SYSTEMIC PHARMACOKINETICS AND
PHARMACODYNAMICS OF
INTRAVITREAL AFLIBERCEPT,
BEVACIZUMAB, AND RANIBIZUMAB
ROBERT L. AVERY, MD,* ALESSANDRO A. CASTELLARIN, MD,* NATHAN C. STEINLE, MD,*
DILSHER S. DHOOT, MD,* DANTE J. PIERAMICI, MD,* ROBERT SEE, MD,*
STEPHEN COUVILLION, MD,* MA’AN A. NASIR, MD,* MELVIN D. RABENA, BS,*
MAURICIO MAIA, P
HD,† SHERRI VAN EVEREN, PHARMD,† KHA LE, PHD,† WILLIAM D. HANLEY, PHD†
Purpose: To evaluate the systemic pharmacokinetics (PKs) of aflibercept, bevacizumab,
and ranibizumab in patients with neovascular age-related macular degeneration (AMD),
diabetic macular edema (DME), or retinal vein occlusion (RVO).
Methods: Prospective, open-label, nonrandomized clinical trial of patients with AMD,
DME, or RVO who were antivascular endothelial growth factor (VEGF) naïve or had not
received anti-VEGF for $4 months. Patients received 3 monthly intravitreal injections of
aflibercept 2.0 mg, bevacizumab 1.25 mg, or ranibizumab (0.5 mg for AMD/RVO, 0.3 mg for
DME). The main outcome measures were serum PKs and plasma free-VEGF concentra-
tions after the first and third injections.
Results: A total of 151 patients were included. In AMD/DME/RVO, systemic exposure to
each drug was highest with bevacizumab, then aflibercept, and lowest with ranibizumab.
Ranibizumab cleared from the bloodstream more quickly than bevacizumab or aflibercept.
Aflibercept treatment resulted in the greatest reductions in plasma free-VEGF relative to
baseline levels, whereas ranibizumab treatment resulted in the smallest decreases in
plasma free-VEGF.
Conclusion: The three anti-VEGF treatments examined in this analysis demonstrated
notable differences in systemic PKs. Generally, the reduction in plasma free-VEGF levels
correlated with elevated levels of circulating anti-VEGF agents, with the reduction in free-
VEGF levels greatest with aflibercept and least with ranibizumab.
RETINA 37:1847–1858, 2017
V
ascular endothelial growth factor (VEGF) is a crit-
ical mediator of physiological angiogenesis and
pathological angiogenesis.
1
Several ophthalmic dis-
eases, including neovascular (wet) age-related macular
degeneration (AMD), diabetic macular edema (DME),
and macular edema after retinal vein occlusion (RVO),
are characterized by abnormal angiogenesis and
increased vascular permeability in the retina, which
ultimately can lead to vision loss if left untreated.
2
The three most commonly prescribed anti-VEGF ther-
apy options available in clinical practice for the treat-
ment of AMD, DME, and RVO are aflibercept,
bevacizumab, and ranibizumab.
3–12
These 3 anti-VEGF treatments differ in their
molecular structure and properties. Aflibercept is
a fusion protein (115 kDa) comprising the second Ig
domain of human VEGFR1, the third Ig domain of
human VEGFR2, and the Fc region of a human
IgG1.
13
Aflibercept binds multiple isoforms of human
VEGF-A, VEGF-B, and placental growth factor.
11
The reported estimated terminal elimination half-life
of free aflibercept in plasma is 5 days to 6 days after
intravenous administration of doses ranging from
2 mg/kg to 4 mg/kg.
12
The vitreous half-life of afli-
bercept in rabbits is 3.63 days.
14
At the time of this
publication, there are no data available on the half-life
of aflibercept in the human eye. Ranibizumab
and bevacizumab bind to all human VEGF-A
isoforms.
15–17
Bevacizumab is a full-length monoclo-
nal antibody (149 kDa) that was developed for cancer
therapy.
16
In human nonvitrectomized eyes, the aque-
ous half-life of bevacizumab was 9.82 days.
18
1847
Ranibizumab is an anti-VEGF-A affinity-matured
monovalent monoclonal antibody fragment (48 kDa)
that was designed for ocular use.
15
It does not contain
the Fc antibody region, and hence, it is cleared from
the bloodstream more rapidly and has a short systemic
elimination half-life of 2 hours.
19
Using a population
pharmacokinetic (PK) analysis from 674 patients with
wet AMD, Xu et al
19
estimated the vitreous half-life of
ranibizumab to be 9 days. This approach, however,
is not applicable to bevacizumab and aflibercept
because upon entering the systemic circulation they
undergo recycling via FcRn receptor.
19
In primates,
the vitreous elimination half-life of ranibizumab is re-
ported to be 3 days.
20
The aqueous half-life of intra-
vitreal ranibizumab 0.5 mg is 7.19 days in human
nonvitrectomized eyes.
21
The systemic PK values of
ranibizumab in patients with RVO and DME were
shown to be similar to those in patients with AMD;
thus, systemic exposure of ranibizumab is not consid-
ered dependent on disease state.
22
Concerns have been raised regarding potential
adverse effects resulting from the systemic suppression
of VEGF from intraocular anti-VEGF treatments.
23
From clinical experience in oncology, there are known
adverse effects related to blocking VEGF in the sys-
temic circulation.
24
These adverse effects include car-
diovascular and arterial thromboembolic effects (ATE),
renal and gastrointestinal (GI) effects, and wound-
healing complications. The majority of comparative
safety data for intravitreal use of anti-VEGF therapeu-
tics come from large clinical trials in neovascular AMD
patients.
25–30
In patients with AMD, greater decreases
in serum and plasma free-VEGF have been observed
with bevacizumab and aflibercept compared with rani-
bizumab.
31–35
Additionally, several studies have
explored the impact of anti-VEGF therapeutics on
serum
36
and plasma
37,38
VEGF levels in patients with
AMD. However, studies comparing the systemic levels
of aflibercept, bevacizumab, and ranibizumab and their
relative effects on circulating VEGF in patients with
DME and RVO are lacking. Because systemic safety
concerns have recently been raised in the non-AMD
populations,
39
it is important to assess systemic PK of
intravitreal anti-VEGF agents in these groups as well.
The objective of the current prospective study was
to evaluate and compare serum drug concentrations
and plasma free-VEGF concentrations in patients with
AMD, DME, or RVO receiving intravitreal injections
of aflibercept, bevacizumab, or ranibizumab. Addi-
tionally, values were compared across indications to
identify if disease impacts any differences observed.
Methods
Study Design
The study protocol was Institutional Review Board
approved and was conducted in accordance with U.S.
Food and Drug Administration Good Clinical Practice
guidelines. Written informed consent was provided by
all patients for study participation. This study was
From the *California Retina Consultants, Santa Barbara, Califor-
nia; and †Genentech, Inc, South San Francisco, California.
Supported by Genentech, Inc, and California Retina Research
Foundation, Santa Barbara, CA. These sponsors participated in the
design and conduct of the studies; data collection, analysis, and
interpretation of results; and preparation, review, and approval of
the manuscript.
Portions of these data have been presented at the American
Society of Retina Specialists, Toronto, ON, Canada, August
24–28, 2013; American Academy of Ophthalmology, New
Orleans, LA, November 16–19, 2013; Associ ation for Research
in Vision and Ophthalmology, Orlando, FL, May 4–8, 2014;
Macula Society, Key Largo, FL, February 19–22, 2014.
R. L. Avery reports grants and nonfinancial support from Gen-
entech, Inc, during the conduct of the study; grants, personal fees,
and nonfinancial support from Genentech, Inc; personal fees from
Iridex; personal fees from Alcon; grants and personal fees from
Allergan; personal fees from Alimera; grants, personal fees, and
nonfinancial support from Regeneron; personal fees and nonfinan-
cial support from Replenish; grants and personal fees from Oph-
thotech; personal fees and nonfinancial support from Novartis;
personal fees from Johnson and Johnson; grants and personal fees
from QLT; and personal fees from Bausch and Lomb outside the
submitted work. In addition, R. L. Avery has a patent Intravitreal
Drug Delivery licensed to Replenish. D. J. Pieramici reports grants
and nonfinancial support from Genentech, Inc, during the conduct
of the study; grants and personal fees from Genentech, Inc; grants
and personal fees from Allergan; personal fees from Santen; and
personal fees from Alimera outside the submitted work.
N. C. Steinle reports grants and nonfinancial support from Genen-
tech, Inc during the conduct of the study; personal fees from
Regeneron; and research funding from Zeiss outside the submitted
work. A. A. Castellarin reports grants and nonfinancial support
from Genentech, Inc, during the conduct of the study; grants and
personal fees from Genentech, Inc; and consulting fees from Aller-
gan and Alimera outside the submitted work. S. Couvillion reports
grants and nonfinancial support from Genentech, Inc, during the
conduct of the study; grants and personal fees from Genentech, Inc,
outside the submitted work. R. See and M. A. Nasir report grants
and nonfinancial support from Genentech, Inc, during the conduct
of the study. D. S. Dhoot reports grants and nonfinancial support
from Genentech, Inc, during the conduct of the study and speaker
and research grants from Regeneron outside the submitted work.
M. D. Rabena reports grants and nonfinancial support from Gen-
entech, Inc, during the conduct of the study. He was an employee of
California Retina Consultants when the study was conducted and is
currently an employee of Genentech, Inc. S. Van Everen was an
employee of Genentech, Inc, at the time the study was conducted
and is currently an employee of REGENEXBIO. W. D. Hanley and
M. Maia are employees of Genentech, Inc. K. Le was an employee
of Genentech, Inc, at the time of the study and is currently an
employee of Agios Pharmaceuticals.
This is an open-access article distributed under the terms of the
Creative Commons Attribution-Non Commercial-No Derivatives
License 4.0 (CCBY-NC-ND), where it is permissible to download
and share the work provided it is properly cited. The work cannot
be changed in any way or used commercially without permission
from the journal.
Reprint requests: Robert L. Avery, MD, California Retina
Consultants, 525 East Micheltorena Street, Suite A, Santa Barbara,
CA 93103; e-mail: bobave@gmail.com
1848 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES
2017
VOLUME 37
NUMBER 10
Health Insurance Portability and Accountability Act
compliant and adhered to the tenets of the declaration
of Helsinki. This study is registered at clinicaltrials.
gov (NCT02118831).
This prospective study enrolled patients from several
offices of a single private practice (California Retina
Consultants, Santa Barbara, CA) who were naive to
anti-VEGF therapy or had not received treatment with
anti-VEGF for at least 4 months. Patients were sorted
into 3 groups based on eye disease (neovascular AMD,
DME, or RVO), with at least 15 patients per disease
state for each drug group. Patients received 3 monthly
intravitreal injections (re-treatment could be given
within 21–35 days from previous injection) of afliber-
cept 2.0 mg, bevacizumab 1.25 mg, or ranibizumab
(0.5 mg for AMD and RVO patients and 0.3 mg for
DME patients). Patients were excluded from the study
if they were unwilling to receive three monthly injec-
tions of an anti-VEGF agent, had been treated with
a systemic anti-VEGF agent for cancer in the past year,
were currently undergoing dialysis, or if they had
received a vitrectomy in the treated eye.
Sample Collection and Bioanalytical Methods
Blood samples were collected at screening, 3 hours
after injection, and at Days 1, 3, 7, and 28 after Dose 1
and Dose 3 for PK and systemic VEGF analyses.
Analyses of serum drug levels and plasma concen-
trations of free-VEGF have been described in detail
previously.
32
CTAD (citrate, theophylline, adenosine,
and dipyridamole) tubes were used for the collection of
plasma samples because of their ability to preserve
platelets and prevent activation.
40,41
VEGF was mea-
sured in plasma samples, as opposed to serum, to pre-
vent or minimize release of VEGF from platelets.
42
Serum levels of aflibercept, bevacizumab, and ranibi-
zumab were analyzed using solution phase enzyme-
linked immunosorbent assays (ELISA). The lower lim-
its of quantitation (LLOQ) for aflibercept, bevacizu-
mab, and ranibizumab were 1,000 pg/mL, 313 pg/
mL, and 15.0 pg/mL, respectively. Plasma concentra-
tions of free-VEGF were determined using the Quanti-
kine ELISA kit, with an LLOQ of 10 pg/mL (R&D
Systems, Minneapolis, MN). As noted by the manufac-
turers of this kit, this assay is not suited to measure
total VEGF in the presence of anti-VEGFs, because of
the binding properties of the capture and detection re-
agents used in the assay.However, this assay is com-
monly used for measuring free-VEGF levels in samples
from patients treated with anti-VEGF therapeutics.
Target-therapeutic complexes are in a state of binding
equilibrium, which we expect to be minimally affected
by the 1:2 sample dilution performed in this assay.
PK Analyses
PK parameters were analyzed using noncompartmen-
tal analysis, and descriptive statistics were used to
summarize the results. Because this was a PK study,
a formal sample size calculation was not done. Non-
compartmental PK analysis was used to evaluate drug
concentration data and determine area under the curve
(AUC) over 28 days (AUC
0–28
[Dose 1], AUC
60–88
[Dose 3]), peak concentration (C
max
), trough concentra-
tion (C
min
), and serum half-life for each anti-VEGF. To
better estimate the serum half-lives of bevacizumab and
aflibercept (which are estimated to have longer systemic
half-lives because of their Fc portion), a protocol
amendment was implemented in the middle of the study
to collect PK data at later time points after the third dose
if the patient had not received another injection the next
month for his or her eye disease. As a result, only
a small fraction of patient data was available for an
accurate half-life estimation, and the serum half-life
data were pooled over all three indications. Serum drug
concentrations of aflibercept, bevacizumab, and ranibi-
zumab and plasma free-VEGF levels that measured
lower than the LLOQ were assigned a value equal to
50% of the LLOQ (rather than a value of 0).
Results
Patients
A total of 151 patients were enrolled in this study,
57 in the AMD group, 46 in the DME group, and 48 in
the RVO group (Table 1).
Systemic Exposure of Aflibercept, Bevacizumab,
and Ranibizumab in Patients With AMD, RVO,
and DME
PK data for patients with AMD have been previously
published.
32
Mean systemic exposure data for the AMD,
DME, and RVO groups are summarized in Tables 2–4.
Subsequent to the initial injection (Day 0), C
max
was
reached at median time to C
max
(t
max
)of1,7,and1
day for aflibercept, bevacizumab, and ranibizumab,
respectively, for patients with AMD, DME, and RVO.
Based on the Dose 3/Dose 1 geometric mean ratios
of C
min
, C
max
, and AUC
0–28
, ranibizumab did not
demonstrate systemic accumulation between Dose 1
and Dose 3 as evidenced by accumulation ratios close
to 1 (Table 5); however, bevacizumab and aflibercept
seemed to exhibit systemic drug accumulation. Mean ±
SD serum half-lives after intravitreal administration
for aflibercept, bevacizumab, and ranibizumab were
SYSTEMIC PK OF ANTI-VEGFS IN AMD/DME/RVO
AVERY ET AL 1849
11.4 ± 4.8 days (n = 39), 18.7 ± 5.8 days (n = 7), and
5.8 ± 1.8 days (n = 43), respectively.
Systemic exposure for each anti-VEGF therapeutic
did not seem to differ by indication and was consistently
highest with bevacizumab, followed by aflibercept, and
lowest with ranibizumab (Figure 1). After Dose 3,
systemic exposure to aflibercept ranged 43- to 107-
fold, 7- to 13-fold, and 13- to 19-fold higher than sys-
temic exposure to ranibizumab, whereas that of bevaci-
zumab was 399- to 788-fold, 21- to 30-fold, and 67- to
91-fold higher than that of ranibizumab based on C
min
,
C
max
, and AUC, respectively, over the 3 indications.
Mean serum concentrations of aflibercept were higher
than its reported half-maximal inhibitory concentration
(IC
50
) for VEGF inhibition (0.068 nM)
43
at most time
points after Dose 1 and Dose 3 (Figure 1). Mean serum
concentrations of bevacizumab were also above its
reported IC
50
(0.668 nM)
43
at most time points after
Dose 3 for all indications (Figure 1). For Dose 1 and
Dose 3, mean serum concentrations of ranibizumab met
or exceeded its reported IC
50
(0.060 nM)
43
at the 3-hour
and 24-hour time points postinjection, but fell below the
IC
50
for the remaining time points.
Systemic Plasma Free-VEGF Levels
Mean free-VEGF levels in plasma were balanced at
baseline for each indication and were comparable with
values reported previously.
35,44
Mean baseline VEGF
levels are summarized in Table 1. It is important to
note that individual patient data for plasma free-VEGF
levels at baseline ranged widely, from below the as-
say’s LLOQ (10 pg/mL) to 264 pg/mL in patients with
AMD, from ,10 pg/mL to 558 pg/mL in patients with
DME, and from ,10 pg/mL to 615 pg/mL in patients
with RVO (Figure 2).
Mean plasma free-VEGF profiles over time after
intravitreal administration of aflibercept, bevacizumab,
and ranibizumab in the AMD, DME, and RVO groups
are shown in Figure 3. For Dose 1 and Dose 3, the
greatest decreases in plasma free-VEGF levels
were observed with aflibercept for all 3 indications
(Figure 3). Mean plasma VEGF levels in patients
who received aflibercept fell below the LLOQ 3 hours
postinjection and remained below the LLOQ at the
Day 1, 3, and 7 time points for all 3 indications.
Patients who received bevacizumab had notable de-
creases from baseline in free-VEGF levels; however,
in patients with DME and RVO, mean free-VEGF
levels remained above the LLOQ at all time points.
In patients with AMD, free-VEGF levels were below
the LLOQ after Dose 3 at the Day 1, 3, and 7 time
points. Patients who received ranibizumab experi-
enced the least amount of change in mean free-
VEGF levels. Overall, there were no notable changes
in mean and median free-VEGF levels from baseline
for ranibizumab (Figures 2 and 3) over all the 3
indications.
Discussion
This study provides evidence that aflibercept, bev-
acizumab, and ranibizumab exhibit different systemic
exposures and effects on systemic VEGF after intra-
vitreal injection. Systemic exposure of each anti-
VEGF drug did not seem to differ by indication and
was consistently highest with bevacizumab and lowest
with ranibizumab. Systemic ranibizumab concentra-
tions remained below its IC
50
(0.06 nM)
43
at most
observed time points for all 3 indications. However,
after the third doses, systemic levels of aflibercept and
bevacizumab exceeded their respective IC
50
for VEGF
inhibition for all 3 indications. Ranibizumab demon-
strated no systemic accumulation between Doses 1 and
3, whereas bevacizumab showed substantive drug
accumulation after Dose 3 compared with Dose 1,
which ranged from 30% to 95% accumulation based
on C
min
, C
max
, or AUC across indications (Table 5).
The only exception was when the accumulation ratio
was calculated using C
min
in patients with DME,
which showed no accumulation for any of the three
Table 1. Patient Demographics and Baseline Characteristics
Characteristic AMD (n = 57) DME (n = 46) RVO (n = 48)
Age, mean, years 76.9 59.7 67.3
Male, n (%) 28 (50.0) 24 (51.1) 27 (57.4)
Treatment group, n
Aflibercept 22 15 15
Bevacizumab 15 15 15
Ranibizumab 20 16 18
Baseline free-VEGF level, mean, pg/mL
Aflibercept 19.2 20.2 18.9
Bevacizumab 22.5 22.8 23.5
Ranibizumab 17.0 20.9 22.6
1850 RETINA, THE JOURNAL OF RETINAL AND VITREOUS DISEASES
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VOLUME 37
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Table 2. Mean Systemic Exposures of Aflibercept, Bevacizumab, and Ranibizumab in Patients With AMD
32
Parameter Aflibercept Bevacizumab Ranibizumab
Geometric Mean Ratio
(AFL/RBZ) [95% CI]
Geometric Mean Ratio
(BEV/RBZ) [95% CI]
First dose
C
max
, nM, mean (SD) 0.45 (0.29), n = 21 0.76 (0.31), n = 15 0.11 (0.13), n = 20 4.65 [3.07–7.05] 8.80 [5.59–13.87]
C
min
, nM, mean (SD) 0.05 (0.02), n = 20 0.45 (0.16), n = 15 0.002 (0.002), n = 19 37.28 [23.72–58.6] 321.61 [197.5–523.7]
AUC
0–28
,nMh, mean (SD) 4.32 (1.77), n = 20 16.10 (5.75), n = 15 0.46 (0.24), n = 19 9.49 [7.37–12.22] 35.73 [27.2–46.94]
Third dose
C
max
, nM, mean (SD) 0.58 (0.52), n = 21 1.47 (0.55), n = 15 0.08 (0.06), n = 18 6.74 [4.46–10.18] 20.97 [13.37–32.89]
C
min
, nM, mean (SD) 0.07 (0.03), n = 21 0.70 (0.29), n = 14 0.002 (0.002), n = 18 52.92 [33.83–82.8] 500.31 [304.51–822.03]
AUC
60–88
,nMh, mean (SD) 5.38 (1.77), n = 21 29.12 (10.35), n = 14 0.52 (0.59), n = 18 12.58 [9.33–16.96] 67.24 [48.26–93.68]
AFL, aflibercept; BEV, bevacizumab; RBZ, ranibizumab.
Table 3. Mean Systemic Exposures of Aflibercept, Bevacizumab, and Ranibizumab in Patients With DME
Parameter Aflibercept Bevacizumab Ranibizumab
Geometric Mean Ratio
(AFL/RBZ) [95% CI]
Geometric Mean
Ratio (BEV/RBZ) [95% CI]
First dose
C
max
, nM, mean (SD) 0.53 (0.39), n = 15 0.75 (0.24), n = 15 0.12 (0.19), n = 15 7.32 [4–13.4] 12.01 [6.57–21.97]
C
min
, nM, mean (SD) 0.04 (0.02), n = 15 0.35 (0.18), n = 15 0.0006 (0.0008), n = 15 79.75 [44.99–141.36] 844.04 [476.18–1496.1]
AUC
0–28
,nMh, mean (SD) 3.41 (1.32), n = 15 14.26 (4.67), n = 15 0.24 (0.08), n = 15 13.96 [10.44–18.67] 59.45 [44.47–79.47]
Third dose
C
max
, nM, mean (SD) 0.57 (0.33), n = 15 1.18 (0.45), n = 14 0.04 (0.03), n = 15 13.59 [9.63–19.18] 30.29 [21.34–43.01]
C
min
, nM, mean (SD) 0.045 (0.024), n = 12 0.42 (0.25), n = 14 0.0007 (0.0009), n = 15 107 [52.27–219.03] 787.55 [390.1–1589.93]
AUC
60–88
,nMh, mean (SD) 4.35 (1.36), n = 14 20.39 (6.83), n = 14 0.23 (0.08), n = 15 19.44 [14.66–25.76] 90.52 [68.3–119.97]
AFL, aflibercept; BEV, bevacizumab; RBZ, ranibizumab.
SYSTEMIC PK OF ANTI-VEGFS IN AMD/DME/RVO
AVERY ET AL 1851