Pomalidomide is nonteratogenic in chicken and
zebrafish embryos and nonneurotoxic in vitro
Chris Mahonya, Lynda Erskinea, Jennifer Nivenb, Nigel H. Greigc, William Douglas Figgd, and Neil Vargessona,1
Schools of aMedical Sciences and bMedicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; cIntramural
Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224; and dIntramural Research Program, National Cancer
Institute, National Institutes of Health, Bethesda, MD 20892
Edited* by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved June 13, 2013 (received for review April 23, 2013)
Thalidomide and its analog, Lenalidomide, are in current use clinically
for treatment of multiple myeloma, complications of leprosy and
cancers. An additional analog, Pomalidomide, has recently been
licensed for treatment of multiple myeloma, and is purported to be
clinically more potent than either Thalidomide or Lenalidomide.
Using a combination of zebrafish and chicken embryos together
with in vitro assays we have determined the relative anti-
inflammatory activity of each compound. We demonstrate that
in vivo embryonic assays Pomalidomide is a significantly more potent
anti-inflammatory agent than either Thalidomide or Lenalidomide. We
tested the effect of Pomalidomide and Lenalidomide on angiogenesis,
teratogenesis, and neurite outgrowth, known detrimental effects of
Thalidomide. We found that Pomalidomide, displays a high degree
of cell specificity, and has no detectable teratogenic, antiangiogenic
or neurotoxic effects at potent anti-inflammatory concentrations. This
is in marked contrast to Thalidomide and Lenalidomide, which had
detrimental effects on blood vessels, nerves, and embryonic develop-
ment at anti-inflammatory concentrations. This work has implications
for Pomalidomide as a treatment for conditions Thalidomide and
Lenalidomide treat currently.
cytoskeleton | Cox2 | CPS49 | drug screening
T halidomide, originally marketed as a nonaddictive non-
barbiturate sedative, was responsible in the 1950s and 1960s
for birth defects in more than 10,000 children (1, 2). Thalidomide
exhibits potent antiangiogenic and immunomodulatory effects, and
is currently used around the world to treat a range of conditions,
most notably multiple myeloma and erythema nodosum leprosum
(ENL; an inflammatory complication of leprosy) (2, 3). However,
long-term use of Thalidomide in adult patients can result in det-
rimental side effects, such as peripheral neuropathy (4, 5), and
carries the risk of inducing birth defects. Indeed, despite careful
controls on Thalidomide use and distribution, a large number of
babies with Thalidomide embryopathy have been born in the last
two decades in Brazil where it is administered mainly for the
treatment of leprosy and its complications (6, 7). Conse-
quently, there is intense interest in developing a more potent
anti-inflammatory form of Thalidomide with enhanced clinical
benefit, but fewer side effects.
Lenalidomide, a structural analog of Thalidomide licensed for
use around the world, is clinically more potently anti-inflammatory
than Thalidomide, with potentially fewer side effects (8, 9). How-
ever, long-term treatment with Lenalidomide can result in pe-
ripheral neuropathy (10, 11) and, as yet, its teratogenic properties
are uncertain, with only limited studies performed to date (12).
Another Thalidomide analog, Pomalidomide, has shown promising
treatment of relapsed and refractory multiple myeloma in phase I
and phase II clinical trials, including in patients that have been
treated previously with Thalidomide and Lenalidomide (13–15).
However, whether Pomalidomide is neurotoxic and/or teratogenic
is understudied and is the focus of this study.
Using a combination of in vivo and in vitro assays for terato-
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genesis, inflammation, angiogenesis, and neurotoxicity, we have
compared the relative activities of Thalidomide, Lenalidomide,
www.pnas.org/cgi/doi/10.1073/pnas.1307684110
and Pomalidomide. We found that Pomalidomide was potently
anti-inflammatory in embryonic and in vitro assays at signifi-
cantly lower concentrations than either Thalidomide or Lenali-
domide. Moreover, at potently anti-inflammatory concentrations
Lenalidomide was also teratogenic, antiangiogenic, and neuro-
toxic, whereas Pomalidomide was not. We further found that
Pomalidomide, but not Lenalidomide, was cell specific, inhibiting
inflammatory response cells but not endothelial cells. We con-
clude that our unique series of in vitro and in vivo assays provides
a fast and effective mechanism for determining drug efficacy and
potential side effects, and that Pomalidomide likely represents
a more effective and potentially safer option for treatment of
multiple myeloma, ENL, and other conditions.
Results
Pomalidomide Has Potent Anti-Inflammatory Activity at Significantly
Lower Concentrations than Thalidomide or Lenalidomide in in Vivo
Assays. Previous studies using in vitro assays have indicated
Pomalidomide is more potently anti-inflammatory than Thalid-
omide and Lenalidomide (16–19). Pomalidomide’s actions in
vivo and its potential to cause adverse side effects are unclear.
Here the immunomodulatory potency of Thalidomide and ana-
logs was investigated using both in vivo and in vitro approaches.
First we made use of Tg(MPO::EGFP)114 transgenic zebrafish,
which express GFP-tagged myeloperoxidase, a marker of neu-
trophils (20). To induce an inflammatory response, a small cut
was made in the tail fin at 72 h postfertilization (hpf), and the
number of fluorescent neutrophils at the wound site quantified
24 h later (right side of line in Fig. 1 A–F). Compounds were
added at the time of injury. In the presence of vehicle alone
(0.1% DMSO), cutting the tail induced a significant increase in
the number of neutrophils throughout the embryo, with (mean ±
SEM) 10.33 ± 0.67 cells at the wound site (Fig. 1 A–C; n = 21).
Treatment with Thalidomide (Fig. 1D; n = 32), Lenalidomide
(Fig. 1E; n = 42), or Pomalidomide (n = 105; Fig. 1F) inhibited
the injury-induced induction of neutrophils in a dose-dependent
manner (Fig. 1G). With Thalidomide exerting 50% activity
(AC50) at 200 μg/mL (775 μM), Lenalidomide AC50 at 140 μg/
mL (542 μM), and Pomalidomide AC50 at 35 μg/mL (128 μM).
We found Thalidomide and Lenalidomide were maximally ef-
fective (Emax) at 250 μg/mL (968 μM) and 200 μg/mL (772 μM),
respectively, reducing the number of fluorescent cells at the
wound site to 2.10 ± 0.75 (n = 7) and 4.6 ± 0.86 (n = 12), re-
DEVELOPMENTAL
spectively. Pomalidomide, however, induced a comparable re-
BIOLOGY
duction in neutrophil number (3.66 ± 0.96; n = 28) at a three- to
fourfold lower concentration (60 μg/mL; 219 μM; Fig. 1G). These
findings establish concentrations of Pomalidomide, Thalidomide,
Author contributions: N.V. designed research; C.M., L.E., J.N., and N.V. performed re-
search; J.N., N.H.G., and W.D.F. contributed new reagents/analytic tools; C.M., L.E., W.D.F.,
and N.V. analyzed data; and C.M., L.E., and N.V. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: n.vargesson@abdn.ac.uk.
PNAS | July 30, 2013 | vol. 110 | no. 31 | 12703–12708

and Lenalidomide to induce anti-inflammatory responses in em-
bryonic in vivo assays.
Thalidomide exerts its anti-inflammatory effects, at least in
part through inhibition of TNF-α and COX2 production (16,
21). To provide an additional, independent measure of the
anti-inflammatory properties of each compound, we used lip-
opolysaccharides (LPS) to induce TNF-α and COX2 expression
in a macrophage cell line, RAW 264.7, and quantified the effect
of drug application using a TNF-α ELISA or qRT-PCR (Fig. 1 I–
K). Thalidomide, Lenalidomide, and Pomalidomide all inhibited
the induction of TNF-α and COX2 by LPS in a dose-dependent
manner (Fig. 1 H and J), without any effect on cell viability (Fig. 1).
In this assay, the AC50 of TNF-α inhibition by Thalidomide was
125 μg/mL (484 μM), Lenalidomide was 40 μg/mL (155 μM), and
Pomalidomide was 20 μg/mL (73 μM). The Emax of Pomalidomide
(60 μg/mL; 219 μM) was substantially lower than Thalidomide
(200–250 μg/mL; 968 μM) or Lenalidomide (200 μg/mL; 772 μM).
Lenalidomide, but Not Pomalidomide, Is Teratogenic. Thalidomide
has well-characterized teratogenic effects, inducing severe de-
velopmental defects in limbs, eyes, and other organs in humans
and animal models (1, 2, 22–28). To investigate whether Lena-
lidomide and Pomalidomide are also teratogenic, we tested each
compound in our established chicken and zebrafish models of
Thalidomide-induced teratogenesis (26). Each compound was
tested at its maximal anti-inflammatory concentration, de-
termined from our in vivo embryonic assays (Lenalidomide, 200
μg/mL; Pomalidomide, 60 μg/mL). Chicken embryos were trea-
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ted at Hamburger and Hamilton (HH) St17-18 (day 2.5), a stage
of rapid limb and head development, incubated for 6 d and the
12704 | www.pnas.org/cgi/doi/10.1073/pnas.1307684110
Fig. 1. Identification of potent anti-inflammatory
concentrations, in embryological assays, of Thalid-
omide, Lenalidomide, and Pomalidomide. Follow-
ing tail fin transsection (line in tail fin in A), Tg
(MPO::EGFP)114 zebrafish (72 hpf) were incubated
with DMSO/compound. (B and C) Embryos were
imaged 24 h after operation. (C) Fin cut with DMSO
incubation showed cell migration to the wound site.
(D–F) Thalidomide (Thal), Lenalidomide (Len), and
Pomalidomide (Pom) reduce the number of migrat-
ing cells. (G) Graphical representation of the number
of migrating cells following drug incubation; note
Thalidomide and Lenalidomide require a higher
concentration to induce the same response as
Pomalidomide. (H) In vitro quantification of TNF-
α production, from LPS-stimulated RAW 264.7
cells, by ELISA. Dotted line marks the maximal level
of TNF-α reduction by Pomalidomide, which is not
reached by Lenalidomide and Thalidomide until 200
and 250 μg/mL (772 and 968μM) respectively. (I) Cell
viability, measured using trypan blue and acid
phosphatase, was not altered. (J) The qRT-PCR
showed a significant decrease in COX2 expression
following Thalidomide, Lenalidomide, and Pomali-
domide treatment. Graphs represent mean ± SEM.
Statistics: (H) statistical significance was analyzed
using Student’s t test. (I and J) Statistical significance
was analyzed using one-way ANOVA with Tukey’s
post hoc test; *P < 0.05; **P < 0.005; ***P < 0.0005,
****P < 0.0001; NS (not significant), P > 0.05. (Scale
bars: 100 μm.)
impact upon craniofacial and forelimb development analyzed
(Fig. 2 A–I). In embryos treated with Lenalidomide, a normally
patterned proximal-to-distal forelimb skeletal pattern developed
(Fig. 2 G, I). However, compared with vehicle alone (0.1%
DMSO; n = 14), the length of the humerus, hand plate and digits
was decreased significantly (Fig. 2G; n = 14). The size of the eyes
also was significantly smaller (Fig. 2 B–C′ and E; n = 6/14), with
anophthalmia (no eyes) occurring in some cases. Beak defects
were also observed (Fig. 2B′; n = 5/14). In contrast, Pomalido-
mide has no significant effect on the development of these or any
other structures in the embryos at the maximal anti-inflammatory
concentration tested (Fig. 2 D–D′, H, and I; n = 16).
Similar results were obtained using zebrafish. Fish embryos at
24 hpf, a stage of rapid development and organogenesis, were
incubated with compounds for 72 h, then fixed and analyzed.
Neither Lenalidomide (200 μg/mL, n = 10) nor Pomalidomide
(60 μg/mL, n = 10) had any effect on pectoral fin length (Fig. 2 K′
and L′). However, compared with vehicle alone (0.1% DMSO,
n = 10) Lenalidomide, but not Pomalidomide, induced micro-
phthalmia (small eyes; n = 6/10; Fig. 2 K′ and L′). We conclude
that at potent anti-inflammatory concentrations Lenalidomide, but
not Pomalidomide, is teratogenic in chicken and zebrafish embryos.
Lenalidomide, but Not Pomalidomide, Is Antiangiogenic. Thalido-
mide exerts its teratogenic effects through inhibition of blood
vessel development (26). To test whether Lenalidomide and
Pomalidomide also are antiangiogenic, we used a combination of
in vivo and in vitro assays. First, we used the Fli1:EGFP trans-
genic zebrafish line, in which blood vessels are labeled with eGFP
(29), to study the effects of Lenalidomide and Pomalidomide on
Mahony et al.

angiogenesis in vivo (26). As a control, CPS49, a well-character-
ized antiangiogenic analog of Thalidomide (26), also was used in
these experiments. Drugs were added to 24 hpf embryos and the
effect on intersomite blood vessel development analyzed 24 h
later. In agreement with our previous results (26), we found that
CPS49 (10 μg/mL; n = 10) completely blocked blood-vessel de-
velopment, resulting in a significant decrease in both stalk num-
ber and length compared with vehicle alone (0.1% DMSO)
controls (Fig. 3 B–B′, E, and F; n = 10). Lenalidomide (200 μg/
mL) had a similar, but less severe, impact on blood-vessel for-
mation, decreasing stalk length, but not stalk number (Fig. 3 C–C′,
E, and F; n = 10). In contrast, in embryos treated with Pomali-
domide at its maximally anti-inflammatory concentration (60 μg/
mL), blood-vessel formation was indistinguishable from controls
(Fig. 3 D–D′, E, F; n = 10).
Because Lenalidomide, but not Pomalidomide, inhibits blood
vessel formation, next we asked if endothelial cells are differ-
entially sensitive to these compounds. To address this question,
we used the human umbilical vein endothelial cell (HUVEC)
tube assembly assay (26). HUVECs were plated on an ECMatrix
gel, incubated for 18 h in the presence of compounds, then im-
aged and analyzed. We found that CPS49 (10 μg/mL; n = 13) and
Lenalidomide (200 μg/mL; n = 15) inhibited completely vascular
lattice formation (Fig. 3 H–I and K). In contrast, angiogenic
growth occurred normally in the presence of Pomalidomide (60
μg/mL; n = 14; Fig. 3 J and K). Previously we have shown that the
antiangiogenic activity of CPS49 correlates with organizational
changes of endothelial cytoskeletal elements important for cell
migration and proliferation (26). Thus, CPS49 inhibits α-tubulin
and increases F-actin assembly (Fig. 3 M–M′ and P–Q). We found
Lenalidomide induced similar changes in HUVEC cytoskeletal
organization (Fig. 3 N–N′ and P–Q), whereas Pomalidomide had
no detectable effect on either α-Tubulin or F-actin assembly
(Fig. 3 O–O′ and P–Q).
Because COX2 is important for both inflammation and an-
giogenesis (16), next we tested the effect of each compound on
the up-regulation of COX2 in HUVEC in response to stimula-
tion with VEGF-A (10 ng/mL; Fig. 3R). We found that, com-
pared with the DMSO control (n = 6), Lenalidomide (n = 8)
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abrogated the VEGF-A–induced stimulation of COX2 (Fig. 3R).
However, in marked contrast to macrophage cells in which
Mahony et al.
Fig. 2. Teratogenic action of Lenalidomide and
Pomalidomide at potent, anti-inflammatory con-
centrations. Chicken embryos were treated at HH
St17-19, incubated and then fixed at E9. Head, eye,
and limb growth was analyzed. (A–A′) Control head
region following DMSO treatment. (B–C′) Lenali-
domide 200 μg/mL (772 μM) treatment results in
severe head defects, particularly beak (B–C′) and
eye defects (C′). (D–D′) Pomalidomide 60 μg/mL (219
μM) does not affect head development. (E) Eye di-
ameter is unchanged following DMSO, and Poma-
lidomide treatment, but is decreased following
Lenalidomide treatment. (F–H) Cartilage stains 6 d
after drug application. (I) Limb element length
quantification. Pomalidomide treated limbs are
unaffected. Zebrafish embryos were incubated with
compound/DMSO at 24 hpf, and then imaged at
96 hpf. Fins, eyes, and otic vesicles were measured.
(J–J′) DMSO control embryos. (K–K′) Lenalidomide-
treated embryos. (L–L′) Pomalidomide-treated em-
bryos. (M–N) Quantification of cartilage structures.
Statistical significance was analyzed using one-way
ANOVA with Tukey’s post hoc test. (J–L′). Graphs
represent mean ± SEM *P < 0.05; **P < 0.005; ***P <
0.0005, ****P < 0.0001; NS, P > 0.05. Scale bars: A–D′
and F–H′, 1,000 μm; J–L′, 200 μm.
Pomalidomide inhibited COX2 expression (Fig. 1J), Pomalidomide
(n = 8) had no effect on COX2 expression in HUVEC (Fig.
3R). We conclude that endothelial cells are selectively sensitive to
Lenalidomide but not Pomalidomide at the concentrations tested.
Neuronal Outgrowth Is Inhibited by Thalidomide and Lenalidomide,
but Not Pomalidomide. A major adverse side effect of long-term
Thalidomide or Lenalidomide clinical use is peripheral neurop-
athy (4, 5, 10, 11). However, whether Thalidomide, or any of its
analogs, has direct effects on neurons has not been established in
previous studies. To test this idea, we used an established mouse
retinal explant outgrowth assay (30). Explants were prepared
from embryonic (E) 14.5 mouse retina and explanted in collagen
gels in presence of compounds diluted in serum-free medium.
After 18 h, the cultures were fixed, stained with a neuron-specific
anti–β-tubulin antibody, and the area covered by the retinal
axons, a measure of both axon length and number, quantified
(30). We found that both Thalidomide (250 μg/mL; n = 42) and
Lenalidomide (200 μg/mL; n = 42), decreased significantly the
extent of neural outgrowth compared with vehicle alone (0.1%
DMSO) controls (n = 62), with only a few short axons extending
from each explant (Fig. 4 A–C and E). In contrast, profuse
outgrowth occurred in the presence of Pomalidomide (60 μg/mL;
n = 25; Fig. 4D), with no significant difference in the extent of
outgrowth relative to controls (Fig. 4E). We conclude that at po-
tent anti-inflammatory concentrations Thalidomide and Lenali-
domide, but not Pomalidomide, have direct inhibitory effects on
neurite outgrowth.
DEVELOPMENTAL
BIOLOGY
Discussion
Pomalidomide has been shown to be effective at reducing TNF-α
in vitro (18, 19). However, the in vivo actions of Pomalidomide
upon the embryo, blood vessels, and nerves—all of which are
targets of Thalidomide—remain unclear. Using in vivo embryonic
assays and in vitro cell cultures we have compared and contrasted
the functions and activities of Thalidomide, Lenalidomide, and
Pomalidomide. We have demonstrated that Pomalidomide has no
detectable effects upon blood-vessel formation, neurite growth,
or embryonic development at maximally anti-inflammatory con-
centrations, unlike Thalidomide and Lenalidomide. Our in vivo
and in vitro assays also demonstrate that Pomalidomide is highly
PNAS | July 30, 2013 | vol. 110 | no. 31 | 12705

anti-inflammatory at substantially lower concentrations than
Thalidomide or Lenalidomide, the drugs licensed currently for
clinical use. Moreover, in marked contrast to Thalidomide and
Lenalidomide, at its maximally effective anti-inflammatory con-
centration, Pomalidomide was selective for macrophage cells.
Thus, Pomalidomide does not inhibit COX2 production in vas-
cular cells following angiogenic stimulation, but is a potent
COX2 inhibitor in inflammatory response cells at the same
concentration. A similar effect has been demonstrated in
monocyte cell lines (16) suggesting Pomalidomide targets in-
flammatory cells specifically. Together these in vivo and in
vitro findings demonstrate that Pomalidomide represents a po-
tent, cell-selective immunomodulatory drug, with a potential
for fewer adverse side effects.
The concentrations of each compound tested and identified
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as the most potent in reducing TNF-α production has also
been demonstrated in other in vitro studies monitoring TNF-α
12706 | www.pnas.org/cgi/doi/10.1073/pnas.1307684110
Fig. 3. Analysis of antiangiogenic effect of Thalidomide,
Lenalidomide, and Pomalidomide at potent, anti-inflammatory
concentrations. Effects upon angiogenesis was analyzed, using
24 hpf Fli1:EGFP zebrafish, following incubation for 3 and
24 h with each compound, or DMSO. (A–A′) DMSO control
embryos exhibit normal angiogenesis. (B–B′) CPS49 10-μg/mL
treatment abolishes blood vessels. (C–C′′) Lenalidomide 200 μg/
mL (772 μM) treatment consistently showed areas of angiogenic
inhibition. (D–D′′) Pomalidomide 60 μg/mL (219 μM) treated
embryos showed normal angiogenesis and no significant
changes in mean stalk length (E) or Mean stalk number (F). To
confirm angiogenic sensitivity, HUVEC were plated on ECMatrix
and incubated for 18 h. (G–J) Angiogenic growth following
compound or DMSO incubation indicates Pomalidomide has no
effect as quantified by measuring area of filopodia outgrowth
(K). (L–O′) HUVEC were treated with drug or DMSO, then ana-
lyzed for α-tubulin (green) and F-actin (red). DNA was visualized
with Hoechst dye (blue). HUVEC were treated with 0.1% DMSO,
CPS49 10 μg/mL, Lenalidomide 200 μg/mL, or Pomalidomide
60 μg/mL for 4 h, then fixed and stained. (P and Q) Fluorescence
intensity (FI) for F-actin and α-tubulin was measured. F-actin was
up-regulated following CPS49 treatment and α-tubulin was
down-regulated with antiangiogenic drugs. (R) Here, qRT-PCR
showed a significant decrease in COX2 expression following
Lenalidomide, but not Pomalidomide treatment. (E and F) Sta-
tistical significance was analyzed using two-way ANOVA with
Bonferroni posttest or (K, P–R) one-way ANOVA with Tukey’s
post hoc test; *P < 0.05; **P < 0.005, ***P < 0.0005; ****P <
0.0001; NS, P > 0.05, FI represent fluorescent intensity. (Scale
bars: 100 μm.)
inhibition in LPS-stimulated RAW 264.7 cells or peripheral
blood mononuclear cells (17, 18, 21, 31). In fact a broad range of
concentrations have been tested in vitro to study actions of
Pomalidomide (18, 19, 32). In chicken and zebrafish embryos,
Pomalidomide activity has not previously been tested, but con-
centrations used are similar to the concentrations of Thalido-
mide used in other teratogenic studies (23, 24, 26, 28, 33).
Pomalidomide has been shown to exert its pharmacological
properties through the parent molecule, with breakdown prod-
ucts showing little activity (34). In contrast, Thalidomide is
known to require breakdown into active byproducts (3), and
previous studies have shown that it is the antiangiogenic break-
down products of Thalidomide that cause teratogenic defects
(26). Pomalidomide has been shown to be weakly antiangiogenic
in a VEGF-stimulated HUVEC assay (35). However, the effect
was subtle and observations upon the endothelial cell and its
cytoskeleton were not demonstrated. In contrast we demonstrate
Mahony et al.
 at highly anti-inflammatory concentrations, determined from in
vivo embryonic assays, Pomalidomide has no effect on angio-
genesis in vivo zebrafish embryos nor in vitro HUVEC cultures,
unlike Lenalidomide. Moreover we observed no changes in the
actin cytoskeleton of endothelial cells following Pomalidomide
exposure, unlike Lenalidomide, which suggests that Pomalidomide
does not target endothelial cells at the anti-inflammatory con-
centrations tested in this study. The cell-selective properties of this
drug corroborate studies demonstrating Pomalidomide inhibits
immune-system cells and not endothelial cells (16). The selective
activity of Pomalidomide in this study could be due to an absence
of antiangiogenic breakdown products. Conversely structural
changes in Pomalidomide relative to Thalidomide may prevent the
drug from interacting with noninflammatory cells. Determining the
molecular targets and signaling pathways facilitating the cell-spe-
cific action will shed light on the mechanisms of action of each of
these compounds. Many molecular targets have been suggested
but the precise molecular pathways involved in anti-inflammation
as well as teratogenesis remain to be elucidated (2, 22, 23, 27,
36, 37). Irrespective of the underlying mechanisms, our find-
ings demonstrate that Pomalidomide, but not Thalidomide and
Lenalidomide, is specific for inflammatory response cells.
Lenalidomide has been reported previously to be nonteratogenic
based on studies in rabbits (12). Our studies show a clear ter-
atogenic effect of Lenalidomide on both chicken and zebrafish
embryos at the concentration required to induce an anti-
inflammatory response. Our studies further demonstrate that
Lenalidomide, in contrast to Pomalidomide, is not selective for
inflammatory cells and also affects blood vessels and nerves.
Although the limb defects induced by Lenalidomide were less
severe than those resulting from exposure to Thalidomide or its
antiantiangiogenic analog CPS49 (26), comparable eye defects
were observed as well as facial defects not seen with the other
compounds. In vitro approaches and in vivo observation in
adult rats have demonstrated that Lenalidomide can be anti-
angiogenic (35, 38), and we have further confirmed that this is
also true in vivo in the embryo. The teratogenic effects of Tha-
lidomide are mediated, at least in part, through inhibition of
blood-vessel formation (26). Thus, the antiangiogenic activity of
Lenalidomide may also explain the detrimental effects of this
drug on embryonic development.
Both Lenalidomide and Pomalidomide are used to treat
multiple myeloma (8–10). Pomalidomide is particularly effective
Fig. 4. Neurotoxicity of Thalidomide, Lenalidomide, and Pomalidomide at
potent, anti-inflammatory concentrations. Ventral temporal pieces of reti-
nas from E14.5 mice embryos were cultured in the presence of DMSO/com-
pound. (A–D) Following anti–β-tubulin staining and fluorescence imaging,
the area of neurite outgrowth was analyzed following DMSO, Thalidomide
250 μg/mL (968 μM), Lenalidomide 200 μg/mL (772 μM), or Pomalidomide
60 μg/mL (219 μM) treatment. (E) Quantification of neurite outgrowth, in-
dicating Pomalidomide has no effect on neurite outgrowth. Statistical sig-
nificance was analyzed one-way ANOVA with Tukey’s post hoc test. Graphs
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represent mean ± SEM *P < 0.05; **P < 0.005; ***P < 0.0005; ****P <
0.0001; NS, P > 0.05. (Scale bar: 300 μm.)
Mahony et al.
in patients experiencing relapsed multiple myeloma (13–15). The
precise mechanisms underlying the causes of multiple myeloma
remain unclear, although an involvement of angiogenesis and
inflammation are widely supported (39, 40). Our data suggests,
in some patients, that the potent anti-inflammatory action of
Pomalidomide could be critical in the treatment/management of
this condition.
A major side effect of Thalidomide and Lenalidomide long-
term clinical use in adults is peripheral neuropathy. However, it
is unclear whether the compounds have direct effects on neurons,
or if the neuronal damage occurs secondary to, for example, vas-
cular damage (5, 41). In contrast to Pomalidomide, our findings
demonstrate a direct, detrimental effect of both Thalidomide and
Lenalidomide on neurons. Neuronal growth was potently inhibi-
ted, with explant death occurring in some cases. This strongly
suggests that direct neurotoxic effects underlie the peripheral
neuropathy suffered by patients taking Thalidomide or Lenalido-
mide for long-term therapies, although further studies will be re-
quired to confirm this.
Nerves and blood vessels display remarkable similarities in the
cellular mechanisms controlling their development. In particular,
endothelial tip cells and neuronal growth cones, the structures
essential for driving vascular and neuronal growth, respectively,
both contain a highly dynamic cytoskeleton essential for loco-
motion and directionality (42). Our findings demonstrate a direct
effect of Lenalidomide, but not Pomalidomide, on organization
of the cell cytoskeleton, including a loss of tubulin. Loss of tu-
bulin likely underlies the effect of Lenalidomide on vascular and
neuronal development. Previous work has demonstrated Tha-
lidomide and CPS49 also reduce tubulin in endothelial cell cy-
toskeleton (26, 33). These findings suggest that effects on the cell
cytoskeleton represent a final common path of action of Lena-
lidomide and Thalidomide on susceptible cells.
Our findings in zebrafish and chicken embryos indicate that at
highly anti-inflammatory concentrations, Pomalidomide is not
teratogenic. A recent study discussed regulatory guidelines issued
by the US Food and Drug Administration, which has indicated
that Pomalidomide (and Lenalidomide) may be teratogenic in
some mammalian species (32). It is well known that Thalidomide
exhibits species-specific differences in teratogenesis (1, 2). The
basis of these species-specific differences remains unclear, and
requires further detailed analysis, but could shed light on mo-
lecular targets and perhaps to safer and more targeted forms of
these compounds. In summary, our studies, in chicken and
zebrafish embryos, indicate Pomalidomide is more potently anti-
inflammatory in vivo at lower doses than Lenalidomide and Tha-
lidomide with no adverse side effect. Our findings, and assays, also
validate the approach of making structural analogs of Thalidomide
to identify more potent and tissue-specific forms of the drug with
minimal or reduced side effects.
Materials and Methods
Compounds. Thalidomide and Pomalidomide were purchased from Sigma.
Lenalidomide was obtained from Sequoia Research Products, United King-
DEVELOPMENTAL
dom Compounds were dissolved in DMSO—final working DMSO concen-
BIOLOGY
tration was 0.1% and applied as described previously (26).
Zebrafish Embryology. Tg(MPO::EGFP)114 (72 hpf; obtained from the Uni-
versity of Sheffield) zebrafish were sedated, tail fin wounded, then in-
cubated with compound or DMSO (dissolved in aquarium water) for 24 h as
established (20). Fish were imaged and the number of fluorescent cells
(neutrophils) was counted. Effects of compounds on vessels were carried out
on Fli1:EGFP embryos (obtained from the Zebrafish International Resource
Center) as previously described (26, 29).
Chicken Embryology. Fertilized white leghorn chicken embryos (Henry
Stewart Ltd., Herefordshire, United Kingdom) were staged as described
previously (26).
PNAS | July 30, 2013 | vol. 110 | no. 31 | 12707
 Retinal Explant Cultures. Experiments were performed using E14.5 C57BL/6J
embryos from an in-house breeding colony. Noon on the day a vaginal plug
was found was considered E0.5. Retinal explants were prepared/analyzed as
described previously (30). Results are the mean (± SEM) from at least three
independent experiments for each condition. All animal research was
licensed, approved and carried out following guidelines issued by the UK
Home Office and University of Aberdeen Ethics Research Committee.
Cell Culture. Mouse leukemic macrophage (RAW 264.7, ATCC) cells were plated
at 1 × 105 cells per well in a 24-well plate (Cellstar), with 0.5 mL of medium
per well, left overnight and stimulated with 1 ng/mL LPS (Sigma) for 3 h.
Supernatant was collected and frozen at −80 °C immediately. TNF-α capture
ELISA (BD Biosciences) was used to quantify TNF-α production. Plates were
read at 450 nm and TNF-α levels were calculated per milliliter of medium.
HUVECs (Lonza) were cultured as previously described (26). In brief, cells were
plated in a 96-well plate (Nunc) at 5 × 104 cells per well with 100 μL of me-
dium per well, and grown on ECMatrix (Millipore) for 18 h. In some conditions
VEGF (10ng/mL) was applied to each well following 1 h preincubation with
drug or DMSO. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay (Sigma) was used to determine cell viability. The plate was
read at 570 nm, with a background absorbance of 690 nm (26). For analysis of
cytoskeletal protein behavior FITC conjugated α-tubulin (Sigma; 1:50) and
Phalloidin (Molecular Probes; 1:100) were used.
Imaging and Analysis. All measurements and analyses were performed using
Adobe Photoshop, Graphpad Prism, and Image J. Statistical comparisons were
made using ANOVA analyses or two-tailed Student’s t test. AC50 values were
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ACKNOWLEDGMENTS. We thank past and current members of the N.V.
laboratory for discussions on this project; Dr. J. Martin Collinson for statistics
advice and comments on the manuscript; Dr. Isobel Crane for RAW cells;
Prof. Ruth Ross and Dr. Cindy Chau for helpful discussions and feedback on
this project; Dr. Stephen Renshaw (University of Sheffield) for the kind gift
of Tg(MPO::EGFP)114 fish. Fli1-EGFP fish were obtained from the Zebrafish
International Resources Center. C.M. was funded by a University of Aber-
deen PhD studentship. This work was funded in part by small grants from
the Kosterlitz Centre of Therapeutics; and the Cell, Developmental Biology,
and Cancer Theme of the University of Aberdeen (to N.V.).
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