Anti-angiogenic thalidomide analogs: A determination of their teratogenic potential using a chicken egg embryo model

Michelle AbramowskiYork College of Pennsylvania

RCK01-01Embryos Harvested Day 16 (8/2/01)

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Development of an in vivo Rapid Screening Method for Teratogenic Effects of Thalidomide Analogs

Michelle AbramowskiYork College of Pennsylvania

INTRODUCTIONIn the United States, over 1.2 million people

are diagnosed with cancer each year (1). The genomic instability and rapid growth of tumors allows them to become resistant to traditional treatment protocols, through a variety of mechanisms (2). This has prompted researchers to explore other treatment possibilities. Tumors require a large blood supply, because of their rapid growth. They must form new blood vessels from pre-existing ones to acquire a blood supply, a process known as angiogenesis. Attacking the tumor’s blood supply appears to be more advantageous than conventional treatments.

Thalidomide (-N-phthalidioglutarimide), a synthetic derivative of glutamic acid, has been found to reduce tumor growth by blocking angiogenesis (3). However, thalidomide has been found to be highly teratogenic, causing congenital anomalies and malformations in fetuses, which has limited its clinical usefulness. Work has been done to begin synthesizing and characterizing anti-angiogenic analogs of thalidomide, that are more effective than the parent compound. While many analogs with increased anti-angiogenic activity have been described (4), little is known about their possible teratogenic effects. Since the developing chicken egg has been previously used as a teratogenic screening model (5), we accessed the effect thalidomide has on this system. This study used the parent thalidomide compound to define an in vivo teratogenic screening model for thalidomide analogs.

 OVERALL GOAL OF PROJECTTo develop a rapid, cost-effective, and reproducible in vivo teratogenic screening model for thalidomide analogs that have greater anti-angiogenic activity.

METHODS and MATERIALSModel System

•Fertile white leghorn chicken eggs were maintained in a 37C incubator.•A hole (2 x 2 cm) was made above air sac for injections (Scheme 1).

Injection Protocol•Thalidomide was activated with rat or human microsomes for 30 minutes at 37C in 0.9 % NaCl. •Injections (50 L) were made through CAM into yolk sac (Scheme 1).

•Exp. #1: Injections days: 3-6 •Exp. #2: Injections days: 7-10

Embryo Harvest•Embryos were harvested.

•Exp. 1: day 16•Exp. 2: day 14

•Observations and measurements:•Weight (g) •Limb length (cm)•Viability

Statistical Analysis•Data were analyzed for statistical significance by ANOVA using INSTAT version 3.0 software.

RESULTS

 

  

   

Figures 2. Affect of thalidomide treatment on embryo viability. Figure 2A: Affect of rat microsome activated thalidomide on embryo viability. Figure 2B: Affect of human microsome activated thalidomide on embryo viability. Bars represent the percent of viable embryos at harvest. Groups: A control, B microsome control, C (2A) 75 g rat-thalid, C (2B) 100 Mol/kg human-thalid, D (2A) 100 Mol/kg rat-thalid, D (2B) 1000 Mol/kg rat-thalid, and E (2A) 1000 Mol/kg rat-thalid.

Figure 1. Chicken embryo at 16 days. Scheme 1. Internal structures of chicken embryo.

Work Cited1. Kyle, R.A., and Rajkumar, S.V. 2001. Therapeutic applications of thalidomide

in multiple myeloma. Seminars in Surgical Oncology 6: 583-587.2. Kerbel, R.S. 1991. Inhibition of tumor angiogenesis as a strategy to circumvent

acquired resistance to anti-cancer therapeutic agents. BioEssays 13: 31-36.3. Baidas, S.M. 2000. Phase II evaluation of thalidomide in patients with

metastatic breast cancer. Journal of Clinical Oncology 20: 2710-2717.4. Moreira, A.L. Thalidomide and analog inhibit endothelial cell proliferative in

vitro. 1999. Journal of Neuro-Oncology 43: 109-114.5. Vesela, D. Embryotoxicity in chick embryo of thalidomide hydrolysis products

following metabolic activation of rat liver homogenate. Functional and Developmental Morphology 4: 313-316.

Acknowledgement Dr. Kaltreider

CONCLUSIONS•There was a substantial difference in microsomes used to activate thalidomide on viability (Figure 2 and 3).•3 injections of 100 Mol/kg of rat-thalid, and 3 injections of 100 Mol/kg of human-thalid appeared to be toxic to the embryo (Figure 2).•No decrease in limb length and weight was observed with thalidomide treatment (Figure 2 and 3, Table 1 and 2). •Human microsomes substantially decreased viability while rat microsomes did not (Figure 2).•Substantial toxicity of 50 g rat activated thalidomide observed in day 10 of treatment (Figure 3).

FUTURE PROJECTSWhile this system was unable to detect teratogenic potential of thalidomide, it may be useful for rapidly screening for embryotoxicity.

Figure 3. Affect of rat microsome activated thalidomide treatment on embryo viability at day 14. Bars represent the percent of viable embryos injected with 50 g rat-thalid on day 7, 8, 9, or 10 of development.

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