Immunomodulatory Effects Associated with Cladribine Treatment

Cladribine; Flow-cytometry; Multiple sclerosisCladribina; Citometría de flujo; Esclerosis múltipleCladribina; Citometria de flux; Esclerosi múltipleCladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.This work was supported by Merck. This study was sponsored by Merck (CrossRef Funder ID: 10.13039/100009945)

Specific Patterns of Immune Cell Dynamics May Explain the Early Onset and Prolonged Efficacy of Cladribine Tablets: A MAGNIFY-MS Substudy.

BACKGROUND AND OBJECTIVES Cladribine tablets cause a reduction in lymphocytes with a predominant effect on B-cell and T-cell counts. The MAGNIFY-MS substudy reports the dynamic changes on multiple peripheral blood mononuclear cell (PBMC) subtypes and immunoglobulin (Ig) levels over 12 months after the first course of cladribine tablets in patients with highly active relapsing multiple sclerosis (MS). METHODS Immunophenotyping was performed at baseline (predose) and at the end of months 1, 2, 3, 6, and 12 after initiating treatment with cladribine tablets. Assessments included lymphocyte subtype counts of CD19+ B cells, CD4+ and CD8+ T cells, CD16+ natural killer cells, plasmablasts, and Igs. Immune cell subtypes were analyzed by flow cytometry, and serum IgG and IgM were analyzed by nephelometric assay. Absolute cell counts and percentage change from baseline were assessed. RESULTS The full analysis set included 57 patients. Rapid reductions in median CD19+, CD20+, memory, activated, and naive B-cell counts were detected, reaching nadir by month 2. Thereafter, total CD19+, CD20+, and naive B-cell counts subsequently reconstituted, but memory B cells remained reduced by 93%-87% for the remainder of the study. The decrease in plasmablasts was slower, reaching nadir at month 3. Decrease in T-cell subtypes was also slower and more moderate compared with B-cell subtypes, reaching nadir between months 3 and 6. IgG and IgM levels remained within the normal range over the 12-month study period. DISCUSSION Cladribine tablets induce a specific pattern of early and sustained PBMC subtype dynamics in the absence of relevant Ig changes: While total B cells were reduced dramatically, T cells were affected significantly less. Naive B cells recovered toward baseline, naive CD4 and CD8 T cells did not, and memory B cells remained reduced. The results help to explain the unique immune depletion and repopulation architecture regarding onset of action and durability of effects of cladribine tablets while largely maintaining immune competence. TRIAL REGISTRATION INFORMATION ClinicalTrials.gov Identifier: NCT03364036. Date registered: December 06, 2017

A plain language summary of the impact of vaccines against flu and chickenpox in people with multiple sclerosis treated with cladribine tablets

What is this summary about?This is a summary of an article originally published in the Multiple Sclerosis Journal. Cladribine tablets (MAVENCLAD (R)) are an oral (taken by mouth) medication, approved for the treatment of people with relapsing forms of multiple sclerosis (MS, with episodes of new or worsening symptoms). They are administered for a maximum of 10 days per year, over a period of 2 years. Cladribine tablets work by temporarily reducing the number of lymphocytes, which are immune cells that help to fight off infections. Because of this, people with MS (also called PwMS) may have concerns about the effect of cladribine tablets on vaccines, as these work via immune cells to build protection against infection.What happened in the MAGNIFY-MS study? A study called MAGNIFY-MS investigated how long it takes for cladribine tablets to begin to work in people with a type of MS called highly active relapsing MS. During the study, some participants received their usual vaccinations against flu (influenza) and against the chickenpox virus (also called varicella zoster virus) as part of their routine medical care. The MAGNIFY-MS study gave the researchers an opportunity to look at how cladribine tablets affect the way the flu and chickenpox virus vaccines work in the body.What were the results? Cladribine tablets do not affect how well the body responds to flu and chickenpox vaccines.What do the results mean? PwMS taking cladribine tablets who are vaccinated against chickenpox, flu or both can be protected against these diseases. An animated video describing this study is also available online. Scan this QR code to watch the video

Inhibition of Bruton’s tyrosine kinase interferes with pathogenic B-cell development in inflammatory CNS demyelinating disease

Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties

Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

Deoxycytidine kinase (dCK) and 5' deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA.Graphical Abstrac

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K+ -sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649

Seizures and disturbed brain potassium dynamics in the leukodystrophy MLC

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole cell patch-clamp recordings and K+-sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. This article is protected by copyright. All rights reserved