP alone, and reduces the

duration of the initial therapy, suggesting that it is an effective

measure of treatment for de novo MG patients.

Acknowledgement

This study was supported in part by a grant from the Ministry

of Education, Science and Culture, Japan.

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