Pharmacokinetics/Pharmacodynamics

Pharmacokinetic and Pharmacodynamic Interactions Between Palifermin and Heparin

The Journal of Clinical Pharmacology 2015, 55(10) 1109–1118 © 2015, The American College of Clinical Pharmacology DOI: 10.1002/jcph.516

Bing-Bing Yang, PhD1, Brad Gillespie, PharmD1, Brian Smith, PhD1, William Smith, MD2, Agneta Lissmats, MSc3, Mattias Rudebeck, MSc, BMedSc3, Torbj€ orn Kullenberg, MD3, and Birgitta Olsson, MSc3

Abstract Oral mucositis, a severe complication during chemo- and/or radiotherapy, is prevented with palifermin treatment, a recombinant human keratinocyte growth factor (KGF/FGF-7). The FGF family belongs to the larger family of heparin-binding growth factors. Because it has been shown that heparin modulates binding of KGF to the KGF receptor and subsequently affects cellular proliferation induced by the KGF mitogenic signal, it is critical to understand the drug–drug interactions between palifermin and heparin, particularly because of heparin’s narrow therapeutic margin. Two studies were performed in healthy subjects to characterize the effect of palifermin on the pharmacodynamics of heparin (activated partial thromboplastin time) and evaluate the impact of heparin on the pharmacokinetics and pharmacodynamics (Ki67 staining of buccal mucosal tissue) of palifermin. Results demonstrated a pronounced pharmacokinetic interaction; heparin coadministration increased the palifermin AUC 4- to 5-fold and decreased its halflife by 40%–45%, suggesting an approximate 70%–80% decrease in palifermin clearance and volume of distribution. These changes in the pharmacokinetics of palifermin during coadministration of heparin, however, did not affect the pharmacodynamic effect of palifermin, or the anticoagulant activity of heparin, and did not lead to increased safety findings. Therefore, these results suggest that dose adjustments for heparin and palifermin are not warranted when administered concurrently.

Keywords palifermin, keratinocyte growth factor, heparin, drug–drug interaction, pharmacokinetics/pharmacodynamics

Chemotherapy and/or radiotherapy kills rapidly proliferating tumor cells and frequently damages rapidly dividing normal cells of the gastrointestinal tract, resulting in a clinical condition called mucositis.1,2 Oral mucositis occurs when the epithelial cells that line the mucosa of the oral cavity are injured and undergo physiologic alterations that range from mild atrophy to severe ulceration. This condition can be extremely painful and may limit the ability of a subject to speak, eat, or swallow and may result in weight loss severe enough to require supplemental nutrition. Keratinocyte growth factor (KGF or FGF-7), a member of the fibroblast growth factor (FGF) family, is a naturally occurring epithelial-specific growth factor found in animals and humans that stimulates cell proliferation. It has trophic, differentiative, and cytoprotective effects on the mucosal epithelium of the oral cavity and small intestine of the gastrointestinal tract.3,4 Palifermin (Kepivance; Swedish Orphan Biovitrum AB (publ), Stockholm, Sweden) is a recombinant human KGF identical to an N-terminal truncated endogenous KGF that contains a sequence of 140 amino acids and mimics the in vitro and in vivo activity of the natural protein.5–7 It is indicated to decrease the incidence, duration, and severity of oral mucositis in adult patients with hematological malignancies receiving myelotoxic therapy requiring hematopoietic stem cell support.8

One of the defining features of the FGF family is a strong affinity for heparin; therefore, collectively FGFs are also known as heparin-binding growth factors. In addition, it has been shown that heparin can modulate binding of KGF to the KGF receptor and subsequently affect the cellular proliferation induced by the KGF mitogenic signal.9,10 Because subjects who receive palifermin treatment may also require heparin administration to alleviate the effects of a clotting disorder, it is critical to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) drug–drug interactions between palifermin and heparin, particularly because heparin has a narrow therapeutic margin and potential for significant toxicity. A first study was performed to characterize the effect of heparin on the PK of palifermin and the effect of

1

Amgen Inc., Thousand Oaks, CA, USA New Orleans Center for Clinical Research (NOCCR), University of Tennessee Medical Center, Knoxville, TN, USA 3 Swedish Orphan Biovitrum AB (publ) (Sobi), Stockholm, Sweden 2

Submitted for publication 6 February 2015; accepted 8 April 2015. Corresponding Author: Birgitta Olsson, Swedish Orphan Biovitrum, SE-112 76 Stockholm, Sweden Email: [email protected]

1110

The Journal of Clinical Pharmacology / Vol 55 No 10 (2015)

palifermin on the PD of heparin (study 1). Subsequently, to put these results into perspective, study 2 was conducted to evaluate the impact of heparin on the PD of palifermin.

Methods Study Designs The studies were approved by an institutional review board (Crescent City Institutional Review Board, New Orleans, Louisiana) and conducted at New Orleans Center for Clinical Research (Knoxville, Tennessee) in accordance with the International Conference on Harmonisation Good Clinical Practice regulations/guidelines. Both studies were publicly registered at ClinicalTrials.gov (NCT00361348 and NCT01163097). Healthy men and women between 18 and 45 years with a body mass index of 19 to 30 kg/m2 inclusive were eligible. All subjects provided written informed consent before entering the study. In both studies, heparin was administered as a continuous intravenous infusion beginning at approximately 800 hours on the first day of a titration period. In study 2 the administration was initiated by an intravenous bolus dose. During the titration period individual heparin doses were adjusted for up to 48 hours to achieve an activated partial thromboplastin time (aPTT) that was between 1.5 and 2.0 times the baseline value, which is the target level for heparin therapy for acute thromboembolic events.11 Subjects who successfully titrated to target aPTT continued to receive the heparin infusion at a fixed rate throughout the study. Subjects who could not maintain aPTT within 1.5 to 2.0 times the baseline value throughout the heparin titration period did not continue in the study. Following palifermin administration, the heparin continued up to 48 hours in study 1 and 70 hours in study 2. Study 1. This study was an open-label, 2-part parallel study. In part 1, subjects were randomized in a 3:2 ratio to receive either:
a single intravenous bolus injection of 60 mg/ kg palifermin on day 1 (following the heparin titration) concurrently with unfractionated heparin (cohort 1PþH) or
a single intravenous bolus injection of 60 mg/ kg palifermin on day 1 (cohort 1P). Preliminary laboratory data from part 1 showed that a majority of subjects in cohort 1PþH had transient increases in liver function test (LFT) values. To assess the impact on LFT values of heparin administered alone, subjects in a third cohort (1H) received heparin treatment in the same manner as 1PþH, but without concomitant palifermin administration. Although the intent was to

rechallenge as many of the cohort 1PþH subjects as possible, study-naive subjects were also allowed to enroll in cohort 1H. Study 2. In this open-label parallel study, subjects were randomized in a 20:15:8 ratio to 1 of 3 groups:
Daily intravenous bolus doses of 40 mg/kg/ day palifermin for 3 consecutive days (days 1–3, following the heparin titration) concomitantly with continuous intravenous infusion of unfractionated heparin (cohort 2PþH).
Daily intravenous bolus doses of 40 mg/kg/ day palifermin for 3 consecutive days (cohort 2P).
Control group with no treatment administered (cohort 2Ctrl).

Palifermin Pharmacokinetics (Studies 1 and 2) Serial serum samples for palifermin concentration measurement were collected before and up to 72 hours after palifermin dosing (study 1), or up to 24 hours after the first and the third palifermin doses (study 2). For cohort 1PþH, additional serum samples were collected before and 24 hours after the initiation of the heparin infusion for endogenous KGF concentration measurement. Serum samples for palifermin and endogenous KGF concentrations were analyzed by a validated enzymelinked immunosorbent assay at Pharmaceutical Product Development (Richmond, Virginia). Microtiter wells precoated with anti-KGF monoclonal antibody captured endogenous KGF or palifermin present in the samples. After washing away any unbound substances, a biotinylated anti-KGF antibody was added to the wells. After a wash step to remove unbound conjugate, a mixture of Vectastain ABC reagent (Avidin DH and biotinylated horseradish peroxidase H reagents) from Vector Lab (Burlingame, California) was added to the wells. After the final wash step, a mixture of substrate solution (tetramethylbenzidine and hydrogen peroxide) was added to the wells to initiate the colorimetric reaction. The reaction was stopped with phosphoric acid solution, and the plate was read with a Molecular Devices plate reader (Spectra MAX Plus, Molecular Devices) with the test filter set to 450 nm and the reference filter set to 650 nm. The color generated as measured by optical density in each well was directly proportional to the concentration of endogenous KGF or palifermin in the sample. A log-log regression model was used to fit the standard curve and to interpolate the sample results. The analytical range of the assay was 0.072 (lower limit of quantification) to 1800 ng/mL. The accuracy ranged from 95% to 106%, with 6% to 16% variability. The following palifermin PK parameters were estimated using noncompartmental methods: initial serum

1111

Yang et al

concentration (C0) by back-extrapolation to time 0 using the first 2 observed concentration values, terminal halflife (t1/2,z), area under the serum concentration–time curve from time 0 to infinity (AUC0–1) for study 1 or from time 0 to 24 hours after dosing (AUC0–24) for study 2, clearance (CL), and volume of distribution at steady state (Vss). Heparin Pharmacodynamics (Study 1). For cohorts 1PþH and 1H, blood samples were collected in citratecontaining tubes at approximately 1400, 1600, and 1800 hours on the day before heparin titration, every 6 hours during the heparin maintenance phase, and 2, 4, and 6 hours and then every 6 hours up to 66 hours afterward. Plasma samples were frozen and transferred to a local clinical laboratory (LabCorp, Knoxville, Tennessee) for aPTT assessment. Baseline aPTT, the average of the aPTT values collected before heparin titration, and area under the aPTT–time curve from time 0 to 6 or 24 hours (AUCaPTT, 0–6 and AUCaPTT, 0–24, respectively) were estimated during heparin maintenance and on day 1. Palifermin Pharmacodynamics (Study 2). Ki67 staining of buccal mucosal tissue was used as a biomarker for evaluating palifermin activity, as increases in Ki67 expression are associated with cell proliferation12 and increased ratios of postdose to baseline Ki67-stained area per millimeter with increasing doses of palifermin in a dose-dependent manner has been demonstrated,13 indicating Ki67 to be an appropriate surrogate marker for palifermin’s pharmacodynamic effect. A baseline buccal biopsy sample was collected from all subjects within 3 days before the start of fixed-dose heparin infusion (cohort 2PþH), palifermin administration (cohort 2P), or before setting zero point (time when active treatment was given for other cohorts; cohort 2Ctrl). Another buccal sample was collected from all subjects 72 hours (day 4) after the first palifermin dose (cohorts 2PþH and 2P) or the corresponding time for cohort 2Ctrl.The buccal biopsies were taken from each side of the mouth, and the order of mouth side was randomized. For cohort 2PþH, the heparin infusion was stopped approximately 2 hours before the biopsy procedure on day 4. The tail from the biopsy punch was placed in a container of 10% neutral buffered formalin for 24–30 hours for fixation. Each biopsy was then transferred to a container of ethanol and shipped at ambient temperature to Covance Laboratories Ltd. (North Yorkshire, UK), for Ki67 assessment. Safety. Safety assessments were conducted throughout the studies and included vital signs, physical examinations, electrocardiograms, and blood and urine laboratory tests. Statistical Analyses For both studies, pharmacokinetic parameters of palifermin were compared between treatment with palifermin-heparin and palifermin only using an analysis of

variance, with the natural log of the parameter as the dependent variable and treatment as the independent variable. The geometric means of the treatments, the ratio of geometric means, and the 90% confidence interval (CI) for the ratio were calculated. The same approach was used to compare the 2 treatments with respect to the PD results (aPTT) in study 1. For study 2, mixed-effect analysis of covariance was used to compare the palifermin PD results (Ki67) for the 2 treatments. The dependent variable was log-transformed, and subject was used as a random effect. The logtransformed baseline value was used as a covariate. All data analyses were performed using SAS (SAS Institute Inc., Cary, North Carolina). Pharmacokinetic parameters for study 1 were derived using noncompartmental methods with WinNonlin Professional (Pharsight Corp., Mountain View, California).

Results Study Subjects Study 1. Eighteen subjects were randomized to the palifermin-heparin group (cohort 1PþH) and 12 subjects to the palifermin-only group (cohort 1P). Five subjects from cohort 1PþH, and 9 study-naive subjects were enrolled in cohort 1H. Four subjects from cohort 1PþH discontinued the study before receiving palifermin because of not achieving the target aPTT level during the heparin titration period (3 subjects) or withdrawing consent (1 subject). Because these 4 subjects had received heparin but not palifermin, they were included in the safety analysis for cohort 1H, but not cohort 1PþH. All other subjects (35 of 39) completed the study. All subjects were male and predominantly white (Table 1). The 3 cohorts were generally well-matched. Study 2. A total of 44 subjects were randomized in study 2: 20 subjects in the palifermin-heparin group (cohort 2PþH), 16 subjects in the palifermin-only group (cohort 2P), and 8 subjects in the control group (cohort 2Ctrl). In cohort 2PþH, 5 subjects did not achieve the target aPTT level during the heparin titration period and were discontinued from the study. All other subjects (39 of 44) completed the relevant study assessments. Similar to study 1, all subjects were male and predominantly white, and the 3 cohorts were considered balanced. Subjects in cohort 2P were slightly older than subjects in cohorts 2PþH and 2Ctrl, but this was not thought to affect the study conclusions (Table 1). Palifermin Pharmacokinetics (Studies 1 and 2) No endogenous KGF concentrations were detected before heparin administration or 24 hours after initiation of heparin infusion. Following administration of palifermin alone, the palifermin serum concentration declined more

12 12 (100.0) 7 (58.0) 4 (33.0) 1 (8.0) 0 (0) 25.6 (8.4) 23.0 18, 43 176.0 (7.3) 176.5 159, 189 76.6 (10.9) 78.3 50.8, 90.5 24.6 (2.8) 23.9 20.1, 28.7

18 18 (100.0) 14 (78.0) 3 (17.0) 0 (0) 1 (6.0) 28.8 (7.8) 26.5 18, 43 178.5 (6.5) 179.0 163, 189 83.8 (11.3) 82.6 67.6, 105.3 26.3 (3.2) 27.2 19.0, 30.0

Palifermin Only

Palifermin þ Heparin

26.2 (2.5) 27.4 21.5, 28.9

83.0 (8.6) 84.4 68.3, 94.1

178.1 (7.9) 179.1 163, 188

28.0 (7.7) 24.0 20, 43

12 (86.0) 1 (7.0) 1 (7.0) 0 (0)

14 (100.0)

14

Heparin Only

1H

25.0 (2.1) 25.0 22.1, 28.6

81.2 (8.3) 78.3 68.4, 100.5

180.4 (7.4) 180.2 165, 195

25.9 (5.3) 24.0 19, 40

12 (80.0) 3 (20.0) 0 (0) 0 (0)

15 (100.0)

15

Palifermin þ Heparin

2PþH

SD, standard deviation; BMI, body mass index. Note: 5 subjects who received heparin þ palifermin in cohort 1PþH also received heparin in cohort 1H. These 5 subjects are counted within each group.

n Sex, n (%) Men Race/ethnicity, n (%) White or Caucasian Black or African American Hispanic or Latino Asian Age (y) Mean (SD) Median Min, Max Height (cm) Mean (SD) Median Min, Max Weight (kg) Mean (SD) Median Min, Max BMI (kg/m2) Mean (SD) Median Min, Max

Cohort/Treatment

1P

Study 1 1PþH

Table 1. Baseline Characteristics of Study Subjects

26.1 (2.1) 26.4 22.6, 29.9

81.1 (8.0) 83.3 69.4, 95.0

176.3 (5.5) 176.4 164, 185

32.9 (7.8) 31.5 22, 44

14 (87.5) 2 (12.5) 0 (0) 0 (0)

16 (100.0)

16

Palifermin Only

2P

Study 2

26.0 (3.2) 26.5 20.0, 30.0

81.3 (11.8) 83.1 58.3, 94.6

176.7 (5.1) 176.3 171, 185

22.8 (3.6) 24.0 18, 27

7 (87.5) 1 (12.5) 0 (0) 0 (0)

8 (100.0)

8

No Treatment

2Ctrl

1112 The Journal of Clinical Pharmacology / Vol 55 No 10 (2015)

Yang et al

than 98% within the first 30 minutes after administration (for study 1: from mean  SD 598  300 ng/mL at 2 minutes to 3.86  1.51 ng/mL at 30 minutes). A slight increase and plateau of the palifermin concentration was observed from 1 to 4 hours after palifermin administration, which was followed by a terminal decline phase (Figure 1). During coadministration with heparin, the initial decrease in serum palifermin concentrations was less (for study 1, from 774  426 ng/mL at 2 minutes to 94.7  30.5 ng/mL at 30 minutes), the plateau occurred earlier (approximately at 30 minutes) and was shorter, and the terminal phase showed a more rapid decline in palifermin concentration. Administration of a single dose of palifermin 60 mg/kg together with heparin increased the mean AUC of palifermin approximately 5 times compared with when palifermin was administered alone, suggesting that the mean CL value of palifermin decreased by 80% (Table 2). Coadministration of heparin also decreased the Vss of palifermin by 74%, and reduced the mean t1/2,z of

1113 palifermin by 44% (2.5 versus 4.5 hours). Because of a rapid decline in the palifermin concentration during the first 30 minutes after palifermin administration, greater variability was observed for the C0, which was estimated by back-extrapolation to time 0 using the first 2 observed concentration values. This pharmacokinetic drug–drug interaction between palifermin and heparin persisted after multiple daily administration of palifermin at 40 mg/kg in study 2 (Figure 1). On day 3, in the presence of heparin, the mean palifermin AUC0–24 increased 4.3-fold, the mean clearance decreased by 76%, the mean Vss decreased by 73%, and the mean t1/2,z was shorter (2.1 vs 3.7 hours; Table 2). Heparin Pharmacodynamics (Study 1) The mean aPTT values for a 24-hour period during the heparin maintenance phase and on day 1 for the palifermin-heparin and heparin-only cohorts are presented in Figure 2. In general, the mean aPTT values were similar between these 2 cohorts at each time; the relatively higher mean value at 18 hours for the heparin-only cohort was due to an extreme value in 1 subject (200 seconds). The comparisons of the aPTT AUCs within each cohort and between cohorts are presented in Table 3. The ratios of geometric means were close to 1.0, suggesting that concomitant administration of palifermin did not alter the PD activity (aPTT) of heparin. Palifermin Pharmacodynamics (Study 2) One subject in the palifermin-heparin cohort was excluded from the palifermin PD analysis because of a missing baseline Ki67 result. The Ki67 levels at baseline were comparable among cohorts except for 1 subject in the palifermin-heparin cohort with an exceptionally high baseline Ki67 (470 counts/mm), which contributed to a lower Ki67 day 4/baseline ratio in the palifermin-heparin group (238 counts/mm on day 4). For the control cohort, the Ki67 levels at baseline and on day 4 were similar, whereas a trend of increased Ki67 levels on day 4 relative to baseline was observed in both palifermin treatment cohorts (Figure 3). The individual day 4/baseline Ki67 ratios in both palifermin cohorts were, with few exceptions, higher than 1, with a mean of 1.37 for the palifermin-heparin cohort and 1.55 for the paliferminonly cohort (Figure 4). The ratio (palifermin-heparin versus palifermin only) of geometric means of day 4/ baseline Ki67 ratios was 0.863 (90%CI, 0.759 to 0.982), indicating a slightly lower effect when palifermin is coadministered with heparin compared with when palifermin is administered alone (Table 4).

Figure 1. Mean serum palifermin concentration–time profiles after administration of palifermin with or without heparin coadministration.

Safety In studies 1 and 2, a single intravenous dose (60 mg/kg) or multiple intravenous doses (40 mg/kg per day) of palifermin administered alone or with continuous

1114

The Journal of Clinical Pharmacology / Vol 55 No 10 (2015)

Table 2. Statistical Summary of Palifermin Pharmacokinetic Parameters After Administration of Palifermin With or Without Heparin Coadministration

Parameter

C0 (ng/mL) AUC0–inf (ng  h/mL) CL (mL  h/kg) Vss (mL/kg) t1/2,z (h) C0 (ng/mL) AUC0–24 h (ng  h/mL) CL (mL  h/kg) Vss (mL/kg) t1/2,z (h)

Palifermin þ Heparin (Test)

Palifermin Only (Reference)

Geometric Mean

Geometric Mean

n

n

Ratio (Test/Ref)

Study 1: single intravenous administration of 60 mg/kg palifermin 885.2 14 904.9 12 0.98 402.4 14 81.2 12 4.95 149.1 14 738.7 12 0.20 394.1 14 1,536.8 12 0.26 2.5 14 4.5 12 0.56 Study 2: multiple intravenous administration of 40 mg/kg per day palifermin — study day 3 361.0 15 730.7 15 0.49 328.1 15 86.9 15 3.78 121.9 15 460.4 15 0.27 361.7 15 1017 15 0.36 2.1 15 3.7 16 0.57

intravenous infusion of unfractionated heparin was well tolerated by healthy subjects. No subject discontinued the studies because of adverse events (AEs), and no serious or severe AEs were reported. In study 1, one subject had an elevation of lipase after treatment with palifermin in combination with heparin, an elevation with a severity grade of 1 on day 4 according to the Common Terminology Criteria for Adverse Events (CTCAE) v.4. The value was normalized by the end of the study. There were no increases in amylase seen after treatment with a single intravenous dose of palifermin.

Figure 2. Mean (SD) aPTT values after intravenous infusion of heparin with or without palifermin coadministration (study 1). For the heparin þ palifermin treatment group, pre- and postdose refer to the administration of palifermin. For the heparin-only treatment group, preand postdose refer to the corresponding time, even though no palifermin was given. The relatively higher mean and large SD at 18 hours for the heparin postdose treatment is due to a single outlier value.

90% Confidence Interval Lower

Upper

0.60 3.93 0.16 0.18 0.49

1.60 6.25 0.25 0.37 0.63

0.13 2.77 0.19 0.21 0.48

1.93 5.14 0.36 0.61 0.68

In study 2, reversible, asymptomatic elevations in amylase and lipase occurred in subjects receiving multiple intravenous doses (40 mg/kg per day) of palifermin. Compared with baseline, mean lipase levels increased up to the upper level of normal, with a maximum increase in 2 subjects of CTCAE v.4 grade 3, on treatment with palifermin alone. Maximum increases were seen on day 5 and resolved by day 8. When multiple doses of palifermin were given with heparin a decrease in lipase of approximately 36% compared with baseline was seen. When palifermin alone was given, there was a mean amylase increase within the reference range, with a maximum increase in 1 subject of CTCAE v.4 grade 2 on day 5. A less pronounced increase in amylase, all values within reference limits and also with a maximum on day 5, was seen when palifermin was given in combination with heparin. After day 5, values gradually decreased in both groups. No changes, either in lipase or amylase, were seen in the control group. In study 1, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) values remained within the reference range for all subjects receiving palifermin only. However, AST and ALT values for subjects receiving palifermin concurrently with heparin were higher at most times, but did not differ from those seen in subjects receiving heparin only. Two subjects, 1 receiving palifermin concurrently with heparin and 1 receiving heparin only experienced transient CTCAE v.4 grade 3 elevations of AST, both without associated bilirubin increases. In study 2, reversible, asymptomatic elevations in AST and ALT occurred in subjects receiving palifermin concurrently with heparin. One subject experienced a transient AST elevation of CTCAE v.4 grade 3, in association with a grade 2 elevation of ALT. There was no concomitant bilirubin elevation. Maximum increases

1115

Yang et al Table 3. Statistical Comparisons of Heparin Pharmacodynamic Parameters (Study 1) Within-Group Comparisons Day 1 (Test) Parameter

LSM

Palifermin þ heparin (cohort 1PþH) AUCaPTT,0–6 (s  h) 289.5 AUCaPTT,0–24 (s  h) 1,144.1 Heparin only (cohort 1H) AUCaPTT,0–6 (s  h) 276.6 AUCaPTT,0–24 (s  h) 1,138.6

Heparin Maintenance (Reference)

Ratio

90% Confidence Interval

n

LSM

n

(Test/Ref)

Lower

Upper

14 14

302.2 1,164.8

14 14

0.96 0.98

0.91 0.91

1.01 1.06

0.94 1.01

0.89 0.94

0.99 1.09

14 14

Heparin þ Palifermin (1PþH) (Test)

294.2 14 1,122.0 14 Between-Group Comparisons Heparin Only (1H) (Reference)

Ratio

90% Confidence Interval

Parameter

LSM

n

LSM

n

(Test/Ref)

Lower

Upper

AUCaPTT,0–6 (ratio) AUCaPTT,0–24 (ratio)

0.96 0.98

14 14

0.94 1.01

14 14

1.02 0.97

0.95 0.90

1.10 1.04

LSM, least squares mean. Note: 5 subjects who received heparin þ palifermin (cohort 1PþH) also received heparin alone (cohort 1H).

were seen on day 5, after which the values gradually normalized. In subjects receiving only palifermin there were only isolated elevations of transaminases, no elevation was more than CTCAE v.4 grade 1. The combined study results indicate that elevations in liver tests were predominantly due to heparin.

Discussion Two clinical phase 1 studies were conducted to assess the PK and PD drug–drug interactions between palifermin

Figure 3. Baseline and day 4 Ki67 expression in oral mucosa after multiple-dose administration of palifermin with or without heparin coadministration (study 2). The bottom and top of the boxes are the 25th and 75th percentiles, respectively; the band of the box is the median value; the ends of the whiskers represent the minimum and maximum values.

and heparin. Results demonstrated a pronounced PK drug–drug interaction between palifermin and heparin. Without heparin coadministration, the palifermin PK profiles after a single intravenous dose or multiple daily intravenous doses of palifermin were similar to those reported in the literature.13–15 With heparin coadministration, the serum concentrations of palifermin were generally higher, the palifermin concentration plateau occurred earlier and was shorter, and the terminal phase showed a more rapid decline. These changes resulted in a 4- to 5-fold increase in AUC and a 40%–45% decrease in t1/2,z, suggesting an approximately 70%–80% decrease in CL and Vss. The differences caused by heparin coadministration were similar in these 2 studies as well as after the first and third doses of palifermin. Similar PK drug interactions with heparin have also been reported for other FGF members in animals and humans16–19 and for hepatocyte growth factor (HGF) in rats.20 These PK drug interactions with heparin are not unexpected given the current knowledge about interactions between growth factors and glycosaminoglycans that include heparin and heparan sulfate; the latter can be found ubiquitously in vivo. Two or three heparan sulfate chains are attached to proteins in the extracellular matrix or on the cell surface to form a proteoglycan (HSPG) that binds to a variety of protein ligands, including FGFs and HGF.21 FGFs, once produced and released by cells, are quickly associated with the extracellular HSPGs; this association may serve 2 physiological purposes: protecting the FGFs from degradation by proteases and creating a local reservoir of growth factors.22 FGFs also can interact with cell-surface HSPGs, which has been shown to be essential for FGF interaction with FGF receptors and

1116

The Journal of Clinical Pharmacology / Vol 55 No 10 (2015)

activity of palifermin, as measured by Ki67 expression, was not significantly affected. Similar findings have been reported for HGF; in the presence of heparin, the clearance of HGF was significantly reduced, but the mitogenic activity of HGF was only slightly reduced.20 The authors suggest that heparin mainly inhibits lowaffinity HGF uptake by complexing with HGF, where its effect on the high-affinity binding site is relatively minor. Results from Reich-Slotky et al26 showed that a majority of low-affinity binding sites for KGF are not heparin related; therefore, these low-affinity sites may play a role in increasing the mitogenic activity of KGF, whereas the binding of KGF to heparin may interfere with binding to the heparin-unrelated low-affinity sites. The increased exposure to palifermin during coadministration with heparin also did not have an apparent impact on the PD activity of heparin, as measured by aPTT, or on its safety. The number of subjects who experienced AEs was generally similar across all treatment groups. There were no severe or serious AEs, and no discontinuations of study medication because of AEs. Administration of palifermin concurrently with heparin resulted in increases in liver test values compared with when palifermin was administered alone. These elevations were transient, asymptomatic, and not different from those observed when heparin was administered alone. This suggests that the increases in liver test values in the palifermin-heparin group could be attributed to heparin, a drug known to increase liver test values.30 As also previously reported,13–15,31 treatment with palifermin only in the current studies was associated with transient, asymptomatic, and reversible increases in serum amylase and lipase that were clinically nonsignificant. During coadministration with heparin, an increase in amylase was also seen, albeit somewhat less pronounced than when palifermin was administered alone. This may be explained by the altered distribution of palifermin when coadministered with heparin. In the combined prior studies there were CTCAE grades 3 and 4 elevations of both amylase and lipase. When correcting for the frequency in placebo-treated subjects, the frequency of amylase and lipase increases of CTCAE grades 3 and 4 elevations was 7% and 6%, respectively. In the current studies there were no CTCAE grade 3 or 4 elevations of

Figure 4. Individual day 4/baseline ratios for Ki67 expression in oral mucosa after multiple-dose administration of palifermin with or without heparin coadministration (study 2).

subsequent receptor signal transduction.23–25 The binding of FGF to HSPGs has been described as low affinity and high capacity, whereas the binding to the FGF receptor is considered high affinity and low capacity.26,27 FGFs can be internalized through binding to the FGF receptors or cell-surface HSPGs. It has been shown that the KGF-KGFR complex is internalized through clathrincoated pits and transported to early endosomes, where the complex is sorted to late endosomes and ultimately degraded in lysosomes.28 The high binding capacity by HSPGs explains the dramatic decrease in the serum concentration of palifermin within 30 minutes after intravenous administration, and the slight increase in the palifermin concentration profile around 1–4 hours after intravenous administration is attributed to the dissociation of palifermin from extracellular HSPGs. Several mechanisms have been postulated for the reduced distribution and clearance of growth factors by heparin. First, heparin protects growth factors from proteolysis.29 Second, growth factors form complexes with heparin that are more slowly cleared from circulation than uncomplexed growth factors.18 And, third, heparin competes with the binding of growth factors to the cellsurface HSPG and reduces the binding and cellular uptake of growth factors.16,19 Although coadministration of heparin resulted in a 4to 5-fold increase in the exposure to palifermin, the PD

Table 4. Statistical Comparisons of Palifermin Pharmacodynamic Parameter (Study 2) Palifermin-Heparin (2PþH)

Palifermin Only (2P)

90% Confidence Interval

Parameter

LSM

n

LSM

n

Geometric Mean Ratio

Lower

Upper

Ki67 observed data Ki67 natural log transformed data

1.373 0.267

14 14

1.547 0.414

16 16

0.863

0.759

0.982

LSM: Least square mean.

1117

Yang et al

amylase and only 2 grade 3 increases in lipase, all asymptomatic. Considering the small number of subjects, the slightly lower frequency of amylase increases, and the slightly higher frequency of lipase increases in these studies are considered in line with what could be expected. Coadministration of palifermin and heparin decreased serum lipase, relative to baseline. It is known that heparin releases stored lipase, which is then degraded by the liver. Once stores are exhausted, serum lipase levels are lowered, as the delivery of newly synthesized lipase molecules is relatively slow.32 This effect is a plausible explanation for why lipase levels in the palifermin-heparin group are lowered from baseline to day 5, despite palifermin being known to increase lipase levels. The safety data from study 1 were compared with an earlier phase 1 study13, in which healthy subjects received a single intravenous dose of palifermin ranging from 60 to 250 mg/kg. In both studies palifermin, administered alone at 60 mg/kg, was tolerable, and no skin or oral AEs were observed. In contrast, although administration of palifermin at 60 mg/kg concurrently with heparin yielded an AUC similar to that observed for the palifermin 250 mg/kg cohort, erythema and other skin events observed in the majority of subjects in the 250 mg/kg cohort were not observed in study 1. This indicates that the safety of palifermin at 60 mg/kg is not negatively affected by concomitant use of heparin at an aPTT of 1.5–2.0 times the baseline. In conclusion, coadministration of palifermin with heparin resulted in an increase in the serum concentration of palifermin and a decrease in Vss, clearance and t1/2,z compared with palifermin administered alone. The increase in serum exposure of palifermin in the palifermin-heparin group did not affect the PD effect of palifermin (Ki67 expression) or the anticoagulant activity of heparin and did not increase the number/frequency of safety findings as compared with administration of palifermin only. Most patients treated with palifermin are critically ill, and treatment with heparin can be indicated. Results from these studies suggest that dose adjustments for heparin and palifermin are not warranted when they are administered concurrently. Author Contributions B-B.Y., M.R., B.O., B.G., B.S., W.S., A.L., and T.K. wrote the article. B-B.Y., B.G., B.S., A.L., B.O., and M.R. designed the studies. W.S. performed the studies. B.G. and M.R. oversaw conduction of the study. B.S., A.L., B-B.Y., B.O., B.G., M.R., and T.K. analyzed and interpreted the data.

Declaration of Conflicting Interests Dr. Yang, Dr. Gillespie, and Dr. B. Smith are currently or were previously employed at Amgen Inc and are shareholders of

Amgen Inc. Ms. Lissmats, Mr. Rudebeck, Dr. Kullenberg, and Ms. Olsson are currently or were previously employed at Swedish Orphan Biovitrum and are shareholders of Swedish Orphan Biovitrum. Dr. W. Smith declares no conflicts of interest.

References 1. Keefe DM. Intestinal mucositis: mechanisms and management. Curr Opin Oncol. 2007;19:323–327. 2. Sonis ST. Mucositis: the impact, biology, and therapeutic opportunities of oral mucositis. Oral Oncol. 2009;45:1015–1020. 3. Farrell CL, Bready JV, Rex KL, et al. Keratinocyte growth factor protects mice form chemotherapy and radiation-induced gastrointestinal injury and mortality. Cancer Res. 1998;58:933–939. 4. Farrell CL, Rex KL, Chen JL, et al. The effect of keratinocyte growth factor in preclinical models of mucositis. Cell Prolif. 2002;35:78–85. 5. Blijlevens N, Sonis S. Palifermin (recombinant keratinocyte growth factor-1): a pleiotropic growth factor with multiple biological activities in preventing chemotherapy- and radiotherapy-induced mucositis. Ann Oncol. 2007;18:817–826. 6. McDonnell AM, Lenz KL. Palifermin: role in the prevention of chemotherapy- and radiation-induced mucositis. Ann Pharmacother. 2007;41:86–94. 7. Niscola P, Scaramucci L, Giovannini M, et al. Palifermin in the management of mucositis in hematological malignancies: current evidences and future perspectives. Cardiovasc Hematol Agents Med Chem. 2009;7:305–312. 8. Kepivance1 European Summary of Product Characteristics. Swedish Orphan Biovitrum AB (publ), Stockholm, 2013. 9. Strain AJ, McGuinness G, Rubin JS, Aaronson SA. Keratinocyte growth factor and fibroblast growth factor action on DNA synthesis in rat and human hepatocytes: modulation by heparin. Exp Cell Res. 1994;210:253–259. 10. Hsu YR, Nybo R, Sullivan JK, et al. Heparin is essential for a single keratinocyte growth factor molecule to bind and form a complex with two molecules of the extracellular domain of its receptor. Biochemistry. 1999;38:2523–2534. 11. Carter BL. Therapy of acute thromboembolism with heparin and warfarin. Clin Pharm. 1991;10:503–518. 12. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311–322. 13. Zia-Amirhosseini P, Salfi M, Leese P, et al. Pharmacokinetics, pharmacodynamics, and safety assessment of palifermin (rHuKGF) in healthy volunteers. Clin Pharmacol Ther. 2006;79:558–569. 14. Zia-Amirhosseini P, Hurd DD, Salfi M, Cheah TC, Aycock J, Cesano A. Pharmacokinetics of palifermin administered as the standard dose and a collapsed dose in patients with hematologic malignancies. Pharmacotherapy. 2007;27:1353–1360. 15. Gillespie B, Zia-Amirhosseini P, Salfi M, et al. Effect of renal function on the pharmacokinetics of palifermin. J Clin Pharmacol. 2006;46:1460–1468. 16. Rosengart TK, Kuperschmid JP, Maciag T, Clark RE. Pharmacokinetics and distribution of heparin-binding growth factor I (endothelial cell growth factor) in the rat. Circ Res. 1989;64:227–234. 17. Whalen GF, Shing Y, Folkman J. The fate of intravenously administered bFGF and the effect of heparin. Growth Factors. 1989;1:157–164. 18. Bush MA, Samara E, Whitehouse MJ, et al. Pharmacokinetics and pharmacodynamics of recombinant FGF-2 in a phase I trial in coronary artery disease. J Clin Pharmacol. 2001;41:378–385. 19. Xia X, Babcock JP, Blaber SI, Harper KM, Blaber M. Pharmacokinetic properties of 2nd-generation fibroblast growth

1118

20.

21. 22.

23.

24.

25.

26.

factor-1 mutants for therapeutic application. PLoS One. 2012;7: e48210. Liu KX, Kato Y, Kato M, Kaku TI, Nakamura T, Sugiyama Y. Existence of two nonlinear elimination mechanisms for hepatocyte growth factor in rats. Am J Physiol. 1997;273:E891–E897. Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their receptors, and signaling. Endocr Relat Cancer. 2000;7:165– 197. Pellegrini L, Burke DF, von Delft F, Mulloy B, Blundell TL. Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature. 2000;407:1029–1034. Schlessinger J, Plotnikov AN, Ibrahimi OA, et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell. 2000;6:743– 750. Xu R, Ori A, Rudd TR, et al. Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity. J Biol Chem. 2012;287:40061–40073. Reich-Slotky R, Bonneh-Barkay D, Shaoul E, Bluma B, Svahn CM, Ron D. Differential effect of cell-associated heparin sulfates on the

The Journal of Clinical Pharmacology / Vol 55 No 10 (2015)

27.

28.

29.

30.

31.

32.

binding of keratinocyte growth factor (KGF) and acidic fibroblast growth factor of the KGF receptor. J Biol Chem. 1994;269:32279– 32285. LaRochelle WJ, Sakaguchi K, Atabey N, et al. Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor. Biochemistry. 1999;38:1765–1771. Belleudi F, Ceridono M, Capone A, et al. The endocytic pathway followed by the keratinocyte growth factor receptor. Histochem Cell Biol. 2002;118:1–10. Damon DH, Lobb RR, D’Amore PA, Wagner JA. Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biologic half-life. J Cell Physiol. 1989;138:221–226. Carlson MK, Gleason PP, Sen S. Elevation of hepatic transaminases after enoxaparin use: case report and review of unfractionated and low-molecular-weight heparin-induced hepatotoxicity. Pharmacotherapy. 2001;21:108–113. Spielberger R, Stiff P, Bensinger W, et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med. 2004;351:2590–2598. N€asstr€ om B, Olivecrona G, Olivecrona T, Stegmayr BG. Lipoprotein lipase during continuous heparin infusion: Tissue stores become partially depleted. J Lab Clin Med. 2001;138(3):206–213.