A new tool to study ribavirin-induced haemolysis

Antiviral Therapy 2012; 17:1311–1317 (doi: 10.3851/IMP2308)
Original article
A new tool to study ribavirin-induced haemolysis
Etienne Brochot1,2*, Catherine François1,2, Sandrine Castelain1,2, François Helle 2, Albert Nguyen Van Nhien3,
Isabelle Duchaussoy 4, Dominique Capron1,4, Eric Nguyen-Khac 4, Gilles Duverlie1,2
Department of Virology, Amiens University Medical Center, Amiens, France
EA 4294, Jules Verne University of Picardy, Amiens, France
3
Carbohydrates Laboratory UMR6219, Jules Verne University of Picardy, Amiens, France
4
Department of Hepatogastroenterology, Amiens University Medical Center, Amiens, France
1
2
*Corresponding author e-mail: [email protected]
Background: Today, treatment of chronic hepatitis C is
based on a synergistic combination of pegylated interferon
and ribavirin with antiprotease inhibitors. Haemolytic
anaemia, which is the major side effect of ribavirin treatment, disrupts ribavirin treatment compliance and varies
significantly from one patient to another. There is an individual susceptibility to ribavirin haemolysis. With a view
to studying haemolysis, and thus optimizing the treatment response, we have developed a new in vitro tool for
analysing the ribavirin-induced lysis of red blood cells.
Methods: Resuspended red blood cells were incubated
with isotonic buffer and a range of concentrations of
ribavirin. Haemolysis was quantified by spectrophotometric measurement of the supernatant at 540 nm.
The assay was used to test the effects of various compounds and to investigate the susceptibility of patients
to haemolytic anaemia.
Results: In our assay, the degree of haemolysis is dependent on the ribavirin concentration used and can be
inhibited by the addition of dipyridamole (50% inhibitory concentration [IC50] 30 mM), ATP or glutathione (IC50
1.63 mM and 767 mM, respectively). We observed a strong
decrease in red blood cell haemolysis in the presence of
the ribavirin prodrug viramidine (Taribavirin®). When
testing the performance of this assay with blood from 24
patients before treatment, we observed a strong correlation between in vitro haemolysis before treatment and
the decrease in haemoglobin levels seen in vivo during
subsequent treatment (P<0.001).
Conclusions: With this new tool it is possible to better
evaluate individual susceptibility to ribavirin-induced
haemolysis before the start of treatment. In addition, this
model will enable the mechanism of ribavirin-induced
anaemia to be further explored and allow molecules that
could reduce ribavirin haemolysis to be screened and
tested in vitro. This approach could help optimize current and future therapeutic strategies involving ribavirin
in the treatment of chronic hepatitis C.
Introduction
Approximately 180 million individuals worldwide are
chronically infected with HCV and are at risk of morbidity and mortality from cirrhosis and hepatocellular
carcinoma. Since the addition of ribavirin (RBV) to
interferon therapy response rates have doubled, illustrating the importance of RBV in HCV treatment [1].
The bodyweight-adjusted dose of RBV appears to be
crucial for optimizing virological response rates [2].
The early cessation or dose reduction of RBV can affect
the treatment response [3]. Hence, RBV doses should
be optimal throughout the course of treatment. It is not
possible to increase the RBV dose indefinitely, however,
as haemolytic anaemia (the major side effect of RBV)
will compromise treatment compliance and outcomes.
RBV-induced anaemia remains poorly described and
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limits the clinician’s ability to maximize the treatment
response. RBV becomes concentrated in circulating red
blood cells (RBCs) and induces an ATP deficiency [4].
This phenomenon leads to RBC toxicity through oxidative membrane damage and the inhibition of intracellular energy metabolism [5]. To maximize the patient
benefit of RBV, a balance must be struck between the
drug’s antiviral activity and its treatment-limiting side
effect [6].
During combination therapy, the average drop in
haemoglobin is approximately 2.5–3 g/dl. The drop
is >4 g/dl in about 20% of patients and approximately 10% of patients present with severe anaemia
(defined as an haemoglobin level <10 g/dl) [7]. RBV
lacks a therapeutic window in which effective but
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non-cytotoxic concentrations are reached and maintained. In most cases, toxicity follows on from efficacy.
On an individual patient basis, however, RBV-plasma
concentration is not always associated with a fall in
haemoglobin levels. There is a strong individual susceptibility to haemolysis. Recently, two polymorphisms
(rs1127354 and rs7270101) in the gene coding for inosine triphosphatase (ITPA) have been found to protect
against RBV-induced anaemia [8]. However, only 30%
of the population have ≥1 of these polymorphisms and
a total lack of ITPA activity is found in just 2% of the
population [9]. It is essential to develop strategies for
maximizing treatment compliance and thus improve
outcomes in current and future therapeutic strategies
involving RBV. A better understanding of the mechanism of RBV-induced anaemia could help to counteract this side effect. In addition, a means of predicting
haemolysis prior to treatment initiation would be of
major therapeutic benefit. To this end, we have developed a new in vitro model of RBV-induced anaemia.
With this model, it is now possible to estimate, in part,
the risk of developing severe anaemia before starting
treatment. Moreover, the model will be able to provide
insights into the mechanism of RBV-induced haemolysis and should facilitate the discovery of new drugs to
counteract this effect.
Methods
In vitro red blood cell haemolysis by ribavirin
To measure RBC haemolysis by RBV, blood was first
collected in EDTA-coated tubes and samples centrifuged at 2,500 rpm for 10 min. RBCs were separated
from plasma and the buffy coat and then washed twice
with 4 volumes of phosphate-buffered saline (pH=7.4).
Next, 300 ml of plasma and 300 ml of RBC suspension
were placed in a polypropylene tube. To obtain a haematocrit of 15%, 1,400 ml of a mixture containing RBV
and isotonic buffer (0.125 M sodium chloride, 0.005
M potassium chloride, 0.0012 M magnesium chloride,
0.0012 M potassium phosphate monobasic, 0.24 M
sodium bicarbonate and 0.01 M glucose, as described
by Grattagliano et al. [10]) were added. RBV was
diluted in this buffer to concentrations ranging from 0
to 10 mM. The tubes were then closed and incubated
horizontally with shaking (130 rpm) for various time
intervals at 37°C.
The haemolysis assay
After incubation, tubes were centrifuged at 2,500 rpm
for 10 min. Haemolysis was quantified by spectrophotometric measurement of cyanmethemoglobin in the
supernatant at 540 nm. The percentage haemolysis in
each tube was determined as follows: % haemolysis =
(A/B) ×100, where A is the optical density at 540 nm
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(OD540) in the supernatant for a tube at a given concentration of RBV and B is the OD540 for the supernatant
from a positive control tube (containing water instead
of isotonic buffer with the same haematocrit).
Reagents
Dipyridamole was purchased from Sigma–Aldrich
(St Quentin Fallavier, France). RBV was provided by
Carbosynth (Compton, UK). All compounds were dissolved in the isotonic buffer described above. Viramidine
(Taribavirin®) was synthesized according to the method
described in the literature [11].
Genotyping
Genomic DNA was extracted from whole blood samples using the QIAamp DNA blood minikit (Qiagen,
Courtabeuf, France), according to the manufacturer’s
instructions. Each patient was genotyped (after having
provided their written informed consent) for two ITPA
polymorphisms [8] using an ABI TaqMan allelic discrimination kit and the ABI7900HT sequence detection
system (both from Applied Biosystems, Courtaboeuf,
France). The ITPA activity was determined according
to the method described by Maeda et al. [12].
Patients
Written informed consent was obtained from each
study participant and ethical approval was obtained
from our local investigational review board. A total
of 24 adult patients infected with genotype 1 HCV
were included in the study. Patients began combination therapy with pegylated interferon (PEG-IFN)-a2b
and RBV for chronic hepatitis C infection between
February 2010 and May 2011. Exclusion criteria
included coinfection with HBV and/or HIV, abnormal RBCs (thalassaemia, sickle cell disease, pyruvate
kinase deficiency or glucose-6-phosphate deficiency)
and impaired kidney function. Patients were excluded
if they had reduced the RBV dose during treatment
or if erythropoietin had been administered. Patients
received weekly injections of PEG-IFN-a2b and RBV
was administered orally. The RBV dose was based on
bodyweight and the mean value for the group of 24
patients was 15.04 ±1.51 mg/kg/day.
Four variables were recorded prior to treatment: age,
creatinine clearance, complete blood count data and
HCV RNA concentration. Haemoglobin levels were
measured at four time points during the course of treatment (at weeks 4, 8, 12 and 24).
Statistical analysis
All experiments were performed in triplicate. R software
was used to perform the statistical analysis. Student’s
t-test and Wilcoxon’s test were used to compare
mean values. The Pearson correlation coefficient was
©2012 International Medical Press
25/10/2012 13:45:04
Studying ribavirin-induced haemolysis
Figure 1. Ribavirin-induced in vitro haemolysis of red blood cells
50
24 h
48 h
72 h
96 h
Haemolysis, %
40
30
20
10
0
0
0.5
1
2
5
10
Ribavirin concentration, mM
Resuspended red blood cells were incubated with isotonic buffer and a range of concentrations of ribavirin. Haemolysis was quantified by spectrophotometric measurement
of the supernatant at 540 nm. The percentage haemolysis was determined with respect to a positive control (containing water instead of isotonic buffer). Measurements
were made after 24, 48, 72 and 96 h incubation at 37°C. Each bar represents the average of three experiments. Error bars represent ±1 standard error of the mean.
determined in a linear regression analysis. The threshold
for statistical significance was set to P<0.05.
Results
Ribavirin-induced in vitro haemolysis of red blood cells
The first objective of this study was to develop an in
vitro assay of RBC haemolysis with increasing RBV
concentrations. After various adjustments of the isotonic buffer composition and the haematocrit, the
percentage haemolysis of RBCs was analysed at various incubation times. An RBV concentration range
from 0 to 10 mM was chosen (Figure 1). The RBV
concentration found in RBCs in vivo is approximately
1.5 mM [13]. Haemolysis was measured after 24, 48,
72 and 96 h incubation with RBV (Figure 1). We can
observe that for each measurement point the variability is very low. In the absence of RBV, the percentage haemolysis after 24, 48, 72 and 96 h was 0.92%
±0.17%, 1.26% ±0.26%, 2.01% ±0.07% and 6.24%
±0.57%, respectively. The effect of RBV on RBCs can
be detected from 48 h onwards. The repeatability of
this model of haemolysis is very good with low intraand interassay variability (approximately 5%). For
RBV concentrations ranging from 0–5 mM, the linearity was good (Pearson coefficient correlation 0.952;
five measurement points, data not shown). For the
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first time, these results demonstrated three key points:
RBV causes lysis of RBCs in vitro, this phenomenon
requires time to be triggered, as observed in patients
and the haemolysis observed in vitro by RBV is linear
and repeatable. For all further analyses, we chose to
read haemolysis after 72 h incubation, which provides
a good compromise between a detectable level of haemolysis and an incubation time that is not too long.
Inhibition of haemolysis
To cause haemolysis, RBV must accumulate inside
RBCs. The human equilibrative nucleoside transporter is involved in the cellular import of natural
and synthetic nucleotides and mediates RBV uptake
by RBCs [14]. We found that the human equilibrative
nucleoside transporter inhibitor dipyridamole significantly decreased RBV-induced haemolysis in vitro
(Figure 2A). This effect was concentration dependent;
there was a 50% inhibitory concentration (IC50) of
approximately 30 mM. These results confirm that haemolysis of RBCs in vitro is due to RBV and show that
it is possible to inhibit haemolysis in vitro by disrupting RBV entry into RBCs.
The addition of ATP and glutathione at various concentrations was then tested in our experimental model
in the presence of 2 mM RBV. As shown in Figure 2B,
ATP and glutathione greatly reduce the haemolytic
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Figure 2. Inhibition of in vitro haemolysis
B
20
20
Glutathione
ATP
Haemolysis, %
15
15
a
Haemolysis, %
A
b
10
c
5
b
b
10
c
b
c
c
5
c
c
0
0
Ribavirin
2 mM
1 µM
10 µM
20 µM
50 µM 100 µM
Dipyridamole
Ribavirin 0.5 mM
2 mM
1 mM
2 mM
5 mM
10 mM
ATP or glutathione
(A) Measurement of haemolysis in the supernatant after 72 h incubation at 37°C. Different concentrations (1–100 mM) of dipyridamole were added to the medium
in the presence of 2 mM ribavirin. The haemolysis assay was performed three times for each set of conditions. Error bars represent ±1 standard error of the mean. (B)
Measurement of haemolysis in the supernatant after 72 h incubation at 37°C. Different concentrations (0.5–10 mM) of ATP or glutathione were added to the medium
in the presence of 2 mM ribavirin. The haemolysis assay was performed three times for each set of conditions. Error bars represent ±1 standard error of the mean.
a
P<0.05, bP<0.01 and cP<0.001, when comparing 2 mM ribavirin alone with 2 mM ribavirin plus dipyridamole (A) or with 2 mM ribavirin plus ATP or glutathione (B).
Figure 3. Effect of viramidine on red blood cell haemolysis
in vitro
30
Haemolysis, %
Ribavirin
Viramidine
20
a
10
b
a
a
b
0
0
0.5
1
2
Concentration, mM
5
10
Measurement of haemolysis in the supernatant after 72 h incubation at 37°C
in the presence of ribavirin or viramidine (0–10 mM). The haemolysis assay
was performed three times for each set of conditions. Error bars represent ±1
standard error of the mean. aP<0.01 and bP<0.001, when comparing ribavirin
with viramidine.
effect of RBV (IC50 1.63 mM and 767 mM, respectively). These results clearly demonstrate that the origin of RBV haemolysis is a decrease in the intracellular
energy and antioxidant pool.
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The effect of viramidine on red blood cell
haemolysis in vitro
Viramidine (Taribavirin®, a 3-carboxamidine prodrug of RBV) is associated with a lower incidence
of anaemia because of its liver-targeting properties
[15,16]. We therefore synthesized viramidine [11]
and checked its activity against poliovirus. At a concentration of 1,000 mM, RBV and viramidine both
decreased the poliovirus titre at 24 h by 2.83 log and
2.03 log, respectively (data not shown). When we
used viramidine in the haemolysis assay, significantly
lower haemolysis was observed (relative to RBV) at
all concentrations tested (Figure 3). The mean reduction for viramidine concentrations between 0.5 and
10 mM was 62.4% ±11.4%. These results suggest
that it will be possible to use the haemolysis assay
developed here both to better understand the mechanism of RBV-induced anaemia and to test molecules
that could attenuate haemolysis.
Correlation between in vitro haemolysis before
treatment and in vivo anaemia during treatment
We next investigated the putative link between in vitro
haemolysis and the decrease in haemoglobin levels
observed during standard treatment with PEG-IFNa2b and RBV. The in vitro haemolysis assay was performed three times on samples from 24 patients before
starting treatment. For each patient, the average maximum percentage haemolysis observed in vitro at 72 h
©2012 International Medical Press
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Studying ribavirin-induced haemolysis
Figure 4. Correlation between in vitro haemolysis before treatment and in vivo anaemia during treatment
Decrease in haemoglobin during treatment
divided by ribavirin daily dose
0.4
r=0.7271552
P=0.000188
0.3
0.2
0.1
0
0
10
20
30
40
50
Maximum in vitro haemolysis before treatment, %
For each of the 24 patients, the haemolysis assay was performed three times on the same sample before the initiation of treatment. For a given patient, the average
maximum percentage haemolysis observed in vitro at 72 h was compared with the reduction in haemoglobin levels during treatment (g/dl), relative to daily dose of
ribavirin (mg/kg/day). Pearson’s coefficient and the P-value were calculated.
Table 1. Inosine triphosphatase polymorphisms, in vitro haemolysis and anaemia
Characteristic
ITPA activity ≤30% (n=6)
ITPA activity >30% (n=18)
P-value
Maximum haemolysis in vitro, %
In vitro haemolysis without RBV, %
Reduction in Hb at week 4, g/dl
Reduction in Hb at week 4, %
Maximum reduction in Hb during
treatment, g/dl
29.62 ±7.69
2.38 ±0.51
0.83 ±0.46
5.46 ±3.01
2.77 ±0.89
35.17 ±7.93
3.74 ±1.08
2.23 ±1.22
13 ±8.76
3.38 ±1.27
0.1552
0.0006
0.0024
0.0097
0.22
Data are mean ±sd unless otherwise indicated. Polymorphisms in the inosine triphosphatase (ITPA) gene were determined for each patient. The possible genotypes
for each biallelic polymorphism are as follows: C/C, A/C and A/A (where A is the minor allele) for rs1127354 and A/A, A/C and C/C (where C is the minor allele) for
rs7270101. ITPA activity was determined according to the method described by Maeda et al. [12]. On this basis, two groups of patients were derived (activity ≤30% or
>30%) and compared in terms of their mean values. Hb, haemoglobin; RBV, ribavirin.
was compared with the RBV dose-related decay in haemoglobin levels during treatment (Figure 4). The two
parameters were well correlated, with a Pearson correlation coefficient of 0.727 (P=0.000188). Patients who
subsequently showed a strong decrease in haemoglobin
levels during treatment had the highest pretreatment
levels of in vitro haemolysis in the presence of RBV
(Figure 4). We conclude that the in vitro haemolysis
assay performed prior to treatment can estimate individual susceptibility to RBV-induced anaemia.
ITPA polymorphism, in vitro haemolysis and anaemia
during treatment
Finally, the 24 patients were screened for ITPA polymorphisms. A deficit in the activity of ITPA leads to
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reduced anaemia during RBV treatment. The probable
ITPA activity was also calculated [12]. On this basis,
patients were subdivided into two groups (activity
≤30% or >30%) and their respective in vitro and in
vivo data compared. The group with low ITPA activity
had a lower mean peak haemolysis value in vitro than
the group with >30% activity (Table 1). In addition,
RBCs from patients with low ITPA activity were more
resistant to haemolysis in vitro in the absence of RBV
(P=0.0006) and showed a significantly lower reduction in haemoglobin levels after 4 weeks of treatment
(P=0.0024; Table 1). However, this difference was no
longer statistically significant when we looked at the
reduction in haemoglobin over the course of treatment
as a whole.
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Discussion
To the best of our knowledge this is the first study to
describe the in vitro haemolysis of RBCs in the presence
of RBV and isotonic buffer. The degree of haemolysis
depends on the incubation time and concentration of
RBV. Two previous studies induced haemolysis rapidly
(after 4 h or 8 h) by using RBV in a hypotonic buffer
[10,17]. This approach introduced variability that was
not due to the RBV concentration and makes the technique difficult to use and reproduce. We also incubated
RBCs in the presence of phosphate-buffered saline.
We noted a strong haemolysis without RBV. With a
new buffer, by testing longer incubation times under
isotonic conditions we observed dose-dependent haemolysis from 48 h. De Franceschi et al. [5] have shown
that the pool of ATP in RBCs decreases after 12 h incubation with RBV. This is probably why a delay in the
onset of haemolysis was observed in our assay. After
optimizing the incubation time, we adjusted the haematocrit of the assay to 15%. We obtained a plateau in
haemolysis at 5 mM. This observation might be due
to a saturation of RBV entry and phosphorylation in
RBCs. A study of the metabolism of RBV in mice indicated that the plateau observed in the intra-erythrocyte
concentration of the drug was due to ATP-dependent
kinases [18]. Inside the RBC, RBV is converted to a
triphosphate derivative by an ATP-dependent adenosine kinase [19].
To test the specificity of haemolysis, we supplemented the incubation buffer with the RBV uptake
inhibitor dipyridamole [14]. Under these conditions, we observed strong inhibition of haemolysis
(Figure 2A). Although dipyridamole is a licensed drug
with antiplatelet and coronodilatating properties, it
cannot be used to reduce haemolysis in vivo because
it would also prevent RBV from entering hepatocytes.
Nevertheless, our in vitro observation proves that it
is possible to pharmacologically counter the adverse
effects of RBV on RBCs, as demonstrated with ATP
and glutathione (Figure 2B). Our assay thus constitutes a new tool for the in vitro screening of compounds that might counter the major adverse effect of
RBV treatment.
We synthesized the nucleoside analogue viramidine
from RBV, as described elsewhere [11]. Viramidine is an
oral prodrug of RBV that is currently undergoing Phase
III clinical trials. Viramidine might increase compliance
with treatments of chronic HCV infection by reducing
the need for anaemia-triggered dose reduction. Here,
we demonstrated that haemolysis is much greater for
RBV than for viramidine at a given drug concentration
(Figure 3). These in vitro results confirm for the first
time that viramidine might have fewer adverse effects
in vivo in patients with chronic hepatitis C.
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The pretreatment prediction of haemolysis is a major
challenge in optimization of the treatment response.
We observed a strong correlation between the peak in
vitro haemolysis in our assay (with samples collected
before treatment) and the fall in haemoglobin seen during therapy (Figure 4). Hence, our assay could be used
to screen for individuals at high risk of severe anaemia (typically older patients, patients with abnormal
RBCs and those with renal and cardiovascular disorders). Patients with increased resistance to haemolysis
in vitro (in the presence of RBV) could be treated with
higher initial doses of the drug, offering a greater likelihood of a sustained virological response. Indeed, a
recent meta-analysis has concluded that the addition of
ribavirin was a benefit in the treatment of hepatitis C
patients with thalassaemia [20]. It would be interesting
to use our assay in patients with haemoglobinopathies.
We observed a good correlation between in vitro
haemolysis and the decrease in haemoglobin during
treatment. However, the correlation is not complete
and could be explained by the individual effect of
interferon on bone marrow. Another explanation is
that, despite the fact that doses of RBV were weightadjusted, plasma concentrations can fluctuate greatly.
We did not need to measure RBV levels in vivo. Indeed,
measuring RBV-induced anaemia is useful to detect
low concentrations in non-responders [21,22], there is
no single upper RBV concentration threshold for anaemic toxicity. Hence, anaemia prediction for a patient
before treatment appears to be more relevant.
Lastly, we studied ITPA polymorphisms that
were recently found to protect against anaemia in a
genome-wide association study [8] of 1,602 European/
American patients. However, the resulting difference in
ITPA function explains only 30% of the variability in
quantitative haemoglobin reduction; thus, IPTA activity is not sufficient for fully predicting susceptibility to
anaemia for a given patient. The same researchers also
showed that these ITPA polymorphisms were associated with a decrease in haemoglobin after 4 weeks of
treatment, but not throughout [23]. This same observation was made in our study.
In conclusion, the assay presented here should be of
use in establishing a patient’s susceptibility to anaemia and might facilitate screening for compounds that
could counteract the effect of RBV on RBCs. Novel
anti-HCV drugs (such as the protease inhibitors telaprevir and boceprevir) are likely to be used in combination with current treatments and are associated
with increased rates of anaemia and frequent erythropoietin use [24]. Thus, RBV will remain an important
drug for achieving a sustained virological response in
HCV-infected individuals and the knowledge and prediction of anaemia will continue to be an important
issue in therapy.
©2012 International Medical Press
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Acknowledgements
We are grateful to David Fraser for English corrections.
Disclosure statement
The authors declare no competing interests.
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Accepted 5 March 2012; published online 5 September 2012
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