Azacitidine (Page 4 of 5)

13 NONCLINICAL TOXICOLOGY

13.1 Carcinogenesis, Mutagenesis, Impairment of Fertility

The potential carcinogenicity of azacitidine was evaluated in mice and rats. Azacitidine induced tumors of the hematopoietic system in female mice at 2.2 mg/kg (6.6 mg/m², approximately 8% the recommended human daily dose on a mg/m² basis) administered IP three times per week for 52 weeks. An increased incidence of tumors in the lymphoreticular system, lung, mammary gland, and skin was seen in mice treated with azacitidine IP at 2.0 mg/kg (6.0 mg/m², approximately 8% the recommended human daily dose on a mg/m² basis) once a week for 50 weeks. A tumorigenicity study in rats dosed twice weekly at 15 or 60 mg/m² (approximately 20%-80% the recommended human daily dose on a mg/m² basis) revealed an increased incidence of testicular tumors compared with controls.

The mutagenic and clastogenic potential of azacitidine was tested in in vitro bacterial systems Salmonella typhimurium strains TA100 and several strains of trpE8, Escherichia coli strains WP14 Pro, WP3103P, WP3104P, and CC103; in in vitro forward gene mutation assay in mouse lymphoma cells and human lymphoblast cells; and in an in vitro micronucleus assay in mouse L5178Y lymphoma cells and Syrian hamster embryo cells. Azacitidine was mutagenic in bacterial and mammalian cell systems. The clastogenic effect of azacitidine was shown by the induction of micronuclei in L5178Y mouse cells and Syrian hamster embryo cells.

Administration of azacitidine to male mice at 9.9 mg/m² (approximately 9% the recommended human daily dose on a mg/m² basis) daily for 3 days prior to mating with untreated female mice resulted in decreased fertility and loss of offspring during subsequent embryonic and postnatal development. Treatment of male rats 3 times per week for 11 or 16 weeks at doses of 15-30 mg/m² (approximately 20%-40%, the recommended human daily dose on a mg/m² basis) resulted in decreased weight of the testes and epididymides, and decreased sperm counts accompanied by decreased pregnancy rates and increased loss of embryos in mated females. In a related study, male rats treated for 16 weeks at 24 mg/m² resulted in an increase in abnormal embryos in mated females when examined on day 2 of gestation.

14 CLINICAL STUDIES

Myelodysplastic Syndromes (MDS)

Study 1 was a randomized, open-label, controlled trial carried out in 53 U.S. sites compared the safety and efficacy of subcutaneous Azacitidine plus supportive care with supportive care alone (“observation”) in patients with any of the five FAB subtypes of myelodysplastic syndromes (MDS): refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMMoL). RA and RARS patients were included if they met one or more of the following criteria: required packed RBC transfusions; had platelet counts ≤ 50.0 x 109 /L; required platelet transfusions; or were neutropenic (ANC <1.0 x 109 /L) with infections requiring treatment with antibiotics. Patients with acute myelogenous leukemia (AML) were not intended to be included. Supportive care allowed in this study included blood transfusion products, antibiotics, antiemetics, analgesics and antipyretics. The use of hematopoietic growth factors was prohibited. Baseline patient and disease characteristics are summarized in Table 3; the 2 groups were similar.

Azacitidine was administered at a subcutaneous dose of 75 mg/m2 daily for 7 days every 4 weeks. The dose was increased to 100 mg/m2 if no beneficial effect was seen after 2 treatment cycles. The dose was decreased and/or delayed based on hematologic response or evidence of renal toxicity. Patients in the observation arm were allowed by protocol to cross over to Azacitidine if they had increases in bone marrow blasts, decreases in hemoglobin, increases in red cell transfusion requirements, or decreases in platelets, or if they required a platelet transfusion or developed a clinical infection requiring treatment with antibiotics. For purposes of assessing efficacy, the primary endpoint was response rate (as defined in Table 4).

Of the 191 patients included in the study, independent review (adjudicated diagnosis) found that 19 had the diagnosis of AML at baseline. These patients were excluded from the primary analysis of response rate, although they were included in an intent-to-treat (ITT) analysis of all patients randomized. Approximately 55% of the patients randomized to observation crossed over to receive Azacitidine treatment.

Table 3. Baseline Demographics and Disease Characteristics
Azacitidine (N=99) Observation (N=92)
Gender (n%)
Male 72 (72.7) 60 (65.2)
Female 27 (27.3) 32 (34.8)
Race (n%)
White 93 (93.9) 85 (92.4)
Black 1 (1.0) 1 (1.1)
Hispanic 3 (3.0) 5 (5.4)
Asian/Oriental 2 (2.0) 1 (1.1)
Age (years)
N 99 91
Mean ± SD 67.3 ± 10.39 68.0 ± 10.23
Range 31 — 92 35 — 88
Adjudicated MDS diagnosis at study entry (n%)
RA 21 (21.2) 18 (19.6)
RARS 6 (6.1) 5 (5.4)
RAEB 38 (38.4) 39 (42.4)
RAEB-T 16 (16.2) 14 (15.2)
CMMoL 8 (8.1) 7 (7.6)
AML 10 (10.1) 9 (9.8)
Transfusion product used in 3 months before study entry (n%)
Any transfusion product 70 (70.7) 59 (64.1)
Blood cells, packed human 66 (66.7) 55 (59.8)
Platelets, human blood 15 (15.2) 12 (13.0)
Hetastarch 0(0.0) 1(1.1)
Plasma protein fraction 1(1.0) 0(0.0)
Other 2(2.0) 2(2.2)
Table 4. Response Criteria
RA RARS RAEB RAEB-T CMMoL
Complete Response (CR), duration ≥ 4 weeks Marrow <5% blasts
Peripheral Blood Normal CBC if abnormal at baselineAbsence of blasts in the peripheral circulation
Partial Response (PR), duration ≥ 4 weeks Marrow No marrow requirements ≥50% decrease in blastsImprovement of marrow dyspoiesis
Peripheral Blood ≥50% restoration in the deficit from normal levels of baseline white cells, hemoglobin and platelets if abnormal at baselineNo blasts in the peripheral circulationFor CMMoL, if WBC is elevated at baseline, a ≥75% reduction in the excess count over the upper limit of normal

The overall response rate (CR + PR) of 15.7% in Azacitidine -treated patients without AML (16.2% for all Azacitidine randomized patients including AML) was statistically significantly higher than the response rate of 0% in the observation group (p<0.0001) (Table 5). The majority of patients who achieved either CR or PR had either 2 or 3 cell line abnormalities at baseline (79%; 11/14) and had elevated bone marrow blasts or were transfusion dependent at baseline. Patients responding to Azacitidine had a decrease in bone marrow blasts percentage, or an increase in platelets, hemoglobin or WBC. Greater than 90% of the responders initially demonstrated these changes by the 5th treatment cycle. All patients who had been transfusion dependent became transfusion independent during PR or CR. The mean and median duration of clinical response of PR or better was estimated as 512 and 330 days, respectively; 75% of the responding patients were still in PR or better at completion of treatment. Response occurred in all MDS subtypes as well as in patients with adjudicated baseline diagnosis of AML.

Table 5. Response Rates
Azacitidine (N=89) Observation Before Crossover (N=83)
Response n (%) n (%) P value
Overall(CR+PR) 14 (15.7) 0 ( 0.0) (<0.0001)
Complete (CR) 5 ( 5.6) 0 ( 0.0) (0.06)
Partial (PR) 9 (10.1) 0 ( 0.0)

Patients in the observation group who crossed over to receive Azacitidine treatment (47 patients) had a response rate of 12.8%.

Study 2, a multi-center, open-label, single-arm study of 72 patients with RAEB, RAEB-T, CMMoL, or AML was also carried out. Treatment with subcutaneous Azacitidine resulted in a response rate (CR + PR) of 13.9%, using criteria similar to those described above. The mean and median duration of clinical response of PR or better was estimated as 810 and 430 days, respectively; 80% of the responding patients were still in PR or better at the time of completion of study involvement. In Study 3, another open-label, single-arm study of 48 patients with RAEB, RAEB-T, or AML, treatment with intravenous Azacitidine resulted in a response rate of 18.8%, again using criteria similar to those described above. The mean and median duration of clinical response of PR or better was estimated as 389 and 281 days, respectively; 67% of the responding patients were still in PR or better at the time of completion of treatment. Response occurred in all MDS subtypes as well as in patients with adjudicated baseline diagnosis of AML in both of these studies. Azacitidine dosage regimens in these 2 studies were similar to the regimen used in the controlled study.

Benefit was seen in patients who did not meet the criteria for PR or better, but were considered “improved.” About 24% of Azacitidine -treated patients were considered improved, and about 2/3 of those lost transfusion dependence. In the observation group, only 5/83 patients met criteria for improvement; none lost transfusion dependence. In all 3 studies, about 19% of patients met criteria for improvement with a median duration of 195 days.

Study 4 was an international, multicenter, open-label, randomized trial in MDS patients with RAEB, RAEB-T or modified CMMoL according to FAB classification and Intermediate-2 and High risk according to IPSS classification. Of the 358 patients enrolled in the study, 179 were randomized to receive azacitidine plus best supportive care (BSC) and 179 were randomized to receive conventional care regimens (CCR) plus BSC (105 to BSC alone, 49 to low dose cytarabine and 25 to chemotherapy with cytarabine and anthracycline). The primary efficacy endpoint was overall survival.

The azacitidine and CCR groups were comparable for baseline parameters. The median age of patients was 69 years (range was 38-88 years), 98% were Caucasian, and 70% were male. At baseline, 95% of the patients were higher risk by FAB classification: RAEB (58%), RAEB-T (34%), and CMMoL (3%). By IPSS classification, 87% were higher risk: Int-2 (41%), High (47%). At baseline, 32% of patients met WHO criteria for AML.

Azacitidine was administered subcutaneously at a dose of 75 mg/m² daily for 7 consecutive days every 28 days (which constituted one cycle of therapy). Patients continued treatment until disease progression, relapse after response, or unacceptable toxicity. Azacitidine patients were treated for a median of 9 cycles (range 1 to 39), BSC only patients for a median of 7 cycles (range 1 to 26), low dose cytarabine patients for a median of 4.5 cycles (range 1 to 15), and chemotherapy with cytarabine and anthracycline patients for a median of 1 cycle (range 1 to 3, i.e. induction plus 1 or 2 consolidation cycles).

In the Intent-to-Treat analysis, patients treated with azacitidine demonstrated a statistically significant difference in overall survival as compared to patients treated with CCR (median survival of 24.5 months vs. 15.0 months; stratified log-rank p=0.0001). The hazard ratio describing this treatment effect was 0.58 (95% CI: 0.43, 0.77).

Kaplan-Meier Curve of Time to Death from Any Cause: (Intent-to-Treat Population)

Figure

Key: AZA = azacitidine; CCR = conventional care regimens; CI = confidence interval; HR = Hazard Ratio

Azacitidine treatment led to a reduced need for red blood cell transfusions (see Table 6). In patients treated with azacitidine who were RBC transfusion dependent at baseline and became transfusion independent, the median duration of RBC transfusion independence was 13.0 months.

Table 6. Effect of Azacitidine on RBC Transfusions in MDS Patients

1 A patient was considered RBC transfusion independent during the treatment period if the patient had no RBC transfusions during any 56 consecutive days or more during the treatment period. Otherwise, the patient was considered transfusion dependent.

Efficacy Parameter Azacitidine plus BSC (n= 179) Conventional Care Regimens (n= 179)
Number and percent of patients who were transfusion dependent at baseline who became transfusion independent on treatment1 Number and percent of patients who were transfusion-independent at baseline who became transfusion-dependent on treatment 50/111 (45.0%)(95% CI: 35.6%, 54.8%)10/68 (14.7%)(95% CI: 7.3%, 25.4%) 13/114 (11.4%)(95% CI: 6.2%, 18.7%)28/65 (43.1%)(95% CI: 30.9%, 56.0%)

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