Proc. Nati. Acad. Sci. USA
`Vol. 91, pp. 4082-4085, April 1994
`Medical Sciences
`
`Thalidomide is an inhibitor of angiogenesis
`(fibroblast growth factor/rabbit cornea)
`
`ROBERT J. D'AMATO*, MICHAEL S. LOUGHNAN, EVELYN FLYNN, AND JUDAH FOLKMAN
`Department of Surgery, Children's Hospital, Harvard Medical School, Boston, MA 02115
`Communicated by John A. Glomset, January 3, 1994
`
`Thalidomide is a potent teratogen causing
`ABSTRACT
`dysmelia (stunted limb growth) in humans. We have demon-
`strated that orally administered thalidomide is an inhibitor of
`aniogenesis induced by basic fibroblast growth factor in a
`rabbit cornea micropocket assay. Experiments including the
`analysis of thalidomide analogs revealed that the antanio-
`genic activity correlated with the teratogenicity but not with the
`sedative or the mild immunosuppressive properties of tha-
`lidomide. Electron microscopic examination of the corneal
`neovascularization ofthafidomide-treated rabbits revealed spe-
`cific ultrastructural changes similar to those seen in the de-
`formed limb bud vasculature of thalidomide-treated embryos.
`These experiments shed light on the mechanism of tha-
`lidomide's teratogenicity and hold promise for the potential use
`of thalidomide as an orally adminsered drug for the treatment
`of many diverse dease dependent on angiogenesis.
`
`Thalidomide is a potent teratogen. It was developed by
`Chemie Grunenthal in the 1950s as a sedative that appeared
`so nontoxic in rodent models that a LD50 could not be
`established. In 1961, McBride (1) and Lenz (2) described the
`association between limb defects in babies and maternal
`thalidomide usage. Although humans are exquisitely sensi-
`tive to the teratogenic effects of thalidomide, experiments in
`rodents failed to reveal similar effects (3, 4). Teratogenic
`effects could be experimentally reproduced by the adminis-
`tration of thalidomide to pregnant rabbits at an oral dose of
`100-300 mg per kg per day (5, 6). Over the past 30 years, the
`mechanism of thalidomide's teratogenicity has been exten-
`sively studied but has remained unsolved (7).
`We now postulate that the limb defects seen with thali-
`domide were secondary to an inhibition of blood vessel
`growth in the developing fetal limb bud. The limb bud is
`unique in requiring a complex interaction of both angiogen-
`esis and vasculogenesis during development (8). Vasculo-
`genesis is the formation of a capillary bed from endothelial
`cells that have differentiated from mesenchymal precursors.
`Angiogenesis is the formation of new blood vessels from
`sprouts ofpreexisting vessels. Therefore, the limb bud would
`be a particularly vulnerable target to a teratogen that inhib-
`ited endothelial cell function. We chose to examine the effect
`of thalidomide on growing vasculature in the chicken chori-
`oallantoic membrane and in the rabbit cornea.
`
`MATERIALS AND METHODS
`Chicken chorioallantoic membrane (CAM) assays were per-
`formed as described (9, 10) and the effects on the developing
`vasculature were recorded at 48 h after implantation of the
`0.5% carboxymethylcellulose pellet containing various
`drugs. Corneal neovascularization was induced by an im-
`planted pellet of poly(hydroxyethyl methacrylate) (Hydron;
`Interferon Sciences, New Brunswick, NJ) containing 650 ng
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`ofthe potent angiogenic protein basic fibroblast growth factor
`(bFGF) bound to sucralfate (sucrose aluminum sulfate; Bukh
`Meditec, Copenhagen) (11). The addition of sucralfate to the
`pellet protects the bFGF from degradation (12) and provides
`for its slow release, thus producing consistent aggressive
`angiogenesis that is more pronounced than that induced by
`bFGF/Hydron alone. Release of bFGF from pellets contain-
`ing sucralfate/Hydron could be detected in vitro for up to 4
`days after the pellets were formed compared tojust 1 day for
`pellets with Hydron alone (11). Pellets were made by mixing
`110 pI of saline containing 12 Mg of recombinant bFGF
`(Takeda, Osaka) with 40 mg of sucralfate; this suspension
`was added to 80 pI of 12% (wt/vol) Hydron in ethanol.
`Aliquots (10 Al) of this mixture were then pipetted onto
`Teflon pegs and allowed to dry producing approximately 17
`pellets. A pellet was implanted into corneal micropockets of
`each eye of an anesthetized female New Zealand White
`rabbit, 2 mm from the limbus, followed by a single topical
`application of erythromycin ointment on the surface of the
`cornea. Histologic examination on consecutive days demon-
`strated progressive blood vessel growth into the cornea
`toward the pellet with only rare inflammatory cells seen. This
`angiogenic response was not altered by severe immune
`suppression with total body irradiation, and pellets with
`sucralfate alone did not induce angiogenesis (data not
`shown). Unlike other models of corneal angiogenesis that
`utilize inflammation to stimulate neovascularization, the new
`vessels are primarily induced by the bFGF. The animals were
`fed daily from 2 days after implantation by gastric lavage with
`either drug suspended in 0.5% carboxymethylcellulose or
`vehicle alone. Thalidomide was purchased from Andrulus
`Pharmaceutical (Beltsville, MD) and EM-12 and Supidimide
`were kindly provided by Grunenthal (Stolberg, F.R.G.).
`Immunosuppressed animals received total body radiation of
`6 Gy for 6 min immediately prior to implantation of the
`pellets. This dose of radiation resulted in a marked leukocy-
`topenia with >80%o reduction in the leukocyte count by day
`2 and >90o reduction by day 3, results that are consistent
`with previous reports (13, 14).
`The animals were examined with a slit lamp every other
`day in a masked manner by the same corneal specialist
`(M.S.L.). The area of corneal neovascularization was deter-
`mined by measuring with a reticule the vessel length (L) from
`the limbus and the number of clock hours (C) of limbus
`involved. A formula was used to determine the area of a
`circular band segment: C/12 x 3.1416 [r2 - (r - L)2], where
`r = 6 mm, the measured radius of the rabbit cornea. We have
`utilized various mathematical models to determine the
`amount of vascularized cornea and have found that this
`formula provides the most accurate approximation ofthe area
`of the band of neovascularization that grows toward the
`pellet. Only the uniform contiguous band of neovasculariza-
`
`Abbreviations: bFGF, basic fibroblast growth factor; CAM, chicken
`chorioallantoic membrane; PGA, phthaloylglutamic anhydride; PG
`acid, phthaloylglutamic acid; TNF-a, tumor necrosis factor a.
`*To whom reprint requests should be addressed.
`
`4082
`
`DR. REDDY’S LABS., INC. EX. 1030 PAGE 1
`
`

`

`Medical Sciences: D'Amato et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`4083
`
`genic dose (200 mg/kg) of thalidomide resulted in an inhibi-
`tion of the area of vascularized cornea that ranged from 30 to
`51% in three experiments with a median inhibition of 36%
`(Figs. 2A and 3) (n = 30 eyes; P = 0.0001, two-way ANOVA
`with ranked data). The inhibition of angiogenesis by tha-
`lidomide was seen after only two doses (Fig. 2B). The rabbits
`did not demonstrate obvious sedation and there were no signs
`of toxicity or weight loss. The teratogenic analog EM-12,
`which shares the other properties of thalidomide, was also
`inhibitory, with a median inhibition of 42% (n = 10 eyes; P
`= 0.002, one-way ANOVA with ranked data). Supidimide, a
`nonteratogenic analog that retains the sedative properties of
`thalidomide, exhibited no activity (area 107% of control; n =
`10 eyes; not statistically different from control). Other ana-
`logs, PGA and PG acid, displayed weaker inhibitory effects
`
`M Supidimide
`o Thalidomide
`El EM12
`0 Thalidomide-irradiated
`
`==4"~
`
`Animal treatment groups
`
`2
`
`4
`8
`6
`Days post-implantation
`
`10
`
`12
`
`A
`
`o 100-
`
`80
`
`60-
`
`40
`
`20-
`
`O-
`
`N ,
`
`vQC.)CZ)
`-F
`a)
`
`0
`
`0
`
`B
`
`40 -
`
`N4
`Pi
`
`E c.
`
`° 30-
`
`N C
`
`o
`
`0a 20-
`co
`-a
`a)
`
`0
`
`0 .
`
`: 0'-
`0
`
`(A) Inhibition of bFGF-induced corneal neovasculariza-
`FIG. 2.
`tion by thalidomide and related analogs expressed as percent of
`median control on day 8. Pellets containing bFGF and sucralfate
`were implanted into micropockets of both corneas of rabbits (18).
`Vessel ingrowth into the clear cornea from the limbus was first noted
`on day 2 and treatments (200 mg/kg orally) were begun on this day.
`The area of corneal neovascularization was measured from day 4
`through day 12. Day 8 measurements were used for comparison
`between groups. No regression of vessels and near maximal neovas-
`cularization was seen at this time point. Statistical analysis was
`performed with ANOVA with ranked data to account for interex-
`perimental variation and to guard against a nonnormal distribution of
`data (i.e., outliers) by utilizing a nonparametric method. (b) Time
`course of inhibition of neovascularization with thalidomide. Mean
`areas of corneal neovascularization with standard error bars are
`presented from one experiment that is representative of the three
`experiments performed with thalidomide on nonirradiated animals.
`Data presented from the first time point after administration of the
`drug through the completion of the study are statistically different (n
`= 10 eyes; P < 0.005 for all time points, one-way ANOVA with
`ranked data).
`
`tion adjacent to the pellet was measured. The noncontiguous
`neovascularization, which can be seen superiorly, was not
`quantified due to its irregular shape. These vessels that often
`grow concurrently toward the pellet from the superior limbus
`arise from vessels ofthe superior rectus supplying the limbus,
`are directly induced by the bFGF/sucralfate pellet, and are
`histologically identical to the inferior limbal vessels. How-
`ever, it should be noted that this superior neovascularization
`was commonly seen in control animals and was never seen in
`thalidomide-treated animals. Thus, the total inhibition of
`neovascularization is conservatively underestimated.
`
`RESULTS
`Our initial investigations were performed on the CAM.
`Neither thalidomide nor EM-12, a related teratogenic analog
`(15), exhibited any inhibitory activity on blood vessel growth.
`This result was expected as it has been proposed that
`thalidomide must be metabolized by the liver to form an
`epoxide that may be the active teratogenic metabolite (16).
`Other thalidomide analogs that have been shown to be
`teratogenic in rodents (17), including phthaloylglutamic an-
`hydride (PGA) and phthaloylglutamic acid (PG acid), were
`also analyzed (Fig. 1). Interestingly, weak antiangiogenic
`activity of the developing vasculature was seen with both PG
`acid and PGA when 100 jig of either compound was placed
`on the CAM in a pellet of 0.5% carboxymethylcellulose.
`Despite frequent mild scarring, avascular zones were pro-
`duced in 15% of the CAMs with PGA compared to control
`0.5% carboxymethylcellulose pellets in which no avascular
`zones were seen (data not shown).
`Based on these initial findings, we decided to test tha-
`lidomide's effect on angiogenesis induced by bFGF in the
`rabbit corneal micropocket model. Treatment with a terato-
`
`Thalidomide
`
`0
`
`N
`
`a
`
`N
`
`01
`
`1 C
`
`11
`
`0 0
`
`c
`
`c
`
`0o
`
`N
`
`'o
`
`EM-12
`
`Phthaloyl Glutamic
`Anhydride (PGA)
`
`,c
`,>
`ll, 0 / b
`0
`
`Phthaloyl
`Glutamic Acid
`(PG Acid)
`
`0 C
`
`-OH
`
`e"
`
`OH
`
`01
`
`1 C c
`
`11
`0
`
`01
`
`1
`
`11
`0
`
`01
`
`1
`
`SJCN
`
`02
`
`N
`
`Supidimide
`
`FIG. 1.
`
`Structure of thalidomide and related analogs.
`
`DR. REDDY’S LABS., INC. EX. 1030 PAGE 2
`
`

`

`4084
`
`Medical Sciences: D'Amato et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`than thalidomide (data not shown). The density of vessel
`ingrowth in thalidomide-treated animals was also markedly
`reduced. Due to the lack of an objective grading scale, these
`results are not presented.
`Thalidomide has immunosuppressive properties that might
`have indirectly affected angiogenesis. Recently, thalidomide
`has been used for its immunosuppressive properties in the
`treatment of leproma reactions (19) and chronic graft versus
`host disease (18, 20-23). However, in humans its immuno-
`suppressive properties are weak and delayed with little effect
`in acute graft versus host disease (24). Because the effect of
`thalidomide on the immune system is similar but weaker than
`that of cyclosporin A (25), we tested cyclosporin A at the
`highest tolerated dose of 25 mg/kg. No statistically signifi-
`cant effect was observed compared to control. To investigate
`
`.I]
`
`further the immune interactions, we pretreated the rabbits
`with the maximally tolerated immunosuppressive dose of
`total body irradiation. Immunosuppressed animals re-
`sponded equally well to thalidomide, with a median inhibition
`of neovascularization of 52% (n = 12; P = 0.0001, one-way
`ANOVA with ranked data) as compared to irradiated place-
`bo-treated controls (Fig. 2A).
`Electron microscopic examination of corneas from tha-
`lidomide-treated and control animals revealed ultrastructural
`differences. Vessels in the thalidomide-treated group dem-
`
`ti½*
`
`Ai
`
`A4-
`
`A
`
`B
`
`*L
`
`'o
`'O"
`I.- 1 '4
`
`.:!
`
`f..... ;7 .1
`
`:fsi,
`-..;z ebi,r,4
`
`"",::,
`
`..
`
`Representative corneas at 8 days after implantation of
`FIG. 3.
`bFGF pellets from control (A) and thalidomide-treated (B) rabbits.
`There is prominent corneal neovascularization (arrows) in the con-
`trol with associated corneal clouding, which was demonstrated
`histologically to be stromal edema without inflammation. The tha-
`lidomide-treated animal has markedly less neovascularization with
`minimal corneal edema.
`
`Electron micrographs of corneal neovascularization ob-
`FIG. 4.
`served in a thalidomide-treated rabbit 10 days after implntation of
`a pellet containing bFGF. (A) High-magnification (x40,000) view of
`typical fenestrations (arrow) in an endothelial cell from corneal
`neovascularization in thalidomide-treated rabbit. (B) High-
`magnification (x60,000) view of an area of cell thinning (asterisk)
`adjacent to a cell junction in thalidomide-treated corneal neovascu-
`larization. These changes were not seen in control day 10 corneal
`neovascularization. (Bars = 0.1 amn.)
`
`DR. REDDY’S LABS., INC. EX. 1030 PAGE 3
`
`

`

`Medical Sciences: D'Amato et al.
`
`Proc. Natl. Acad. Sci. USA 91 (1994)
`
`4085
`
`onstrated fenestrations not seen in control animals (Fig. 4A).
`Fenestrations have been previously reported to be specific to
`regressing corneal blood vessels after removal of the angio-
`genic stimulus (26). However, in that model, endothelial cell
`regression was associated with platelet plugging and cellular
`hypoxic changes such as swollen mitochondria, which were
`not seen in the thalidomide-treated animals. Interestingly,
`histologic changes previously described in the vasculature of
`the limb buds from chicken embryos treated with thalidomide
`(27) were also seen in the corneal neovascularization of our
`thalidomide-treated rabbits including vesicular projections
`into the lumen and extreme thinning of cell processes (Fig.
`4B). In general, the corneal neovascularization from thali-
`domide-treated rabbits appeared more immature than that
`observed in control animals with poorly formed cell junc-
`tions, incomplete basement membrane, and fewer associated
`pericytes. These findings support the hypothesis that tha-
`lidomide has a direct effect on the growing vasculature.
`
`DISCUSSION
`Orally administered thalidomide is an inhibitor of angiogen-
`esis induced by bFGF in the rabbit cornea micropocket
`assay. The mechanism by which thalidomide inhibits angio-
`genesis is unknown. Thalidomide has shown no effect on
`bFGF-induced proliferation of endothelial cells in culture
`(data not shown). Current studies are focused on the identi-
`fication of the most active thalidomide metabolite. The
`formation of an active metabolite by the liver in vivo provides
`an explanation of the observation that the effect of tha-
`lidomide on growing vessels is seen only when given sys-
`temically.
`Thalidomide has been shown to suppress tumor necrosis
`factor a (TNF-a) production from macrophages (28). How-
`ever, macrophages were rarely seen in histologic examina-
`tions of our model of corneal neovascularization. Further-
`more, studies examining the role of TNF-a in corneal angio-
`genesis have failed to detect TNF-a production in a model of
`inflammatory corneal angiogenesis in which macrophages
`were prominent (29). TNF-a is only weakly angiogenic in
`vivo. It acts by inducing secondary inflammation in contrast
`to bFGF, which stimulates angiogenesis without inflamma-
`tion (30). Thus, the ability of thalidomide to inhibit angio-
`genesis induced by pharmacologic doses of bFGF supports
`the hypothesis that thalidomide directly inhibits an essential
`component of angiogenesis and does not operate through
`effects on TNF-a production.
`In conclusion, thalidomide is a potent angiogenesis inhib-
`itor in vivo. In this model of corneal angiogenesis, we have
`tested many putative angiogenesis inhibitors (including an-
`timitotic agents, cis-retinoic acid, tamoxifen, and others).
`Thalidomide was the only agent capable of inhibiting angio-
`genesis after oral administration. Evaluation of thalidomide
`analogs demonstrated a correlation between teratogenicity
`and antiangiogenic potential. The weak and delayed immu-
`nosuppressive action of thalidomide when used clinically, its
`inhibition of angiogenesis in radiation-immunosuppressed
`animals, and the lack of effect of the functionally related
`immunosuppressive agent cyclosporin A argue for a direct
`effect of thalidomide on angiogenesis. Further support for
`this hypothesis is derived from the ultrastructural changes
`seen in thalidomide-treated animals. There are clear impli-
`cations for the use of this drug in the treatment of pathologic
`angiogenesis that occurs in diabetic retinopathy, macular
`
`degeneration, and solid tumors. Because antiangiogenic ther-
`apy is likely to be long-term, there is a need for an orally
`efficacious inhibitor.
`
`Special thanks to Klio Chatzistefanou, Geri Jackson, Evelyn
`Gonzalez, Pat D'Amore, Helene Sage, Michael O'Reilly, Michael
`Kaplan, Tony Adamis, Elizabeth N. Allred, Ramzi Cotran, and
`Dianna Ausprunk for their assistance and advice. We also thank E.
`Frankus and K. Zwingenberger of Grunenthal GMBH for providing
`technical information, EM-12, and supidimide. R.J.D. is a Howard
`Hughes Medical Institute physician research fellow. M.S.L. is partly
`supported by the Ruth Rae Davidson corneal fellowship endowment.
`Animal studies were reviewed and approved by the animal care and
`use committee of Children's Hospital and are in accordance with the
`guidelines of the Department of Health and Human Services.
`
`1.
`2.
`3.
`
`4.
`5.
`6.
`
`7.
`8.
`
`9.
`
`McBride, W. G. (1961) Lancet u1, 1358.
`Lenz, W. (1962) Lancet 1, 45.
`Cahen, R., Sautai, M., Montagne, J. & Pessonier, J. (1964)
`Med. Exp. 10, 201-224.
`Christie, G. A. (1962) Lancet ii, 249.
`Helm, F. (1966) Arzneim. Forsch. 16, 1232-1237.
`Stertz, H., Nothduft, H., Lexa, P. & Ockenfels, H. (1987)
`Arch. Toxicol. 60, 376-381.
`Stepens, T. D. (1988) Teratology 38, 229-239.
`Seifert, R., Zhao, B., Christ, B. (1992) Anat. Embryol. 186,
`601-610.
`Auerbach, R., Kubai, L., Knighton, D. & Folkman, J. (1974)
`Dev. Biol. 41, 391-394.
`Crum, R., Szabo, S. & Folkman, J. (1985) Science 230, 1375-
`1378.
`Gonzalez, E., Adamis, T. & Folkman, J. (1994) Invest. Oph-
`thalmol. Vis. Sci. 33 Suppl., 424.
`Folkman, J., Szabo, S., Stovroff, M., McNeil, P., Li, W. &
`Shing, Y. (1991) Ann. Surg. 214, 414-427.
`13. Bogman, M., Cornelissen, I., Berden, J., De Jong, J. & Koene,
`R. (1984) J. Immunol. Methods 70, 31-38.
`Bigelow, C., Adler, L., Appelbaum, F. (1987) Transplantation
`44, 351-354.
`Helm, F., Frankus, E., Friderichs, E., Graudums, I. & Flohe,
`L. (1981) Arzneim. Forsch. Drug Res. 31, 941-949.
`Blake, D. A. & Balasubramanian, V. (1981) Proc. Natl. Acad.
`Sci. USA 78, 2545-2548.
`Kocher-Becker, U., Kocher, W. & Ockenfels, H. (1984) Z.
`Naturforsch. 36, 904-909.
`Vogelsang, G. B., Taylor, S., Gordon, G. & Hess, A. D. (1986)
`Transp. Proc. 18, 904-906.
`Sheskin, J. (1965) Clin. Pharm. Ther. 6, 303-306.
`Field, E. O., Gibbs, J. E., Tucker, D. F. & Hellman, K. (1966)
`Nature (London) 211, 1308-1313.
`Lim, S. H., McWannell, A., Vora, A. J. & Boughton, B. J.
`(1988) Lancet 1, 117.
`McCarthy, D. M., Kanfer, E. J. & Barrett, A. J. (1987)
`Biomed. Pharmacother. 43, 693-697.
`Saurat, J. H., Camenzind, M., Helg, C. & Chapuis, B. (1988)
`Lancet 1, 359.
`McCarthy, D. M., Kanfer, E. J. & Barrett, A. J. (1989)
`Biomed. Pharmacother. 43, 693-697.
`Keenan, R., Eiras, G., Burckart, G., Stuart, R., Hardesty, R.,
`Vogelsang, G., Griffith, B. & Zeevi, A. (1991) Transplantation
`52, 908-910.
`Ausprunk, D., Falterman, K. & Folkman, J. (1978) Lab. Invest.
`38, 284-294.
`Jurand, A. (1966) J. Embryol. Exp. Morphol. 16, 289-300.
`Sampaio, E., Sarno, E., Galilly, R., Cohn, Z. & Kaplan, G.
`(1991) J. Exp. Med. 173, 699-703.
`Sunderkotter, C., Roth, J. & Sorg, C. (1990)Am. J. Pathol. 137,
`511-515.
`Frater-Schroder, M., Risau, W., Hallman, R., Gautschi, P. &
`Bohlen, P. (1987) Proc. Natl. Acad. Sci USA 84, 5277-5281.
`
`10.
`
`11.
`
`12.
`
`14:
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`27.
`28.
`
`29.
`
`30.
`
`DR. REDDY’S LABS., INC. EX. 1030 PAGE 4
`
`