Biodrugs 2009; 23 (1): 43-51 1173-8804/09/0001-0043/$49.95/0

ORIGINAL RESEARCH ARTICLE

ª 2009 Adis Data Information BV. All rights reserved.

Pharmacokinetic and Pharmacodynamic Profile of New Biosimilar Filgrastim XM02 Equivalent to Marketed Filgrastim Neupogen Single-Blind, Randomized, Crossover Trial Heinz Lubenau,1 Peter Bias,2 Anne-Katrin Maly,1 Karl Ernst Siegler3 and Kai Mehltretter4 1 2 3 4

BioGeneriX AG, Mannheim, Germany Merckle GmbH, Ulm, Germany CRS-Mannheim GmbH, Gru¨nstadt, Germany CRS-Mannheim GmbH, Mannheim, Germany

Abstract

Objective: Filgrastim XM02 is a biosimilar non-glycosylated recombinant methionyl form of human granulocyte colony-stimulating factor (r-MetHuG-CSF) expressed in Escherichia coli for subcutaneous and intravenous administration in the treatment of different forms of neutropenia and stem cell mobilization. This study was conducted to compare the pharmacokinetic and pharmacodynamic characteristics of the new biosimilar filgrastim XM02 with the marketed filgrastim (Neupogen). Methods: Two filgrastim doses (5 and 10 mg/kg) of the new biosimilar filgrastim XM02 and the marketed filgrastim were administered either as intravenous infusion or subcutaneous injection in four single-dose, crossover, randomized substudies, conducted in 36 subjects each. Serum concentrations of filgrastim were determined using an enzyme-linked immunosorbent assay test kit on samples taken at intervals up to 48 hours after administration. The CD34+ stem cell count up to 15 days after administration was determined by flow cytometry using a validated CD34+ cell enumeration kit, and the absolute neutrophil count (ANC) up to 96 hours after dosing was determined by the Beckman Coulter AcT differential automated hematology analyzer. The primary pharmacokinetic endpoint was the AUC48h (area under the serum concentration-time curve) of filgrastim serum concentration determined by the linear trapezoidal rule. Equivalence (biosimilarity) between the two filgrastim products was assessed by 90% confidence limits obtained from analyses of variance of log-transformed pharmacokinetic and pharmacodynamic endpoints, applying 80–125% equivalence intervals. Results: The mean serum concentration profiles of filgrastim, ANC and CD34+ cells over time were similar for the two filgrastims. The 90% confidence intervals for all test/reference ratios for pharmacokinetic and pharmacodynamic endpoints lay within the accepted bioequivalence range of 80–125%. Both filgrastims showed similar safety profiles and were well tolerated. Conclusions: Equivalence of the two filgrastims was clearly demonstrated for all four dose/route of administration groups. Equivalence could be demonstrated for the serum concentration profile, for the ANC profile and, even more importantly, for the CD34+ cell count, which is a marker for the ability of the granulocyte colony-stimulating factor to mobilize stem cells.

Background and Objective Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein produced by the endothelium, monocytes, and a

number of other immune cells. It stimulates the bone marrow to produce granulocytes and stem cells and to pulse them into the blood. It also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature

Lubenau et al.

44

neutrophils. Its selective action compared with other cytokines is mediated via a high-affinity G-CSF-specific receptor mechanism.[1] Filgrastim is a bacterially synthesized, non-glycosylated recombinant methionyl form of human G-CSF (r-MetHuGCSF) and was first approved as Neupogen by the US FDA in 1991. Structurally, XM02 is very similar to the active ingredient of the commercially available Neupogen (Escherichia coliderived r-MetHuG-CSF, manufactured by Amgen Inc. and distributed by Amgen, Germany).[2] Filgrastim is indicated for reducing the hematotoxic effects of chemotherapy and in patients undergoing bone marrow transplants, long-term treatment of severe congenital, cyclical or idiopathic neutropenia, or neutropenia associated with advanced HIV infection.[2] Filgrastim is rapidly absorbed following subcutaneous (SC) injection. Peak serum concentrations of the drug are attained generally within 4–5 hours, and the average serum half-life is 3.5 hours. The absolute bioavailability of SC filgrastim was estimated to be 62% and 72% for the 375 and 750 mg doses, respectively.[3] Serum concentrations and the area under the serum concentration-time curve (AUC) of filgrastim both increase linearly with increasing dose. However, data following administration of doses of 300–600 mg/day to healthy subjects showed a nonlinear increase in filgrastim serum concentrations.[4] Filgrastim causes marked increases in the peripheral blood neutrophil count within 24 hours, with minor increases in monocytes. In healthy subjects, mean peak absolute neutrophil counts (ANCs) of 13.8–22.2 · 109/L were reached at approximately 12 hours after the administration of filgrastim 75–300 mg. They returned to pretreatment levels within

48 hours and to normal levels in most cases within 4 days after cessation of filgrastim administration.[5] Administration of filgrastim mobilizes CD34+ stem cells in the peripheral blood, an indirect indicator of the marrowrepopulating ability of peripheral blood progenitor cells. In healthy subjects, the number of CD34+ cells in peripheral blood increased significantly from 38.4 (–23.1)/mL to 250.1 (–79.5)/mL of blood on day 5 after single-dose administration.[6] On cessation of r-MetHuG-CSF, the levels of peripheral blood progenitor cells returned to baseline within 4–6 days.[7] However, the number of CD34+ cells collected varied highly among individuals.[8,9] BioGeneriX AG has clinically developed a biosimilar nonglycosylated r-MetHuG-CSF expressed in E. coli for SC and intravenous (IV) administration in the treatment of different forms of neutropenia and stem cell mobilization. The international nonproprietary name (INN) will be filgrastim after marketing authorization. The present study assessed biosimilarity between filgrastim XM02 and filgrastim (Neupogen) in healthy subjects.

Materials and Methods This phase I, multicenter, single-dose, single-blind, randomized study, with four two-period crossover substudies (two treatments and two sequences) was designed to compare pharmacokinetic and pharmacodynamic properties and the claim of biosimilarity of filgrastim XM02 to the marketed filgrastim, Neupogen, for both SC and IV administration. The study design complied with the published product-specific

Table I. Demographic characteristics of the pharmacokinetic and pharmacodynamic study population Filgrastim dose group (mg/kg)

M/F

Age (years) mean (SD) [max./min.]

Body height (cm) mean (SD) [max./min.]

Bodyweight (kg) mean (SD) [max./min.]

Body mass index (kg/m2) mean (SD) [max./min.]

IV 5 (n = 31)

18/13

35.2 (6.7) [22/45]

173.4 (9.6) [155/189]

73.6 (11.4) [52.0/94.0]

24.4 (2.4) [20.3/31.4]

IV 10 (n = 30)

15/15

30.4 (8.7) [18/45]

170.8 (7.1) [158/186]

71.6 (11.2) [56.0/92.0]

24.5 (3.0) [19.8/31.1]

SC 5 (n = 33)

18/15

31.9 (8.1) [18/45]

174.1 (9.6) [152/193]

74.3 (10.4) [54.5/91.5]

24.5 (2.7) [19.5/29.7]

SC 10 (n = 30)

15/15

32.3 (7.7) [20/45]

171.5 (8.8) [153/188]

70.3 (10.1) [53.0/92.5]

23.9 (2.9) [19.4/29.9]

Total (n = 124)

66/58

32.5 (7.9) [18/45]

172.5 (8.9) [152/193]

72.5 (10.8) [52.0/94.0]

24.3 (2.7) [19.4/31.4]

F = female; IV = intravenous infusion; M = male; max. = maximum; min. = minimum; n = number of subjects; SC = subcutaneous injection; SD = standard deviation. ª 2009 Adis Data Information BV. All rights reserved.

Biodrugs 2009; 23 (1)

Filgrastim Pharmacokinetics and Pharmacodynamics

45

Table II. Subjects with adverse events after 5 or 10 mg/kg intravenous or subcutaneous administration of filgrastim (XM02 or Neupogen) Adverse event (MedDRA preferred terms)a

No. of subjects with adverse events (% subjects treated) reference: Neupogen (n = 131)

test: XM02 (n = 136) mild

moderate

Myalgia

22 (16.2)

3 (2.2)

Back pain

15 (11.0)

3 (2.2)

Bone pain

3 (2.2)

Headache

26 (19.1)

10 (7.4)

Paresthesia

2 (1.5)

1 (0.7)

Dizziness

2 (1.5)

Nausea

8 (5.9)

1 (0.7)

2 (1.5)

1 (0.7)

Toothache Fatigue

severe

1 (0.7)

mild 11 (8.4)

1 (0.8)

20 (15.3)

2 (1.5)

3 (2.3)

1 (0.8)

24 (18.3)

13 (9.9)

severe

5 (3.8)

2 (1.5)

Pyrexia

2 (1.5)

Pharyngolaryngeal pain

4 (2.9)

Nasopharyngitis

3 (2.2)

Hyperhidrosis

2 (1.5)

Decreased appetite

5 (3.7)

Rhinitis

2 (1.5)

5 (3.8)

2 (1.5)

1 (0.7) 3 (2.3)

Arthralgia

2 (1.5)

Pain in extremity

1 (0.8)

Asthenia

4 (3.1)

Injection site burning

1 (0,8)

Pruritus

3 (2.3)

Alopecia

2 (1.5)

Gastrointestinal disorder

3 (2.3)

Hematoma Totalb

moderate

1 (0.8)

1 (0.8)

3 (2.3) 62 (45.6)

18 (13.2)

1 (0.7)

62 (47.3)

19 (14.5)

0 (0)

a Adverse events occurring in >1% of the study subjects. b Overall number of subjects with adverse events per treatment. MedDRA = Medical Dictionary for Regulatory Activities, Version 7.1.

guidance on biosimilar medicinal products containing recombinant G-CSF[10] and the guidelines on bioequivalence.[11] The protocol was approved by the Independent Ethics Committee of the Landesa¨rztekammer Baden-Wu¨rttemberg and the German Federal Institute for Drugs and Devices (Bundesinstitut fu¨r Arzneimittel und Medizinprodukte, BfArM). The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Subjects gave written informed consent to participate in the study. Healthy female and male subjects from the panel of the Department of Recruiting at CRS-Mannheim GmbH, Mannheim, Germany, and the second study center, PHAROS GmbH, Ulm, Germany, were screened for eligibility and ª 2009 Adis Data Information BV. All rights reserved.

enrolment. For inclusion, subjects had to fulfill the following entry criteria: age 18–45 years, bodyweight 55–90 kg, body mass index 18.5–29.9 kg/m2, and healthy condition. Individuals were considered healthy if they had no history of hypersensitivity to the study medication or E. coli-derived products, no present or previous history of any chronic diseases, no febrile or infectious illness for at least 7 days prior to the first administration of the study medication, no symptoms or signs at the physical examination and laboratory tests, and were negative for hepatitis B and C and HIV infection markers in serum. The subjects were not included if they had a known history of prior treatment with blood cell colony-stimulating factors, interleukins or interferons; had participated in another clinical trial Biodrugs 2009; 23 (1)

Lubenau et al.

46

a XM02 5 μg/kg (n = 31) Neupogen® 5 μg/kg (n = 31) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

300 000 250 000 200 000 150 000 100 000

Filgrastim (pg/mL)

50 000 0 0

4

8

12

16

20

24

28

32

36

40

44

48

b XM02 5 μg/kg (n = 33) Neupogen® 5 μg/kg (n = 33) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

70 000 60 000 50 000 40 000 30 000 20 000 10 000 0 0

4

8

12

16

20

24

28

32

36

40

44

48

Time (h)

Fig. 1. Filgrastim serum concentration after (a) intravenous and (b) subcutaneous administration of the recombinant human filgrastim products XM02 and Neupogen.

within the last 90 days before screening; had donated blood or lost blood within the past 3 months; or had received a general anesthetic within 3 months preceding this study. Eligible subjects remained at the clinical site from the morning of the day before dosing until 48 hours following drug administration for each of the two study periods, and returned for further visits as required for blood sampling and evaluation until day 15. A washout period of at least 3 weeks separated the two administrations. During day -1 of period 1, eligible subjects were randomly assigned to one of the four substudies (IV 5 mg/kg and 10 mg/kg, SC 5 mg/kg and 10 mg/kg) and one of the two sequences (test-reference/reference-test) to ascending numbers for the current trial. The test drug, biosimilar filgrastim (XM02, manufactured by Sicor Biotech, Vilnius, Lithuania) was supplied in pre-filled syringes by BioGeneriX (Mannheim, Germany), each containing 0.8 mL of sterile, clear, colorless, preservative-free solution for injection, consisting of 480 mg (48 MIU) of the active ingredient as well as excipients (acidic sodium acetate buffer, sorbitol, polysorbate 80, and water for injection). ª 2009 Adis Data Information BV. All rights reserved.

Neupogen (manufactured by Amgen Inc. and distributed by Amgen Germany) was supplied in pre-filled syringes, each containing 0.5 mL of sterile, clear, colorless, preservative-free solution for injection, consisting of 300 mg (30 MIU) of filgrastim as well as excipients (acidic sodium acetate buffer, sorbitol, polysorbate 80, and water for injection). The manufacturing process of Neupogen was not disclosed to the public. As a prerequisite for the approval of biosimilars, the primary amino acid sequence of XM02 and Neupogen are identical. The expression host (E. coli) of XM02 and Neupogen are identical as well. All other characteristics have to be similar. The following aspects for production can be assumed to be different: the expression vector, fermentation conditions, refolding conditions, and the downstream process. The drug product formulation differs between XM02 and Neupogen in that XM02 uses a higher polysorbate concentration and a higher pH value. Administration was performed by IV infusion after dilution into 5% glucose solution, and by SC injection. Individual doses were calculated from the actual bodyweight measured in the evening before each administration. Dosing Biodrugs 2009; 23 (1)

Filgrastim Pharmacokinetics and Pharmacodynamics

was performed at approximately 8 am on day 1 of each period. Subjects were fasted. SC doses were injected into the back of the upper arm, with subjects in a semi-supine position. IV doses were infused (using a Perfusor syringe pump) into the brachial vein over 30 minutes at a continuous infusion rate per minute, with subjects in a supine position. Blood samples for the determination of filgrastim serum concentrations were taken at 0 (pre-dose), 15, 30, 45, 60, and 90 minutes, and at 2, 3, 4, 6, 8, 10, 12, 16, 24, and 48 hours after the start of the IV infusion (16 samples in each period), and at 0 (pre-dose), 30, 60, and 90 minutes, and at 2, 3, 4, 6, 8, 10, 12, 16, 24, and 48 hours after the SC injection (14 samples in each period). Serum concentrations were determined with the enzyme-linked immunosorbent assay test (ELISA) kit (Quantikine, R&D Systems, Minneapolis, MN, USA) by Cirion Biopharma Research Inc. (Laval, QC, Canada). Blood samples for determination of the ANC were collected at 0 (pre-dose) and 30 minutes, and at 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 32, 48, 72, and 96 hours after the injection/start of the infusion (16 samples in each period). The ANC was determined by the Beckman Coulter AcT (Fullerton, CA, USA) differential automated hematology analyzer.

47

Blood samples for the CD34+ cell count were collected at 0 (pre-dose), 24 (day 2), 48, 72, 96, 120, 144, 168 (day 8), 240 (day 11), and 336 hours (day 15) after the injection/start of the infusion (10 samples in each period). The CD34+ count was determined by flow cytometry using a validated CD34+ Cells Enumeration Kit (DakoCytomation Denmark A/S, Glostrup, Denmark)[12-15] at the Institut fu¨r Immunologie und Genetik am Klinikum Kaiserslautern, Kaiserslautern, Germany. Flow cytometry was performed on a Coulter Epics XL-MCL instrument (Beckman Coulter) equipped with an argon-ion laser tuned at 488 nm. Within each batch of samples, quality control samples for CD34+ were analyzed together with the test samples of the subjects. The acceptance criteria were fulfilled for each batch, demonstrating the reliability of the assay procedure. The screening medical examination within 4 weeks before administration of the first dose consisted of a medical history, a physical examination, measurement of blood pressure and heart rate, a 12-lead electrocardiogram, and clinical laboratory tests. Adverse events were assessed throughout the study. The blood pressure and pulse rate were measured pre-dose and at 2, 4, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, and 336 hours (14 samples in each period) and oral body temperature pre-dose

Table III. Pharmacokinetic parameters after 5 or 10 mg/kg subcutaneous (SC) or intravenous (IV) administration of filgrastim (XM02 or Neupogen) Filgrastim dose (mg/kg)

IV 5 (n = 31)

IV 10 (n = 30)

SC 5 (n = 33)

SC 10 (n = 30)

Filgrastim parameter

Filgrastim (XM02) test geom. mean (geom. SD)

Filgrastim (Neupogen) reference geom. mean (geom. SD)

Obtained from ANOVA ratio test/reference

90% CI for ratio

AUC48h

480 201 (1.25)

470 373 (1.25)

101.65

96.55, 107.01

AUC1

481 103 (1.25)

471 431 (1.25)

101.61

96.54, 106.95

Cmax

129 786 (1.21)

126 124 (1.18)

102.37

97.44, 107.55

t1=2lZ

7.75 (1.60)

8.00 (1.54)

97.71

82.03, 116.37

AUC48h

1 056 472 (1.23)

990 996 (1.25)

106.62

102.14, 111.30

AUC1

1 057 420 (1.23)

991 892 (1.25)

106.62

102.15, 111.29

Cmax

231 142 (1.18)

221 562 (1.20)

104.58

100.88, 108.41

t1=2lZ

6.11 (1.45)

5.98 (1.50)

102.87

88.58, 119.48

AUC48h

157 585 (1.43)

159 426 (1.41)

98.63

92.05, 105.66

AUC1

158 975 (1.42)

160 783 (1.40)

98.66

92.15, 105.64

Cmax

17 976 (1.56)

18 416 (1.56)

97.55

87.22, 109.10

t1=2lZ

7.81 (1.59)

8.94 (1.25)

87.84

77.15, 100.01

AUC48h

471 148 (1.28)

430 717 (1.33)

109.39

104.02, 115.03

AUC1

472 239 (1.28)

431 860 (1.33)

109.35

104.01, 114.96

Cmax

46 239 (1.37)

43 145 (1.47)

107.17

99.30, 115.66

t1=2lZ

5.34 (1.25)

5.67 (1.26)

94.34

87.02, 102.27

AUC = area under the serum concentration-time curve (AUC48h: until 48 h after dosing, AUC1 = extrapolated to infinity) [h concentration (pg/mL); geom. = geometric; SD = standard deviation; t1=2 kZ = terminal elimination half-life (h). ª 2009 Adis Data Information BV. All rights reserved.



pg/mL]; Cmax = maximum serum

Biodrugs 2009; 23 (1)

Lubenau et al.

48

a 40

XM02 5 μg/kg (n = 31) Neupogen® 5 μg/kg (n = 31) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

35 30 25 20 15 10

ANC (103/μL)

5 0 0

6

12

18

24

30

36

42

48

54

60

66

72

78

84

90

96

b 45

XM02 5 μg/kg (n = 33) Neupogen® 5 μg/kg (n = 33) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

40 35 30 25 20 15 10 5 0 0

6

12

18

24

30

36

42

48

54

60

66

72

78

84

90

96

Time (h)

Fig. 2. Absolute neutrophil count (ANC) after (a) intravenous and (b) subcutaneous administration of the recombinant human filgrastim products XM02 and Neupogen.

and at 4, 6, 12, 24, 36, and 48 hours after the injection/start of the infusion (six samples in each period). A follow-up examination was performed. Sample size estimation for the AUC, considered as the primary pharmacokinetic parameter, was based on the assumptions of a test/reference ratio of 90%, an intrasubject coefficient of variation of 18%, statistical power of 80%, and equivalence limits of 80–125%; thus, the required sample size was 28 subjects per subgroup. Assuming a discontinuation rate of 20%, eight spare subjects were included in each subgroup. A total of 4 · 36 = 144 subjects had to be randomized into this study. All statistical analyses were performed with SAS Version 9.1 software (SAS Institute Corp., Cary, NC, USA). The primary pharmacokinetic endpoint was the AUC48h of the filgrastim serum concentration determined by the linear trapezoidal rule. Secondary pharmacokinetic endpoints were the AUC1 (area under the serum concentration-time curve extrapolated to infinity), Cmax (peak serum concentration value), and t1=2 ª 2009 Adis Data Information BV. All rights reserved.

(elimination half-life determined by linear regression on the terminal portion of the concentration-time curve). The equivalence between the test (XM02) and the reference product (Neupogen) was assessed by 90% confidence intervals (CIs) obtained from analyses of variance (ANOVA) of logtransformed pharmacokinetic endpoints; 80–125% equivalence intervals were applied. To evaluate the equivalence of the pharmacodynamic characteristics, the parameters AUCt for the area under the cellcount time curve, and Cmax for the maximum cell count were evaluated for ANC and CD34+ cells. As the ANC and CD34+ are endogenous, estimations of elimination half-lives were not possible and thus were not extrapolated. The ANC Cmax and CD34+ Cmax were obtained directly from the data. The ANC AUC96h and CD34+ AUC336h were determined by the linear trapezoidal rule from time zero to the last sampling time. An ANOVA was applied on ANC parameters (ANC AUC96h, ANC Cmax) and CD34+ cells parameters (CD34+ AUC336h, CD34+ Cmax) as described above. Biodrugs 2009; 23 (1)

Filgrastim Pharmacokinetics and Pharmacodynamics

49

All statistical analyses in this study were supported by descriptive statistics. Results Four of the 144 subjects included in this study withdrew consent before administration without giving reasons. Altogether, 140 subjects had received at least one dose of the test or reference product and were included in the safety analysis. The pharmacokinetic and pharmacodynamic evaluation was based on the 124 subjects who completed both treatment periods without major protocol deviations. The mean age was 32.5 years (range 18–845 years), the mean bodyweight 72.5 kg (52–94 kg), and the mean height 172.5 cm (152–193 cm). The average body mass index was 24.3 kg/m2 (19.4–31.0 kg/m2). The substudies were homogenous according to demographic and baseline data (table I). No deaths or serious adverse events occurred during the study. The subjects presented with mainly mild adverse events, mostly myalgia, back pain, or headache (table II). One subject

was withdrawn because of severe back pain and moderate sweating during infusion of the 5 mg/kg XM02 solution. The two events in this subject were assessed to be probably related to the study medication; both were not serious and resolved without intervention. The other withdrawals due to adverse events were clearly not related to the investigational medicinal products. The safety results of the two filgrastim products were comparable and in line with observations in an earlier trial with XM02[16] and published data on Neupogen.[2] In each of the four substudies, mean serum concentrations of filgrastim (figure 1) showed similar profiles over time for XM02 and Neupogen. Geometric mean values were also similar in the two filgrastims. The ANOVA 90% CIs for the primary pharmacokinetic parameter, AUC48h, and the secondary pharmacokinetic parameters, Cmax and t1=2 , were within the acceptance limits of 80–125% (table III).[11] In each of the four substudies, the geometric mean values of the relevant pharmacodynamic marker for the activity of the recombinant G-CSF,[10] the ANC, and the secondary pharmacodynamic endpoint, CD34+ cell count, were similar

a 24

XM02 5 μg/kg (n = 31) Neupogen® 5 μg/kg (n = 31) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

20 16 12 8

CD34+ (1 μL)

4 0 0

24

46

72

96

120

144

168

192

216

240

264

288

312

336

b 24

XM02 5 μg/kg (n = 33) Neupogen® 5 μg/kg (n = 33) XM02 10 μg/kg (n = 30) Neupogen® 10 μg/kg (n = 30)

20 16 12 8 4 0 0

24

46

72

96

120

144

168

192

216

240

264

288

312

336

Time (h)

Fig. 3. CD34+ cell count after (a) intravenous and (b) subcutaneous administration of the recombinant human filgrastim products XM02 and Neupogen. ª 2009 Adis Data Information BV. All rights reserved.

Biodrugs 2009; 23 (1)

Lubenau et al.

50

Table IV. Absolute neutrophil count (ANC) over time and maximum ANC after 5 or 10 mg/kg subcutaneous (SC) or intravenous (IV) administration of filgrastim (XM02 or Neupogen) Filgrastim dose (mg/kg)

Filgrastim parameter

IV 5 (n = 31)

ANC AUC96h ANC Cmax

IV 10 (n = 30)

ANC AUC96h ANC Cmax

SC 5 (n = 33)

ANC AUC96h ANC Cmax

SC 10 (n = 30)

ANC AUC96h ANC Cmax

Filgrastim (XM02) test geom. mean (geom. SD)

Filgrastim (Neupogen) reference geom. mean (geom. SD)

Obtained from ANOVA ratio test/reference

90% CI for ratio

738.37 (1.45)

776.67 (1.34)

97.25

87.47, 108.13

18.73 (1.30)

19.46 (1.33)

98.21

93.08, 103.61

916.96 (1.61)

958.90 (1.53)

96.56

80.07, 116.45

21.71 (1.30)

22.21 (1.29)

98.97

92.39, 106.03

956.93 (1.44)

983.14 (1.25)

97.58

88.23, 107.92

22.58 (1.27)

21.14 (1.29)

107.27

102.60, 112.16

1305.93 (1.37)

1245.18 (1.57)

104.88

91.22, 120.58

26.94 (1.27)

26.98 (1.26)

99.84

96.10, 103.72

ANC AUC96h = area under the ANC versus time curve until 96 h after dosing (h SD = standard deviation.

over time (figures 2 and 3). The ANOVA 90% CIs of the respective areas under the curve and maximum counts lay within the generally accepted ranges for bioequivalence (tables IV and V).[11] Conclusions Both the pharmacokinetic results and the pharmacodynamic response (ANC, CD34+) of this study clearly demonstrate biosimilarity of XM02 filgrastim to Neupogen, meeting the regulatory acceptance criteria defined by regulatory guidance.[11] The 90% CIs for the test/reference ratios of the AUCt and Cmax of filgrastim serum concentrations, ANC, and



103/mL); ANC Cmax = maximum ANC (103/mL); geom. = geometric;

CD34+ lie within the acceptance limits of 80–125% for both doses and administration routes. Thus, equivalence of the two filgrastims could be demonstrated for all four substudies. The filgrastim serum concentrations and ANC results are also in line with another study comparing SC doses of 5 and 10 mg/kg of XM02 and Neupogen[16] and with the pharmacokinetic profile after SC administration of Neupogen 300 mg.[17] Single doses of XM02 or Neupogen administered via SC injection or IV infusion at doses of either 5 or 10 mg/kg were well tolerated. The safety results also showed comparability between XM02 and Neupogen, and are in line with published data on Neupogen.[2]

Table V. CD34+ count over time and maximum CD34+ count after 5 or 10 mg/kg subcutaneous (SC) or intravenous (IV) administration of filgrastim (XM02 or Neupogen) Filgrastim dose (mg/kg)

Filgrastim parameter

Filgrastim (XM02) test geom. mean (geom. SD)

Filgrastim (Neupogen) reference geom. mean (geom. SD)

IV 5 (n = 31)

CD34+ AUC336h

1451.35 (1.50)

1545.21 (1.51)

93.77

87.02, 101.05

8.56 (1.57)

8.79 (1.61)

96.78

86.61, 108.15

1644.85 (1.65)

1525.62 (1.68)

107.71

101.37, 114.44

10.43 (1.65)

9.68 (1.69)

107.09

97.46, 117.68

1462.63 (1.53)

1448.61 (1.47)

100.96

93.29, 109.26

8.42 (1.64)

8.78 (1.46)

95.67

85.60, 106.92

1860.82 (1.57)

2063.90 (1.50)

90.16

84.79, 95.87

12.23 (1.67)

13.29 (1.70)

92.05

82.42, 102.81

CD34+ Cmax IV 10 (n = 30)

CD34+ AUC336h CD34+ Cmax

SC 5 (n = 33)

CD34+ AUC336h CD34+ Cmax

SC 10 (n = 30)

CD34+ AUC336h CD34+ Cmax

CD34+ AUC336h = area under the CD34+ count versus time curve until 336 h after dosing (h geom. = geometric; SD = standard deviation. ª 2009 Adis Data Information BV. All rights reserved.



Obtained from ANOVA ratio test/reference

90% CI for ratio

1/mL); CD34+ Cmax = maximum CD34+ count (1/mL);

Biodrugs 2009; 23 (1)

Filgrastim Pharmacokinetics and Pharmacodynamics

Based on the results of this study, the two recombinant human filgrastim products, XM02 and Neupogen, can be expected to result in similar efficacy in the clinical setting.

Acknowledgments BioGeneriX AG, Mannheim, Germany, funded both the study and preparation of the manuscript and was responsible for the study design, interpretation of the data, and final approval of both the clinical study report and the manuscript. The study was performed at the Contract Research Organisation CRS-Mannheim GmbH, Mannheim, Germany. The tasks of CRS-Mannheim GmbH included conduct of the study; collection, management, and analysis of the data; and preparation and review of the integrated clinical study report and manuscript. Furthermore, CRSMannheim GmbH gave expert input on the study design and interpretation of the data. Drs Karl Ernst Siegler and Kai Mehltretter are employees of CRS-Mannheim GmbH. Writing assistance was provided by Dr Sabine Stiegler and Nadja Faisst, CRS-Mannheim GmbH, Gru¨nstadt, Germany. Dr Heinz Lubenau and Anne-Katrin Maly are employees of BioGeneriX AG, Mannheim, Germany. Dr Peter Bias is an employee of Merckle GmbH, Ulm, Germany, which belongs to the same holding company (ratiopharm GmbH, Ulm, Germany) as BioGeneriX AG.

References 1. Nagata S, Tsuchiya M, Asano S, et al. Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 1986; 319 (6052): 415-8

51

6. Schwinger W, Mache C, Urban C, et al. Single dose of filgrastim (rhG-CSF) increases the number of hematopoietic progenitors in the peripheral blood of adult volunteers. Bone Marrow Transplant 1993 Jun; 11 (6): 489-92 7. Stroncek DF, Clay ME, Petzoldt ML, et al. Treatment of normal individuals with granulocyte-colony-stimulating factor: donor experiences and the effects on peripheral blood CD34+ cell counts and on the collection of peripheral blood stem cells. Transfusion 1996 Jul; 36 (7): 601-10 8. Roberts AW, DeLuca E, Begley CG, et al. Broad inter-individual variations in circulating progenitor cell numbers induced by granulocyte colonystimulating factor therapy. Stem Cells 1995 Sep; 13 (5): 512-6 9. Watts MJ, Addison I, Long SG, et al. Cross-over study of the haematological effects and pharmacokinetics of glycosylated and non-glycosylated G-CSF in healthy volunteers. Br J Haematol 1997 Aug; 98 (2): 474-9 10. European Medicines Evaluation Agency. Committee for Medicinal Products for Human Use. Guidance on biosimilar medicinal products containing recombinant granulocyte-colony stimulating factor (annex to guideline). London: EMEA, 2005 Jun 11. European Medicines Evaluation Agency. Committee for Proprietary Medicinal Products (CPMP). Note for guidance on the investigation of bioavailability and bioequivalence. London: EMEA, 2001 Jul 12. Brando B, Barnett D, Janossy G, et al. Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. Cytometry 2000 Dec; 42 (6): 327-46 13. Gratama JW, Orfao A, Barnett D, et al. Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis. Cytometry 1998 Jun 15; 34 (3): 128-42 14. Keeney M, Gratama JW, Sutherland DR. Critical role of flow cytometry in evaluating peripheral blood hematopoietic stem cell grafts. Cytometry A 2004 Mar; 58 (1): 72-5 15. Sutherland DR, Anderson L, Keeney M, et al. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother 1996 Jun; 5 (3): 213-26

2. Amgen Ltd. Summary of product characteristics: Neupogen 30MU and 48MU solution for injection – electronic medicines compendium 2007 [online]. Available from URL: http://emc.medicines.org.uk/emc/assets/c/html/DisplayDoc.asp?DocumentID=7907 [Accessed 2008 Apr 1]

16. Lubenau H, Sveikata A, Gumbrevicius G, et al. Bioequivalence of two recombinant granulocyte colony-stimulating factor products after subcutaneous injection in healthy volunteers. Int J Clin Pharm Ther. In press

3. Wang B, Ludden TM, Cheung EN, et al. Population pharmacokinetic-pharmacodynamic modeling of filgrastim (r-metHuG-CSF) in healthy volunteers. J Pharmacokinet Pharmacodyn 2001 Apr; 28 (4): 321-2

17. Hernandez-Bernal F, Garcia-Garcia I, Gonzalez-Delgado CA, et al. Bioequivalence of two recombinant granulocyte colony-stimulating factor formulations in healthy male volunteers. Biopharm Drug Dispos 2005 May; 26 (4): 151-9

4. Borleffs JC, Bosschaert M, Vrehen HM, et al. Effect of escalating doses of recombinant human granulocyte colony-stimulating factor (filgrastim) on circulating neutrophils in healthy subjects. Clin Ther 1998 Jul-Aug; 20 (4): 722-36 5. Bronchud MH, Potter MR, Morgenstern G, et al. In vitro and in vivo analysis of the effects of recombinant human granulocyte colony-stimulating factor in patients. Br J Cancer 1988 Apr; 58 (1): 64-9

ª 2009 Adis Data Information BV. All rights reserved.

Correspondence: Dr Heinz Lubenau, Vaximm GmbH, Chirurgische Klinik, Universita¨ts-Klinik Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany. E-mail: [email protected]

Biodrugs 2009; 23 (1)