Laboratory Investigations
`
`A pilot trial of cladribine (2-chlorodeoxyadenosine)
`in remitting-relapsing multiple sclerosis
`
`Zbigniew Stelmasiak1,2, Janusz Solski3, Jacek Nowicki1, Ewa Porębska-Piwowarczyk1,
`Beata Jakubowska3, Mirosław S. Ryba4, Paweł Grieb4
`1 Clinical and Research Center for Demyelinating Diseases, Lublin, Poland
`2 Department of Neurology, Medical University of Lublin, Lublin, Poland
`3 Department of Clinical Analytics, Medical University, Lublin, Poland
`4 Laboratory of Experimental Pharmacology, Polish Academy of Sciences Medical Research Centre, Warsaw, Poland
`
`key words: multiple sclerosis, cladribine, immunosuppression
`
`SUMMARY
`Lymphocytotoxic nucleoside analog cladribine (2-chlorodeoxyadenosine) has recently been reported to
`favourably alter the clinical course of chronic progressive multiple sclerosis (MS). In the present study 10
`patients with the remitting-relapsing form of MS were treated with six courses of this drug (5 mg subcutaneously
`or 10 mg orally, once daily, repeated on five consecutive days) given once a month, followed by two additional
`courses at three month intervals. The patients were observed for two years after the initiation of the therapy.
`The treatment resulted in the reduction of lymphocyte counts to approx. 40% of the initial value at 6 months,
`with a trend toward recovery evident only at 24 months. Neurological status of the patients (expressed semi-
`quantitatively according to the EDSS scale) showed a significant improvement between 6 and 15 month of the
`study. The number of relapses, compared to the two-year period immediately before the treatment, remained
`unchanged in three patients, and was markedly reduced (almost five times on average) in the remaining seven
`patients. Patients who experienced the reduced relapse rate also seemed to show longer and more pronounced
`improvement in their neurological status.
`
`Med Sci Monit, 1998; 4(1): 4-8
`
`INTRODUCTION
`
`According to the prevailing theory, multiple sclero-
`sis (MS) is an autoimmune disease in which abnor-
`malities in immune regulation lead to the lympho-
`cyte-dependent demyelination process in the cen-
`tral nervous system [1,2]. The most common thera-
`peutic approach to autoimmune diseases is based
`on the use of drugs producing general immunosup-
`pression. Clinical trials with available immunosup-
`pressants (such as cyclophosphamide, metothre-
`xate, or cyclosporin A) evidenced at most a modest
`and transient benefit to MS patients
`[3,4].
`However, until more specific treatments are dis-
`covered, general immunosuppression is considered
`a theoretically justified therapeutic option in this
`disease.
`
`Cladribine (2-chlorodeoxyadenosine) is a purine
`analog with a potent and clinically useful activity
`against some indolent leukemias and lymphomas
`[5,6]. The drug displays a highly selective toxicity
`toward malignant lymphocytes, and normal lym-
`phocytes are also subject to the cytotoxic effect of
`the drug. Cytotoxicity is mediated by cladribine
`phosphorylation by deoxycytidine kinase, the
`enzyme located predominantly in lymphoid cells
`[7]. Cladribine phosphates which accumulate in
`lymphoid cells trigger cellular events resembling to
`some extent the effects of irradiation, namely the
`accumulation of DNA strand breaks leading to pro-
`grammed cell death [8].
`
`Cladribine-induced mmunosuppression is a side
`effect in the therapy of lymphoid malignancies.
`However, immunouppressive activity of the drug
`may be useful in the treatment of diseases of
`
`Received: 97.08.30
`Accepted: 98.01.03
`
`Correspondence address: Pawe∏ Grieb PhD DSc, Laboratory of Experimental Pharmacology, Polish Academy of Sciences Medical Research
`Centre, ul. Pawiƒskiego 5, 02-106 Warsaw, Poland, e-mail: pgrieb@ibbrain.ibb.waw.pl
`
`Hopewell EX1013
`
`1
`
`

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`Stelmasiak Z et al – A pilot trial of cladribine…
`
`Six cladribine courses were given at monthly inter-
`vals, and two additional courses were given at 9
`and 12 or 15 months. Each course consisted of five
`doses of the drug taken once daily on consecutive
`days. Cladribine was given either subcutaneously
`(dose 5 mg per day, six patients) or orally (dose 10
`mg per day, four patients). These dosing regimens
`produce equivalent area under the concentration-
`time curve of the drug [12]. Blood counts and neu-
`rological examinations were taken at prescheduled
`days (preceeding the beginning of each treatment
`course) at monthly intervals during the first 6
`months, and later every three months. The patients
`were instructed to maintain a close contact with
`supervising physicians during the study period, and
`to refer to them immediately in case of relapse,
`infection, or any other unusual event. Steroids
`were allowed, if severe relapse occurred.
`Significance of changes in the EDSS scores were
`assessed by non-parametric statistical methods
`(Friedman ANOVA and post-hoc Wilcoxon matched
`pairs test), using Statistica software package.
`
`RESULTS
`
`Compliance of patients and tolerance of therapy
`was good, and hematological side effects were
`mild. Granulocyte counts remained relatively con-
`stant at about 4 000 per μl on average. Platelet
`counts dropped only slightly (to approx. 200.000
`on average, and not in a single case below
`100 000 per μl). Lymphocyte counts dropped from
`the initial count of 2 336±595 per μl (mean±S.D.)
`to 968±229 per μl at 6 months, and remained at
`approximately 1000 per μl for the next 15 months.
`A tendency toward normalization of lymphocyte
`
`Figure 1. Average lymphocyte counts during and after treatment
`with cladribine.
`
`3
`
`6
`
`9
`
`12
`Months
`
`15
`
`18
`
`21
`
`24
`
`5
`
`3500
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`Lymphocyte counts [1/μl]
`
`autoimmune etiology. Treatment with repeated
`doses of cladribine appeared to halt the progres-
`sion of progressive multiple sclerosis [9], although a
`possibility of hematological toxicity of the drug
`(bone marrow depression) raised some concern
`[10]. In the present study we tested clinical efficacy
`of cladribine in remitting-relapsing form of MS in a
`two-year open-label pilot clinical trial.
`
`MATERIAL AND METHODS
`
`The protocol of the study was approved by the
`Ethical Committee of the Medical University of
`Lublin. Participation in the experimental trial of
`cladribine was offered to a limited group of multi-
`ple sclerosis patients with a definitive remitting-
`relapsing course of the disease, and a certain
`degree of neurological deficit, who were enrolled
`in the outpatient service of the Clinical and
`Research Center for Demyelinating Diseases.
`Clinical diagnosis of MS was additionally confirmed
`by brain MRI scans immediately prior to the start of
`the trial. Patients at relapse, or with evidence of
`secondary progressive course of the disease were
`not admitted. Further exclusion criteria were active
`infections, blood cytopenia of any kind, laboratory
`evidence of kidney or liver dysfunction, and
`hepatitis B antigenemia.
`The study group consisted of 10 patients (eight
`females and two males), aged 21-51 years (medi-
`an: 35 years), body weight 52-75 kg (median: 66
`years). The time from the initial diagnosis of MS
`was 2-16 years (median: 9 years). During the two-
`year period immediately before entering the study
`the number of relapses reported by individual
`patients varied from 2 to 6 (3.8 per patient on
`average). The neurological status prior to the start
`of the therapy, expressed semiquantitatively in the
`EDSS scale [11], ranged from 1 to 6.5 (median:
`4.5). The patients selected to participate in the trial
`were informed about the experimental drug status
`of cladribine, and possible side effects of therapy,
`and gave their written consent. Those in reproduc-
`tive age were instructed to avoid pregnancy, or not
`to father a child, for at least two years from the
`beginning of the therapy.
`Cladribine (2-CdA) used in the present study was
`synthesized and supplied free of charge by the
`Foundation for the Development of Diagnostics
`and Therapy (Warsaw, Poland). The drug was pre-
`pared as sterile solution in isotonic saline, 1 mg/ml
`for oral use and 2.5 mg/ml (phosphate-buffered at
`pH 7.4) for subcutaneous injections.
`
`2
`
`

`

`tions responded to standard antibiotic therapy, cre-
`ating no significant threat.
`Analysis of the data for the whole group revealed
`that EDSS scores were significantly reduced during
`the treatment compared to the initial values
`(Friedman ANOVA (cid:114)2(N=6, df=8)=18.07,
`p<0.02, and that the decreases observed at the
`third to fifteenth month were significant by
`Wilcoxon matched pairs test (Table 1). The total
`number of relapses reported by the patients during
`the two years immediately preceeding the initia-
`tion of the treatment was 38, whereas it was only
`15 during the two-year study period.
`Further inspection of the data revealed that, from
`the point of view of the apparent efficacy of the
`treatment, the group under study can be divided
`into two sub-groups, the ‘responsders’ and the
`‘non-responders’. The ‘responders’ sub-group con-
`sisted of seven patients who reported a total of 29
`relapses during the two preceeding years (average
`relapse rate 2.07 per patient per year), and a total
`of 6 relapses during the two-year study period
`(average relapse rate 0.43 per patient per year).
`Two of the responders reported no relapses for the
`entire two-year period of treatment and post-treat-
`ment follow-up, whereas during the two preceed-
`ing years one of them experienced three, and the
`other one four relapses. The ‘non-responders’ sub-
`group consisted of three patients whose relapse
`rate was the same during the two-year periods
`prior to and after the initiation of the therapy (total
`of 9 relapses, average relapse rate 1.5 per patient
`per year). Averaged EDSS scores for ‘responders’
`and ‘non-responders’ are shown in Fig. 2. One
`patient who did not respond to treatment dis-
`played only a slight and transient reduction in lym-
`phocyte count during the treatment, but the other
`two showed the reduction of approximately the
`same magnitude as in some of the ‘responders’.
`The average body weight was the same in both
`sub-groups.
`
`DISCUSSION
`
`Clinical trials of general immunosuppressants in
`multiple sclerosis produced disappointinging re-
`sults. The reason may be that the depth of
`immunosuppression required for successful modifi-
`cation of the natural history of MS cannot be
`achieved with ‘conventional’ immunosuppressive
`drugs because of systemic side effects. Indeed, it
`has been shown in a double-blind placebo-con-
`trolled trial with cyclosporin A in relapsing-remit-
`
`Laboratory Investigation
`
`counts became evident only at the end of the 2-
`year observation period (Fig. 1). The magnitude of
`lymphocyte count reduction varied among the
`patientswas different in particular cases, ranging
`from virtually no effect in one patient to a transient
`drop by 80% of the initial value (to less than 500
`per μl) in another one. However, there was no cor-
`relation between the depth of lymphocyte nadir,
`or the magnitude of lymphocyte count reduction
`from the initial value, and the drug dose expressed
`per kg of body weight (normalized for oral vs. sub-
`cutaneous route of administration). Two out of four
`patients taking cladribine orally complained tran-
`siently of upper abdominal pain, but the associa-
`tion of this effect with drug intake could not be
`ascertained. During the study period 17 infection
`episodes (most of them upper respiratory tract or
`urinary tract infections) occurred in the whole
`group (including eight in a single patient). All infec-
`
`Table 1. Average EDSS scores during and after the treatment.
`
`p
`
`–
`0.01
`0.02
`0.02
`0.01
`0.02
`0.26
`0.14
`0.34
`
`Month after the initiation
`of treatment
`
`EDSS (mean±SD)
`
`4.3±1.8
`3.4±1.5
`2.9±1.7
`2.7±1.9
`2.6±1.6
`2.4±1.7
`3.3±2.4
`3.4±2.5
`4.3±2.0
`
`0 3 6 9 1
`
`2
`15
`18
`21
`24
`
`Figure 2. Average EDSS scores „responders” (filled bars) and „non-
`responders” (open bars).
`
`6
`
`4
`
`2
`
`0
`
`6
`
`0
`
`6
`
`12
`Months
`
`18
`
`24
`
`3
`
`

`

`ting MS [13] that beneficial effect of the therapy
`(reduction in relapse rate) could be achieved only
`when high (and toxic) doses of the drug were
`given, so that severe concomitant side effects
`(hypertension, renal insufficiency and anemia) pre-
`cluded such treatment.
`
`The present trial was performed with a small num-
`ber of patients, and was not placebo-controlled. Its
`results concerning the efficiacy of the treatment
`shall, therefore, be interpreted with great caution.
`Nevertheless, we consider them potentially impor-
`tant. The therapy appeared to be effective in seven
`patients who reported a very marked (almost five-
`fold on average) reduction in the relapse rate dur-
`ing the 2 years after the initiation of the treatment,
`while it seemed ineffective in the remaining three.
`The ‘responders’ showed also a somewhat more
`pronounced improvement in their neurological sta-
`tus as measured by EDSS scores. We were unable
`to identify any factor which would differentiate
`between the ‘responders’ and the ‘non-respon-
`ders’. There was no indication that a ‘good re-
`sponse’ is related to the actual cladribine dose per
`body weight. Although the reduction of lympho-
`cyte count was only minute and transient in one
`‘non-responder’, in the other two the lymphocyte
`drops were within the range of reductions obser-
`ved in the ‘responders’.
`
`When side effects of the therapy are considered,
`cladribine favourably compares with other immu-
`nosuppressants. In the treatment of lymphoid ma-
`lignancies, besides an increased incidence of infec-
`tions, the only relatively frequent side effect is
`thrombocytopenia (which, at least in some cases,
`may be related to the marrow involvement in the
`disease process), but there is virtually no non-
`hematologic toxicity at the doses up to 0.1 mg/kg
`daily i.v. for five to seven days days [5,6]. The limit-
`ed toxicity of cladribine is attributed to the con-
`finement of the drug-activating enzyme (deoxycyti-
`dine kinase) to the lymphoid cells. Although dan-
`gerous marrow depression were reported in some
`multiple sclerosis patients treated with cladribineby
`Beutler et al [10], our experience (extended
`already to a larger group of patients [14]) indicates
`that in this clinical setting a two- to three-fold
`reduction of blood lymphocyte count can be
`achieved by the drug given subcutaneously without
`clinically significant hematological side effects.
`
`There are some aspects of cladribine pharmacoki-
`netics and pharmacodynamics which may be
`responsible for its beneficial activity in MS. The
`drug is able to cross the blood-brain barrier result-
`
`Stelmasiak Z et al – A pilot trial of cladribine…
`
`ing in CSF: plasma ratio of 25% [15,16], so that it
`may exert some toxicity also toward lymphocyte
`clones inhabitating the CNS. It displays, at least in
`the in vitro conditions, immunosuppressive effects
`not related to the simple reduction of lymphocyte
`counts: it inhibits T and B cell activation and the
`response of T cells to co-stimulation by proteins of
`the extracellular matrix [17,18]. Its property of
`stimulating the activity of the NK cells in vitro [19]
`and restoring their impaired activity in vivo
`(observed in leukemic patients by Lauria et al [20])
`may also be of some significance, because in
`relapsing-remitting MS new MRI-visible lesions
`were reported to appear only during the periods of
`reduction of NK functional activity [21].
`
`CONCLUSIONS
`
`In patients with remitting-relapsing multiple sclero-
`sis treatment with cladribine decreases lymphocyte
`counts in peripheral blood, to 1/3 of the initial val-
`ues on average. The relapse rate in some (but not
`all) patients is impressively decreased. Side effects
`of the therapy are mild. The reason why some
`patients do not respond to the treatment remains
`to be identified. Along with clinical evaluation in
`MS, the detailed pattern of cladribine-induced
`immunosuppression in clinical conditions deserves
`further study.
`
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