RWJ 26251

Cladribine: mechanisms and mysteries in multiple sclerosis

Benjamin Meir Jacobs,1 Francesca Ammoscato,1 Gavin Giovannoni,1,2 David Baker,1 Klaus Schmierer1,21The Blizard institute (Neuroscience), Queen Mary University of London, Barts and the London School of Medicine and Dentistry, London, UK 2emergency Care and Acute Medicine Clinical Academic Group Neuroscience, Barts Health NHS Trust, The Royal London Hospital, London, UK

Objectives The aims of this manuscript were to review the evidence for the efficacy and safety of cladribine
in multiple sclerosis (MS) and to review the molecular and cellular mechanisms by which cladribine acts as a disease-modifying therapy in MS.
Methods This is a narrative review of the available clinical and preclinical data on the use of cladribine in MS.
Deficiency of adenosine deaminase (ADA) causes a severe combined immunodeficiency characterised by profound lymphopaenia. Carson and Beutler discovered that lymphocyte death in this condition results from the accumulation of toxic levels of deoxyadenosine triphosphate (dATP).10 11 2-Chlo- rodeoxyadenosine (2-CdA, cladribine), a compound structurally similar to deoxyadenosine but rela- tively resistant to ADA-mediated degradation,

Correspondence to results Clinical trial data argue strongly that cladribine was developed to exploit the lymphocyte-specific

Dr Benjamin Meir Jacobs, Centre for Neuroscience and Trauma, Blizard institute, Barts and The London School of Medicine and Dentistry, London e1 2AT, UK; ben.meir.jacobs@

To cite: Jacobs BM, Ammoscato F, Giovannoni G, et al. J Neurol Neurosurg Psychiatry epub ahead of print: [please include Day Month Year]. doi:10.1136/

is a safe and effective therapy for relapsing MS and that it may also be beneficial in progressive MS. The
pharmacology of cladribine explains how it is selectively toxic towards lymphocytes. immunophenotyping
studies show that cladribine depletes lymphocyte populations in vivo with a predilection for B cells. in vitro studies demonstrate that cladribine also exerts immunomodulatory influences over innate and adaptive immunity.
Conclusions Cladribine is a safe and effective form of induction therapy for relapsing MS. its mechanism of benefit is not fully understood but the most striking action is selective, long-lasting, depletion
of B lymphocytes with a particular predilection for memory B cells. The in vivo relevance of its other immunomodulatory actions is unknown. The hypothesis that cladribine’s action of benefit is to deplete
memory B cells is important: if correct, it implies that selective targeting of this cell population and sparing of other lymphocytes could modify disease activity without predisposing to immunosuppression-related complications.


Cladribine is a synthetic purine nucleoside analogue initially developed as a treatment for haematolog- ical malignancies.1 It is now used for a range of indi- cations including hairy cell leukaemia, low-grade B cell lymphomas, Langerhans cell histiocytosis, acute myeloid leukaemia, refractory coeliac disease and multiple sclerosis (MS).2–8
MS is a common, disabling, inflammatory demy- elinating and degenerative disease of the central nervous system (CNS). The effectiveness of immu- nosuppressive and immunomodulatory treatments for MS demonstrates that autoreactive lymphocytes play a role in the pathogenesis of MS. However, the precise cellular, humoral and cytokine mechanisms that underpin the development and progression of the disease are not fully understood.9
toxicity of dATP to achieve therapeutic depletion of lymphocytes.

discussion/observations Pharmacokinetics
Cladribine can be administered parenterally or orally as it has an estimated bioavailability of 40%.12 It is largely excreted unchanged in the urine and has good CNS penetration with the cerebrospinal fluid concentration estimated to be around 25% of plasma concentration in healthy subjects.12 Blood- brain-barrier disruption, a common pathological finding at all stages of the MS disease process, may predispose to greater CNS penetration.12–14

Clinical efficacy Phase I/II trials
Initial evidence of efficacy in people with MS (pwMS) emerged from studies on advanced, progressive MS. In the MS-Scripps trial, 51 people with progressive MS of >2 years duration received placebo or intravenous cladribine 0.1 mg/kg/day during the first year of the study,15 followed by a crossover arm in year 2. Disability, measured by the expanded disability status scale (EDSS), progressed during the first year in the placebo group but was stable in the cladribine group.15 An EDSS plateau of shorter duration was also seen in the initial placebo arm when they received active treatment in year
2.EDSS progression resumed in the initial active treatment arm once they crossed over to placebo. While around 50% of participants had evidence of gadolinium (Gd)-enhancing lesions on T1-weighted MRI brain at enrolment, only 1/42 participants continued to have such lesions at 24 months. The time course of conversion to radiological absence of Gd-enhancing lesions closely followed cladribine treatment, suggesting that cladribine prevented the development of new Gd-enhancing lesions.15 In the MS-001 trial, 159 people with progressive MS were randomised to receive placebo, low-dose subcuta- neous cladribine or high-dose cladribine.16 At 12
months of follow-up, cladribine treatment had no effect on EDSS progression, but it did minimise radiological progression as evidenced by new T1-Gd lesions and T2-weighted lesions.16 There are several possible explanations for the lack of clinical benefit seen in this trial. The MS-001 population had a very high baseline EDSS (median of 6.0), were followed up for a relatively short period of time (1 year) and had a high proportion of people with primary progressive MS (30%). The disconnect between the radiological and clinical findings probably represents a lack of response among the people with primary progressive disease, as the bulk of the radiological benefit was seen in the people with SPMS. These data do not provide definitive evidence of clinical benefit in progressive MS, but are sufficient to justify a phase III trial powered to detect delays in disability progression over a feasible timescale (2 years or more) and using outcome tools that measure functions with persistent reserve capacity.17
There is stronger evidence for cladribine in relapsing MS. The ONWARD study assessed the efficacy of oral cladribine (3.5 mg/
kg) as an adjunct to Interferon (IFN)-beta in active relapsing MS. Participants were randomised to receive IFN-beta alone or IFN-beta plus. Over 2 years of follow-up, cladribine was associ- ated with a lower annualised relapse rate (0.12 vs 0.32) but had no effect on EDSS progression. The mean numbers of new T1 Gd-enhancing lesions and active T2-weighted lesions were lower in the cladribine group.18 The MS-Scripps-C trial randomised 52 people with relapsing MS to receive placebo or subcutaneous cladribine. Over 18 months of follow-up, cladribine was associ- ated with an improvement in relapse rate, relapse severity and MRI measures of disease activity.19 Another phase II randomised 84 people with relapsing MS to subcutaneous cladribine or placebo for 1 year, followed by a crossover in year 2.20 Cladribine was associated with a reduction in the annualised relapse rate (0.15 vs 0.42 in year 1) and some evidence of residual benefit persisting after treatment discontinuation; the relapse rate in the initial active-treatment arm was 0.42 in year 2 compared with 0.86 at baseline.20
Phase III trials
Two phase III trials were carried out to confirm the clinical effectiveness of cladribine in early and established relapsing MS. In the Oral Cladribine for Early MS (ORACLE-MS) trial, participants were enrolled if they had experienced a first clinical demyelinating event within 75 days, two or more clinically silent lesions on T2-weighted MRI and an EDSS of 5.0 or less.8 A total of 616 participants were randomised to receive placebo, 5.25 mg/kg of oral cladribine or 3.5 mg/kg of oral cladribine.8 There was a statistically significant reduction in the risk of conversion to Poser criteria-defined MS within the 96-week study period in both cladribine groups compared with placebo.21 Cladribine was also associated with a significantly lower burden of Gd-en- hancing lesions on T1-weighted MRI and a reduction in the number of new or enlarging lesions on T2-weighted scans.8 Participants who had CDMS entered an open-label maintenance period during which they received treatment with IFN. Of 109 participants in this arm, the annualised relapse rate was lower in both groups initially treated with cladribine compared with placebo. These results suggest a long-term benefit to cladribine therapy after discontinuation of the drug despite progression to CDMS.22
The CLARITY trial (CLAdRIbine Tablets treating MS orallY) recruited participants with relapsing-remitting MS who had had at least one previous relapse in the preceding 12 months and an EDSS of 5.5 or less.7 A total of 1326 participants were

randomised to 5.25 mg kg-1 of oral cladribine, 3.5 mg kg-1 of oral cladribine or placebo. The annualised relapse rate at 96 weeks of follow-up was significantly lower in the cladribine groups versus placebo. Cladribine improved the proportion of participants who did not have any relapses during the study period, the time to the first relapse and the odds of requiring rescue, and the risk of 3-month sustained disability progression in the cladribine groups.7 Furthermore, at 96 weeks of follow-up, oral cladribine improved the proportion of participants who remained free from disease activity, a combined metric including freedom from relapses, disability and MRI evidence of disease activity; 44% of the low-dose group, 46% of the high-dose group and 16% of the placebo group achieved sustained freedom from disease activity at the end of the study period.23 Cladribine treatment was asso- ciated with an increase in the proportion of participants who remained free from Gd-enhancing lesions and new T2 lesions at the end of the 96-week study period.24 There was also a statis- tically significant reduction in annual brain volume loss in the cladribine treatment groups compared with placebo.25 The posi- tive clinical effects were further underpinned by positive Quality of life data also indicated significant improvement in one out of the two metrics used (EQ-5D) in the cladribine versus placebo groups.26 Notably, the recently published 2-year blinded, place- bo-controlled extension study to CLARITY demonstrated that there was no significant difference in terms of relapse rate or disability progression between the groups receiving 2 years total and 4 years total of cladribine.27
In summary, there is level 1B evidence that, in relapsing MS, induction treatment with cladribine reduces the risk of conver- sion from a first demyelinating event to CDMS, reduces the relapse rate, the risk of disability progression and MRI defined disease activity. It is also associated with a significant probability of achieving no evident disease activity and improved quality of life.
On the basis of this evidence, the National Institute for Clin- ical Excellence (NICE) now recommends the use of cladribine tablets (TA493) for people with high active MS if the person has ‘rapidly evolving severe relapsing-remitting MS, that is, at least two relapses in the previous year and at least one T1 Gd-en- hancing lesion at baseline MRI’ or ‘relapsing-remitting MS that has responded inadequately to treatment with disease-modifying therapy (DMT), defined as one relapse in the previous year and MRI evidence of disease activity’.28 Although the evidence is best for relapsing disease, the ONWARD trial suggests that the use of cladribine may be useful as an adjunct in ‘secondary progres- sive’ disease, for people who are still having relapses as well as gradual disability progression.18 Furthermore, we feel that there is sufficient phase I and II trial evidence15 16 to make the case for a phase III trial of cladribine in advanced progressive MS to assess whether cladribine could improve metrics of progression beyond the EDSS, such as upper limb function17 and cognition, as has been successfully demonstrated for natalizumab.29
In both ORACLE-MS and CLARITY, the most common adverse events were headache, nasopharyngitis and lymphopaenia. The proportion of participants experiencing severe adverse events was slightly higher in the cladribine groups in both studies. Opportunistic infections were more common in the cladribine groups of both studies. In CLARITY, three cases of malignant neoplasms were reported in the cladribine group, including a melanoma, pancreatic carcinoma and ovarian cancer. In addition, one choriocarcinoma was reported in the high-dose cladribine
group 9 months after study discontinuation.7 No neoplasms were reported in the placebo group. Furthermore, the incidence of serious infection was higher in the cladribine groups (2.6% vs 1.6%). The apparent increase in cancer risk seen in CLARITY is likely due to an abnormally low cancer incidence in the placebo population (n=0), as the cancer incidence in the treatment arms was comparable to treatment and placebo groups in other phase III trials of DMTs for MS.30
Cladribine is presumed to be highly teratogenic based on its mechanism of action and results from animal models. However, the long-term effects of cladribine on fertility are unknown. Preclinical safety studies reported by the manufacturer suggest that cladribine does not affect fertility. Successful pregnancy has been reported following cladribine treatment for other condi- tions.31 32 The summary of product characteristics recommends exclusion of pregnancy before each cycle of cladribine and addi- tional contraceptive caution for at least 6 months following the last dose.. For male patients, contraceptive precautions are recommended for at least 6 months following the last dose of cladribine. Breastfeeding is contraindicated during treatment and for 1 week after the last dose.33









Figure 1 Schematic of adenosine and cladribine metabolism. Orange boxes indicate sites of inhibition by cladribine. ADA, adenosine deaminase; AMPK, AMP kinase; dAdo, deoxyadenosine; dAM/D/
TP, deoxyadenosine mono/di/triphosphate; DCK, deoxycytidine kinase; dino, deoxyinosine; NDPK, nucleoside diphosphate kinase; 5-NT,
5-nucleotidase; RNR, ribonucleotide reductase.
Cellular mechanisms
Cladribine is actively transported into cells via specific trans- membrane nucleoside transporters and extruded by ATP-binding cassette C4 exclusion pump.34 35 Whereas deoxyadenosine is vulnerable to ADA-mediated conversion to deoxyinosine, cladribine is resistant (but not insensitive) to the action of ADA due to the substitution of a chlorine atom for a hydrogen at position 2 of the purine ring.36 The initial phosphorylation of cladribine to cladribine-monophosphate is catalysed predomi- nantly by deoxycytidine kinase (DCK), although there is some contribution from mitochondrial deoxyguanosine kinase.34 35 The action of DCK is opposed by the phosphorylase activity of cytosolic 5’-nucleotidases (5-NT). The cytosolic 5-nucleotidase isozymes c-NT1A and c-NT1B are the predominant enzymes involved in cladribine metabolism given their specificity for adenosine metabolites.37 38 DCK catalyses the rate-limiting step in the nucleotide salvage pathway, which is involved in replen- ishing the deoxynucleoside-triphosphate (dNTP) pool for DNA synthesis. The relative levels of enzymatic activity of DCK and 5-NT appear to determine the sensitivity (or resistance) of the cell to cladribine toxicity: a high DCK:5-NT ratio renders the cell more susceptible to cladribine toxicity in cell lines and in patient-derived peripheral blood lymphocytes.38–46 Sequential phosphorylation of cladribine by DCK, AMP kinase and nucleo- side diphosphate kinase generates cladribine-triphosphate (CdA- TP), the active form that mediates cellular cytotoxicity (figure 1).
Cladribine is toxic to both resting and proliferating lympho- cytes through a variety of mechanisms. During DNA synthesis, CdA-TP is incorporated into the 3’ end of the growing DNA strand, inhibits further chain elongation by DNA polymerase II and therefore leads to the accumulation of single-strand breaks, promoting p53-dependent and independent apoptosis.47 Simi- larly, during DNA repair, CdA-ATP competes with dATP for incorporation into the repair site and prevents functional DNA repair. In addition to these effects on DNA synthesis and repair, CdA-TP inhibits ribonucleotide reductase (RNR), and thereby depletes the size of the dNTP pool available for DNA synthesis.48 The relative deficiency of available dATP produced by RNR inhibition favours the incorporation of CdA-TP into DNA, perpetuating the cytotoxic effects of the drug. Once CdA-TP is incorporated into DNA, it impairs transcription, leading to

a reduction in full-length mRNA transcripts which presumably dysregulates gene expression and disrupts protein synthesis.49 CdA-TP also promotes apoptosis via a direct effect on mito- chondrial permeability; it induces mitochondrial release of cyto- chrome c and apoptosis-inducing factor, leading to downstream activation of the caspase 3 pathway and subsequent apoptosis.50
The relative importance of these various mechanisms is not fully understood but clearly depends on the abundance and distribution of intracellular nucleoside pools, the cell type, stage of the cell cycle and the relative expression levels of the enzymes involved in cladribine metabolism and targeted by cladribine. The relevant mechanism of cytotoxicity probably differs substan- tially between proliferating and non-proliferating cells.

Effects on circulating lymphocyte populations
Cladribine may modify the disease course in MS by depleting circulating autoreactive B and T lymphocytes which are thought to drive inflammatory demyelination, axonal degeneration and neuronal loss seen in lesional and non-lesional CNS.51 Anal- ysis of lymphocyte populations from clinical trials in MS have shed further light on the magnitude, kinetics and specificity of lymphocyte depletion produced by cladribine.
Immunophenotyping of 309 CLARITY participants revealed that cladribine administration leads to rapid, profound and long- lasting lymphocyte depletion.52 The mean number of lympho- cytes reached a nadir with the end of the second cycle and was sustained throughout the study period. Maximum depletion was approximately 45% (low-dose) and 33% (high-dose) of baseline. The same magnitude and kinetics of depletion were mirrored in CD4+ and CD8+ T cells. Both CD45RA+ naïve and CD45RA– memory T cell subsets were depleted in a similar fashion.
While both T cells and B cells were depleted by cladribine, the magnitude and kinetics of depletion between them was very different. T cells were depleted early, remained suppressed at a plateau of ~50% of baseline and were depleted in a dose-depen- dent manner. B cells on the other hand were depleted by around 90% after the first cycle, repopulated within 40 days to 80% of pretreatment levels and were again depleted rapidly following the second cycle to around 20% of baseline levels. The magni- tude and kinetics of B cell depletion were similar between the two doses of cladribine, with the only notable difference being












Figure 2 Representative RNAseq data extracted from the primary cell atlas meta-analysis of published transcriptomic atlases on bioGPS. mRNA expression levels as extracted from the meta-analysis are shown for the three primary genes involved in cladribine metabolism, DCK and the two cytosolic 5-nucleotidase isoforms most involved in adenosine metabolism. Data are shown for B and T lymphocytes. DCK, deoxycytidine kinase.


that participants receiving the lower dose repopulated faster, but reached the same proportion of baseline, following the first cycle of treatment.52
Similar findings were reported from the Scripps trials; cladribine was associated with rapid, prolonged lympho- cyte depletion. Of note, the CD3 +T lymphocyte count was suppressed to <25% of baseline within 6 months, reached a nadir and remained at a plateau despite crossover to placebo.15 This effect was mirrored in the CD4+ and CD8+ populations as expected, although the extent of CD4+ depletion was greater than for the CD8+ population, with the CD4+ count reaching around 15% of baseline at 3–6 months. The CD19+ count was depleted significantly following treatment to a nadir of around 10% of baseline levels, repopulated partially and did not recover to baseline levels at the end of the study despite the crossover washout period of 12 months.15
These data indicate that pulsed ‘induction’ therapy with cladribine leads to long-lasting lymphocyte depletion which persists well beyond drug administration. In addition, while all lymphocyte subsets appear vulnerable to cladribine-mediated cytotoxicity, the B cell population is depleted to the greatest extent, although with faster recovery compared with T cells.
However, the magnitude and kinetics of depletion vary substantially within the B cell population. Converging lines of evidence suggest that memory B cells (CD19+CD27+) may be particularly susceptible to cladribine. First, publicly available gene expression profiles indicate that the ratio of DCK:5-NT is higher at almost all stages of B cell differentiation than T cell differentiation (figure 2) ( Further- more in addition to elevated DCK,46 B cells express lower levels of ADA protein than T cells.53 The DCK:5-NT ratio is particularly high in germinal centre B cells, the precursor to canonical class-switched T-cell-dependent memory B cells. Proteomic data indicate that in lymphoid organs where B cell maturation takes place, DCK is highly expressed whereas c-5NT1a and c-5NT1b proteins are almost undetectable ( The vulnerability of lymphoid follicle-resident lymphocytes to cladribine toxicity suggests that multiple stages of the germinal centre reaction may be
inhibited by cladribine. Second, depletion of memory B cells may be a unifying feature of effective DMTs for MS9; there is a strong correlation between the extent of memory B cell depletion and the effectiveness of a particular DMT. As cladribine has demonstrated comparable efficacy with the most potent DMTs available, we would anticipate that it would deplete memory B cells. Third, in CLARITY, although cladribine produced profound, long-lasting depletion of B cells, the CD19+ counts at any individual time point did not differ significantly between those who experienced at least one relapse and those who remained relapse-free. One explanation for these data is that lack of access to full clinical dataset reduced the power of the analysis. Another explana- tion is that using the total CD19 count masks the contribution of the CD19+CD27+ memory population, which constitutes roughly 30% of the circulating CD19+ population.54 Further- more, depletion of lymphoid-resident lymphocytes may not be reflected in analysis of peripheral blood lymphocytes. Fourth, we have demonstrated that, at 1-year postcycle, cladribine treatment is associated with a profound reduction in periph- eral unswitched memory B cells (IgD+CD27+) and class- switched memory B cells (IgD-CD27+) compared with healthy controls and untreated pwMS.55 Given the recent observation that the abundance of some subsets of antigen-experienced B cells (IgD-CD27+ switched memory cells and plasma cells) within the CSF is correlated with disease activity,56 it is plau- sible to suggest that cladribine reduces disease activity in MS by depleting circulating memory B cells and thus reducing the influx of these cells into the CNS.
From depletion to modulation
The influence of cladribine on the immune system probably extends beyond lymphocyte depletion in vivo. First, cladribine also depletes various innate immune cells including NK cells and monocytes, although to a lesser extent than lympho- cytes.50 52 In the CLARITY dataset, cladribine led to rapid deple- tion of CD56+ NK cells consistent with their DCK expression52 (figure 2). Second, cladribine penetrates the CNS and may
deplete CNS-resident immune cells in vivo. In vitro, cladribine induces apoptosis in cultured rat microglia via dissipation of the mitochondrial membrane potential and subsequent caspase
3.activation.57 Neither phagocytosis nor LPS-induced NO or TNFα release were affected by cladribine, suggesting that the drug may affect microglia by inducing apoptosis, rather than by modulating microglial signalling.57
Third, cladribine may influence the cytokine milieu inde- pendently of its leucocyte-depleting action. In a study of 50 pwMS, subcutaneous cladribine was associated with a rise in the plasmacytoid:myeloid dendritic cell ratio.58 In healthy human monocyte-derived DCs, cladribine reduced endocytosis of FITC-dextran.59 Interestingly, when murine DCs loaded with a MOG peptide were incubated with or without cladribine in the presence of MOG-specific naïve T cells, cladribine was associated with a higher proportion of T cells developing into IL-10-producing cells and a lower proportion developing into IFNγ and TNFα-producing cells. These data suggest that cladribine inhibits DC phagocytosis, polarises DCs towards a tolerogenic phenotype and thus promotes differentiation of naïve T cells into Treg cells.59
Notably, cladribine also inhibits T cell activation without affecting cell viability. Overnight incubation of healthy lympho- cytes with cladribine reduced stimulation-induced IFNγ and TNFα secretion by around 50%.60 Adding deoxycytidine, a competitive antagonist for DCK-dependent phosphorylation, prevented cell death but did not normalise stimulation-induced cytokine secretion or T cell activation. These results indicate that the T cell-modulating effects of cladribine are at least partly independent of lymphocyte toxicity and are partly independent of DCK.60 This effect is long-lasting; when healthy PBMCs were incubated with cladribine for 72 hours and subsequently cultured for up to 58 days in the presence of IL-2, there were no long- lasting effects of cladribine exposure on proliferation. However, there were long-lasting effects on the cytokine response to TCR crosslinking. Cladribine led to an increase in production of IL-4, IL-5, IL-10, with no change in other cytokines.61 It is possible that cladribine may bias T cell differentiation towards a tolero- genic phenotype: this effect could explain the observation that, in CLARITY, clinical benefit outstripped and outlasted the rela- tively mild and short-lived T cell depletion.52
From modulation to migration
Cladribine may also inhibit the recruitment of circulating leucocytes to the CNS compartment and thereby limit the degree of CNS inflammation in MS. Using an in vitro Boyden Chamber assay, Kopadze et al assessed chemotaxis of PBMCs from five pwMS and five controls in the presence and absence of cladribine.62 Cladribine reduced the proportion of unstimu- lated PBMCs which successfully migrated from 8% to 1.4%.62 Chemotaxis in vivo is partly dependent on chemokine signal- ling. Interestingly, cladribine incubation reduced the produc- tion of chemokines by monocyte-derived human DCs.59 There was a reduction in the amount MIP-1a, MIP-1b and MIG and a commensurate decrease in monocyte chemotaxis in vitro.59 Furthermore, in a study of 25 pwMS, cladribine treatment reduced serum and CSF levels of the chemokine RANTES while only reducing CSF, but not serum, IL-8 (a neutrophil chemoat- tractant).63 Thus, cladribine reduces chemokine production in vitro and in vivo and reduces chemotaxis in vitro. It will be inter- esting to determine whether pwMS treated with cladribine have evidence of reduced immune cell infiltration into the CNS asso- ciated with treatment, analogous to the effect of natalizumab.

Cladribine is a safe and effective treatment for relapsing forms of MS. Its mechanisms of benefit are unclear but complex. Cladribine selectively depletes lymphocytes and has a predilec- tion for B lymphocytes. Memory B lymphocytes appear partic- ularly vulnerable to this depletion. Aside from its pro-apoptotic effects, cladribine promotes immune tolerance and reduces immune cell infiltration into the CNS. Further work is required to clarify which B cell subsets are most affected, the relationship between lymphocyte depletion and disease activity, the bene- fits of cladribine in progressive MS and the in vivo effects of cladribine on BBB permeability to immune cells.

Contributors BMJ, DB, GG and KS planned and discussed the idea for the manuscript. BMJ wrote the initial draft. BMJ, KS, DB, FA and GG edited the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interest DB reports being a founder and consultant to Canbex Therapeutics and receiving research funds from Canbex Therapeutics, Sanofi- Genzyme and Takeda in the past 3 years. GG reports receiving fees for participation in the advisory board for Abbvie Biotherapeutics, Biogen, Canbex, ironwood, Novartis, Merck, inc, Merck Serono, Roche, Sanofi Genzyme, Synthon, Teva and vertex; speaker fees from Abbvie, Biogen, Bayer HealthCare, Genzyme, Merck Serono, Sanofi-Aventis and Teva and research support from Biogen, Genzyme, ironwood, Merck, inc, Merck Serono and Novartis. KS reports being a principal investigator of trials sponsored by Novartis, Roche, Teva and Medday; involved in trials sponsored by Biogen, Sanofi-Genzyme, BiAL, Cytokinetics and Canbex and receiving speaking honoraria for lecturing and advisory activity and/
or meeting support from Biogen, Merck, inc, Merck Serono, Novartis, Roche, Sanofi-Genzyme and Teva. All authors have presented posters at the european Congress for Treatment and Research in Multiple Sclerosis (eCTRiMS) annual congress.
Patient consent Not required.
Ethics approval This work is strictly a review and so no ethical approval was required.
Provenance and peer review Not commissioned; externally peer reviewed. © Article author(s) (or their employer(s) unless otherwise stated in the text of the
article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
1Beutler e. Cladribine (2-chlorodeoxyadenosine). Lancet 1992;340:952–6.
2Piro LD, Carrera CJ, Carson DA, et al. Lasting remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine. N Engl J Med 1990;322:1117–21.
3Jäger G, Neumeister P, Brezinschek R, et al. Treatment of extranodal marginal zone
B-cell lymphoma of mucosa-associated lymphoid tissue type with cladribine: a phase ii study. J Clin Oncol 2002;20:3872–7.
4Donadieu J, Bernard F, van Noesel M, et al. Salvage Group of the Histiocyte Society. Cladribine and cytarabine in refractory multisystem Langerhans cell histiocytosis: results of an international phase 2 study. Blood 2015;126:1415–23.
5Holowiecki J, Grosicki S, Giebel S, et al. Cladribine, but not fludarabine, added to daunorubicin and cytarabine during induction prolongs survival of patients with acute myeloid leukemia: a multicenter, randomized phase iii study. J Clin Oncol 2012;30:2441–8.
6Al-Toma A, Goerres MS, Meijer Jw, et al. Cladribine therapy in refractory celiac disease with aberrant T cells. Clin Gastroenterol Hepatol 2006;4:1322–7.
7Giovannoni G, Comi G, Cook S, et al. A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis. N Engl J Med 2010;362:416–26.
8Leist TP, Comi G, Cree BA, et al. effect of oral cladribine on time to conversion to clinically definite multiple sclerosis in patients with a first demyelinating event (ORACLe MS): a phase 3 randomised trial. Lancet Neurol 2014;13:257–67.
9Baker D, Marta M, Pryce G, et al. Memory B cells are major targets for effective immunotherapy in relapsing multiple sclerosis. EBioMedicine 2017;16:41–50.
10Carson DA, Seegmiller Je. effect of adenosine deaminase inhibition upon human lymphocyte blastogenesis. J Clin Invest 1976;57:274–82.
11Carson DA, Kaye J, Seegmiller Je. Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: possible role of nucleoside kinase(s). Proc Natl Acad Sci U S A 1977;74:5677–81.
12Liliemark J. The clinical pharmacokinetics of cladribine. Clin Pharmacokinet 1997;32:120–31.
13Cramer SP, Simonsen H, Frederiksen JL, et al. Abnormal blood-brain barrier permeability in normal appearing white matter in multiple sclerosis investigated by MRi. Neuroimage Clin 2014;4:182–9.
14Yates RL, esiri MM, Palace J, et al. Fibrin(ogen) and neurodegeneration in the progressive multiple sclerosis cortex. Ann Neurol 2017;82:259–70.
15Beutler e, Sipe JC, Romine JS, et al. The treatment of chronic progressive multiple sclerosis with cladribine. Proc Natl Acad Sci U S A 1996;93:1716–20.
16Rice GP, Filippi M, Comi G. Cladribine and progressive MS: clinical and MRi outcomes of a multicenter controlled trial. Cladribine MRi Study Group. Neurology 2000;54:1145–55.
17Dubuisson N, Baker D, Thomson A, et al. Disease modification in advanced MS: focus on upper limb function. Mult Scler 2017;23:1956–7.
18Montalban X, Cohen B, Leist T, et al. efficacy of cladribine tablets as add-on to
iFN-beta therapy in patients with active relapsing MS: final results from the phase ii ONwARD study (P3.029). Neurology 2016;86.
19Romine JS, Sipe JC, Koziol JA, et al. A double-blind, placebo-controlled, randomized trial of cladribine in relapsing-remitting multiple sclerosis. Proc Assoc Am Physicians 1999;111:35–44.
20Stelmasiak Z, Solski J, Nowicki J, et al. effect of parenteral cladribine on relapse rates in patients with relapsing forms of multiple sclerosis: results of a 2-year, double-blind, placebo-controlled, crossover study. Mult Scler 2009;15:767–70.
21Poser CM, Paty Dw, Scheinberg L, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13:227–31.
22Comi G. Cladribine tablets in the ORACLe-MS study open-label maintenance period: analysis of efficacy in patients after conversion to clinically definite multiple sclerosis (CDMS) (P6.349). Neurology 2017;88:P6.349.
23Giovannoni G, Cook S, Rammohan K, et al. Sustained disease-activity-free status in patients with relapsing-remitting multiple sclerosis treated with cladribine tablets in the CLARiTY study: a post-hoc and subgroup analysis. Lancet Neurol 2011;10:329–37.
24Comi G, Cook SD, Giovannoni G, et al. MRi outcomes with cladribine tablets for multiple sclerosis in the CLARiTY study. J Neurol 2013;260:1136–46.
25De Stefano N, Giorgio A, Battaglini M, et al. Reduced brain atrophy rates are associated with lower risk of disability progression in patients with relapsing multiple sclerosis treated with cladribine tablets. Mult Scler 2018;24:222–6.
26Afolabi D, Albor C, Zalewski L, et al. Positive impact of cladribine on quality of life in people with relapsing multiple sclerosis. Mult Scler 2017:135245851772638.
27Giovannoni G, Soelberg Sorensen P, Cook S, et al. Safety and efficacy of cladribine tablets in patients with relapsing-remitting multiple sclerosis: Results from the randomized extension trial of the CLARiTY study. Mult Scler 2017:135245851772760.
28Guidance TA. Cladribine tablets for treatingCladribine tablets for treating relapsing– remitting multiple sclerosisrelapsing–remitting multiple sclerosis. .
29Kapoor R, Ho PR, Campbell N, et al. ASCeND investigators. effect of natalizumab on disease progression in secondary progressive multiple sclerosis (ASCeND): a phase 3, randomised, double-blind, placebo-controlled trial with an open-label extension. Lancet Neurol 2018;17:405–15.
30Pakpoor J, Disanto G, Altmann DR, et al. No evidence for higher risk of cancer in patients with multiple sclerosis taking cladribine. Neurol Neuroimmunol Neuroinflamm 2015;2:e158.
31Barete S, Lortholary O, Damaj G, et al. Long-term efficacy and safety of cladribine (2-CdA) in adult patients with mastocytosis. Blood 2015;126:1009–16.
32Orlowski RZ. Successful pregnancy after cladribine therapy for hairy cell leukemia. Leuk Lymphoma 2004;45:187–8.
33Mavenclad, iNN-cladribine. library/EPAR_-_Product_Information/human/004230/WC500234561.pdf.
34Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 2002;3:415–24.
35Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia 2001;15:875–90.
36Scheible H, Laisney M, wimmer e, et al. Comparison of the in vitro and in vivo metabolism of Cladribine (Leustatin, Movectro) in animals and human. Xenobiotica 2013;43:1084–94.
37Hunsucker SA, Mitchell BS, Spychala J. The 5’-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther 2005;107:1–30.
38Leist TP, weissert R. Cladribine: mode of action and implications for treatment of multiple sclerosis. Clin Neuropharmacol 2011;34:28–35.

39Kawasaki H, Carrera CJ, Piro LD, et al. Relationship of deoxycytidine kinase and cytoplasmic 5’-nucleotidase to the chemotherapeutic efficacy of
2-chlorodeoxyadenosine. Blood 1993;81:597–601.
40Lorkova Let al. Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs fludarabine, gemcitabine and cladribine, but not to other classes of anti-lymphoma agents. Blood 2013;122:3835–3835.
41Lotfi K, Månsson e, Chandra J, et al. Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide. Br J Haematol 2001;113:339–46.
42Månsson e, Flordal e, Liliemark J, et al. Down-regulation of deoxycytidine kinase in human leukemic cell lines resistant to cladribine and clofarabine and increased ribonucleotide reductase activity contributes to fludarabine resistance. Biochem Pharmacol 2003;65:237–47.
43Månsson e, Spasokoukotskaja T, Sällström J, et al. Molecular and biochemical mechanisms of fludarabine and cladribine resistance in a human promyelocytic cell line. Cancer Res 1999;59:5956–63.
44Orr RM, Talbot DC, Aherne wG, et al. 2’-Deoxycytidine kinase deficiency is a major determinant of 2-chloro-2’-deoxyadenosine resistance in lymphoid cell lines. Clin Cancer Res 1995;1:391–8.
45Schirmer M, Stegmann AP, Geisen F, et al. Lack of cross-resistance with gemcitabine and cytarabine in cladribine-resistant HL60 cells with elevated 5’-nucleotidase activity. Exp Hematol 1998;26:1223–8.
46Spasokoukotskaja T, Arnér eS, Brosjö O, et al. expression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues. Eur J Cancer 1995;31A:202–8.
47Parker wB, Bapat AR, Shen JX, et al. interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. Mol Pharmacol 1988;34:485–91.
48Cardoen S, van Den Neste e, Smal C, et al. Resistance to 2-chloro-2’-deoxyadenosine of the human B-cell leukemia cell line eHeB. Clin Cancer Res 2001;7:3559–66.
49Genini D, Adachi S, Chao Q, et al. Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. Blood 2000;96:3537–43.
50Singh v, Prajeeth CK, Gudi v, et al. 2-Chlorodeoxyadenosine (cladribine) induces apoptosis in human monocyte-derived dendritic cells. Clin Exp Immunol 2013;173:288–97.
51Compston A, Coles A. Multiple sclerosis. Lancet 2008;372:1502–17.
52Baker D, Herrod SS, Alvarez-Gonzalez C, et al. Both cladribine and alemtuzumab may effect MS via B-cell depletion. Neurol Neuroimmunol Neuroinflamm 2017;4:e360.
53Massaia M, Ma DD, Sylwestrowicz TA, et al. enzymes of purine metabolism in human peripheral lymphocyte subpopulations. Clin Exp Immunol 1982;50:148–54.
54Morbach H, eichhorn eM, Liese JG, et al. Reference values for B cell subpopulations from infancy to adulthood. Clin Exp Immunol 2010;162:271–9.
55Ceronie B, Jacobs BM, Baker D, et al. Cladribine treatment of multiple sclerosis is associated with depletion of memory B cells. J Neurol 2018;265:1199–.
56eggers eL, Michel BA, wu H, et al. Clonal relationships of CSF B cells in treatment- naive multiple sclerosis patients. JCI Insight 2017;2.
57Singh v, voss ev, Bénardais K, et al. effects of 2-chlorodeoxyadenosine (Cladribine) on primary rat microglia. J Neuroimmune Pharmacol 2012;7:939–50.
58Mitosek-Szewczyk K, Tabarkiewicz J, wilczynska B, et al. impact of cladribine therapy on changes in circulating dendritic cell subsets, T cells and B cells in patients with multiple sclerosis. J Neurol Sci 2013;332(1-2):35–40.
59Kraus SH, Luessi F, Trinschek B, et al. Cladribine exerts an immunomodulatory effect on human and murine dendritic cells. Int Immunopharmacol 2014;18:347–57.
60Laugel B, Borlat F, Galibert L, et al. Cladribine inhibits cytokine secretion by T cells independently of deoxycytidine kinase activity. J Neuroimmunol 2011;240-241,:52–7.
61Korsen M, Bragado Alonso S, Peix L, et al. Cladribine exposure results in a sustained modulation of the cytokine response in human peripheral blood mononuclear cells. PLoS One 2015;10:e0129182.
62Kopadze T, Döbert M, Leussink vi, et al. Cladribine impedes in vitro migration of mononuclear cells: a possible implication for treating multiple sclerosis. Eur J Neurol 2009;16:409–12.
63Bartosik-Psujek H, Belniak e, Mitosek-Szewczyk K, et al. interleukin-8 and RANTeS levels in patients with relapsing-remitting multiple sclerosis (RR-MS) treated with cladribine. Acta Neurol Scand 2004;109:390–2.RWJ 26251

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