Therapeutic Plasma Exchange in Multiple Sclerosis Patients with Abolished Interferon-beta Bioavailability

Background

Material and methods

STUDY DESIGN:

An open-label, single-center proof of concept study was initiated in 2013 at the Neurology and Neurosurgery Clinic, Faculty of Medicine at Vilnius University, Vilnius University Hospital Santariskiu Clinics, Department of Neurology. The study protocol was approved by the Vilnius Regional Bioethics Committee (Trial registration number 158200-13-644-191) and written informed consent was signed by each subject.

STUDY POPULATION:

The participants were MS patients treated at Vilnius University Hospital Santariskiu Clinics. We included 6 patients in the study and the study was ended earlier than initially planned because the results were so evident that it was considered unethical to continue the study.

The trial population consisted of subjects with RRMS according to the following inclusion and exclusion criteria.

EXCLUSION CRITERIA:

The mean age of these patients at the screening onset was 43.7 years (range, 36–54 years). Four patients were women (66.7%) and 2 patients were men (33.3%). The average treatment duration of immunomodulatory therapy prior to the screening was 4.5 (±1.8) years. The mean disability score on the EDSS was 3.7 (±0.9) (Table 1). All patients were clinically stable during the study.

THE PROCEDURE:

Six patients met the inclusion criteria and were included over a 4-month recruitment period. Patients underwent 4 separate plasma exchange sessions of 2.0–2.5 plasma volumes over a 5–8 day span. After induction plasmapheresis, in order to sustain the bioavailability of IFN-β, the patients were transferred to the maintenance plasmapheresis – 3 sessions were performed in total, with 1 plasma exchange session per month. Donor plasma was used for plasma replacement. All participants received IFN-β-1b (Betaferon) 250 μg subcutaneously every other day during the entire study. One patient after the first incomplete plasma exchange session (1100 ml plasma volume was replaced) was switched to the centrifugal plasmapheresis due to adverse reactions to donor plasma; the patient underwent 6 centrifugal plasmapheresis sessions of 310–380 ml volumes every day (Table 2). Premedication with clemastine IM and dexamethasone IV before TPE was given to avoid hypersensitivity reactions and calcium gluconate was infused IV during the TPE to avoid hypocalcemic symptoms.

IFN-β BIOACTIVITY MEASUREMENT:

Blood from MS patients was drawn into PAXgene Blood RNA Tube (PreAnalytiX GmbH, Hombrechtikon, Switzerland) before and 12 hours after injection of INF-β. Total RNA was extracted using Purlink FFPE Total RNA Isolaton Kit (Life Technologies, Carlsbad CA, USA) according to the manufacturer‘s protocol. Total RNA was subsequently reverse-transcribed to cDNA using RevertAid™ M-MULV reverse transcriptase and random hexamers (Thermo Fisher Scientific, Vilnius, Lithuania). Previously published sequences for MxA and GAPDH primers and probes were used in duplex qPCR reactions [35]. qPCR reactions were performed in a 20-μl reaction volume containing Maxima® Probe qPCR Master Mix supplemented with Uracil-DNA glycosylase (Thermo Fisher Scientific, Vilnius, Lithuania), 300-nmol/l final concentration of each primer, 200 nmol/l of each probe, and 2-μl cDNA solution. qPCR was performed on a RotorGene 6000 analyzer (Qiagen, Hilden, Germany) using the following cycling conditions: 2 min at 50°C followed by 5 min at 95°C and 45 amplification cycles at 95°C for 20 s, 60°C for 15 s, and 72°C for 20 s. Relative quantification of MxA expression was calculated by the ΔΔCq method. Peripheral blood cDNA from a pool of healthy donors was used as a calibrator in ΔΔCq calculations and was run in parallel with patient samples. This calibrator was assigned the normalization ratio 1. MS patient samples before and after INF-β injection were always analyzed in the same qPCR run. Two cut-off values for INF-β bioactivity were determined based on MxA expression results of 30 untreated MS patients, as described previously [36].

THE DEFINITION OF BIOLOGICAL RESPONDERS, POOR BIOLOGICAL RESPONDERS, AND BIOLOGICAL NON-RESPONDERS:

Biological responders, poor biological responders, and biological non-responders were defined based on the absolute values of their MxA expression/induction indicators regarding 2 cut-off values established before the study. Biological responders were defined as the patients whose MxA expression values were above both threshold levels. In case only 1 threshold was reached, a patient was assigned to the group of poor biological responder. If neither of the 2 thresholds was reached, a patient was defined as a biological non-responder. MxA mRNA expression cut-off of <0.586 before INF-β injection and MxA mRNA expression cut-off of <3.84 after INF-β injection were considered as negative.

Results

TPE EFFECT ON THE BIOAVAILABILITY OF IFN-β:

A sharp increase of MxA mRNA expression and induction was found in 4 patients after induction plasmapheresis (in 1 case after centrifugal plasmapheresis); the patients regained an in vivo MxA response to IFN-β therapy and became “biological responders” (Figure 1). In 2 patients, an increase of MxA mRNA expression or induction was found but the values persisted below the expression and induction cut-off and the patients remained as “poor biological responders”. In the patients who became “biological responders”, MxA mRNA expression values after the maintenance plasmapheresis sessions reverted to the baseline levels after 1 or 2 months (Figure 2), in 2 patients after the first maintenance plasmapheresis and in the other 2 patients after the second session.

THE EFFECT OF TPE ON THE BABS:

The mean BAbs titer in all patients before TPE was 528 BTU (±335.0 BTU). In 4 patients, a decrease of BAbs titer was found after induction TPE; a decrease by 403.8 BTU on average was found. In the first and in the second patient, an increase of BAbs titer by 610 and 377 BTU, respectively, was found. In contrast to increased BAbs titers, these 2 patients regained an in vivo MxA response to IFN-β therapy after the initial course of plasmapheresis. The mean titer of BAbs in all patients after induction plasmapheresis was 423.8 BTU (±459.5 BTU) and 396.8 (±354.0 BTU) in 5 patients after the maintenance plasmapheresis (Figure 3).

SAFETY AND TOLERABILITY OF THE TPE:

One of the 6 patients after the first incomplete plasma exchange session was switched to the centrifugal plasmapheresis due to the excessive itchy rashes. The rashes persisted after the additional treatment with dexamethasone and clemastine. One patient during the first and the second sessions of induction plasmapheresis had urticaria, which regressed completely after the treatment with antihistamines. One patient after the induction plasmapheresis sessions underwent only 2 procedures of maintenance plasmapheresis; the third session was canceled due to abdominal pain. The pain appeared 3 weeks later after the last (second) maintenance plasmapheresis, and appendicitis was diagnosed. After the surgery, plasma exchange was not performed because the patient refused to continue to participate in the study.

Discussion

Most RRMS patients treated with IFN-β preparations do not develop persistent and high-titer NAbs associated with reduced measures of radiographic and clinical efficacy. For those that do, however, NAbs is a significant clinical problem, especially if other therapies have already been used, are not tolerated, or are not available. There is no consensus on prevention of NAb development or management of NAb-positive MS patients except for switching them to non-interferon therapy. However, NAb reversion cannot be accelerated by the concomitant treatment with corticosteroids or by the combined treatment with azathioprine or oral low-dose methotrexate and corticosteroids [29,31,32].

Because TPE is effectively used to remove serum proteins from the circulation, we decided in the present study to perform plasma exchange sessions to restore and then sustain the recovered bioavailability of IFN-β. All the patients in this study had been treated with high-dose IFN-β for more than 18 months and did not have an in vivo MxA response to IFN-β. We used the in vivo response to IFN-β for IFN-β bioavailability because MxA mRNA levels inversely correlate with NAb titers. Furthermore, several studies have shown that high titers of NAbs invariably are associated with decreased IFN-β bioactivity but low NAbs titers are correlated poorly with expression of IFN-β induced genes or gene products [37–39].

The present data support the hypothesis that induction TPE may promote the recovery of in vivo biological response to IFN-β, because 4 patients became “biological responders” after induction plasmapheresis. It should be noted that 2 patients who did not regain the ability of IFN-β to induce the MxA mRNA were the oldest (54 and 49 years old) and had with the longest treatment duration (6 and 7 years, respectively). In contrast, the mean age of all others was 39.8 years and mean treatment duration was 3.5 years (Table 1). However, in other studies, which assessed the effect of immunosuppression on NAbs, no data about the impact of immunosuppression in the different age groups or in the groups with different treatment duration were evaluated.

Unfortunately, the effect of maintenance plasmapheresis on the IFN-β bioavailability was transient – even during the maintenance plasmapheresis sessions when one TPE session was performed per months, the biological activity of IFN-β returned to baseline levels after 1 or 2 months.

On the contrary, the effect of TPE on BAbs was different from the effect on the MxA mRNA expression values. Whereas BAbs levels markedly increased after TPE (Figure 3) in patients with restored IFN-β bioavailability, they did not change in patients in who low availability of IFN-β remained (Figure 1).

In chronic diseases, TPE use is more problematic than in acute diseases due to the phenomenon of rebound antibody production. Therefore, TPE alone is not effective for persistent reduction of NAbs and sustaining of IFN-β bioavailability. Antibodies are effectively removed after plasmapheresis in various autoimmune diseases (e.g., myasthenia gravis and chronic inflammatory demyelinating polyneuropathy), but the additional immunosuppressive therapy after plasmapheresis courses is required for long-term management to prevent the re-synthesis of antibodies. It is possible that the same combination of plasmapheresis and immunosuppression must be used in NAb-positive patients to achieve and maintain adequate bioavailability of IFN-β.

The results of the present study demonstrate that there is still no optimal methodology to restore or improve the markers of IFN-β bioactivity in abolished IFN-β ability or to prevent the development of NAbs in IFN-β-treated RRMS patients. Consequently, to tailor the best treatment for MS patients and to prevent the development of NAbs, the neurologist must carefully consider the results of clinical trials and the particular individual clinical and prognostic characteristics of each patient, including susceptibility to NAbs and clinical effects of therapy.