The recombinant fusion protein linking the human coagulation factor IX (FIX) to recombinant human albumin (rIX-FP) is currently investigated in clinical.
Quantitative whole body autoradiography (QWBA) study on the effect of albumin fusion on the biodistribution of recombinant factor rFIX Eva Herzog1, Stephen Harris2, Andrew McEwen2, Ingo Pragst1, Gerhard Dickneite1, Stefan Schulte1 and Sabine Zollner1 1CSL
Behring GmbH, Preclinical Research and Development, 35041 Marburg, Germany. 2Quotient Bioresearch, Metabolic Chemistry, Rushden, UK.
INTRODUCTION
RESULTS
The recombinant fusion protein linking the human coagulation factor IX (FIX) to recombinant human albumin (rIX-FP) is currently investigated in clinical phase II/III trials (PROLONG-9FP) for prophylaxis and on-demand treatment of bleeding in haemophilia B patients. However, to date, there is only limited information available on the tissue distribution of FIX products following intravenous administration.
Figure 1: Radiochemical purity of FIX activity of rIX-FP following [3H]-labelling
Figure 3: QWBA images obtained at 0.25 to 24 h after [3H]-rIX-FP dosing
Figure 4: % total radioactive dose per tissue at 0.25 and 24 h after [3H]-rIX-FP, [3H]-rFIX, or [3H]-albumin dosing
Characterisation of labelled protein •
Plasma analysis •
% Specific activity
120
AIM
100
60 40
•
0 rIX-FP
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(BeneFIX®, Pfizer Inc.) or [3H]-albumin (Recombumin®, Novozymes Biopharma) via the tail vein. Following dosing, a single animal was selected for quantitative whole-body autoradiography (QWBA) at the following time points: [3H]-rIX-FP and [3H]-albumin: 0.25, 1, 3, 8, 24, 72, 120 and 240 h; [3H]-rIX: 0.25,
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The animals selected for autoradiography at 240 h ([3H]-rIX-FP; [3H]-albumin) or
b
a
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Overall recovery of radioactivity was approximately 81% of the total [3H]rIX-FP dose within 240 h (Table 1).
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The major route of elimination of [3H]-rIX-FP, [3H]-rFIX and [3H]-albumin was urinary (Table 1)
A terminal blood sample was used for pharmacokinetic (PK) analysis.
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The animals were then perfused with phosphate buffered saline and sections of the snap-frozen carcass moulded in 1% carboxymethylcellulose were analyzed at up to five different sectioning levels.
Accounting for 73% of [3H]-rIX-FP elimination within 240 h
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Accounting for 51% of [3H]-rFIX elimination within 24 h
more detailed analysis of the knee joint.
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Liquid scintillation counting (LSC) was used to determine the radioactivity concentrations of dose formulation, plasma, urine, cage wash and oxidized faeces.
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Proportions of radioactivity in purified [3H]-labelled protein solutions (formulation,
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plasma, urine) were determined and quantified by size exclusion and high performance liquid chromatography (HPLC).
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Quantification of tissue concentrations of radioactivity were calculated based on a
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Series of [3H]-blood standards prepared using control blood and [3H]-glycine (target range 10 – 10000 nCi/g) exposed to phosphor bio-image plates alongside whole body sections followed by bio-image analysis using a Fuji FLA-5100 Fluorescent Image Analyser.
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Longest retention of [3H]-rlX-FP and [3H]-rFIX was seen in the bone endosteum with [3H]-rlX-FP signals detectable up to 240 h (Figure 3).
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Although most of the radioactivity in systemic circulation by 240 h after dosing could not be attributed to the intact, unchanged protein, the tissue distribution profile remained similar to that seen at earlier time points, suggesting that the signals may still be attributable to unchanged protein
50.0
55.0
45.0
50.0 45.0
40.0
40.0
35.0
35.0
30.0
30.0
Albumin
LMW component
25.0 20.0
20.0
15.0
10.0
10.0
5.0
5.0
0.0 0.00
0.0 5.00
10.00
15.00
20.00 mins
5.00
0.00
15.00
10.00
20.00min
0.25 hours
Whole body tissue distribution comparison •
The highest total dose per tissue was observed for the liver accounting for up to 20% of the [3H]-rlX-FP and [3H]-rFIX derived radioactivity (data not shown) and an average dose of 17% and 11% over 24 h, respectively (Figure 4).
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Whereas initial tissue radioactivity concentrations of [3H]-rlX-FP or [3H]rFIX were comparable (Figure 4, left column), tissue retention of [3H]rlX-FP and [3H]-albumin was prolonged compared to [3H]-rFIX, reaching plateau levels only after 3 - 8 h after dosing, resulting in elevated tissue concentrations at 24 h post dosing (Figure 4, right column).
Figure 6: QWBA images of the knee joint tissue obtained at 0.25 and 24 h after [3H]-rIX-FP or [3H]-rFIX dosing Table 1: Recovery of radioactivity in excreta (% of dose administered)
Sample Urine
Faeces
Cage Total ∑
Time (h)
[3H]-rIX-FP
[3H]-rFIX
[3H]-albumin
0-8
13.5
11.7
10.7
8-24
26.4
39.3
36.8
24-48
16.9
-
28.7
48-240
16.1
-
37.2
Subtotal
72.9
51.0
113.4
0-24
0.9
8.9
3.7
24-48
0.8
-
2.0
48-240
2.6
-
6.9
Subtotal
4.3
8.9
12.5
Subtotal
3.7
2.9
0.7
80.8
62.8
126.5
Figure 5: QWBA images obtained at 0.25 and 24 h after or [3H]-albumin dosing
[3H]-rIX-FP, [3H]-rFIX,
Chromatographic analysis determined that all radioactivity in urine was associated with a LMW component. This was true for [3H]-rIX-FP, [3H]rFIX and [3H]-albumin (data not shown).
After a single IV administration of [3H]-rlX-FP or [3H]-rFIX to rats, the tissues exhibiting the highest radioactivity concentration included the adrenal gland, kidney, bone endosteum, spleen, lung, liver, bone marrow, intestinal mucosa, myocardium, peri-dontal membrane, and glandular tissues (Figure 3 and 5)
55.0
[3H]-rIX-FP
60.0
In addition, one hind leg from each animal was used for separate sectioning and a
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•
•
•
[3H]-rIX-FP
c ps
cps
24 h ([3H]-rIX) QWBA were returned to glass metabolism cages to enable the separate collection of urine and faeces for estimation of excretion balance.
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Pharmacokinetic analysis confirmed a similar plasma half-life of [3H]-rIXFP and [3H]-albumin which was > 3 x longer compared to [3H]-rFIX (data not shown).
Whole body tissue distribution of [3H]-rlX-FP
1, 3 and 24 h.
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At 120 h, proportion of intact [3H]-rIX-FP had fallen < 1% (data not shown).
Eight albino rats received a single IV dose of 320-420 μCi/kg of [3H]-rIX-FP, [3H]-rFIX
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•
•
•
15.0
•
At 24 h, 73% of the [3H]-rIX-FP radioactivity could still be assigned to intact protein compared to only 44% of the [3H]-rFIX signal (data not shown).
MATERIAL AND METHODS
25.0
•
•
Excretion balance
[3H]-rIX-FP
Figure 2: Plasma profile of [3H]-rIX-FP at 8 and 24 h post dosing
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Up to 8 h post dose, all radioactivity detected in plasma corresponded to intact [3H]-rIX-FP; from 24 h on, additional peaks were observed corresponding to albumin (8.5 min) and a LMW component (13.4 min) (Figure 2).
80
20
The present study was designed to explore the biodistribution of recombinant Factor IX (rFIX) and how albumin fusion may affect it.
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[3H]-rIX-FP maintained its purity and biological activity following [3H]-NSP labelling (Figure 1)
Knee joint tissue distribution profile of [3H]-rlX-FP •
Detailed analysis of the knee joint showed high concentrations of radioactivity in the zone of calcified cartilage within the growth plate region, the bone endosteum (inner membranous lining) and the periosteum (outer membranous lining) shortly after dosing (Figure 6)
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The initial knee joint distribution profile of [3H]-rlX-FP or [3H]-rFIX was comparable (Figure 6, left panel)
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Retention of [3H]-rlX-FP within the knee joint was prolonged (Figure 6, right panel) with radioactivity signals deteable up to 240 h post dosing (data not shown).
CONCLUSION
The study shows that rIX-FP exhibits equal biodistribution compared to a marketed rFIX product (BeneFIX®), but clearly distinguishes itself by a prolonged presence in plasma and tissues allowing a reduction in dosing frequency leading to increased therapeutic convenience and compliance in patients.
