Cladribine Injection - Pharmaceutical Information, Clinical Trials, Detailed Pharmacology, Toxicology
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Cladribine Injection - Scientific Information

Manufacture: Fresenius Kabi USA, LLC
Country: United States
Condition: Hairy Cell Leukemia
Class: Antimetabolites
Form: Liquid solution, Intravenous (IV)
Ingredients: Cladribine, Sodium chloride, Phosphoric acid, Sodium phosphate, Dibasic

Pharmaceutical Information

Drug Substance

Proper name: Cladribine
Chemical name: 2-chloro-6-amino-9-(2-deoxy-β-D-erythropento-furanosyl)purine
Molecular formula and molecular mass: C10H12N5O3Cl                  285.7
Structural formula:


Physicochemical properties: Cladribine is a white nonhygroscopic, crystalline powder.
Cladribine Injection is available in single-use vials containing 10 mg (1 mg/mL) of cladribine, a chlorinated purine nucleoside analog. Each millilitre of Cladribine Injection contains 1 mg of the active ingredient, cladribine, and 9 mg (0.15 mEq) of sodium chloride as an inactive ingredient.

Cladribine is a synthetic antineoplastic agent for continuous intravenous infusion. It is a clear, colourless, sterile, preservative-free, isotonic solution.

The solution has a pH range of 5.5 to 8.0. Phosphoric acid and/or dibasic sodium phosphate may have been added to adjust the pH to 6.3 ± 0.3.

Clinical Trials

Two single-centre open studies of cladribine have been conducted in patients with Hairy Cell Leukemia with evidence of active disease requiring therapy. In the study conducted at the Scripps Clinic and Research Foundation (Study A), 89 patients were treated with a single course of cladribine given by continuous intravenous infusion for 7 days at a dose of 0.09 mg/kg/day. In the study conducted at the M.D. Anderson Cancer Center (Study B), 35 patients were treated with a 7-day continuous intravenous infusion of cladribine at a comparable dose of 3.6 mg/m2/day.

A complete response (CR) required clearing of the peripheral blood and bone marrow of hairy cells and recovery of the hemoglobin to 12 g/dL, platelet count to 100 x 109 L, and absolute neutrophil count to 1,500 x 106/L. A good partial response (GPR) required the same hematologic parameters as a complete response, and that fewer than 5% hairy cells remain in the bone marrow. A partial response (PR) required that hairy cells in the bone marrow be decreased by at least 50% from baseline and the same response for hematologic parameters as for complete response. A pathologic relapse was defined as an increase in bone marrow hairy cells to 25% of pre-treatment levels. A clinical relapse was defined as the recurrence of cytopenias, specifically decreased in hemoglobin ≥ 2 g/dL, ANC ≥ 25% of pre-treatment levels or platelet counts ≥ 50 x 109/L. Patients who met the criteria for a complete response but subsequently were found to have evidence of bone marrow hairy cells (< 25% of pre-treatment levels) were reclassified as partial responses and were not considered to be complete responses with relapse.

Among patients evaluable for efficacy (n = 106), using the hematologic and bone marrow response criteria described above, the complete response rates were 65% and 68% for Study A and Study B, respectively, yielding a combined complete response rate of 66%. Overall response rates (i.e., complete plus partial responses) were 89% and 86% in Study A and Study B, respectively, for a combined overall response rate of 88%.

Using an intent-to-treat analysis (n = 123) and further requiring no evidence of splenomegaly as a criterion for CR (i.e., no palpable spleen on physical examination and ≤ 13 cm on CT scan), the complete response rates for Study A and Study B were 54% and 56%, respectively, giving a combined CR rate of 54%. The overall response rates (CR + GPR + PR) were 90% and 85%, for Studies A and B respectively, yielding a combined overall response rate of 89%.

Table 1: Response Rates to Cladribine Treatment in Patients with Hairy Cell Leukemia
Complete Response Overall Response
Evaluable Patients (n = 106) 66% 88%
Intent-to-treat Population (n = 123) 54% 89%

In these studies, 60% of the patients had not received prior chemotherapy for Hairy Cell Leukemia or had undergone splenectomy as the only prior treatment and were receiving cladribine as a first-line treatment. The remaining 40% of the patients received cladribine (as a second-line treatment, having been treated previously with other agents, including α-interferon and/or deoxycoformycin. The overall response rate for patients without prior chemotherapy was 92%, compared with 84% for previously treated patients. Cladribine is active in previously treated patients; however, retrospective analysis suggests that the overall response rate is decreased in patients previously treated with splenectomy or deoxycoformycin and in patients refractory to α-interferon.

Table 2: Overall Response Rates (CR + GPR + PR) to Cladribine Treatment in Patients with Hairy Cell Leukemia
Overall Responsen = 123 NR + Relapse
No Prior Chemotherapy 68/74
92%
6 + 4
14%
Any Prior Chemotherapy 41/49
84%
8 + 3
22%
Previous Splenectomy 32/41⚹
78%
9 + 1
24%
Previous Interferon 40/48
83%
8 + 3
23%
Interferon Refractory 6/11⚹
55%
5 + 2
64%
Previous Deoxycoformycin 3/6⚹
50%
3 + 1
66%

NR = No Response

⚹ P < 0.05

After a reversible decline, normalization of peripheral blood counts (Hemoglobin ≥12.0 g/dL. Platelets ≥ 100 x 109/L, absolute neutrophil count (ANC) ≥ 1,500 x 106/L) was achieved by 92% of evaluable patients. The median time to normalization of peripheral counts was 9 weeks from the start of treatment (Range: 2 to 72). The median time to normalization of platelet count was 2 weeks, the median time to normalization of ANC was 5 weeks and the median time to normalization of hemoglobin was 8 weeks. With normalization of platelet count and hemoglobin, requirements for platelet and RBC transfusions were abolished after Months 1 and 2, respectively, in those patients with a complete response. Platelet recovery may be delayed in minority of patients with severe baseline thrombocytopenia. Corresponding to normalization of ANC, a trend toward a reduced incidence of infection was seen after the third month, when compared to the months immediately preceding cladribine therapy. (See also Warnings and Precautions and Adverse Reactions.)

Table 3: Cladribine Treatment in Patients with Hairy Cell Leukemia: Time to Normalization of Peripheral Counts
Parameter Median Time to Normalization
of Count⚹
Platelet Count 2 weeks
Absolute Neutrophil Count 5 weeks
Hemoglobin 8 weeks
ANC, Hemoglobin and Platelet Count 9 weeks

⚹ Day 1 = First day of infusion

For patients achieving a complete response, the median time to response (i.e., absence of hairy cells in bone marrow and peripheral blood together with normalization of peripheral blood parameters), measured from treatment start, was approximately 4 months. Since bone marrow aspiration and biopsy were frequently not performed at the time of peripheral blood normalization, the median time to complete response may actually be shorter than that which was recorded. At the time of the data cut-off, the median duration of complete response was greater than 8 months and ranged to 25+ months. Among 93 responding patients, 7 had shown evidence of disease progression at the time of the data cut-off. In 4 of these patients, disease was limited to the bone marrow without peripheral blood abnormalities (pathologic progression), while in 3 patients there were also peripheral blood abnormalities (clinical progression). Seven patients who did not respond to a first course of cladribine therapy received a second course of therapy. In the five patients who had adequate follow-up, additional courses did not appear to improve their overall response.

Deaths

Of the 196 patients with Hairy Cell Leukemia entered in the two trials, there were 8 deaths following treatment. Of these, 6 were of infectious etiology, including 3 pneumonias, and 2 occurred in the first month following cladribine therapy. Of the 8 deaths, 6 occurred in previously treated patients who were refractory to α-interferon.

Detailed Pharmacology

Nonclinical Pharmacodynamics

In Vitro

The following table summarizes the data for the effects of cladribine on human cell lines and peripheral blood cells. The IC50 or ID50 values (CEM cells) may vary with experimental protocols and duration of drug exposure.

Table 4: Inhibition of the Growth of Various Human Cells Treated with Cladribine
Cell Line or CellsCell TypesIC50 or ID50 (nM)
CEMT-lymphoblast14
MOLT-3T-lymphoblast24
MOLT-4T-lymphoblast55
HL60Myeloid20
THP-1Myeloid24
U937Myeloid23
RAJIB-lymphoblast27
SBB-lymphoblast-
K562Myeloid Progenitor256
WI-38Fibroblast-
CCRF-CEMT-lymphoblast3
WI-L2B-lymphoblast35
WI-L2 (AKase deficient)B-lymphoblast35
WI-L2 (dCKase deficient)B-lymphoblast>2,000
Monocytes ⚹Monocyte27
Lymphocytes ⚹Lymphocyte20
GM 01380Fibroblast-

Akase – adenosine kinase

dCKase – deoxycytidine kinase

- – No concentration inhibited 50%

⚹ – Isolated Peripheral Blood Cells

Freshly isolated human peripheral blood monocytes and lymphocytes and normal human GM 01380 fibroblasts were cultured for 5 days with various concentrations of cladribine. Viable monocytes and fibroblasts were measured by the MTT[3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazoliumbromide] reduction assay, and viable lymphocytes were enumerated by dye exclusion. The data indicate that lymphocytes and monocytes are sensitive to the cytotoxic effects of cladribine, in vitro, at nanomolar concentrations, whereas the fibroblast line, GM 01380, is unaffected. This cytotoxicity of both peripheral blood cell populations is substantially prevented by deoxycytidine. However, contrasting lymphocytes, monocyte lysis is not prevented by nicotinamide or 3-aminobenzamide, inhibitors of poly (ADP ribose) synthetase, suggesting that the cytotoxic mechanism differs in these two cell types.

Utilizing a human tumour colony forming assay, the cytotoxic activity of cladribine toward several human solid tumours was assessed. Overall, cladribine, at concentrations of 1.0 and 10.0 µg/mL, reduced the tumour survival rate (defined as < 50% survival of tumour colony-forming units) by 8% and 23% respectively, when given in a 1-hour pulse; and by 11% and 31% respectively when given as a continuous exposure. The data indicate that cladribine is much less active against solid tumours than against leukemic lymphoblasts.

Whereas lymphoblasts are sensitive to nanomolar concentrations of cladribine, the solid tumours required at least 100-fold greater concentrations. The data suggest that some solid tumours may respond to cladribine therapy in vivo, but the concentration of drug required to kill these tumour cells may be considerably higher than the concentrations required to kill lymphoid cells.

Cladribine has been examined in vitro for cytotoxic effects against normal bone marrow and a number of human leukemia and lymphoma cells. In these studies the effects of the cladribine on spontaneous thymidine uptake by 40 leukemic or 20 normal human bone marrow suspensions were monitored. Twelve of 20 acute lymphoblastic leukemia (ALL) cell suspensions bearing the common acute lymphocytic leukemia antigen (CALLA+) and 4 of 5 T and pre-T acute lymphoblastic leukemia cell preparations were more sensitive to the inhibitory effects of cladribine (ID50 ≤ 5nM) than any normal bone marrow. The pre-B-cell acute lymphocytic leukemias and the acute myelocytic leukemias (AML) varied greatly in their sensitivity to cladribine (2 nM to > 50 nM). These studies indicate that the CALLA-positive ALL specimens and the T and pre-T ALL specimens are significantly more sensitive to cladribine than normal bone marrow. The data suggest that cladribine inhibits the proliferation and survival of malignant T and non T, non-B lymphocytes at concentrations that spare normal bone marrow cells and other cell types.

Cladribine showed curative therapeutic activity (50% of mice were cured; i.e., survived > 60 days) in mice bearing L1210 leukemia, when administered at 15 mg/kg every 3 hours on days 1, 5, and 9 after tumour inoculation. Increase in the life-span of dying mice was dose-dependent. Cladribine was most effective when administered via a multiple dosage schedule on days 1, 5, and 9 after tumour implantation. Neither single treatment nor a daily dose of 50 mg/kg over 6 days produced cures (survival beyond 60 days).

The degree of cladribine binding to plasma proteins has been investigated in normal rat (male; Sprague-Dawley), dog (female; Beagle), monkey (male; cynomolgus) and human (male; fasted, no caffeine consumption for 24 hours prior to blood donation and not on any medication) plasma. For all species, heparin was used as the anticoagulant. In humans, the degree of cladribine binding to serum proteins was also investigated. Cladribine solutions (spiked with 3H-2-CdA) were added to plasma/serum to achieve concentrations of 6.1 ng, 61.1 ng or 6.1 µg/mL and dialyzed to equilibrium at 37 °C.

Cladribine was minimally bound to plasma proteins in all species (~ 10 to 20%) at each drug concentration tested. At the same cladribine concentrations, human plasma and serum yielded similar results, indicating that the anticoagulant (heparin) did not compete with cladribine binding sites.

Nonclinical Pharmacokinetics

In Vivo

In a pilot study, female Sprague-Dawley rats had cannulas implanted in both the femoral (administration) and jugular (sampling) veins. Cladribine (spiked with 3H-2-CdA) was administered, at 1 mg/kg, either by bolus (2 rats) or by a constant 1-hour infusion (2 rats). Immediately following bolus dosing, the total plasma radioactivity concentration was ~ 1.2 µg-eq/mL. Since this sample was drawn immediately postdose, the radiolabelled concentration is likely to equate to the cladribine concentration. In this case, cladribine would distribute into an initial volume ~ 0.8 L/kg. At 1 hour, circulating radioactivity concentrations were ~ 0.5 µg-eq/mL and remained essentially constant at that concentration for 96 hours. Following the end of the constant infusion, the plasma radioactivity concentration was ~ 0.6 µg-eq/mL, and, as from bolus administration, showed minimal decline over 96 hours.

Since the above concentrations are based on radioactivity measurements, it can be assumed that this elimination profile does not reflect the actual decline of cladribine from rat plasma. In man, following a 2-hour infusion, the terminal half-life of cladribine has been estimated at ~ 5.4 hours.

In a pilot study in rats treated with radiolabelled cladribine, approximately 41 to 44% of the administered label was recovered in the urine in the first 6 hours from a 1 mg/kg bolus or infusion. Only small amounts of radioactivity were recovered after 6 hours. Less than 1% of the administered radioactivity was excreted in the feces following a bolus dose to rats. From preliminary profiling of the 0 - 6 hour urine in one rat, it would appear that three of the radioactivity peaks were associated with intact cladribine, 2-CdAMP and 2-CA. Since it is recognized that 2-CA can be a degradation product of cladribine, it is possible that its detection in rat urine may be an artifact, as the samples were stored at -20 °C for about 4 to 6 weeks prior to assay. Quantitatively, ~ 37 to 46% of the urinary radioactivity was associated with 2-CdA, which would infer that in the Sprague-Dawley rat cladribine does undergo biotransformation to some extent.

In addition, in an earlier pilot study, < 1% of the administered radioactivity was excreted in the feces following bolus injection.

The metabolism of 2-chloro-2'-3'-dideoxyadenosine (2-CddA) has been investigated in mice. The total urinary excretion of unchanged CddA for 24 hours, after exposure to 24 mg/kg, was 3.4 percent of the delivered dose. At least two possible CddA metabolites were detected in mouse urine which did not co-elute with 2-chloro-2'-3'-dideoxyinosine, 2-CA or 2-chlorohypoxanthine.

Toxicology

Carcinogenesis and Mutagenesis

No animal carcinogenicity studies have been conducted with cladribine. However, its carcinogenic potential cannot be excluded based on demonstrated genotoxicity of cladribine. Cladribine induced chromosomal effects when tested in both an in vivo bone marrow micronucleus assay in mice and an in vitro assay using CHO-WBL cells.

As expected for compounds in this class, the actions of cladribine yield DNA damage. In mammalian cells in culture, cladribine caused the accumulation of DNA strand breaks. Cladribine was also incorporated into DNA of human lymphoblastic leukemia cells. Cladribine was not mutagenic in vitro (Ames and Chinese hamster ovary cell gene mutation tests) and did not induce unscheduled DNA synthesis in primary rat hepatocyte cultures. However, cladribine was clastogenic both in vitro (chromosome aberrations in Chinese hamster ovary cells) and in vivo (mouse bone marrow micronucleus test).

As expected for compounds in this class, the actions of cladribine have been shown to yield DNA damage. The mutagenicity studies are summarized below.

Table 5: Mutagenicity Studies with Cladribine
Assay Species/Cell Line Dose Levels Control Groups Results
Ames Test Salmonella typhimurium strain TA98, TA100, TA1535, TA1537, TA1538; Escherichia coli strain WP2uvrA 10, 50, 100, 250, 500, and 1,000 µg/plate, +/-S-9 mix Vehicle: saline positives1: MNNG, 9-aminoacridineXHCl, 2-anthramine, sodium azide Cladribine negative for inducing mutations in bacteria
DNA Repair Assay Primary rat hepatocyte cultures
(in vitro assay)
1, 5, 10, 50, 75, 100, 150, and 200 µg/mL Vehicle: saline positive: 2-AAF Cladribine negative for inducing unscheduled DNA synthesis
DNA Synthesis Inhibition CCRF-CEM Cells (Human Lympho-blastic Cells) 0.3 µM - 2 Cladribine incorporated into DNA; 90% reduction in DNA synthesis (0.3 ΦM); Decreased levels of dNTPs
dNTP Imbalance: DNA Strand Breaks Mouse mammary tumour FM3A cells (F28-7) 0.5, 1.0, 5.0, and 20.0 µM - 2 Intracellular dNTP imbalance; double strand DNA breaks; cell death
DNA Strand Breaks: NAD depletion Human peripheral blood lymphocytes 0.1, 1, and 10 µM - 2 DNA strand breaks; inhibition of RNA synthesis; reduced intracellular NAD levels; reduced ATP pool; cell death3
DNA Repair Inhibition Human peripheral blood lymphocytes 0.1, 1, 10, and 100 µM γ-radiation Blocked γ-radiation-induced unscheduled DNA synthesis (DNA repair)

1Choice of positive control dependent on strain and/or presence or absence of S-9 mix.

2No control groups identified.

3Effects prevented by deoxycytidine a competitive inhibitor of cladribine phosphorylation; nicotinamide (NAD precursor) and 3-aminobenzamide [both inhibitors of poly (ADP-ribose) synthetase] also protected the cells from cladribine toxicity.

KEY: S-9 mix = Aroclor 1254-included rat liver S-9 mix; MNNG = N-methyl-N-nitro-N'-nitrosoguanidine; 2-AAF= 2-acetylaminofluorene, dNTP = deoxynucleotide triphosphate

Toxicology Studies

Table 6: Acute Toxicity Studies with Cladribine
Strains/Species (# sex/group) Age/Weight Administration Route Dose Groups Observations Results
Crl:CD-1 (ICR)BR, VAF/Plus mice (5M, F/group) 6 weeks/
M: 26 - 32 g
F: 20 - 25 g
i.v. 25 mg/kg 2-CdAa
25 mg/kg 2-CdA +
2-chloroadenineb

Vehicle: 0.9% NaCl
Clinical signs and symptoms.
Mortality, body weight changes
No mortality.
No treatment–induced signs of toxicity
CDF1 mice
(3 or 4 F/group)
4 – 6 weeks/Wt
NA
i.p. 30, 45, 60, 75, 90, 120, 150, 180, and 210 mg/kgc
Vehicle: 0.9% NaCl
Mortality MTD = 120 mg/kg
LD50 = 150 mg/kg
LD90 ≤ 180 mg/kg

a1.0 mg/mL solution of 2-CdA in 0.9% NaCl at a dose volume of 25 mL/kg.

b 1.0 mg/mL 2-CdA + approximately 75 µg/mL 2-chloroadenine (breakdown product observed in clinical formulation under some storage conditions) in 0.9% NaCl at a dose volume of 25 mL/kg.

c Administered as a 0.1% solution in 0.9% NaCl.

KEY: NA = not available, i.v. = intravenous; i.p = intraperitoneal; MTD = maximum tolerated dose; Wt = weight.

Table 7: Repeated Dose Toxicity Studies with Cladribine (Subacute)
Strains/Species
(# sex/group)
Age/Weight Duration of Trt. Admin. Route Dose Groups Observations Results
CDF1/Mice
(4 F/group)
4 - 6 weeks/ Wt NA5 daysi.p.50, 75, 100, and 125 mg/kg/daya

Vehicle = 0.9% NaCl
Clinical signs, mortalityMTD = 50 mg/kg/day
LD50 = 75 mg/kg/day
LD90 # 100 mg/kg/day
Cynomolgus
Monkey/Macaca fascicularis
(1 M/dose)
Age & Wt NA7 - 10 daysi.v. cont. infusion#1: 1.0, 2.0 mg/kg/dayb #2: 1.0 mg/kg/day (10 days)Mortality, clinical signs, serum chemistry, necropsy and histopathology.Moribund condition & mortality observed 2 - 3 days after end of treatment.
Major signs of toxicity: anorexia, nausea & vomiting, seizures, ataxia, suppression of rapidly dividing tissues.
Cynomolgus
Monkey/ Macaca fascicularis (4 M, F/grp; 2 M, F/0.1 mg/kg grp)
Age NA/1.8 – 4.9 kg14 days followed by 6-week recovery periodi.v. cont. infusionc0.1, 0.3 & 0.6 mg/kg/day

Vehicle: saline
Mortality, clinical signs, body wt, food consumption, ECG, ophthalmoscopy, neurological exam, hematology & serum chemistry, FACS, necropsy and histopathology.No mortality. Major signs of toxicity in 0.6 mg/kg/day group: body wt loss, reduced motor activity, diarrhea in males; reduction in red & white cells (including lymphocyte & monocyte subsets).
Marked suppression of proliferating tissues; cellular depletion of lymphoid tissues and bone marrow. Effects reversed following 6 weeks of recovery.
0.3 mg/kg/day group: leukopenia
0.1 mg/kg/day group: no toxicity

aAdministered as a 0.1% solution in 0.9% NaCl

b7 days for each, separated by a 7-day recovery period

cInfusion rate of 7.5 mL/kg/day

KEY: i.v. = intravenous; i.p. = intraperitoneal; Wt = weight; #/group = number of animals per group; Admin. = administration; Trt = Treatment; NA = not available; cont. = continuous; wks = weeks; M = male; F = female; ECG = electrocardiogram

Table 8: Reproductive Toxicity Studies with Cladribine
Strain/Species (3/group)a Route/Duration of Administration Dose Groups Observations Results
Mouse/Crl:CD-1 (ICR)(BR) (30/group) i.v./Days 6-15 of gestationb 0.5, 1.5 & 3.0 mg/kg/day

Vehicle: saline
Maternal body weight and clinical signs.
Number of corporea lutea/ implantations and early/late resorptions; fetal survival, fetal weight/sex; fetal alterations
3 mg/kg/day: Mean maternal body weight sign. reduced, attributed to a sign. increase in number of resorptions and concomitant reduced number of the live fetuses.
Increases in incidence of fetal variations and malformations.
1.5 mg/kg/day: Increase in skeletal variations.
0.5 mg/kg/day: No effect on fetal development.
Rabbit/New Zealand White (Hra: (NZW/SPF) (18/group) i.v./Days 7-19 of gestationc 0.3, 1.0 & 3.0 mg/kg/day
Vehicle: saline
Maternal body weight, food consumption & clinical signs.
Number of corporea lutea/ implantations and early/late resorptions; fetal survival, fetal weight/sex, fetal alterations
3 mg/kg/day: Mean fetal weight. sign. reduced. Abnormalities of head, limbs and palate.
1.0 and 0.3 mg/kg/day: No effect on fetal development.

aAll animals were female.

bDose volume of 10 mL/kg at an infusion rate of 1 mL/min.

cAdministered as a bolus injection in dose volume of 2.5 mL/kg.

KEY: i.v. = intravenous; sign. = significant(ly); #/group = number of animals per group