Fludarabine Phosphate Injection - Pharmaceutical Information, Clinical Trials, Detailed Pharmacology, Toxicology.
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Fludarabine Phosphate Injection - Scientific Information

Manufacture: Fresenius Kabi USA, LLC
Country: Canada
Condition: CLL (Chronic Lymphocytic Leukemia), Leukemia, Chronic Lymphocytic (Chronic Lymphocytic Leukemia)
Class: Antineoplastics
Form: Liquid solution, Intravenous (IV), Powder
Ingredients: Fludarabine phosphate, mannitol, sodium hydroxide

Pharmaceutical Information

Drug Substance

Proper name: fludarabine phosphate
Chemical name: 2-fluoro-9-(5-O-phosphono-β-D-arabinofuranosyl)-9H-purin-6-amine
Structural formula:


Molecular Formula: C10H13FN5O7P
Molecular Weight: 365.2
Solubility: Soluble in water (9 mg/mL) and insoluble in ethanol.
Physico-chemical
Properties:
Fludarabine phosphate is a white powder. It has pKa values of 3.2 ± 0.1 and 5.8 ± 0.1 and melts with decomposition between 195 0C and 202 0C.
pH: 2.0 (9 mg/mL in H2O)
Partition Coefficient
(n-octanol/water): log POW = - 2.1 (pH 2)
(flask-shaking method): log POW = < -3.0 (pH 5)
log POW = < -3.0 (pH 8)
log POW = < -3.0 (pH 9)

Clinical Trials

Two single-arm open-label studies of fludarabine phosphate have been conducted in patients with CLL refractory to at least 1 prior standard alkylating-agent-containing regimen. In a study conducted at M.D. Anderson Cancer Center (MDACC), 48 patients were treated with a dose of 22 - 40 mg/m2 daily for 5 days every 28 days. Another study conducted by the Southwest Oncology Group (SWOG) involved 31 patients treated with a dose of 15 - 25 mg/m2 for 5 days every 28 days. The overall objective response rates were 48% and 32% in the MDACC and SWOG studies, respectively. The complete response rate in both studies was 13%; the partial response rate was 35% in the MDACC study and 19% in the SWOG study. These response rates were obtained using standardized response criteria developed by the National Cancer Institute CLL Working Group and achieved in heavily pretreated patients. The ability of fludarabine phosphate to induce a significant rate of response in refractory patients suggests minimal cross-resistance with commonly used anti-CLL agents.

The median time to response in the MDACC and SWOG studies was 7 weeks (range of 1 to 68 weeks) and 21 weeks (range of 1 to 53 weeks), respectively. The median duration of disease control was 91 weeks (MDACC) and 65 weeks (SWOG). The median survival of all refractory CLL patients treated with fludarabine phosphate was 43 weeks and 52 weeks in the MDACC and SWOG studies, respectively. Normalized lymphocyte count, one measure of disease regression, occurred at a median of 2 weeks (complete responders), 2 weeks (partial responders) and 22 weeks (non-responders).

Rai stage improved to stage II or better in 7 of 12 MDACC responders (58%) and in 5 of 7 SWOG responders (71%) who were stage III or IV at baseline. In the combined studies, mean hemoglobin concentration improved from 9.0 g/dL at baseline to 11.8 g/dL at the time of response in a subgroup of anemic patients. Similarly, average platelet count improved from 63500/mm3 to 103300/mm3 at the time of response in a subgroup of patients who were thrombocytopenic at baseline.

Detailed Pharmacology

Mechanism of Action

The biological activity of 2F-ara-A was assessed in a number of models. 2F-ara-A has been shown to inhibit DNA synthesis in cultured mouse leukemia L1210 cells and in an in vivo mouse L1210 leukemia model. Total RNA synthesis in vitro was not inhibited by treatment with 2F-ara-A; however, protein synthesis was reduced substantially. It has been shown that 2F-ara-A is not deaminated by adenosine deaminase, contributing to the stability of the compound.

The activity, metabolism and toxicity of 2F-ara-A in the human lymphoblastoid T-cell line (CCRF-CEM) were compared with 9-β-D-arabinofuranosyl-adenine (ara-A). Inhibition of cell growth was equivalent for these two agents, provided that ara-A was protected from deamination. Similar studies conducted with CCRF-CEM showed that ara-A and 2F-ara-A exerted early killing effects preferentially during the S-phase of cell proliferation. Both compounds were converted to the triphosphate form, which accumulated intracellularly and inhibited DNA synthesis. This nucleoside metabolite, 2F-ara-ATP, was also shown to inhibit DNA polymerase α and, to a lesser extent, ribonucleotide reductase in mouse leukemia cells (L1210), human epithelial cells (HEp-2), and HeLa cells.

In the systems tested, 2F-ara-ATP is the active metabolite which acts by inhibiting DNA polymerase α.and ribonucleotide reductase thus preventing DNA synthesis. In addition, in vitro studies have shown that exposure of CLL lymphocytes to 2F-ara-A triggers extensive DNA fragmentation and apoptosis.

Antitumor Activity

The effects of schedule and route of administration on the antitumor activity of fludarabine phosphate were examined using an in vivo mouse leukemia model (implanted L1210 leukemia cells). The drug was active following intraperitoneal administration on all treatment schedules. Antitumor activity increased almost three fold when the number of drug treatments was increased. In addition, the administration of several doses in one day was more effective than administration of one larger dose.

A single administration (900 mg/kg) on day 1 produced an increased life span (ILS) of 42% while administration of a smaller dose (250 mg/kg) 3 times a day on day 1 (total dose 750 mg/kg) gave a 98% ILS. This pattern of increased activity with administration of several doses in a day was also observed with the intermittent treatment schedule. A single administration on each of 3 days (total dose 2010 mg/kg) produced an ILS of 122% while administration of a smaller dose 3 times a day over 3 days (total dose 1125 mg/kg) produced the greatest activity, a 525% ILS with 6 long-term survivors (50 days) among the tumor-bearing mice.

With the administration of the drug 3 times a day on day 1, negative animal weight differences (body weight change over 5 days for test animals minus that for controls) of more than 4 grams at the highest dose evaluated suggests some acute drug toxicity. Based on equivalent total doses, administration of 3 smaller doses per day at 3-hour intervals was much more effective than a single administration for each day of treatment using the in vivo mouse leukemia model.

A single oral administration of fludarabine phosphate on day 1 was not effective against the L1210 leukemia. However, when given as 5 daily oral doses, the highest nontoxic dose of the drug, defined as the dose which results in at least 7 or 8 50-day survivors among the normal mice (800 mg/kg daily on days 1 – 5), was effective in a maximal ILS of 50%.

When the drug was administered i.v., it was more effective with daily administration for 5 days than it was with a single injection on day 1. Daily treatment for 5 days at a non-toxic dose level increased the life span of tumor-bearing mice by 71% and a higher, more toxic treatment for 5 days produced an ILS of 95%; in contrast, a single i.v. treatment on day 1 produced a maximum ILS of 28%.

The intraperitoneally (i.p.) implanted L1210 leukemia was less sensitive to fludarabine phosphate when the drug was given either intravenously (i.v.) or orally compared to i.p. administration. A maximal ILS value of 122% was produced following i.p. administration of 266 mg/kg on days 1 - 5. This same dose given by i.v. administration on days 1 - 5 produced an ILS value of 95%. However, with both i.p. and i.v. administration, the dose that produced the maximum ILS value was toxic to the non-tumored animals.

Fludarabine phosphate also demonstrated activity against the intraperitoneally implanted P388 leukemia. In two different experiments, the drug increased the life span of mice bearing the P388 leukemia by 115% and 53% following i.p. administration of 200 and 100 mg/kg injections, respectively, on days 1 - 9.

Cytotoxicity of Fludarabine Phosphate

Fludarabine phosphate has demonstrated significant antitumor activity against intraperitoneally (i.p.) implanted murine L1210 leukemia and the human LX-1 lung tumor xenograft. The drug has shown moderate activity against the murine subcutaneously (s.c.) implanted CD8F1 mammary epithelioma and the i.p. implanted P388 lymphocytic leukemia. Fludarabine phosphate was not active against the i.p. implanted B16 melanoma, the s.c. implanted colon tumor or the intravenously (i.v.) implanted Lewis lung epithelioma, nor was it effective against the human CX 1 colon or MX-l mammary xenografts in the subrenal assay.

Effects on Bone Marrow Survival and Tumor Cell Sensitivity

Fludarabine phosphate was tested in an in vitro human bone marrow cell survival assay and tumor cell sensitivity assay. The sensitivity of normal human granulocyte-macrophage colony-forming units in culture (GM-CFUC) showed a simple negative exponential curve characterized by a logarithmic decrease in survival as a function of drug concentration. Fludarabine phosphate exhibited an LD63 of 0.51 μg/mL for normal human granulocyte-macrophage colony-forming units in culture (GM-CFUC). In the tumor sensitivity assay, fludarabine phosphate demonstrated an LD40 and LD78 of 0.26 and 0.77 μg/mL, respectively.

Blood and bone marrow samples obtained from patients with relapsed leukemia and lymphoma after treatment with a single dose of 20 - 125 mg/m2 of fludarabine phosphate revealed that the area under the concentration-time curves for 2F-ara-A and 2F-ara-ATP were increased in proportion to the product dose. There was a high correlation between 2F-ara-ATP levels in circulating leukemic cells and those in bone marrow cells aspirated at the same time. DNA synthetic capacity of leukemic cells was inversely related to the associated 2F-ara- ATP concentration. 2F ara-ATP concentrations were three times higher in bone marrow cells from patients with lymphomatous bone marrow involvement than from those without evidence of marrow disease.

A dose-response relationship between fludarabine phosphate concentration and inhibition of DNA synthesis in leukemia cells and bone marrow cells in culture was obtained.

Bone marrow progenitor cells from a normal subject and 10 patients with solid tumors, whose bone marrow was free of metastases, were treated with fludarabine phosphate and other cytotoxic drugs, using a bilayer soft agar culture. The in vitro effect of the drugs on bone marrow progenitor cells was not as toxic as expected relative to the myelosuppressive potency observed in vivo. In the case of fludarabine phosphate, it has been postulated that these findings might be related to incomplete in vitro phosphorylation to the triphosphate, 2F-ara-ATP.

Lymphocytotoxicity in Humans

Fludarabine phosphate was assessed for its lymphocytotoxicity in 11 patients receiving the investigational drug for treatment of nonhematologic cancers refractory to standard treatment. Fludarabine phosphate was administered by intravenous infusion at doses ranging from 18 mg/m2/day to 40 mg/m2/day, with each dose given on a 5-day dosing regimen.

Lymphocyte subsets were determined prior to treatment and on day 5 of treatment, 4 hours after the infusion. Observations indicated that lymphocytopenia developed rapidly but was reversible. Total T-lymphocyte counts fell during all treatment regimens, with a 90% decrease in mean absolute T-cell count. All major T-lymphocyte subsets were affected. B-lymphocyte counts decreased by 50% on average. Recoveries of total mononuclear cells, total T-cells and non-T, non-B cells were reduced substantially by fludarabine phosphate treatment. B-cell recovery was not affected.

These results indicate that T-cells are more sensitive than B-cells to the cytotoxic effects of fludarabine phosphate.

Modulation of T-Cell Function by Fludarabine Phosphate

The effects of fludarabine phosphate on the growth and function of bone marrow and peripheral blood mononuclear cells (PBMC) from cancer patients were evaluated. Drug toxicity was dependent on time of incubation and concentration of fludarabine phosphate tested. After a 3-hour incubation of PBMC with 1 μg/mL of fludarabine phosphate, there was no effect on cell number whereas, after 48 hours, the cell count was 59% of control, untreated cells. In contrast, a 3-hour or 48-hour incubation of PBMC with 100 μg/mL of fludarabine phosphate reduced cell number to 65.7% or 63% of control, respectively.

Lymphocyte subpopulations of normal PBMCs were evaluated after treatment in vitro with fludarabine phosphate for 72 hours. A dose-dependent decrease in total T-cell number was noted. Incubation with 1 μg/mL of fludarabine phosphate reduced T-cells by 16.7%; 100 μg/mL reduced T-cells by 42%. The subset of T-cells predominantly affected was T-helper cells, reduced by 53.5% after incubation with 100 μg/mL of fludarabine phosphate. B-cells, monocytes and natural killer cells were not reduced, but rather increased relative to control. Fludarabine phosphate also inhibited the response of PBMC to mitogens in a dose-and time-dependent manner.

In Vitro Testing of Fludarabine Phosphate in Glioma Cell Cultures

Fludarabine phosphate was tested for growth inhibitory effects on human glioma cells isolated from patient specimens. Cells were treated with 1 - 10 μM of fludarabine phosphate beginning 4 days after cells were plated. After 3 more days of incubation, cell number was determined.

Inhibition of cell growth was dose-dependent and approximately equal to inhibition seen after treatment with the same concentrations of 5-fluorouracil. Dose-dependent growth inhibition was also observed when interferon-beta (1 - 1000 IU/mL) was incubated with glioma cell cultures. Although the combination of fludarabine phosphate and 5- fluorouracil or interferon-beta produced additive inhibitory effects, no synergistic effects were observed.

Pharmacokinetics (Animals)

Fludarabine phosphate and its metabolites have been studied in mice, dogs, miniature pigs and monkeys to elucidate their pharmacokinetic, distribution and excretion profiles.

In the mouse, dog and monkey, the pharmacokinetics of fludarabine phosphate and its major metabolite, 2F- ara-A, generally exhibited bicompartmental characteristics after intravenous administration, with rapid clearance and relatively large volumes of distribution.

The pharmacokinetic parameters of fludarabine phosphate and its metabolites are presented in Table 1 and Table 2, located on the following pages.

Tissue Distribution, Metabolism and Excretion in Animals

Tissue distribution and excretion studies were conducted with fludarabine phosphate in mice, dogs and monkeys at doses between 30 and 500 mg/m2.

Fludarabine phosphate is metabolized to 2F-ara-A and, to a lesser extent, 2F-ara-HX in the mouse and monkey, while in the dog, 2F-ara-A and 2F-ara-HX are both major metabolites. The majority of the administered compound is metabolized and then eliminated in the urine within 24 hours after dose administration.

Preclinical data in rats demonstrated a transfer of fludarabine phosphate and/or metabolites through the feto-placental barrier.

The metabolism, distribution and excretion information is presented in Table 3 located on the following pages.

Lactation

There is evidence from preclinical data after intravenous administration to rats that fludarabine phosphate and/or metabolites transfer from maternal blood to milk. In a peri-/postnatal developmental toxicity study, fludarabine phosphate was intravenously administered to rats during late gestation and the lactation period at dose levels of 1, 10, and 40 mg/kg/day. The offspring of the high-dose group showed a decrease in body weight gain and viability and a delay in skeletal maturation on day 4 postpartum. However, it should be taken into account that the dosing period covered also the late prenatal development.

Table 1: Pharmacokinetic Parameters of Fludarabine Phosphate and 2F-ara-A
STUDY DETAILS RESULTS
Species Dose of Test Article
(mg/m2)
Route of
Admin.
Metabolite t1/2α t1/2β Vd
(mL)
Clearance
(mL/min)
Comments
Mouse (BDF1)
18 - 25 grams
40 2F-ara-AMP i.v. 2F-ara-AMP 0.7 min 21.2 min 73.4 2.40 In mice, 2F-ara-AMP was rapidly dephosphorylated to 2Fara-A. 2F-ara-HX was also present in serum. HPLC (Waters Associates model) and TLC were used.
2F-ara-A 31.1 min 113.9 min 60.6 0.37
500 2F-ara-AMP i.v. 2F-ara-AMP 2.5 min 26.9 min 309.1 7.97
2F-ara-A 35.7 min 184.9 min 88.0 0.33
Dog (Beagle)
7.8 - 10.8 kg
40 2F-ara-AMP i.v. 2F-ara-AMP 5.3 min 30.5 min 142960.0 3254.0 In dogs, 2F-ara-AMP was rapidly dephosphorylated to 2Fara- A. A larger percentage of the metabolite 2F-ara-HX was found in dog serum when compared to mice. HPLC (Waters Associates model) and TLC were used.
2F-ara-A 15.7 min 96.6 min 9552.7 68.5
2F-ara-HX 113.5 min --- --- 115.5
500 2F-ara-AMP i.v. 2F-ara-AMP 9.2 min 51.5 min 196520.0 2646.0
2F-ara-A 4.6 min 90.3 min 7243.5 55.6
2F-ara-HX 112.5 min --- --- 111.2
Dog (Beagle)
2 dogs
260 2F-ara-AMP i.v. 2F-ara-A 13 min 96 min 0.712 L/kg VdSS 5.4 mL/min/kg Total plasma clearance was more than 2-fold greater in dogs than in man. The steady-state volume of distribution in man is approximately 70% larger than in dogs. The terminal slope of 2F-ara-HX decay parallels the 2F-ara-A decay. Standard chromatographic and spectral assays were used.
Monkey
(3 animals)
20 2F-ara-AMP i.v. 2F-ara-AMP (plasma) 56 min --- --- --- 2F-ara-A crossed the blood-brain barrier with a lag time of 0.5 to 2.0 hours and accumulated in the CSF. To quantify the metabolites, HPLC was used.
2F-ara-A (plasma) 2.5 - 3.1 h 21.3 - 35.6 h --- ---
2F-ara-A (CSF) 1.1 - 1.8 h 20.4 - 29.8 h --- ---
Mouse (BDF1)
25 - 31 grams
30 2F-ara-A i.v. 2F-ara-A 17 min 72 min --- --- Standard chromatographic and spectral assays were used.
Metabolites 30 min 124 min --- ---
Dog (Beagle)
9.7 - 10.3 kg
30 2F-ara-A i.v. 2F-ara-A < 5 min 112 min --- --- Standard chromatographic and spectral assays were used.
400 2F-ara-A i.v. 2F-ara-A 130 min --- --- ---
Monkey (Rhesus)
3.9 - 4.6 kg
30 2F-ara-A i.v. 2F-ara-A 26 min 125 min --- --- 12 - 14% of 2F-ara-A became serum protein bound.
400 2F-ara-A i.v. Phosphate Metabolites 131 min --- --- ---
2F-ara-A 15 min 6.7 h --- ---

HPLC: high performance liquid chromatography
TLC: thin layer chromatography

Table 2: Pharmacokinetic Parameters of Fludarabine Phosphate and Metabolites
STUDY DETAILS RESULTS
Species/Test
Model
Test
Article
Dose
Route
of
Admin.
Metabolite t1/2 Time to Cmax Cmax Comment
Mouse (BD2F1)
P388 tumor cell model
1485 mg/kg
2F-ara-AMP
i.p. 2F-ara-AMP 1.2 h ascites fluid --- --- After separation of nucleotides by HPLC, metabolites were quantified by UV or radioactivity.
2F-ara-A 2.1 h ascites fluid 4 h (ascites) ---
2F-ara-A 3.8 h plasma 1 - 6 h (plasma) > 1 mM
2F-ara-HX 3.0 h plasma 4 h (plasma) ≈ 0.4 mM
2F-ara-HX --- 4 h (ascites) ---
Mouse (BD2F1)
P388 tumor cell model
1485 mg/kg
2F-ara-AMP
i.p. --- --- --- --- After separation of nucleotides by HPLC, metabolites were quantified by UV or radioactivity.
2F-ara-ATP 4.1 h (intracellular, P388 cells) 6 h (intracellular, P388 cells) 1036 μM
2F-ATP 3.7 h (intracellular, P388 cells) 6 h (intracellular, P388 cells) 27 μM
Miniature swine (5 animals)
14 - 16.5 kg
10, 16, 25 mg/m2
2F-ara-AMP
i.p. 2F-ara-A --- 5 - 140 min (peritoneal fluid) 7.7 - 18 μg/mL (peritoneal fluid) HPLC was used.
120 - 240 min (plasma) 0.15 - 0.46 μg/mL (plasma)

Cmax: maximal concentration
i.p.: intraperitoneal

Table 3: Metabolism, Distribution and Excretion of Fludarabine Phosphate
Species Design Compound
Administered
Dose
(mg/m2)
Metabolism and Distribution Elimination Metabolites
Mouse
(BDF1)
i.v. administration 2F-ara-AMP 40
500
The major metabolite was 2F-ara-A in mice. The liver, spleen and kidney were the major organs containing the metabolites. Elimination occurred exponentially from tissue, although the rate of elimination from serum was faster. All metabolites were excreted in the urine. 2F-ara-A
2F-ara-AMP
2F-ara-HX
2F-A
Polyphosphorylated derivatives
Mouse i.v. administration 2F-ara-AMP 40
500
2F-ara-AMP underwent dephosphorylation to 2F-ara-A in mice. Elimination of 2F-ara-A from tissue occurred exponentially Serum:
2F-ara-A
2F-ara-HX
Tissue:
2F-ara-A
2F-ara-HX
2F-A
2F-ara-AMP
2F-ara-ADP
2F-ara-ATP
Mouse
(BD2F1)
P388 tumor cell implant model
i.p. administration 2F-ara-AMP 1485 (mg/kg) Peak 2F-ara-A ascites conc. occurred at 4 h
Peak 2F-ara-HX ascites conc. occurred at 4 h
Peak 2F-ara-A plasma conc. ($1mM) occurred at 1 - 6 h
Peak 2F-ara-HX plasma conc. (≈0.4mM) occurred at 4 h
2F-ara-A t1/2 = 2.1 h (ascites)
----
2F-ara-A t1/2 = 3.8 h (plasma)
2F-ara-HX t1/2 = 3 h (plasma)
2F-ara-A (ascites & plasma)
F-ara-HX (ascites & plasma)
2F-ara-ATP (intracellular)
2F-ara-AMP (intracellular)
Mouse
(BD2F1)
P388 tumor cell implant model
i.p. administration 2F-ara-AMP 1485 (mg/kg) The peak concentration (1,036 μM) of the primary intracellular metabolite, 2F-ara-ATP, was reached 6 h post drug administration in P388 cells.
Peak levels of 2F-ara-ATP were reached at 4 - 6 h in bone marrow and intestinal mucosa with 2F-ara-ATP accumulated 20 times less than in P388 cells. 2F-ara-ATP has been determined the active metabolite.
2F-ara-ATP t½ = 4.1 h (in P388 cells)
2F-ara-ATP t½ = 2 h (in host tissue)
----
----
Mouse
P388 tumor cell implant model
i.p. administration 2F-ara-AMP 1485 (mg/kg) 930 μM 2F-ara-ATP was the peak intracellular concentration observed in P388 cells.
Peak 2F-ara-ATP concentrations of 34 nmol/μmol of DNA accumulated in bone marrow.
Peak 2F-ara-ATP concentrations of 23 nmol/μmol of DNA accumulated in the intestinal mucosa.
The metabolite 2F-ara-A passed rapidly from ascites to blood in concentrations proportional to the dose.
DNA synthesis was inhibited to 1% of controls at 6 h.
2F-ara-ATP disappeared from P388 cells with an intracellular half-life of 4.1 h. 2F-ara-ATP disappeared from bone marrow and intestinal mucosa with a half-life of 1.5 h 2F-ara-A exhibited a plasma half-life of 3.5 h. 2F-ara-A
2F-ara-ATP
Dog (Beagle) i.v. administration 2F-ara-AMP 40
500
The dog metabolized a greater % of the compound to 2F-ara-HX than the mouse. 2F-ara-A, 2F-ara-HX, and 2F-A were all excreted in urine. 2F-ara-A
2F-ara-HX
2F-A
Dog (Beagle) i.v. administration 2F-ara-AMP 40
500
2F-ara-AMP underwent dephosphorylation to 2F-ara-A in dogs. ---- 2F-ara-A
Dog (Beagle) i.v. administration 2F-ara-AMP 260 Tissue binding of 2F-ara-A compared to plasma protein binding was substantially greater in the dog when compared to humans. 2 F-ara-AMP was metabolized by dephosphorylation to 2F-ara-A with subsequent deamination to 2F-ara-HX. 2F-ara-A
2F-ara-HX
Miniature swine i.p. infusion 2F-ara-AMP 10
16
25
Peak i.p. levels of 2F-ara-A occurred at 5 - 140 minutes. Peak serum levels of 2F-ara-A occurred 120 - 240 minutes. ---- 2F-ara-A
Monkey i.v. administration 2F-ara-AMP 20 Peak 2F-ara-A plasma levels occurred at 7 - 14 minutes. Peak 2F-ara-A CSF levels occurred at 31 - 127 minutes. 2F-ara-A crossed the blood-brain barrier accumulating in the CSF with a lag time of 0.5 - 2 h. ---- 2F-ara-A
Mouse
(BDF1)
i.v. administration 2F-ara-A 30 42% of radioactivity found in the liver, 20% in the spleen, pancreas, and colon, and 15% in the lung and small intestine was a phosphorylated derivative of 2F-ara-A. ≥59% of the drug was excreted in urine as 2F-ara-A at 24 h. 12% of dose was excreted as a metabolite at 24 h. 2F-ara-AMP
2F-ara-ADP
2F-ara-ATP
Mouse
P388 tumor cell implant model
i.p. administration 2F-ara-A 234 (mg/kg) 560 μM of 2F-ara-ATP was the peak intracellular concentration observed.
2F-ara-A passed rapidly from ascites to blood in concentrations proportional to the dose.
2F-ara-ATP disappeared with an intracellular half-life of 2.9 h.
2F-ara-A exhibited a plasma halflife of 2.2 h.
2F-ara-ATP
Dog (Beagle) i.v. administration 2F-ara-A 30 Dogs consistently metabolized greater portions of 2F-ara-A with higher levels detected in the serum and urine when compared to mice. 27% of the drug was excreted unchanged in the urine at 24 h.
53% of the drug was excreted as metabolites in urine at 24 h.
----
Dog (Beagle) i.v. administration 2F-ara-A 400 Dogs consistently metabolized greater portions of 2F-ara-A with higher levels detected in the serum and urine when compared to mice. 18% of the drug was excreted unchanged in the urine at 24 h.
70% of the drug was excreted as metabolites in the urine at 24 h.
----
Monkey (Rhesus) i.v. administration 2F-ara-A 30 ---- 50% of the drug was excreted unchanged in 24 h.
26% of the drug was excreted as metabolites at 24 h.
----
Monkey (Rhesus) i.v. administration 2F-ara-A 400 ---- 58% of the drug was excreted unchanged at 24 h.
25% of the drug was excreted as metabolites at 24 h.
----
Rat (Sprague Dawley) i.v. administration 3H-2F-ara-AMP 60 (10 mg/kg) After intravenous administration of 3H-2Fara-AMP to lactating rats, levels of radioactivity in milk was about 30% of that in maternal blood. Thus, 2F-ara-AMP and/or metabolites are transferred into milk. The half-life of disposition of radioactivity from blood is about 2 h. This mirrored by the estimated half-life of 3 h calculated for excretion in milk. ----
Rat (Sprague Dawley) i.v. administration 3H-2F-ara-AMP 60 (10 mg/kg) 3H-2F-ara-AMP and/or metabolites cross the feto-placental barrier and reached levels in fetus similar to as in maternal blood. No long-lasting retention of the 3H-labelled substances could be observed in fetus and in maternal tissues examined ----

Pharmacokinetics (Humans)

The pharmacokinetics of fludarabine phosphate given intravenously have been determined in adult patients undergoing Phase I clinical trials at the University of Texas Health Science Center at San Antonio (UT), the University of Texas System Cancer Center at the M.D. Anderson Cancer Center (MDACC) and at Ohio State University (OSU). In addition, the pharmacokinetics of intraperitoneal fludarabine phosphate were also determined at UT and the pharmacokinetics of intravenous fludarabine phosphate in pediatric patients with leukemias and solid tumors were determined at the Children's Hospital of Los Angeles, the National Cancer Institute (NCI) and the Mayo Clinic.

Preliminary nonclinical and Phase I human studies demonstrated that fludarabine phosphate is rapidly converted to 2F-ara-A within minutes after intravenous infusion and then phosphorylated intracellularly by deoxycytidine kinase to the active triphosphate, 2F-ara-ATP. Consequently, clinical pharmacology studies have focused on 2F-ara-A pharmacokinetics.

Described on the following pages are three principal pharmacokinetic studies that characterize the pharmacokinetic parameters of 2F-ara-A. Despite the differences in dosage and dosing schedules between these various studies discussed on the following pages, several consistent results were obtained. For the infusion studies, a mean terminal half-life of 9.2 hours was found in the population of patients studied at UT and a median terminal half-life of approximately 8 hours was observed in the patients studied at MDACC. These values compare favorably to the 10.16-hour mean terminal half-life reported by the OSU investigators following large intravenous bolus injections. The terminal half-life of 2F-ara-A does not appear to be dose-dependent, as the doses used in these studies ranged from 18 to 260 mg/m2.

The discrepancies between the studies regarding the biphasic or triphasic elimination patterns appear to be due to differences in sampling schedules and duration of intravenous administration. In addition, sampling duration has an impact upon the calculated value of the terminal half-life (t1/2γ). The majority of pharmacokinetic studies use a blood sampling duration of 24 to 30 hours, which gives a calculated terminal half-life (t1/2γ) of 8 - 10 hours. However, when the sampling duration is increased to 72 hours, the additional time points give a calculated t1/2γ of up to 31 hours. Because the plasma concentration of 2F-ara -A declines more than 50-fold from the peak concentration before this long elimination phase, the consequences of the relatively low 2F-ara-A concentration remaining in the plasma after 24 hours (< 0.1 pmol/L) remain uncertain as far as drug scheduling is concerned.

In addition, both the UT and OSU investigators found a positive correlation between area under concentration-time curves and degree of neutropenia, reinforcing the assertion that toxicity (myelosuppression) is dose-related.

Phase I-II Study of Fludarabine in Hematologic Malignancies (Study No. T83-1275) conducted at the University of Texas, San Antonio

Methods

The pharmacokinetic parameters of the principal metabolite of fludarabine phosphate, 2F-ara-A, were determined in 7 adult patients (6 males; 1 female) who received fludarabine phosphate at doses of 18 or 25 mg/m2/day as a 30-minute intravenous infusion daily for 5 consecutive days. Blood and urine samples were analyzed by HPLC for concentrations of 2F-ara-A.

The plasma concentration-time data, which were determined by HPLC, were analyzed by non-linear least squares regression analysis (NONLIN) using a zero order infusion input with first order elimination from the central compartment. Both a two-and a three-compartment model were tested and the data fitted the two-compartment open model.

Pharmacokinetic Parameters

Peak plasma concentrations of 2F-ara-A ranged from 0.199 to 0.876 μg/mL and appeared to be related to the dose and rate of infusion. Mean plasma concentrations of 2-fluoro-ara-A on days 1 and 5 in patients receiving 18 mg/m2/day were 0.39 and 0.51 μg/mL, respectively. Mean plasma concentrations of 2F-ara-A on days 1 and 5 in patients receiving 25 mg/m2/day were 0.57 and 0.54 μg/mL, respectively. There was no drug accumulation during the 5-day treatment period.

The pharmacokinetic parameters derived from this study are presented in Table 4.

Table 4: 2F-ara-A Kinetic Parameters
Patient BSA
(m2)
Dose Duration of
infusion (min)
Peak conc.
(μg/mL)
Clearance rates
(L/h/m2)
Volumes of
distribution
(L/m2)
t1/2 (h)
mg/m2 mg Day 1 Day 5 Day 1 Day 5 Plasma Tissue VdSS Vd α β
1 1.57 18 27 32 30 0.285 0.285 13.43 28.3 115.4 48.6 0.59 7.0
2a 1.74 18 31 25 30 0.199 0.377 1.51 28.1 1629.9 75.3 1.69 787.5
3 1.62 18 29 38 30 0.693 0.856 4.35 19.8 59.8 16.1 0.37 10.7
4 1.90 25 48 30 30 0.876 0.611b 10.38 23.8 91.9 22.9 0.39 7.8
5 1.94 25 48 35 30 0.509 0.550 8.30 5.1 86.4 46.8 1.99 10.6
6 1.74 25 43 33 30 0.550 --c 5.28 9.9 88.6 37.0 1.26 13.9
7 2.06 25 51 30 30 0.336 0.458b 12.71 33.8 135.2 55.2 0.59 8.44
Mean
SD
9.1
3.8
20.1
10.9
96.2
26.0
37.8
15.4
0.60d
-
9.24d
-
a Patient omitted from calculation of mean and SD
b Day 5 levels drawn on day 4
c Day 5 levels not studied
d Harmonic mean half-life

The mean central compartment volume of distribution (Vd) was 37.8 L/m2 with a mean steady-state volume of distribution (Vdss) of 96.2 L/m2. The mean tissue clearance was 20.1 L/h/m2 and the mean plasma clearance was 9.1 L/h/m2 . Plasma concentrations declined bi-exponentially with a harmonic mean initial half-life (t1/2α) of 0.6 hour and a harmonic mean terminal half-life (t1/2β) of 9.2 hours. As presented in Table 8, approximately 24% of the parent compound, fludarabine phosphate, was excreted in the urine as 2F-ara-A during the 5-day treatment period.

Table 5: Urinary Excretion of 2F-Ara-A
Patient % Dose in Urine Creatinine Clearance
(mL/min)
Day 1 Day 2 Day 3 Day 4 Day 5 5-day
Average
1

2

3

4

5

6

7

Mean
14

72

28

25

20

14

17

27
25

16

29

12

20

23

25

21
31

19

29

20

14

27

35

25
7

14

24

38

20

18

45

24
53

9

7

-

13

35

8

21
26

25

24

24

17

23

26

24
76

73

37

77

59

50

73

63
S.D. 21 6 7 13 19 3 15

Correlation of Pharmacokinetic Parameters with Clinical Parameters

As presented in Table 6, a correlation was observed between decreasing absolute granulocyte count and the area under the concentration-time curve (AUC). The Spearman rank correlation coefficient between absolute granulocyte count and AUC was -0.94 which was statistically significant (p < 0.02). The Spearman rank correlation coefficient was also calculated between absolute granulocyte count and total plasma clearance (TPC). Here the correlation coefficient was 0.94 which was also statistically significant (p < 0.02). The correlation coefficient between creatinine clearance and TPC was 0.828 (0.05 < p < 0.1). No correlation was observed between TPC and any of the liver function measurements.

Table 6: Comparison of AUC with Absolute Granulocyte Nadir and Creatinine Clearance
Patient Dose
(mg/m2 per day X 5)
AUCa
(mg.h/L)
AGCb Creatinine Clearance
(mg/mL)
1 18 6.4 3999 76
7 25 9.73 1916 73
4 25 12.2 624 77
5 25 14.9 608 59
6 25 23.4 299 50
3 18 20.5 176 37

a Days 0 - 5
b Absolute granulocyte count

Summary and Conclusions

Intravenous doses of 18 and 25 mg/m2/day for 5 days exhibited bi-exponential decay with a mean initial half- life (t1/2α) of 0.6 hour and a mean terminal half- life (t1/2β) of 9.2 hours. The mean plasma clearance was 9.1 L/h/m2 and the mean tissue clearance was 20.1 L/h/m2. The mean Vd ss was 96.2 L/m2, which is approximately twice body weight, suggesting that tissue binding of the drug occurs. In addition, there was a significant inverse correlation between AUC and absolute granulocyte count (r = -0.94, p < 0.02) suggesting that myelosuppression is dose related.

Phase I-II Study of Fludarabine in Hematologic Malignancies (Study No. T83-1275) conducted at the M.D. Anderson Cancer Center

Methods

The pharmacokinetic parameters of the fludarabine phosphate metabolite, 2F-ara-A, were determined in 19 adult patients (12 males; 7 females) who received the drug as a 30-minute intravenous infusion daily for 5 consecutive days. Ten of the patients were diagnosed as having lymphoma and 9 as having leukemia. In this study, 5 patients received doses of 20 mg/m2/day, 5 patients received doses of 25 mg/m2/day, 1 patient received 30 mg/m2/day, 4 patients received 50 mg/m2/day, 2 patients received 100 mg/m2/day, and an additional 2 patients received 125 mg/m2/day. Pharmacokinetic profiles were generally determined after the first dose of fludarabine phosphate. Plasma concentrations of 2F-ara-A and intracellular concentrations of 2F-ara-ATP were determined by HPLC. Intracellular concentrations were determined for mononuclear cells obtained from blood and bone marrow samples. The incorporation of 2F-ara-ATP into nucleic acids was determined using HPLC and liquid scintillation counting methods.

Pharmacokinetic Parameters

Plasma concentrations of fludarabine phosphate were undetectable at the times when the first samples were obtained. Of the patients receiving 20 or 25 mg/m2 /day, only 2 had detectable peak 2F-ara-A concentrations (1.4 and 2.2 μM) and, in this group of patients, 2F-ara-A levels were completely undetectable 3 hours after the completion of infusion of fludarabine phosphate.

At fludarabine phosphate dose levels of 50 - 125 mg/m2/day, the disappearance of 2F-ara-A was biphasic and independent of dose with a median initial half-life (t1/2α) of 1.41 hours and a median terminal half-life (t1/2β) of approximately 8 hours. Plasma pharmacokinetic parameters for patients with relapsed leukemia (N = 8, patient nos. 5 - 12) are presented in Table 7.

Table 7: Pharmacological Characteristics for 2F-ara -A in the Plasma of Patients with Relapsed Leukemia
Patient Fludarabine
Phosphate Dose
(mg/m2)
2F-ara-A Parameters
t1/2αa (h) t1/2βb (h) AUCc (μM.h)
5

6

7

8
50

50

50

50
3.30d

0.49

1.42

1.25
23.90

>24.00

7.77

7.76
14

28

10

16
Median 50 1.34 7.76e 15
9

10

11

10
100

100

125

125
1.40

1.87

0.93d

2.20
8.90

6.88

13.00

6.22
15

37

94

37
Median 112.5 1.64 7.89 37

a Initial rate of elimination
b Terminal rate of elimination
c Area under the concentration-time curve calculated to 24 h
d As the 2-h sample was the earliest obtained, this value is based on extrapolation of the line to 30 minutes
e The median value excluding patients 5 and 6 whose elevated creatinine levels may signal impaired renal function and thus a longer t1/2β

A wide range of variation of pharmacokinetic parameters of 2F-ara-ATP in circulating leukemic cells was observed; however, when the median peak 2F-ara-ATP concentrations of 24-hour AUC values were compared at each dosage increment (20 or 25 mg/m2, 50 mg/m2, and 100 or 125 mg/m2 ), a clear dose-dependence emerged (Table 8). Cellular elimination was not dose-dependent, with a half-life of approximately 15 hours at all dose levels. There was a strong correlation between the 2F-ara-ATP levels in leukemic cells obtained from peripheral blood and those found in bone marrow (r = 0.84, p = 0.01), suggesting that there were no pharmacological barriers in the bone marrow. Those patients with bone marrow involvement had the highest 2F-ara-ATP levels. In addition, intracellular 2F-ara-ATP levels in circulating leukemic cells at 12 - 14 hours after fludarabine phosphate infusion were inversely related to the DNA synthetic capacity of the cells relative to pretreatment. DNA synthesis remained maximally inhibited (> 80%) until cellular concentrations of 2F-ara-ATP fell below 90 μM.

Table 8: Pharmacological Characteristics of 2F-ara-ATP In Circulating Leukemic Cells
Patient Diagnosis Fludarabine
Phosphate
Dose (mg/m2)
2F-ara-ATP Parameters
Peak (μM) t1/2a (h) AUCb (μM.h)
1

2

3

4
CLLc

DWDLd

DLCLe

NMCLf
20

20

25

25
42

51

15

24
13.3

16.8

13.7

>24.0
600

840

220

480
Median 22.5 33 15.3 540
5

6

7

8
AMMLg

AMLh

AML

ALLi
50

50

50

50
58

47

147

105
10.7

>24.0

14.1

12.8
780

700

2060

1340
Median 50 82 13.5 1060
9

10

11

12
AML

CML-BCj

ALL

ALL
100

100

125

125
112

1

747

226
>24.0

6.0

5.2

>24.0
2560

10

3470

6050
Median 112.5 169 15.0 3015

a Elimination half-life
b Area under the concentration-time curves calculated to 24 h
c Chronic lymphocytic leukemia
d Diffuse, well-differentiated lymphoma
e Diffuse, large cell lymphoma
f Nodular mixed cell lymphoma
g Acute myelomonocytic leukemia
h Acute myeloblastic leukemia
i Acute lymphoblastic leukemia
j Chronic myelogenous leukemia in blast crisis

Summary and Conclusions

Intravenous doses of 20 - 125 mg/m2/day exhibited bi-exponential decay in plasma with a median initial half-life (t1/2α) of 1.41 hours and a median terminal half-life (t1/2β) of approximately 8 hours for 2F-ara-A. The median intracellular half-life for 2F-ara-ATP was approximately 15 hours. The terminal half-lives of both 2F-ara-A and 2F-ara-ATP were not dependent on the dose of fludarabine phosphate. In addition, there was a high correlation between 2F-ara-ATP levels in circulating leukemic cells and bone marrow cells aspirated at the same time. DNA synthetic capacity of leukemic cells was inversely related to intracellular 2F- ara-ATP levels. Finally, 2F-ara-ATP levels were approximately 3 times higher in bone marrow cells from patients with bone marrow involvement than from those patients without evidence of bone marrow disease, suggesting that tumor cells may have a greater capacity to accumulate and retain nucleoside analogue triphosphates than do normal cells.

Phase I -Pharmacokinetic Study of Fludarabine (NSC-312887) (Study No. W83-328) conducted at Ohio State University

Methods

Twenty-six patients participated in this study, in which fludarabine phosphate was administered as a rapid intravenous (IV) infusion of 2 - 5 minutes duration. Seven patients received fludarabine phosphate at a dose of 260 mg/m2, 1 patient received a dose of 160 mg/m2, 8 patients received a dose of 120 mg/m2, 4 patients received 100 mg/m2, and an additional 6 patients received 80 mg/m2. Plasma concentrations of fludarabine phosphate could not be detected 5 minutes after the discontinuation of the infusion. Plasma concentrations of 2F-ara-A, the principal metabolite of fludarabine phosphate, were determined by HPLC over a time period of 0 - 30 hours post dosing. The plasma concentration-time data were analyzed by the NONLIN computer program and fitted a 3-compartment open model with first-order elimination from the central (blood) compartment, using the equations for rapid intravenous infusion.

Pharmacokinetic Parameters

Harmonic mean half-lives, mean residence time and total body clearance of 2F-ara-A for each of the dose levels are shown in Table 9. This metabolite exhibited a very short initial half-life (mean t1/2α) of 5.42 minutes, followed by an intermediate half-life (mean t1/2β) of 1.38 hours and a terminal half-life (mean t1/2γ) of 10.16 hours. In the 26 patients, the terminal half- lives ranged from 4.92 to 19.7 hours. The harmonic mean residence time (Vd ss/ClT) was 10.4 hours and total body clearance (ClT) ranged from 26.5 to 120.4 mL/min/m2 with a mean of 68.98 mL/min/m2.

Table 9: 2F-ara-A Harmonic Mean Half-lives,Mean Residence Time, and Total Body Clearance In Patients
Dose
(mg/m2)
No. of
Patients
t1/2α
(min)
t1/2β
(hour)
t1/2γ
(hour)
MRT
(hour)
ClT
(mL/min/m2)
260

160

120

100

80
7

1

8

4

6
6.85

4.87

4.12

5.77

6.41
1.67

1.52

1.20

1.15

1.55
9.86

9.03

11.77

8.26

10.44
9.26

8.76

12.55

9.30

10.49
72.34

66.50

58.33

85.11

68.93
Mean of all patients 26 5.42 1.38 10.16 10.36 68.98
C.V. (%) - - - - - 33.7

C.V.: coefficient of variation
MRT: mean residence time

Table 10: 2F-ara-A Mean Volume Pharmacokinetic Parameters
Dose
(mg/m2)
No. of
Patients
V1
(L/m2)
V2
(L/m2)
V3
(L/m2)
VdSS
(L/m2)
Vd γ
(L/m2)
260

160

120

100

80
7

1

8

4

6
7.97

6.63

6.28

7.73

7.73
12.83

10.15

10.79

14.14

11.98
20.87

18.17

26.54

27.69

26.27
41.68

34.96

43.61

49.55

45.97
61.95

52.00

60.45

64.99

65.11
Mean of all patients 26 7.30 12.11 24.81 44.22 62.30
C.V. (%) 31.9 25.1 40.7 25.7 28.0

The mean volume parameters for each dosage level are shown in Table 10. The central compartment volume of distribution was approximately 20% of body weight (V1 = 7.30 L/m2). The steady-state volume of distribution indicated significant binding of the drug to tissue components (Vdss = 44.22 L/m2). The smallest of the microscopic rate constants was k31, indicating release of the drug from the deep tissue compartment to be the rate-determining step in the elimination of 2F-ara-A from the body. Table 11 lists the microscopic rate constants for the first 9 patients studied.

Table 11: 2F-ara-A Microscopic Rate Constants (N = 9)
Patient Dose
(mg/m2)
k12
(min-1)
k21
(min-1)
k13
(min-1)
k31
(min-1)
k10
(min-1)
W.Y. 260 0.0402 0.0341 0.00650 0.00333 0.00786
R.E. 260 0.0940 0.0418 0.00375 0.00176 0.01644
H.W. 260 0.0470 0.0360 0.00588 0.00268 0.00632
E.P. 260 0.0556 0.0379 0.01102 0.00299 0.00733
N.R. 120 0.0421 0.0314 0.00708 0.00204 0.00828
M.M. 80 0.0786 0.0301 0.00909 0.00327 0.01580
J.B. 80 0.0621 0.0401 0.00917 0.00289 0.01296
R.D. 80 0.0867 0.0414 0.01239 0.00323 0.00692
E.K. 80 0.0107 0.0213 0.00240 0.00160 0.00340
Mean 0.0574 0.0349 0.00748 0.00264 0.00948
C.V. (%) 45.6 18.9 43.7 25.4 47.6

Correlation of Pharmacokinetic Parameters with Clinical Parameters

Upon completion of the pharmacokinetic studies, a multivariate correlation analysis was undertaken of all pharmacokinetic parameters with the following clinical parameters: bilirubin, serum creatinine, creatinine clearance, BUN, SGOT, SGPT, LDH, alkaline phosphatase, hemoglobin, hematocrit, baseline WBC, baseline platelets, WBC nadir, platelet nadir, WBC toxicity grade, platelet toxicity grade, nausea and vomiting grade, age and sex. Pearson correlation coefficients were substantiated by Spearman correlations. Despite the small number of patients, total body clearance correlated well with creatinine clearance and serum creatinine indicating that renal excretion is important for the elimination of the drug from the body. The volume parameters, particularly VdSS and Vdγ, correlated with creatinine clearance and serum creatinine (p ≤ 0.011). A positive correlation of CT with hemoglobin and hematocrit was observed (p ≤ 0.035) and may be due to the metabolism of 2F-ara -A in the RBC. In addition, apparent correlations of Vdγ with WBC toxicity (p = 0.025) and γ with hematocrit (p = 0.035) were observed. Tables 12 and 13 list the correlation coefficients and p values for the above correlations.

Table 12: Correlation of 2F-ara-A Pharmacokinetic Parameters with Creatinine Clearance and Serum Creatinine
Pharmacokinetic
Parameter
Correlation
Coefficient (r)a
p
value
N
Creatinine

Clearance
ClT

V3

VdSS

Vdγ
0.71

0.62

0.72

0.77
0.002

0.011

0.002

< 0.001
16

16

16

16
Serum

Creatinine
ClT

V1

VdSS

Vdγ
-0.48

-0.44

-0.49

-0.67
0.013

0.025

0.011

< 0.001
26

26

26

26

a Pearson correlation coefficients which were substantiated by Spearman correlations

Table 13: Correlation of 2F-ara-A Pharmacokinetic Parameters with Other Clinical Parameters
Pharmacokinetic
Parameter
Clinical
Parameter
Correlation
Coefficient (r)a
p
value
N
ClT

ClT

ClT

Vdγ

Vdγ

γ
BUN

Hgb

Hct

BUN

WBC tox.

Hct
-0.48

0.42

0.46

-0.39

-0.46

0.41
0.012

0.035

0.017

0.050

0.025

0.035
26

26

26

26

24

26

a Pearson correlation coefficients which were substantiated by Spearman correlations

A rank ordering of the areas under the plasma concentration- time curve (AUC) for the first 9 patients enrolled in the study showed good agreement with the corresponding severity of neutropenia developed by each patient (Table 14). Thus, the capacity of the compound to depress hematopoiesis appears to be dose-related.

Table 14: Areas Under the Plasma Concentration-Time Curve And Neutropenia Grade
Patient Dose
(mg/m2)
AUC.
(μM min x 10-3)
Neutropenia
Grade
H.W. 260 13.29 3
E.P. 260 13.19 3
R.E. 260 8.16 2
W.Y. 260 7.41 3
N.R. 120 5.58 0
R.D. 80 5.08 0
E.K. 80 4.57 1
M.M. 80 2.65 2
J.B. 80 2.54 0

Toxicology

Toxicology information from acute toxicity (Table 15 and Table 16, long-term toxicity (Table 17), mutagenicity (Table 18) and reproductive studies (Table 19) is presented in the following pages.

The results from intravenous embryotoxicity studies in rats and rabbits indicated an embryolethal and teratogenic potential of fludarabine phosphate as manifested in skeletal malformations, fetal weight loss, and postimplantation loss.

In view of the small safety margin between teratogenic doses in animals and the human therapeutic dose, as well as in analogy to other antimetabolites which are assumed to interfere with the process of differentiation, the therapeutic use of fludarabine phosphate is associated with a relevant risk of teratogenic effects in humans (see section WARNINGS AND PRECAUTIONS).

Table 15: Acute Toxicity Studies - Mouse
Study Type/
Route of
Administration
Animal
Information
Number of
Animals
Dosage
(mg/kg/day)
Results
Single-dose lethality
Intravenous injection
Study no. SIB 6101.2
Mouse
(CD2F1)
Age: 6 - 8 weeks
Wt.: 18.3 - 23.6 g
180
(90 males, 90 females)
0
800
967
1170
1414
1710
2068
2500
No treatment
Dose-related decrease in motor activity (reversible in survivors), tonic spasms and death. Lethal dose estimates (mg/kg) were:

LD10 LD50 LD90
M 979.2 1404.2 2013.6
F 780.2 1235.6 1956.9
M&F 874.4 1321.1 1995.9
Five daily doses lethality
Intravenous injection
Study No. SIB 6101.3
Mouse
(CD2F1)
Age: 6 - 8 weeks
Wt.: 17.1 - 23.8 g
270
(135 males, 135 females)
0
325
412
523
664
843
1070
1358
No treatment
Dose-related decrease in motor activity (reversible in survivors) and death. Lethal dose estimates (mg/kg) were:

LD10 LD50 LD90
M 404.6 593.3 870.0
F 355.4 496.8 694.5
M&F 372.5 542.7 790.7
Single dose toxicity
Intravenous injection
Study No. SIB 6101.7
Mouse
(CD2F1)
Age: 6 - 8 weeks
Wt.: 18.6 - 23.2 g
100
(50 males, 50 females)
Males:
0
490 a
979 b
1404 c
No treatment

Females:
0
390 a
780 b
1236 c
No treatment
Dose-dependent effects on nervous, hematopoietic, GI, renal and male reproductive systems. LD50: lethal to males and females, with females more acutely affected than males. LD10: mildly toxic to renal and hematopoietic systems, with decreased mean relative testicular weights. 1/2LD10: decrease in motor activity in a few mice, decreased mean relative testicular weights.
Five daily doses toxicity
Intravenous injection
Study No. SIB 6101.4
Mouse
(CD2F1)
Age: 6 - 8 weeks
Wt.: 17.3 - 22.2 g
100
(50 males, 50 females)
Males:
0
203 a
405 b
593 c
No treatment

Females:
0
178 a
355 b
497 c
No treatment
Dose-dependent effects on hematopoietic, GI, renal and male reproductive systems. LD50: lethal to male and female mice. LD10: delayed toxicity to the testes (decreased mean relative testicular weight). 1/2LD10: can be considered safe in the mouse.

a = 1/2LD10
b = LD10
c = LD50

Table 16: Acute Toxicity Studies – Rat and Dog
Study Type/
Route of
Administration
Animal
Information
Number of
Animals
Dosage
(mg/kg/day)
Results
Single-dose toxicity
Intravenous injection
Study No. TBT03-008
Rat
(Sprague-Dawley)
Age: 8 - 11 weeks
Wt.: 200 - 269 g
24
(15 males, 9 females)
800
1400
2000
Dose-dependent signs of toxicity were hypoactivity, rough fur, squinted eyes, hypothermia, gross findings in lymph nodes, thymus, heart, lungs and stomach, and death. Estimated LD50 values were 910 mg/kg (males) and 1050 mg/kg (females).
Single dose toxicity
Intravenous injection
Study No. SIB 6101.5
Dog
(Beagle)
Age: 8 - 10 months
Wt.: 7.0 - 11.6 kg
20
(10 males, 10 females)
13.1 a
131.2 b
262.4 c
393.6 d
524.8 e
Dose-dependent signs of toxicity included changes in clinical status and adverse effects on the hematopoietic, gastrointestinal, renal and hepatic systems. In addition, male dogs receiving 4 x MELD10 had pancreatic and reproductive toxicity, and were sacrificed moribund. The 1/10 MELD10 and MELD10 doses were considered safe, as effects seen were minimal and readily reversible.
Five daily doses toxicity
Intravenous injection
Study Nos. SIB 6101.6 and 6101.6c
Dog
(Beagle)
Age: 8 - 9 months
Wt.: 6.5 - 11.7 kg
24
(12 males, 12 females)
0
5.59 a
55.85 b
111.76 c
167.7 d
223.52 e
Dose-dependent signs of toxicity included alterations in clinical status and adverse effects on the hematopoietic, renal, gastrointestinal and hepatic systems resulting in moribund sacrifice or death by day 8 for all 4 x MELD10 animals, as well as one female at the 3 x MELD10 dose level. The 1/10 MELD10 and MELD10 dose levels were considered safe, as effects seen were minimal and readily reversible.

MELD = Mouse Equivalent Lethal Dose
a = 1/10 MELD10
b = MELD10
c = 2 x MELD10
d = 3 x MELD10
e = 4 x MELD10

Table 17: Subchronic Studies - Intravenous 13-Week Toxicity Studies in Rats and Dogs
Study Type/
Route of
Administration
Animal
Information
No. of
Animals
Dosage
(mg/kg/day)
Results
13-week subchronic toxicity
Intravenous
Study No. TBT03-003
Rat
(Sprague-Dawley)
Age: 8 - 14 weeks
Wt.: 215 - 312 g
160
(80 males, 80 females)
0, 1, 10, 50 There were 9 mortalities across all dose groups throughout the 13 weeks. None were attributable to the test article. At 50 mg/kg/day, toxicity was expressed as increased physical activity during dosing, increased incidence of piloerection, effects on body weights, food consumption, water consumption and clinical chemistry parameters, and decreases in red blood cell parameters. Organ weight changes included decreased absolute testes weights (males) and increased (relative to body weight) adrenal, kidney, liver and spleen weights in both sexes at this dose. There were correlated gross pathologic and histologic abnormalities in most of these organs. Fludarabine phosphate given intravenously to rats for 91 consecutive days at doses of 1 and 10 mg/kg/day was well tolerated.
13-week subchronic toxicity
Intravenous
Study No. TBT03-002
Dog
(Beagle)
Age: 12 - 16 months
Wt.: 7.1 - 17.9 kg
16
(8 males, 8 females)
0, 1, 10, 50 One male dog in the 50 mg/kg/day group died on day 42. Signs of toxicity noted in the 50 mg/kg/day group included weight loss, decreases in some red and white blood cell parameters, possible decrease in testicular weight, lymphoid depletion of the thymus and chronic inflammation of the stomach. For the male that died during the study, additional findings included hemorrhage in numerous tissues. The only test article-related change in the 10 mg/kg/day group was mild lymphoid depletion of the thymus in one male, although testicular weights may have been slightly decreased. The ❝no toxic effect❞ dose level was 10 mg/kg/day in female dogs and 1 mg/kg/day in male dogs.
Table 18: Mutagenicity Studies
Study Type System Used Concentration Range Results
Ames mutagenesis assay
Study No. TBT03-009
Salmonella typhimurium
Strains
TA 98
TA 100
TA 1535
TA 1537
Activated and non-activated assays:
0.0015; 0.005; 0.015; 0.05;
0.15; 0.5 mg/plate
Non-activated assay
Fludarabine phosphate, at concentrations of 0.0015 - 0.15 mg/plate, did not increase the mean number of revertants per plate over the negative control value for each of the four strains of bacteria tested. The highest concentration tested, 0.5 mg/plate, was toxic to all strains of bacteria utilized.

Activated assay
At concentrations of 0.0015 to 0.15 mg/plate, the mean number of revertants per plate was not increased over the control value for any of the four strains of bacteria tested. At 0.5 mg/plate, fludarabine phosphate was toxic to one strain (TA 1537).

Fludarabine phosphate was non-mutagenic to S. typhimurium strains tested, under both activated and non-activated conditions.
Sister chromatid exchange assay
Study No. TBT03-010
Chinese hamster ovary cells
(CHO)
Non-activated assay:
10; 15; 30; 50; 100; 150; 300; 500 μg/mL

Activated assay:
50; 125; 250; 500; 1000; 1500; 2000; 2500 μg/mL
Non-activated assay
A significant increase in sister chromatid exchanges (SCEs) was seen in cells exposed to fludarabine phosphate at a concentration of 50 μg/mL with higher concentrations precluded from analysis due to cellular toxicity. Concentrations of 15 and 30 μg/mL did not cause statistically significant increases in SCEs.

Activated assay
Concentrations of 500 and 1000 μg/mL caused significant increases in SCEs per cell. Concentrations of 125 and 250 μg/mL did not increase SCEs per cell. Concentrations higher than 1000 μg/mL were toxic to cells and thus precluded from analysis.

Fludarabine phosphate has been demonstrated to cause significant increases in SCEs under both activated and non-activated assay conditions.
CHO/HGPRT
Mammalian cell mutagenesis assay
Study No. TBT03-012
Chinese hamster ovary cells
(CHO)
Non-activated assay:
0.3; 1; 3; 10; 30; 100; 300; 500 μg/mL

Activated assay:
3; 10; 30; 100; 300; 1000; 1500; 2000; 2500 μg/mL
Non-activated assay
At concentrations of 1 to 300 μg/mL, fludarabine phosphate was nonmutagenic as indicated by mean mutation frequencies not significantly different from the negative (solvent) control values. A concentration of 500 μg/mL produced significant cellular toxicity and could not be analyzed.

Activated assay
Mean mutation frequencies were not significantly different from the solvent control value at fludarabine phosphate concentrations ranging from 3 to 1000 μg/mL. Higher concentrations were not selected for analysis due to toxicity to cells.

It was concluded that fludarabine phosphate was non-mutagenic under both non-activated and activated conditions in the CHO/HGPRT system.
Chromosome aberration assay
Study No. TBT03-011
Chinese hamster ovary cells
(CHO)
Non-activated assay:
2.6, 4.5, 9, 13, 26, 45, 90, 130, 260 μg/mL

Activated assay:
30, 50, 100, 150, 300, 500, 1000, 1500, 2000 μg/mL
Non-activated assay
The concentrations of fludarabine phosphate analyzed, 9, 26, and 90 μg/mL, did not increase the percentage of aberrant cells (both excluding and including gaps). Concentrations of 130 and 260 μg/mL were toxic to cells.

Activated assay
A significant increase in the percentage of cells with chromosomal aberrations (both excluding and including gaps) were detected at concentrations of 1500 and 2000 μg/mL. No significant increases in aberrant cells were noted at the other two concentrations analyzed, 150 and 500 μg/mL.

Fludarabine phosphate has been demonstrated to increase chromosome aberrations under activated conditions but did not increase chromosome aberrations under non-activated conditions in this assay.
Mouse micronucleus test
Study No. PHRR AD76
Mouse, NMRI (SPF) 0; 100; 300; 1000 mg/kg body weight

cyclophosphamide (30 mg/kg) positive control
One day after application at the toxic dose level of 1000 mg/kg, 3/20 mice showed moderate apathy, while on day 2, 2/20 died.

In the 1000 mg/kg dose group, a significant increase in the micronucleated polychromatic erythrocyte (PCE) and normochromatic erythrocyte (NCE) counts was observed at both sampling times. Additionally, in the mid-dose group, a significant increase in micronucleated PCE counts was observed 24 hours after administration. Furthermore, bone marrow depression was observed in all treatment groups at 24 hours post-administration and in the high- and mid-dose groups at 48 hours post-administration.

The positive control gave the expected increase in the micronucleated cell counts. A significant decrease in the PCE/NCE ratio was also observed.
Dominant lethal test
Study No. PHRR AV36
Mouse, NMRI, BR (SPF) 0; 100; 300; 800 mg/kg body weight

cyclophosphamide (120 mg/kg) positive control
Only the highest dose tested (800 mg/kg) was clearly toxic after single administration as demonstrated by a mortality rate of approximately 40%.

Fludarabine phosphate showed no potential to induce germ cell mutations in male mice at any germ cell stage over a complete spermatogenic maturation. No biologically relevant positive response for any of the parameters evaluated (number of total implantations and those resulting in death per pregnant female, pre-implantation losses and fertility index) were observed at any mating interval at any dose level.

The positive control gave the expected mutagenic response demonstrating the sensitivity of the test system.
Table 19: Reproductive Studies - Intravenous Developmental Toxicity Studies of Fludarabine Phosphate
Study Type/
Route of
Administration
Animal
Information
No. of
Animals
Dosage
(mg/kg/day)
Results
Range-finding developmental toxicity
Intravenous injection (gestation days 6 - 15)
Study No. TBT03-004
Rat
(Sprague Dawley)
Age: 12 weeks
Wt.: 227 - 266 g
30 females 0
4
10
40
100
400
Mortality was 100% at the 400 mg/kg/day dose level; all other animals survived to scheduled sacrifice. Signs of toxicity in the 40, 100 and 400 mg/kg/day groups included lethargy, hypothermia, changes in the feces, decreased body weight gain or body weight loss, and decreased food consumption. Postimplantation loss was 100% and 30% at the 100 and 40 mg/kg/day dose levels respectively. Ten fetuses in two litters in the 40 mg/kg/day group had fetal malformations, which included omphalocele and various limb and tail anomalies. The 4 and 10 mg/kg/day dose levels produced no signs of maternal or developmental toxicity. The No Observable Adverse Effect Level (NOAEL) was 10 mg/kg/day.
Developmental toxicity
Intravenous injection (gestation days 6 - 15)
Study No. TBT03-006
Rat
(Sprague Dawley)
Age: 12 months
Wt.: 208 - 299 g
100 females 0
1
10
30
No treatment-related deaths occurred during the study, nor were there any clinical signs of toxicity. Mean maternal body weight gain was slightly decreased early in the dosing phase and mean fetal weight was low for the 30 mg/kg/day group. The small number of malformations seen were considered not test article-related, due to a lack of dose response; however, the 10 and 30 mg/kg/day groups showed dose-related increases in the incidence of several skeletal variations (rib and vertebrae anomalies), indicating developmental toxicity at both dose levels. A dose level of 1 mg/kg/day was considered No Observable Adverse Effect Level (NOAEL).
Range-finding developmental toxicity
Intravenous injection (gestation days 6 - 18)
Study No. TBT03-005
Rabbit
(New Zealand White)
Age: 6 months
Wt.: 3.0 - 3.9 kg
30 females 0
1
5
10
25
50
Mortality was 100% for the 50 and 25 mg/kg/day groups. Signs of toxicity in the 10, 25, and 50 mg/kg/day groups included ataxia, lethargy, labored respiration, changes in the feces, maternal body weight losses and decreased food consumption. The 5 mg/kg/day group also had slightly decreased food consumption early in the dosing phase. Postimplantation loss was slightly increased in the 10 mg/kg/day group. In addition, 30 of 35 fetuses in this group had external malformations, consisting primarily of craniofacial and/or limb and digit defects. The No Observable Adverse Effect Level (NOAEL) was considered to be 1 mg/kg/day.
Developmental toxicity
Intravenous injection (gestation days 6 - 18)
Study No. TBT03-007
Rabbit
(New Zealand White)
Age: 6 months
Wt.: 3.1 - 4.2 kg
80 females 0
1
5
8
Maternal survival was not affected and no clinical signs of toxicity were apparent in any group. The 5 and 8 mg/kg/day groups showed dose-related inhibition of maternal body weight gain and food consumption. Post-implantation loss was increased and mean fetal body weight was low, at the 8 mg/kg/day dose level. External and skeletal malformations, generally specific to the head, limbs, digits and tail, were increased in the 8 mg/kg/day group. In addition, diaphragmatic hernia (a soft tissue malformation) was noted at a low frequency but in a dose-related pattern (3, 1 and 1 fetuses in the 8, 5 and 1 mg/kg/day groups, respectively). The incidence of skeletal variations was also increased in a dose-related manner in the 5 and 8 mg/kg/day groups. A dose level of 1 mg/kg/day was considered the No Observable Adverse Effect Level (NOAEL) for maternal toxicity but equivocal for fetal developmental toxicity because of the appearance of a single fetus with diaphragmatic hernia at this dose level.
Reproduction Toxicity (Peri-/Postnatal Study)
Intravenous injections (gestation days 15 to 21 postpartum)
Rat
(Jcl:Sprague Dawley)
96 Females 0
1
10
40
Following daily i.v. administration during late gestation and the lactation period, fludarabine phosphate was well tolerated at dose levels of 1 and 10 mg/kg/d with no relevant changes observed in dams or offspring. Signs of maternal toxicity (decreased body weight gain and food consumption, soft feces/diarrhea and piloerection) occurred in the 40 mg/kg/group. The offspring of the high dose group showed a decreased viability index on day 4 postpartum, a decreased weaning index and a reduced body weight gain. The skeletal maturation was delayed (reduced ossification of phalanges and vertebrae) in pups of the high dose group sacrificed on day 4 postpartum. In postnatal behavioural and learning tests, no drug related effects were observed. No relevant changes in the incidence of external and internal malformations in F2 fetuses were observed. The general toxicological no-effect dose level in this peri-/postnatal reproduction toxicity study was estimated to be 10 mg/kg/d.