Archivum [mmunologiae et Therapiae Experimentalis, 1995, 43, 323—327
`PL ISSN 0004-069X
`
`Effect of Repeated Treatments with Cladribine
`(2-Chlorodeoxyadenosine) on Blood Counts
`in Multiple Sclerosis Patients
`
`Pawet Griep!. ZBIGNIEW STELMASIAK2,
`MirostawRypa!
`
`JANUSZ SOLSKI37,
`
`JACEK Nowicki", BEATA JAKUBOWSKA®? and
`
`Medical Research Centre, Polish Academy of Sciences, Dworkowa3, 00-784 Warsaw, * Clinical and Research Centre for Demyelina-
`ling Diseases and * Department of Clinical Analytics, Medical Academy, Krasnicka 100, 20-718 Lublin, Poland
`
`Abstract. We report the results of blood morphology monitoring of || remitting-relapsing multiple sclerosis
`patients whoreceived repeated treatments with cladribine (2-chlorodeoxyadenosine). The drug was given once,
`daily, subcutaneously(5 mg) or orally (10 mg) for 5 consecutive days, as 6 monthly courses followed by one or
`two additional courses at 3 or 6 monthintervals. The treatments were well tolerated, although manypatients
`suffered from incidental upper respiratorytract infections, most of which occured during the last 6 months of
`the observation period. One patient had recurrent infections, including an episode ofurosepsis. All infections
`responded to standard therapywith antibiotics. Progressive lymphocyte reduction to 1000/,1 on average, and
`clear but clinically insignificant drop in thrombocytes, was observed. Granulocyte counts were sometimes
`markedly elevated. A few patients developed macrocytosis, but none required transfusion. With our dosing
`and schedule, cladribine seems relatively safe in multiple sclerosis patients.
`
`Key words: 2-chlorodeoxyadenosine; blood counts; toxicity.
`
`
`Introduction
`
`is
`Cladribine (2-chlorodeoxyadenosine, 2-CdA)
`a deoxyadenosine analog selectively toxic against hu-
`man lymphocytes (both resting and proliferating) and
`monocytes®**,
`It
`is also highly active against many
`leukemiacell lines in vitro'!*, The drug proved to be
`a remarkablyeffective treatment of hairy cell leuke-
`mia (response rate > 90%after a single course of trea-
`tment)??°, and promising results have been repor-
`ted in other indolent
`lymphoproliferative diseases,
`mainly in B cell chronic lymphocytic leukemia
`(B-CLL) and low grade non-Hodgkin lymphomas’.
`Its immunosuppressive potential has been recognized
`in
`an early study when a
`severe autoimmune
`
`hemolytic anemia, concomitant with a diffuse lym-
`phoma, was abated following treatment”; this preli-
`minary observation was confirmed in further clinical
`trials*. A recent report** suggests that cladribine may
`favourably influence the natural history of progres-
`sive multiple sclerosis, and our preliminary data sug-
`gest also some beneficial effect in remitting-relapsing
`type of this disease '':?3, Future clinical evaluation of
`this drug also in other autoimmune disorders seems
`warranted,
`Cladribine displays a marked toxicity toward bone
`marrowprogenitor cells in vitro*:'?. In agreement with
`these observations, main dose-limiting side effects of the
`drug in patients with both lymphoid? and non-hemato-
`logical?!
`tumors are thrombocytopenia and,
`less fre-
`
`1
`
`Hopewell EX1023
`
`Hopewell EX1023
`
`1
`
`

`

`
`
`324
`
`P. Grieb et al.: Effect Cladribine in MS Patients
`
`quently, anemia and granulocytopenia (with conse-
`quential infections). Cytopenias, usually more severe
`in heavily pretreated patients with advanced lympho-
`id malignancies '?, could be attributed to bone mar-
`row involvement in a disease process and/or cumula-
`tive toxicity of the drug with previous chemotherapy.
`However, marrow suppression and prolonged macro-
`cytosis following repeated doses of cladribine was ob-
`served also in multiple sclerosis patients, rising con-
`cern of a delayed and long lasting hematologic toxici-
`ty of the drug even when bone marrowis unaffected *.
`In a pilot study of cladribine in remitting-relap-
`sing multiple sclerosis we have monitored blood mor-
`phology of patients taking repeated treatments with
`the drug over 18 months.
`
`Materials and Methods
`
`Cladribine (purity > 98% by HPLC) was synthe-
`sized by Z. Kazimierczuk (Department of Biophysics,
`University of Warsaw). The drug was prepared as
`isotonic saline solution, | mg/kg for oral use, and 2.5
`mg/kg (phosphate-buffered at pH 7.4) for subcutane-
`ous injections. Eleven patients (8 females and 3 males,
`age 21—51, body weight 62+8 SD kg, range 52—75
`kg) suffering from remitting-relapsing multiple sclero-
`sis, with normal blood counts, and normal kidney
`and liver function tests, were treated with multiple
`courses of the drug. Each course consisted of 5 daily
`doses, 10 mg orally in seven patients and 5 mg sub-
`culaneously in the remaining four. The patients recei-
`ved 6 monthly courses, and additional courses were
`given at 9, and (in some patients) also at 12 or 15
`months. Blood morphology was monitored before
`each treatment course, and later every three months.
`Statistical significance of deviations from control va-
`lues was assessed by t-test for paired data and as-
`sumed to bestatistically significant if p < 0.05.
`
`Results
`
`The treatments were well tolerated. Although two
`study participants taking cladribine orally complai-
`ned of upper abdominalpain, its relationship to the
`treatment cannot be ascertained, since none of a con-
`siderable numberof patients treated with cladribine
`given orally for B-CLL reported such complaints
`(J. Liliemark, Department of Clinical Pharmacology,
`Karolinska Hospital, Stockholm, personal communi-
`cation). Several patients reported increased frequency
`of upper respiratory tract infections, most of which
`
`occurred during the last 6 months of the observation
`period. Onepatient suffered from recurrentinfections,
`including one episodeof urinary tract infection which
`occured during the third month of the study. All in-
`fections were succesfully treated with standard anti-
`biotic therapy.
`Averaged hematological data of all patients are
`displayed in the graphs as means + | SEM. Follo-
`wing treatments, lymphocyte counts decreased sharp-
`ly, reaching the nadir after the sixth course (Fig. 1).
`
`3000
`
`2000
`
`[Vul]
`
`1000
`
`oO
`
`3
`
`6
`
`9
`Months
`
`12
`
`15
`
`18
`
`Fig. 1. Average lymphocyte counts during treatment with cladribi-
`ne, The decrease is statistically significant from the second month
`of therapy
`The degree of lymphocytopenia (its measure being the
`nadir of lymphocyte counts after 6 months ofthe stu-
`dy) was not related to the dose of the drug per body
`weight (Fig. 2), which mayreflect differing individual
`susceptibility of lymphocytes to the cytotoxiceffect of
`the drug. There was no appreciable drop in hemo-
`globin content (data not shown). Platelet counts dec-
`reased significantly (Fig. 3), but never dropped below
`100000/ul. A slight macrocytosis developed in some
`patients, but was not significant in the whole group,
`except at 18 months (Fig. 4). Averaged granulocyte
`count did not significantly deviate from the control
`level, except of the drop at 18 months (Fig. 5), but
`transient granulocytosis appeared in some patients
`(Figs. 6, 7).
`
`Discussion
`
`Until recently 2-chlorodeoxyadenosine had been
`given by continuous 7-day intravenous infusions and
`repeated monthly, if required. This mode of admini-
`
`
`
`2
`
`

`

`P. Grich et al.; Fffect Cladribine in MS Patients
`
`1600
`
`1400
`
`1200
`
`800
`
`
`
`
`
`lymphocytecountsatsixmonths[1/jl]
`
`600 400
`100
`
`65
`
`-
`70
`
`:
`.
`90
`85
`80
`75
`daily cladribine dose [}.g/kg]
`
`95
`
`100
`
`Fig. 2. The lack of correlation between the lymphocyte counts at
`6 months of therapy and dailycladribine dose per kg body weight.
`Squares — the drug taken subcutancously; circlets — drug taken
`orally (dose normalized to subcutancous route assuming 50% oral
`bioavailability). Data available for 10 paticnts
`
`400
`
`350
`
`300
`
`250
`
`200
`
`150
`
`[1000/:!}
`
`Months
`
`Fig. 3. Average platelet counts during treatment with cladribine.
`The decrease is statistically significant from the third month of
`therapy
`
`stration was established on the basis of the in vitro
`studies suggesting that multi-day exposure to nano-
`molar concentrations of the drug is required to kill
`resting humanperipheral blood lymphocytes*. In this
`mode of administration a single course of 0.09 mg/kg
`daily(i.c., total dose 0.63 mg/kg) is usually well tolera-
`ted by leukemia patients? *, but
`the probability of
`cytopenia increases when treatments are repeated.
`
`105
`
`100
`
`95
`
`90
`
`B5
`
`80
`
`75
`
`[H"]
`
`Months
`
`Fig. 4. Average MCVduring treatment with cladribine. The mse is
`statistically significant at 18 months
`
`70
`2000 1000
`
`least as effective as week-long intravenousinfusions '?.
`
`7000
`
`6000
`
`5000
`
`4000
`
`[Vul]
`
`3000
`
`0
`
`3
`
`6
`
`9
`Months
`
`12
`
`15
`
`18
`
`Fig. 5. Average granulocyte counts during treatment with cladribi-
`ne. The changeis statistically significant only at 18 months
`
`Recent pharmacokinetic studies have shownthat
`the area under the plasma concentration versus time
`curve (AUC), which is a measure of the exposure of
`target cells (peripheral blood lymphocytes)
`to the
`drug, is practically of the same size when the doseis
`given as continuous 24 h i.y. infusion, and whenit is
`given once daily, either as an i.v. infusion lasting 2 h,
`or subcutaneously, or when a doubled dose is gi-
`ven orally (oral bioavailability is approximately
`50%)'*'*. In the treatment of lymphoid malignan-
`cies intermittent dosing schedules proved to be at
`
`3
`
`

`

`
`
`326
`
`#000
`
`7000
`
`6000
`
`5000
`
`» 4000
`[ul]
`
`3000
`
`2000
`
`1000
`
`Months
`
`Fig. 6 Lymphocyte and granulocyte counts of the patient no. 9
`(a female, the drug given subcutaneously, daily dose 0.07 mg kg)
`
`5000
`
`4000
`
`3000
`
`2000
`
`=
`
`\
`
`\
`\ o~\ ~~
`
`-
`
`im
`
`ey
`
`granulocytes
`gee ss
`
`1000
`
`Tn :
`hg
`
`1
`
`eea
`lymphocytes
`
`0
`3
`6
`9
`12
`15
`18
`Months
`
`Fig. 7. Lymphocyte and granulocyte counts of the patient no. 4
`(a female, the drug given orally, daily dose 0.18 mg/kg)
`
`Cladribine is a pro-drug requiring intracellular
`enzymatic phosphorylation to exert cytotoxicity *. In
`B-CLL patients at the end of 2 hiv. infusion intracel-
`lular concentration of CdA-phosphates in lymphocy-
`tes was two orders of magnitude higher than the peak
`plasma level of the drug.
`Its decay had t,,, of ap-
`proximately 24 h, and lymphocytic intracellular
`AUC’s of CdA phosphatesafter intermittent and con-
`tinuous infusion were similar'®:'7. Although no data
`are available on the intracellular kinetics of CdA-
`phosphates in non-leukemic lymphocytes in vivo, one
`may expect it to be similar to that
`in B-CLL cells.
`
`P. Grieb et al.: Effect Cladribine in MS Patients
`
`Therefore, intermittent (e.g, subcutaneous, or oral)
`dosing of cladribine should also be effective for reduc-
`tion of lymphocyte population and induction of im-
`munosuppression. The results of the present study
`suggest, that it may also produce lower hematologic
`toxicity.
`In the Scripps study, chronic progressive multiple
`sclerosis patients were treated with 4 to 6 monthly
`courses of cladribine, dose 0.087 —0.1 mg/kg per day
`for 7 days, given as continuousi.v. infusion. The nadir
`of lymphocyte counts (ca. 500/,i1) occurred after the
`last treatment. and the rebound was slow and incom-
`plete (even during more than 3 years), While severe
`hematologic toxicity was observed in only 2 out of
`more than 20 patients and it could have beenrelated
`to prior or concomitant intake of other marrow-de-
`pressing drugs, all patients on average displayed mar-
`ked signs of marrowtoxicity: a progressive, although
`moderate and reversible fall
`in hemoglobin concen-
`tration, a distinct macrocytosis and a moderate throm-
`bocytopenia (these changes were not reversing after
`the treatment was discontinued), and a modest, but
`noticeable drop in granulocyte counts. Those hema-
`tological side effects were markedly more severe than
`these observed in our patients.
`of
`that
`study and
`The design our present
`Scripps*:?? differ in some points: 1) patients suffered
`fromdifferent form of the disease (in our group it was
`remitting-relapsing, while in the Scripps study it was
`chronic progressive multiple sclerosis); 2) while the
`daily doses of the drug received by our patients
`(0.067 —0,096 mg/kg) were similar, the total dose in
`our study was lower on average by approximately
`30% because the course of treatmentlasted 5 instead
`of 7 days: 3) we used intermittent dosing (once daily
`orally or subcutaneously) instead of a week-long con-
`tinuous infusion. We hypothesise that the last factor
`is
`the most
`important one, ie. that hematological
`side effects of cladribine depend onits dosing schedu-
`le and are more severe when the drug is given in
`continuous infusion. A possible explanation maybe
`that
`the entry of cladribine to the marrowcells is
`slower than to lymphocytes, and reducing exposure
`to 5 days andutilizing intermittent drug delivery may
`to some extent spare marrow progenitors, while still
`acting on lymphocytes. In agreement with this hypot-
`hesis are the data of PETzeR et al.'” who showed that
`the inhibition by cladribine of proliferation of bone
`marrow progenitors in vitro is achieved at high con-
`centrations (> 300 nM) and it develops over a few
`days. In some of our patients granulocyte counts in-
`creased markedly
`(supposedly
`as
`a
`response
`
`4
`
`

`

`P. Grich et al: Effect Cladribine in MS Patients
`
`327
`
`10.
`
`13.
`
`
`
`Hiescu-GinsapreG C., Hun Y. O. Stass 8. and FREIREICH
`to infections), which clearly indicates that with our
`E. (1992): Treatment of hairy cell
`leukemia with 2-chlorodeo-
`dosing and schedule the drug is toxic neither to gra-
`xyadenosine (2-CdA), Blood, 79, 882— 887.
`nulocytes nor to granulocyte progenitors.
`Gorski A. and Grier P. (1994): Cladnbine (2-chloro-2'-deoxy-
`The reduction of lymphocyte counts in our study
`adenosine); new perspectives in clinical
`immunosuppression.
`was on average only about half of that observed in
`Arch. Immunol. Ther. Exp., 42, 11—12.
`. Gries P., RyBA M., STFLMASIAK Z.. NowIcCk! J.. Sorski J. and
`the Scripps study. Would the severity of lymphocyto-
`JakunowskKa B. (1994); Cladribine treatment of multiple scle-
`penia be an index of therapeutic effictacy of cladribi-
`rosis. Lancet, 344, 538.
`ne, one might expect
`the Scripps protocol be more
`. HICKISH
`T., SERAFINOWSK1 P., CUNNINGHAM D,, Oza A,
`effective. However, the depletion of lymphocytes, whi-
`Dortann F., Jupsosn LL. Mittar B, C., Lister T. A, and
`le certainly being a sort of measure of the effect of
`Rotban A.
`(1993): 2-chlorodeoxyadenosine: Evaluation of
`a novel predominantly lymphocyte-selective agent in lymphoid
`cladribine on the lymphatic component of the immu-
`malignancies. Br. J. Cancer, 67, 139 —143.
`ne system, may not be the only factor contributing to
`Junivsson G. and Littemark J. (1994); Survival of previously
`therapeutic activity of this drug in autoimmune disea-
`treated patients with chronic lymphoevtic leukemia following
`ses. Jn vitro cladribine, acting by a yet unknown me-
`Ly. cladribine (2-chlorodcoxyadenosine, CdA), Abstract. Blood.
`chanism, is a potent inhibitor of lymphocyte activa-
`84 (suppl. 1), 462.
`. LILieMaRK J, ALBERTIONT F., HASSAN M. and JULIUSSON G.
`tion'®, and this property maywell contribute to its
`(1992): On the bioavailability of oral and subcutaneous 2-chlo-
`immunosuppressive efficiacy in vive, Functional pro-
`ro-2'-deoxvadenosine in humans: alternative routes of adminis-
`perties of lymphocytes following in vire treatment
`tration. J. Clin, Oncol., 10, 1514— 1518.
`with the drug remain be described.
`. LIneMARK J. and Juniusson G. (1991): On the pharmacokine-
`tics of 2-chloro-2'-deoxyadenosine in humans. Cancer Res., 51,
`$570—5572.
`References
`
`. Liuemarnk J. and Jutmsson G. (1994): 2-Chloro-2’-deoxvade-
`nosine — clinical, bhochemical and pharmacokinetic considera-
`tions. Arch. Immunol. Ther. Exp., 42, 7— 10.
`7. LitieMark J., Perrerson B, and Jutrmusson G. (1991): The rela-
`tionship between plasma 2-chloro-2'-deoxyadenosine (CdA)
`and cellular CdA nucleotides (CdAN) after intermittent and
`continuous iV.
`infusion in patients with chronic lymphocytic
`leukemia. Abstract. Blood, 78 (suppl. 1) 33.
`. NAGOURNEY R. A., Evans S.S., MESSENGER J. C,, ZHUANG St
`BeuTLer F, (1994): New chemotherapeutic agent: 2-chlorodeo-
`Y.ound WeIsexruan $. M. (1993): 2-chlorodeoxvadenosine ac-
`xyadenosine, Semin. Hematol, 31, 40
`—45.
`3. BFUTLER E., Kozrow J. A.. MCMILLANR., Sipe J. C., ROMINE J.
`lvily and cross resistance patterns in primary cultures of hu-
`man hematologic neoplasms. Br. J. Cancer, 67, 10—14,
`S.and Carrera C. J, (1994): Marrow suppression produced by
`. Perzrrk A. L., Biccurs BR. ZitaAN U.. Haun M., Grisen F. HL,
`repeated doses of cladribine. Acta Haematol., 91, 10-15.
`PRAGNELL. L, BRAUNSTEINER FH. and Konwattnka G, (1991):
`4. BRUTLER F., Prro L. D., SAVEN AL. KAY ALC. MCMILLAN R.,
`LONGMIRE R.. CARRERA C. J., Morin P. and Carson D. A.
`Theinhibitory eflect of 2-chlorodeoxyadenosine on granulocy-
`lic, erythroid and To
`lymphocytic colony growth. Blood. 78,
`(1991): 2-Chlorodeoxvadenosine (2-CdA). A potent chemothe-
`2583—2587.
`rapeutic and immunosuppressive nucleoside. Leuk. Lympho-
`20. Piro L. D., Carrera C.J, Carso~n D. A, and Brutter E.
`ma, 5,
`§—
`14
`5. BrnGert R., PETZER A. L., ZIIAN UL. GRISEN FF. H., SCHTRMER
`(1990): Lasting remissions in hairy ecll
`leukemia induced by
`a single infusion of 2-chlorodeoxyadenosine. N, Engl. J. Med.
`M.. Haun M. and Konwattnka G.
`(1993): Effect of deaxyeyti-
`322, 1117-1121.
`dine on 2-chlorodeoxyadenosine-mediated growth inhibition
`21. SAVEN A., KAWASAKI H.. CARRERA C. J. WALTZ T., CopELAND
`of normal humanerythroid and myeloid progenitor cells. Exp.
`B., ZYROFF J.. Kosty M., Cakson D. A., BEUTLER F. and Piro
`Hematol., 21, 432 —437.
`6. Carrera C. J.. Tenar C., Lorz M., Curb J. G., Prro L. D.,
`L. D. (1993): 2-Chlorodeoxyadenosine dose escalation in non-
`7 hematologic malignancies. J. Clin. Oncol, 11, 671—678.
`BeuTLER F. and Carsow D. A. (1990); Potent toxicity of 2-chlo-
`. SPF J.. ROMINI
`JOS. Roziow J. A. MCMILLAN R.. ZYROEFFJ.
`rodeoxyadenosine against human monocytes in vitro and in
`vivo, J, Clin, Invest., 86, 1480 — 1488.
`(1994): Cladribine treatment of chronic pro-
`and BEUTLER EF.
`gressive multiple sclerosis. Lancet, 344, 9—
`13,
`7. Carson D. A. Wasson D. B. and BeuTtLer b. (1984): Antileu-
`23.
`STULMASIAK 4., SOLSKI J, Nowick! JL, JakKuBowskaA B, Rypa
`kemic and immunosuppressive activity of 2-chloro-2’-deoxya-
`denosine. Proc, Natl. Acad. Sci. USA, 81, 2232—2236.
`M. and Gri-e P. (1994): Short
`term effects of 2-chlorodeaxy-
`8, Carson D. A, Wasson D, B,, Tartte R. and Yu A. (1983):
`adenosine (2-CdA)
`in remitting-rclapsing multiple sclerosis
`(MS). Abstract. J. Neurol, 241 (suppl. 1), 90,
`Specific toxicity of 2-chlorodeoxyadenosine toward resting and
`proliferating human lymphocytes. Blood, 62, 737-743.
`9. Estpy B. H.. Kur‘ock R.. KRANTARIIAN H. M,, O'BRIEN S. M.,
`McCrrpie K. B., Brean M., Konner C., Katina M.
`J..
`
`Li. Rena J. E. LummM W. C. Harwoop F. C.,
`lL. Avery T.
`SANTANA WV, M. and Bianuiy R. L. (1989): Biochemical phar-
`macology of 2-chlorodeoxyadenosine in malignant human he-
`matopoicticcell lines and therapeutic effects of 2-bromodeoxy-
`adenosine in drug combinations in mice. Cancer Res., 49.
`4972 —4975
`
`bo
`
`
`
`Received in March 1995
`Accepted in May 1995
`
`Archiwum Immunologtac
`
`36:95
`
`
`
`5
`
`