A prospective, open-label phase III study was carried out to evaluate the efficacy and tolerability of a preparation of virus-inactivated normal human intravenous immunoglobulin. Fifteen adult patients suffering from chronic idiopathic thrombocytopenic purpura (ITP), (basal platelet count <20x109/l), received 0.4 g/kg daily for 5 days. The increase in platelet count, reduction in hemorrhagic events, duration of platelet response, and incidence of adverse events were recorded. Fourteen out of 15 patients achieved a platelet count ?50x109/l (response rate was 93.3%), and only 2 out of 10 patients with positive baseline Rumple-Leed test remained positive after treatment (p =0.0022). Interestingly, a close inverse correlation between platelet counts and haemoglobin levels was observed. No side effects were recorded. This study provides evidence of therapeutic efficacy and the good tolerability of the immunoglobulin preparation we used in this trial, and suggests the role of haemolysis as a mechanism for the increase in platelet count

Balestri, Francesca

Fiorentino, Bruno

Grossi, Alberto

Guarnieri, Chiara

Guazzini, Stefano

Minichilli, Fabrizio

Scarpellini, Manuela

Tognoni, Daniela

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567
INTRAVENOUS IMMUNOGLOBULIN IN ITPCopyright ' Hellenic Society of Haematology
HAEMA (`ßìÆ) ÉSSN: 1108-2682
www.mednet.gr/eae/haema
Haema 2006; 9(4): 567-571
Original article
Intravenous immunoglobulin therapy in the treatment of the acute
phase of chronic idiopathic thrombocytopenic purpura in adults
Alberto Grossi1, Francesca Balestri1, Daniela Tognoni1, Fabrizio Minichilli2, Bruno Fiorentino3,
Chiara Guarnieri3, Manuela Scarpellini3, Stefano Guazzini3
1U.O. Haematology, Azienda Ospedaliera Universitaria Careggi, Florence, 2Sezione di Epidemiologia e Ricerca
sui Servizi Sanitari, IFC-CNR, Pisa, 3Kedrion SpA, Castelvecchio Pascoli, Italy
Abstract. A prospective, open-label phase III study was carried out to evaluate the efficacy and
tolerability of a preparation of virus-inactivated normal human intravenous immunoglobulin. Fifteen
adult patients suffering from chronic idiopathic thrombocytopenic purpura (ITP), (basal platelet count
<20x109/l), received 0.4 g/kg daily for 5 days. The increase in platelet count, reduction in hemorrhagic
events, duration of platelet response, and incidence of adverse events were recorded. Fourteen out of
15 patients achieved a platelet count ‡50x109/l (response rate was 93.3%), and only 2 out of 10 pa-
tients with positive baseline Rumple-Leed test remained positive after treatment (p =0.0022). Inter-
estingly, a close inverse correlation between platelet counts and haemoglobin levels was observed. No
side effects were recorded. This study provides evidence of therapeutic efficacy and the good tolera-
bility of the immunoglobulin preparation we used in this trial, and suggests the role of haemolysis as
a mechanism for the increase in platelet count.
Key words: intravenous immunoglobulin    idiopathic thrombocytopenic purpura
* Correspondence: Alberto Grossi, MD, Istituto Leonardo da Vinci-Haematology, via Colletta 22/r,
Florence, Italy, Tel.:+393385200467, Fax: +300552487230, e-mail: alberto grossi@libero.it
INTRODUCTION
Autoimmune thrombocytopenia is caused by an au-
toantibody against patients own platelets. It may present
either as idiopathic thrombocytopenic purpura (ITP) or
secondary immune thrombocytopenia as a manifestation
of other autoimmune or neoplastic diseases, especially non
Hodgkin lymphomas. Intravenous immunoglobulins
(IVIG), which were first used in 19811, correct or amelio-
rate thrombocytopenia in about 80% of patients with
chronic ITP2, and the rapid increase in platelet count is
obtained as early as 2 days from the beginning of treat-
ment, peaking after 5-7 days and lasting for about 2 weeks.
Long-term efficacy has only been observed occasionally3,4,
thus this therapy is especially useful when a rapid increase
in platelet count is required, such as in the risk or event of
severe bleeding, and for the preparation of patients for
surgical procedures or delivery. Intravenous immunoglo-
bulin administration is generally safe and headache, skin
rashes and fever are the infrequent (12-23% of patients)
and usually mild side effects5. Haemolysis due to the pres-
ence of ABO haemolysins has also been described6, al-
though in a different setting than ITP. Current prepara-
tions of IVIG do not contain aggregates that would pre-
vent their intravenous use, and latest generation products
contain intact IgG molecules that retain their functions.Received: November 29, 2005; Accepted: May 5, 2006
J. Meletis et al
568
A. Grossi t l
In this study, a virus-inactivated preparation of immuno-
globulin for intravenous administration was used in a
group of patients with chronic ITP, previously treated with
steroids and/or IVIG.
PATIENTS AND METHODS
The Ethic Committee of Azienda Ospedaliera and
University Careggi, Florence, Italy, approved the study
protocol. The study was carried out according to the Dec-
laration of Helsinki and subsequent amendments, and
according to Good Clinical Practice (GCP) and Good
Laboratory Practice (GLP) regulations. In compliance
with the regulations of GCP, all patients gave their writ-
ten informed consent to participate in the study before
entering the study.
Inclusion criteria were as follows: a) chronic ITP di-
agnosed according to the criteria of George et al (1996)7;
b) patients with the age between 18 to 80 years; c) pa-
tients with good response to previous treatment with IVIG
or steroids; d) platelet count of £20x109/l before adminis-
tration of investigational therapy on the day of the first
infusion (a wash-out period of at least 2 weeks was per-
mitted); e) adequate contraception (if the patient was a
woman of childbearing age) for the whole duration of the
study.
Patients with severe heart or renal failure, pregnancy
or lactation, positive tests for anti-HIV, anti-HCV, Hb-
sAg, risky behaviour for blood-transmitted viral infections
such as drug abuse or risky sexual relations, a history of
severe adverse transfusion reactions with blood or blood
derivatives or intolerance to other IVIG preparations,
especially in very rare cases of IgA deficiency and hyper-
sensitivity to any component of the investigational medi-
cation were excluded.
Fifteen patients (13 female, 2 male, with a median age
of 51 years, range 22 to 73 years) entered the study. Pa-
tients were given one cycle of the investigational drug (Ig
VENA, Kedrion, Italy) 2 g/Kg body weight subdivided into
5 infusions of 0.4 g/kg, daily on 5 consecutive days. In un-
responsive cases, a second cycle of 0.4-1 g/kg for 5 days
was administered within 14 days. Treatment with IVIG
other than Ig VENA or other blood derivatives was not
allowed throughout the study.
The primary efficacy end-point was defined as the pro-
portion of patients who achieved a platelet count ‡50x109/l.
Secondary efficacy variables were: I) the time to
achieve a platelet count of ‡50x109/l, II) the duration of
platelet count of ‡50x109/l until the day on which one of
the following events occurred such as: requirement for
the administration of additional treatment either with the
investigational preparation or with another medication for
ITP; documentation of a platelet count below 50x109/l;
the last determination of platelet count (14th day), III) the
maximum platelet counts, IV) results of the Rumpel-Leed
test (petechiae count): difference in the number of newly
formed petechiae between day 5 and day 0 (day of 1st in-
fusion).
Statistical Analysis
Petechiae count (Rumpel-Leed test) and platelet
count were summarized for each day considering mean,
standard deviation (SD), median and InterQuartile Range
(IQR): 25th percentile-75th percentile.
Both Rumpel-Leed test and comparison between
median platelet counts of each day versus the baseline
values were evaluated with non parametric Wilcoxon
matched-pairs signed-rank test.
The comparison between mean platelet counts each
day versus baseline values were evaluated with paramet-
ric paired t test.
Median and mean trend of platelet counts over time
were evaluated by median spline functions.
The cumulative probability of platelet response with a
platelet count of ‡50x109/l was estimated using a non para-
metric Kaplan-Meyer survival procedure. Spearmans
Rank Order Correlation Coefficient was used for analy-
sis of correlation between platelet count and haemoglo-
bin. Simple regression analyses for repeated counts (plate-
let counts were determined on 8 different days for each
patient) using the method of Generalized Estimating
Equations (GEE) were used to assess the association be-
tween platelet counts and haemoglobin values. All statis-
tical analyses were performed using STATA 8 SE soft-
ware.
 Safety and Tolerability
Adverse events were recorded during the 24 hours
following administration of the investigational product,
and during the subsequent three days. Vital signs (blood
pressure, heart rate, body temperature and respiratory
rate) were recorded every 30 minutes during the infusion
period of IVIG and one hour after the end of infusion.
Routine laboratory tests included complete blood count,
serum creatinine, urinalyses and anti-A, anti-B and anti-
D haemagglutinins.
569
INTRAVENOUS IMMUNOGLOBULIN IN ITP
RESULTS
All the patients achieved a platelet count of ‡50x109/l
(93.3%), except one who did not achieve the target plate-
let count (maximal count 47x109/l) even after a second
cycle of therapy. Platelet counts started to increase on day
2 of treatment, and at this time 3/15 (20%) patients
reached a normal platelet count. At the end of treatment
(day 5) more than half of the patients (8/15  Normaliza-
tion Rate: 53%) achieved normal platelet counts which
were maintained up to 5 days later in all patients except
one.
Cumulative probability of success estimate (Kaplan-
Meier) was 40% for day 1, 67% for day 2, 87% for day 3
and 93% for day 4, and then remained stable up to day 7
(Figure 1). Out of the 14 patients reaching the target plate-
let count, only three showed a decrease of platelet counts
below 50x109/l before the end of the study, as described in
Figure 2.
Median platelet value on day 1 was significantly high-
er than on day 0 (p=0.0007) (Table 1), but the target plate-
let count value was achieved on day 2 and continued to
increase up to day 5 and then started to decrease (Figure
3). On day 14 median platelet value was also significantly
higher than baseline value (p=0.0009). The peak of me-
dian platelet count was 159x109/l (IQR: 92-226). Mean
platelet value on day 1 was also significantly higher than
the value on day 0 (p=0.0001) (Table 1). The increase
trends of mean and median platelet counts were very sim-
ilar, but the peak of mean platelet count (Mean–SD,
181.9x109/l –130.2) was higher than median platelet count
peak (159x109/l) (Figure 3).
The mean of maximum platelet count was 219.3x109/l
–167.7 and this value was reached between day 5 and day
10 (Figure 3). A significant inverse correlation between
haemoglobin value and rise of the platelet count was ob-
served (Spearmans rho= -0.35 p <0.0001) (Figure 4): an
increase of 60 x 109/l in platelet count was associated with
Figure 2. Kaplan-Meier cumulative probability of response in
patients treated with Ig VENA 400 mg/kg · 5 days.
Figure 1. Kaplan-Meier cumulative probability of success in
patients treated with Ig VENA 400 mg/kg · 5 days.
Figure 3. The curves represent the behaviour of median and
mean values of platelet counts during the observation period
of patients.
Table 1. Mean and SD of platelet count, Median and IQR of
platelet count by period of observation.
Time Day Mean SD Median IQR
0 0 11.8 6.6 12 5-20
1 1 46.8 26.62 45 26-70
2 2 85.4 59.63 73 47-118
3 3 126.1 106.5 99 59-167
4 4 160.5 128.6 126 85-198
5 5 181.9 130.2 159 92-226
6 10 168.7 132.5 130 58-258
7 14 131.8 156.6 76 46-155
J. Meletis et al
570
A. Grossi t l
DISCUSSION
The results of this study show that IVIG preparation
we used was effective in adult patients with ITP and se-
vere thrombocytopenia (baseline £20x109/l). The response
rate was 93.3 % vs 75-80% reported in the literature, while
normalization rate was similar (53 % vs 50-60%) as well
as time to response and course of values2, and the differ-
ences could be ascribed to the small number of patients
of our study. It is worth pointing out that normalization
was evaluated according to the conservative lower refer-
ence value limit set in the central laboratory of our insti-
tution (140x109/l). In published studies the value is often
set at 100x109/l, which would pose the rate of this param-
eter at 66%, among the highest values reported in the lit-
erature. Duration of response is generally reported to be
3 to 4 weeks. In this study, the observation of patients was
stopped after 2 weeks since at this time 80% of patients
showed both the platelet count and the course of median
values similar to those reported in literature. Furthermore,
as expected, it was shown that the increase in platelet count
was associated with the correction of bleeding tendency,
as demonstrated by the normalization of Rumpel-Leed
test in the majority of patients (86.7%).
A positive test result for the presence of anti-D isoag-
glutinins was observed in some of the patients treated with
Ig VENA. Passive transmission of antibodies to erythro-
cyte antigens A, B and D has been described and may
interfere with some serological tests (e.g. Coombs test).
Since all Ig VENA batches are tested for the presence of
anti-D antibodies and released only if found negative, the
absence of clinical signs of haemolysis could lead us to
conclude that this finding is transient and can be ascribed
to test artefacts. Another possible explanation could be
the presence of anti-idiotype antibodies in the IVIG prep-
aration mimicking secondary anti-D Ig response.
Furthermore, the same patients have been re-tested
on occasion of normal controls and found anti-D nega-
tive.
Several hypotheses have been proposed to explain the
efficacy of IVIG treatment in ITP: a) blocking of mac-
rophage Fc receptors that would prevent the removal of
autoantibody-coated platelets8; b) a possible long-lasting
change in immune response with suppression of autoan-
tibodies synthesis in the (few) patients that present a sus-
tained response after treatment9,10, which cannot be ex-
plained by phagocytic cell blockade; c) a correction of
megakaryocyte production and maturation due to the in-
Figure 4. Haemoglobin values vs platelet counts. The figure
indicates a strict inverse correlation between the two parame-
ters (Spearmans rho = -0.35 p<0.0001).
a decrease in haemoglobin of 1 g/dl.
Rumpel-Leed Test
At baseline 10 patients (66.6%) had a positive Rumpel-
Leed test, and the number of petechiae ranged from 0 to
15 (mean – SD: 5.2–4.7; median: 5, I QR: 0-10). On day
5, after the completion of treatment, only 2 patients
(13.3%) had a positive Rumpel-Leed test and the num-
ber of petechiae diminished in both of them (from 15 to 5
and from 7 to 3). The reduction in the number of petechi-
ae was statistically significant (p=0.0022).
Anti-D Test
Haemagglutinins that were present at baseline per-
sisted in all patients and their titre did not undergo major
changes. At baseline 2 out of 15 patients were anti-D pos-
itive, 3 patients became anti-D positive after 1 day of treat-
ment, and 2 additional cases at day 2. Therefore the per-
centage of anti-D positive patients increased from 13.3%
to 46.6%. Anti-D positivity persisted until the end of treat-
ment in all the 7 patients.
Safety
No adverse events were reported and no relevant
changes in laboratory tests were reported, in particular
no clinical or laboratory evidence of haemolysis in patients
with anti-D haemagglutinins was observed.
571
INTRAVENOUS IMMUNOGLOBULIN IN ITP
hibitory effect of autoantibodies11,12; d) prevention of plate-
let phagocytosis due to coating of erythrocytes by infused
immunoglobulin, as the number of red blood cells is much
higher than that of circulating platelets. The last hypoth-
esis proposed by Salama et al.13 seems to be supported by
the results of the present study, in which an inverse corre-
lation was observed between platelet count and haemo-
globin. However it seems likely that a combination of
mechanisms cooperate in correcting thrombocytopenia in
ITP patients treated with IVIG.
This study indicates that the IVIG preparation we used
enhances a rapid increase in platelet count of the majori-
ty of our patients population, without significant adverse
events. The therapeutic characteristics of the IVIG pre-
paration make it eligible for the treatment of severe throm-
bocytopenia episodes in patients with chronic ITP. Large
scale, prospective clinical trials with long follow-up peri-
ods using this IVIG preparation should also be done.
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Intravenous immunoglobulin therapy in the treatment of the acute phase of chronic idiopathic thrombocytopenic purpura in adults

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Intravenous immunoglobulin therapy in the treatment of the acute phase of chronic idiopathic thrombocytopenic purpura in adults

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