Cladribine Research Articles

Interferon Beta-1a treatment promotes SARS-CoV-2 mRNA vaccine response in multiple sclerosis subjects

BackgroundSeveral concerns exist on the immunogenicity of SARS-CoV-2 vaccines in multiple sclerosis (MS) subjects due to their immunomodulating disease modifying therapies (DMTs). Here we report a comparison of the humoral response to BNT162b2-mRNA coronavirus (COVID)-19 vaccine and the immunological phenotype in a cohort of 125 MS subjects undergoing different DMTs, with no history of SARS-CoV-2 infection. MethodsWe collected serum and blood samples at the first day of vaccine (T0) and 21 days after the second vaccine dose (T1) from 125 MS subjects, undergoing eight different DMTs. Sera were tested using the Elecsys anti-SARS-CoV-2-IgG assay for the detection of IgG antibodies to SARS-CoV-2 spike protein. The anti-spike IgG titres from MS subjects were compared with 24 age- and sex-matched healthy controls (HC). Percentage and absolute number of B and T lymphocytes were evaluated by cytofluorimetric analysis in the same study cohort. ResultsWhen compared with SARS-CoV-2 IgG levels in HC (n = 24, median 1089 (IQR 652.5–1625) U/mL), we observed an increased secretion of SARS-CoV-2 IgG in interferon-beta 1a (IFN)-treated MS subjects (n = 22, median 1916 (IQR 1024–2879) U/mL) and an impaired humoral response in MS subjects undergoing cladribine (CLAD) (n = 10, median 396.9 (IQR 37.52–790.9) U/mL), fingolimod (FTY) (n = 19, median 7.9 (IQR 4.8–147.6) U/mL) and ocrelizumab (OCRE) (n = 15, median 0.67 (IQR 0.4–5.9) U/mL) treatment. Moreover, analysis of geometric mean titre ratio (GMTR) between different DMT's groups of MS subjects revealed that, when compared with IFN-treated MS subjects, intrinsic antibody production was impaired in teriflunomide (TERI)-, natalizumab (NAT)-, CLAD-, FTY- and OCRE-, while preserved in DMF- and GA-treated MS subjects. ConclusionHumoral response to BNT162b2-mRNA-vaccine was increased in IFN-treated MS subjects while clearly blunted in those under CLAD, FTY and OCRE treatment. This suggests that the DMTs could have a key role in the protection from SARS-CoV-2 related disease and complication in MS subjects, underlying a novel aspect that should be considered in the selection of the most appropriate therapy under COVID-19 pandemic.

Cladribine Alters Immune Cell Surface Molecules for Adhesion and Costimulation: Further Insights to the Mode of Action in Multiple Sclerosis.

Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.

Phase II Study of Venetoclax Added to Cladribine (CLAD) and Low Dose AraC (LDAC) Alternating with 5-Azacytidine (AZA) in Older and Unfit Patients with Newly Diagnosed Acute Myeloid Leukemia (AML)

Phase II Study of Venetoclax Added to Cladribine (CLAD) and Low Dose AraC (LDAC) Alternating with 5-Azacytidine (AZA) in Older and Unfit Patients with Newly Diagnosed Acute Myeloid Leukemia (AML)

Pre-Existing Humoral Immunological Memory Is Retained in Patients with Multiple Sclerosis Receiving Cladribine Therapy.

Cladribine (CLAD) is a lymphodepleting agent approved for active relapsing multiple sclerosis (MS). The impact of CLAD on the adaptive humoral immune system has not sufficiently been studied. This study aimed to assess the influence of CLAD treatment on specific antibody titers to common pathogens. We included 18 MS patients treated with CLAD. Serum IgG antibody levels to measles, mumps, rubella, hepatitis B and varicella zoster virus (VZV), as well as diphtheria and tetanus toxins, were measured prior to the initiation of treatment and at 12 and 24 months after first CLAD administration. Moreover, specimens were longitudinally analyzed regarding absolute blood concentrations of IgG and main lymphocyte subsets. No reduction in antibody levels against measles, mumps, rubella, VZV, hepatitis B, diphtheria toxin and tetanus toxin associated with CLAD treatment was observed. Loss of seroprotection occurred in <1%. We found no significant impact of CLAD on absolute serum IgG levels. Absolute lymphocyte counts were significantly reduced at the end of each treatment year (p < 0.00001 and p < 0.000001). This study suggests that CLAD does not interfere with the pre-existing humoral immunologic memory in terms of pathogen-specific antibody titers.

Exit-strategies in Natalizumab-treated RRMS at high risk of progressive multifocal leukoencephalopathy: A multicentre comparison study

The main aim of the study is to evaluate the efficacy and safety profile of Ocrelizumab (OCR), Rituximab (RTX), and Cladribine (CLA), employed as Natalizumab (NTZ) exit-strategies in Relapsing-Remitting Multiple Sclerosis (RRMS) patients at high-risk for progressive multifocal leukoencephalopathy (PML).

Exit Strategies in Natalizumab-Treated RRMS at High Risk of Progressive Multifocal Leukoencephalopathy: a Multicentre Comparison Study

The main aim of the study is to evaluate the efficacy and safety profile of ocrelizumab (OCR), rituximab (RTX), and cladribine (CLA), employed as natalizumab (NTZ) exit strategies in relapsing–remitting multiple sclerosis (RRMS) patients at high-risk for progressive multifocal leukoencephalopathy (PML). This is a multicentre, retrospective, real-world study on consecutive RRMS patients from eleven tertiary Italian MS centres, who switched from NTZ to OCR, RTX, and CLA from January 1st, 2019, to December 31st, 2019. The primary study outcomes were the annualized relapse rate (ARR) and magnetic resonance imaging (MRI) outcome. Treatment effects were estimated by the inverse probability treatment weighting (IPTW), based on propensity-score (PS) approach. Additional endpoint included confirmed disability progression (CDP) as measured by Expanded Disability Status Scale and adverse events (AEs). Patients satisfying predefined inclusion and exclusion criteria were 120; 64 switched to OCR, 36 to RTX, and 20 to CLA. Patients from the 3 groups did not show differences for baseline characteristics, also after post hoc analysis. The IPTW PS-adjusted models revealed that patients on OCR had a lower risk for ARR than patients on CLA (ExpBOCR 0.485, CI 95% 0.264–0.893, p = 0.020). This result was confirmed also for 12-month MRI activity (ExpBOCR 0.248 CI 95% 0.065–0.948, p = 0.042). No differences were found in other pairwise comparisons (OCR vs RTX and RTX vs CLA) for the investigated outcomes. AEs were similar among the 3 groups. Anti-CD20 drugs were revealed to be effective and safe options as NTZ exit strategies. All investigated DMTs showed a good safety profile.

Long-term peripheral immune cell profiling reveals further targets of oral cladribine in MS.

ObjectivesTo expand the knowledge about the immunological consequences of cladribine (CLAD), a pulsed immune reconstitution therapy approved for active multiple sclerosis (MS), beyond the known short‐term effects on peripheral immune cell subsets.MethodsIn this study, we characterized depletion and restitution kinetics as well as cytokine profiles of peripheral immune cell subsets in 18 patients with MS following treatment with oral CLAD. The methods involved blood collection prior to CLAD and every three months over a period of 24 months, and extensive characterization of various immune cells subsets by multiparametric flow cytometry.ResultsWe found a selectivity of CLAD towards central memory T cells and memory B cells and detected a hyper‐repopulation of maturing B cells. Counts of classical (−65%) and various nonclassical TH17 cells (−84% to −87%) were markedly reduced 24 months after treatment start, and were comparable with depletion rates of class‐switched memory B‐cell phenotypes (−87% to −95%). The nadir of TH cells was more pronounced in the second treatment year. We observed a proportional surge of CD20 T‐cell subsets and an expansion of regulatory T, B and NK cells. Natural killer T cells (NKT) were only depleted in year two and did not recover.InterpretationPeripheral immune cell profiling revealed more differentiated insights into the immunological effects of CLAD. While some immune cell subsets expanded, we also observed additive depleting effects after the second treatment course. Further studies are required to elucidate whether these changes are paramount for the consistent and prolonged disease‐modifying effect of CLAD.

A Rare Case of Systemic Mastocytosis With Associated Clonal Hematological Non-Mast Cell Lineage Disease That Transformed to Acute Leukemia With IDH2 Mutation

An elderly 72-year-old man presented with anemia, thrombocytopenia, monocytosis, splenomegaly and lymphadenopathy. Bone marrow biopsy was consistent with mast cell neoplasm with positive CD117, CD25, CD34 myeloblasts and polymerase chain reaction (PCR) revealed mutation of D816V. He developed bilateral femoral neck fractures and biopsy confirmed that he has systemic mastocytosis (SM). He received cladribine and midostaurin with stable disease for 21 months. His SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD) transformed to acute myelogenous leukemia with isocitrate dehydrogenase 2 ( IDH2 ) mutation. A trial of enasidenib was given for 5 months but without any response. Patient decided to go with home hospice and died afterwards. J Med Cases. 2020;11(10):317-319 doi: https://doi.org/10.14740/jmc3552

Antileukemic activity of novel adenosine derivatives

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

Two-Year Extension Study of Cladribine for Multiple Sclerosis

The original CLARITY study demonstrated an improvement in clinical and radiological outcomes in patients with relapsing–remitting multiple sclerosis

Both cladribine and alemtuzumab may effect MS via B-cell depletion.

Objective:To understand the efficacy of cladribine (CLAD) treatment in MS through analysis of lymphocyte subsets collected, but not reported, in the pivotal phase III trials of cladribine and alemtuzumab induction therapies.Methods:The regulatory submissions of the CLAD Tablets Treating Multiple Sclerosis Orally (CLARITY) (NCT00213135) cladribine and Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis, study one (CARE-MS I) (NCT00530348) alemtuzumab trials were obtained from the European Medicine Agency through Freedom of Information requests. Data were extracted and statistically analyzed.Results:Either dose of cladribine (3.5 mg/kg; 5.25 mg/kg) tested in CLARITY reduced the annualized relapse rate to 0.16–0.18 over 96 weeks, and both doses were similarly effective in reducing the risk of MRI lesions and disability. Surprisingly, however, T-cell depletion was rather modest. Cladribine 3.5 mg/kg depleted CD4+ cells by 40%–45% and CD8+ cells by 15%–30%, whereas alemtuzumab suppressed CD4+ cells by 70%–95% and CD8+ cells by 47%–55%. However, either dose of cladribine induced 70%–90% CD19+ B-cell depletion, similar to alemtuzumab (90%). CD19+ cells slowly repopulated to 15%–25% of baseline before cladribine redosing. However, alemtuzumab induced hyperrepopulation of CD19+ B cells 6–12 months after infusion, which probably forms the substrate for B-cell autoimmunities associated with alemtuzumab.Conclusions:Cladribine induced only modest depletion of T cells, which may not be consistent with a marked influence on MS, based on previous CD4+ T-cell depletion studies. The therapeutic drug-response relationship with cladribine is more consistent with lasting B-cell depletion and, coupled with the success seen with monoclonal CD20+ depletion, suggests that B-cell suppression could be the major direct mechanism of action.

Efficacy and safety of cladribine in treatment of patients with refractory/relapsed acute myeloid leukemia

Objective To investigate the efficacy and safety of cladribine in the treatment of patients with refractory/relapsed acute myeloid leukemia (AML). Methods The data of 8 patients with refractory/relapsed AML treated with regimens containing cladribine at a dose of 5 mg/m2 per day for 5 consecutive days were retrospectively analyzed. The efficacy and adverse reactions were observed during treatment. Results Among the 8 patients, 5 patients achieved complete remission (CR), 1 patient achieved partial response (PR), and 2 patients obtained non remission (NR). The adverse reactions could be tolerated. Conclusion Regimen containing cladribine is an effective treatment procedure for the patients with refractory/relapsed AML, and its adverse reactions can be tolerated, which requires further clinical study. Key words: Leukemia, myeloid, acute; Cladribine; Recurrence; Refractory

Synthesis of Anti-leukemia Drug Cladribine

Synthesis of Anti-leukemia Drug Cladribine

Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

Treatment of childhood refractory Langerhans cells histiocytosis

The definition of refractory Langerhans cells histiocytosis(LCH)is risk organs(bone marrow, liver, spleen)involvement and the first-line treatment failure.It has a very poor prognosis.The survival depends on timely and effective salvage treatment.Currently the treatment for refractory LCH includes isolated 2-chlorodeoxyadenosine, 2-chlorodeoxyadenosine combined with high-dose cytarabine, indomethacin, hematopoietic stem cell transplantation and targeted therapy.In this paper, the advances in treatment of the refractory LCH in children were reviewed. Key words: Refractory Langerhans cell histiocytosis; Therapy; 2-chlorodeoxyadenosine; Indomethacin; Children

Oligomeric Regulation of Ribonucleotide Reductase (RNR) by Antileukemic Nucleotides

Human RNR (hRNR) is the rate‐limiting enzyme for the de novo dNTP synthesis required for DNA replication and repair. hRNR activity is positively correlated with cancer cell proliferation and nucleotide analogs that can suppress hRNR activity such as gemcitabine and clofarabine (ClF) are proven effective in chemotherapy. However, the precise mechanisms by which many of the nucleoside prodrugs inhibit hRNR are unknown in most cases. The active hRNR is minimally an α2β2 holocomplex. Leukemia drug ClF is shown to inhibit hRNR through persistent hexamerization of α (α6) without requiring β. We hypothesize that α hexamerization is a universal mechanism by which antileukemic nucleotides inhibit hRNR, ultimately presenting a powerful small‐molecule approach to regulate hRNR. To test this hypothesis, I have sought to establish the mechanisms of hRNR inhibition by two additional clinically used nucleoside prodrugs—cladribine (CLA) and fludarabine (FLU). My studies have revealed that di‐ and triphosphates are active forms of both drugs that reversibly inhibit hRNR. CLA nucleotides [CLAD(T)P] are more potent than FLU nucleotides [FLUD(T)P] (Ki= 0.8, 0.1, 4.9 and 6.6 µM, respectively). In all cases, enzyme inhibition is coupled to α6 formation. We further validate in NIH‐3T3 cells stably expressing wild type α and hexamerization‐defective mutant α that hRNR inhibition by these nucleosides is directly correlated with oligomeric deregulation in cellular environment.

Expression of p53 protein and the histomorphological view of epidermis in experimental animals after cladribine application

Summary Cladribine acts as an antineoplastic, immunosuppressive and anti-inflammatory agent. The aim of this study was to examine the possible impact of this drug on the epidermis of Wistar rats. The control group received standard feed and water. The experimental group I was treated with cladribine in a schema corresponding to the treatment of multiple sclerosis in humans. The material for microscopic and immunohistochemical evaluation was obtained 24 h after administration of the last dose of the drug. The drug in experimental group II was administered in the same way as experimental group I, but test material was downloaded following a 4 week break after the administration of the last dose of the drug. After the completion of the experiment, skin samples were taken from all rats for histomorphological and immunohistochemical studies. An analysis of experimental group I H&E samples showed visible changes in epidermis patterns, and the.tissue layer was found to be much thinner compared to the analogous structures in the control group. The difference in p53 expressions between the control and both experimental groups was found to be statistically significant (P<0.0001). Cladribine administration induces changes in the images of the epidermis of experimental animals. Based on morphological analysis and p53 protein expression, it can be concluded that the 4-week break in drug administration contributed to the regeneration of the rats’ skin epithelial tissue.

Advance in new oral drugs for multiple sclerosis

Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system and represents one of the most common causes of chronic neurologic disability. Until recently, major treatments have relied on agents by injection which can induce injection ⁃related adverse events. The parenteral route of administration may affect the compliance with therapy in multiple sclerosis. Therefore, there is a need for the development of oral agents. The research of five oral drugs (cladribine, fingolimod, teriflunomide, laquinimod and dimethyl fumarate), is carrying out in Phase Ⅲ study. In this article, we review the undergoing clinical trials of these five oral agents. DOI：10.3969/j.issn.1672⁃6731.2012.02.011

Diagnosis and treatment progress of children's acute myeloid leukemia

Acute mveloid leukemia(AML) is a heterogeneous group of leukemias that arise from clonal transformation of hematopoietic precursors.This review summarizes classification of pediatric AML,more precise risk-group stratification,ongoing phase Ⅲ studies and minimal residual diseasse monitoring.In addition,we discuss the opportunities for innovative chemotherapy drugs in refractory AML and relapsed AML,such as gemtuzumab ozogamicin,liposomal daunorubicin,clofarabine,cladribine and lmatinib.Finally,the roles of allogeneic hematopoietic stem cell transplantation in pediatric AML are also discussed. Key words: Children; Acute myeloid leukemia; Diagnosis; Treatment

Composite Endpoint Analyses for Alemtuzumab and for Cladribine in Multiple Sclerosis: Two Reports

In a previous phase II trial of alemtuzumab, researchers reported that multiple sclerosis (MS) patients had a 74% lower relapse rate and a 71% lower