6.

1 ANTIANGIOGENIC ACTIVITY
INTRODUCTION
The chicken embryo chorioallantoic membrane assay has been a model for studying neovascularization since the early 1970s when it was adapted by Folkmann et al.

Assay Description
While there have been many variations developed over the years, the basic assay is performed by implanting a membrane or coverslip containing the compound of interest on the chick embryo chorioallantoic membrane through a hole cut in the egg shell. The subsequent incubation period ranges from 1-3 days, depending on the compound, after which time angiogenesis can be quantified via image analysis or colorimetric detection methods. The CAM assay is quick, technically simple, and inexpensive. However, a major drawback is that it is labor intensive due to the large number of eggs that are required to obtain consistent results. The investigator should note that this is a non-mammalian system which should be taken into consideration when interpreting results.

5.3.1 Test protocol 5.3.1.1 Antiangiogenesis study by chorioallantoin membrane (CAM) assay
CAM assay is routinely used as a preliminary method to determine antiangiogenic effect of a compound (S. Taylor 1982, R. Crum 1985, M.A. Moses 1990). This assay is based upon the formation of a chorioallantoic membrane, in which neovascularization takes place, in fertilized chicken eggs at a certain stage of the development of the embryo. Agarose pellets impregnated with the test compound are placed onto the vascular membrane of opened eggs, and the influence on angiogenesis is evaluated (D. H. Paper 1999). For assay purpose the fertile chicken eggs were procured from Kalchina hatchery, Modinagar. 5.3.1.1.1 Requirements Fertile Chicken Eggs Agarose 70% EtOH -1,4-galactan sulfate

5.3.1.1.2 Procedure Twelve eggs were used per experiment to test one compound as a given dose. The eggs were fertilized at 370C and 80% relative humidity in ideal conditions. The shells of eggs were cleaned with 70% EtOH to avoid infections. After 72 hrs 8-10 ml of albumin was removed with a syringe at the lower side of the egg, and the hole was sealed with tape. Subsequently the upper part of the shell was removed, and the eggs were covered with a plastic film and incubated for another 72 hrs. At this point of time, when the diameter of CAM is between 1.8 and 2.6 cm, the pellets containing the test substances were placed on the CAM. Test substances were dissolved or suspended in a 2.5% agarose solution. After gel formation, the volume of agarose gel corresponding to the dose of the test compound to be applied to the CAM was taken by means of a micropipette for viscous solutions. Therefore the agarose pellets do not have a uniform size. The half-cone-shaped agarose pellets are fixed because they slightly sink into the CAM. After 24 hrs the antiangiogenic effect was measured, either, after addition of cream as a contrast fluid, by means of a stereomicroscope, and observing the avascular zone surrounding the pellet, or, by observing avascular zone surrounding the pellet (if clear) by naked eye. Antiangiogenic activity is expressed as a score where 0 = no or weak effect, 1 = medium effect, and 2 = strong effect (capillary free zone is at least twice as large as the pellet). Also membrane irritation and embryotoxicity can be evaluated. -1,4-galactan sulfate (LuPS S5) with an average molecular weight of 20000 was used as positive control (R. Hoffman 1996) and an agarose pellet as a blank. 5.3.2 Results and discussion Effect of the test compounds on angiogenesis is recorded in Table 5.1. All the compounds were tested at a dose of 10 g/pellet, i.e. having less than 40 nmol/pellet, because at higher dose most of compounds showed a toxic effect. In (Z)-1-phenyl-2-(4-nitrophenyl) ethene analogues, compound 3a, 3d, 5a, 5b, 5c and 5d showed an antiangiogenic score of more than 1. Compound 5a was found to be most potent with a score of 1.6 0.1 which is more than that of standard. Compound 3a also showed good score, equal to that of standard. In (Z)-1(furan-2-yl)-2-(4-nitrophenyl) ethene analogues compounds 10 and 11h showed an antiangiogenic score of more than 1. All other compounds showed antiangiogenic score less than 1. Compound 11h was found to be most potent with a score of 1.3 0.1 which is comparable to that of standard. No compound showed score greater than standard. The result shows that synthesized compounds have significant antiangiogenic activity. The most active analogues 3a, 3d, 5a, 5b, 5c, 5d and 10 have smaller groups like COOH, COOCH3 or COCl as bridge substituents while the least active analogues 6e, 6f, 6g, 6i, 6j, 6m, 11c, 11e, 11g and 11k have comparatively large groups as bridge substituents (Potent activity of compound 11h is exception in this regard). Compounds having aromatic substituents (6b, 6d, 6i, 6j, 6m, 11c-g), were least active. So, we can say that compound possessing the Piperidin-1ylcarbonyl, carboxyl, methoxycarbonyl and chlorocarbonyl moiety on (Z)-1-phenyl-2-(4nitrophenyl) ethene and (Z)-1-(furan-2-yl)-2-(4-nitrophenyl) ethene skeleton are among the

most active compounds in our study. We can say that size of bridge substituents, in the series studied, affect the antiangiogenic activity. In 4,5-disubstituted oxazole derivatives, Compound 15b, 15c, 15d, 15e, 15i and 15m showed potent activity with an antiangiogenic score of more than 1. Other compounds were not as much active. Compound 15d was most active with a score of 1.5 0.1, equal to that of standard. Results show that compounds, having 3,4,5-trimethoxyphenyl group on either position (4 or 5) on oxazole ring, have potent activity. Results further supported already reported data that the three methoxy groups are involved in binding of the molecule with the active site. However, 4,5-bis(3,4,5-trimethoxyphenyl) analog (15a) was not as much active which shows that the part of active site, at which second aromatic ring binds, does not have same amount of affinity for the three methoxy groups, as they have for other one.

In 3-aroylindole analogues, compounds 21d and 21g were found to be most active, and showed an antiangiogenic score of more than 1. All other compounds showed antiangiogenic score less than or equal to 1. In 1-aroylindole analogues, compound 23d, 23e and 23f showed an antiangiogenic score of more than 1. All other compounds showed antiangiogenic score less than or equal to 1. In this series compound 23d was found to be most active with an antiangiogenic score of 1.8 0.1. Activity of compounds 21d, 21g, 23e and 23f was also found to be potent. Results show that 1-aroylindole analogues are more active than their corresponding 3-aroylindole analogues. This may be due to, either presence of dimethylaminomethyl group at 3-position or 1-aroylindole configuration better suited for binding to active site. Furthermore, compounds containing methyl group at 2-position were more active than their corresponding unsubstituted analogues. It reveals that 2-position methyl group plays an important role in binding of the molecule to the active site. In 3-aroylindole analogues, compound containing nitro group at 5-position (21d) was more active than corresponding chloro and bromo group containing analogues (21e and 21f). Good activity of compound 21g showed that triisopropylsilyl group at 1-position increases activity i.e. a bulky group at 1-position can also enhance/retain the activity. In 1-aroylindole analogues, all three 2methyl analogues (23d, 23e and 23f) showed potent activity. 5-Nitro analogue (23d) was most active having a score greater than that of standard.

Table 5.1 Antiangiogenic activity of compounds in the CAM assay Test compound 3a 4a Concentration (g/pellet) nmol/pellet 10 28 10 27 Antiangiogenic scoreb sd (n = no. of experiment) 1.4 0.1 (n =3) 1.0 0.1 (n =2)

5a 6a 6b 6c 6d 6e 3b 4b 5b 6f 3c 4c 5c 6g 3d 4d 5d 6h 6i 6j 6k 6l 6m 6n 8 9 10 11a 11b 11c 11d 11e 11f 11g 11h 11i 11j 11k 15a

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

26 24 22 26 22 21 32 31 30 29 32 30 30 29 33 31 31 28 26 23 27 27 24 20 39 37 36 37 32 28 29 27 30 26 31 31 27 23 25

1.6 0.1 (n =3) 0.9 0.1 (n =2) 0.4 0.2 (n =2) 0.7 0.1 (n =2) 0.3 0.3 (n =2) 0.6 0.1 (n =2) 1.0 0.1 (n =2) 0.8 0.1 (n =2) 1.2 0.1 (n =2) 0.6 0.1 (n =2) 0.8 0.1 (n =2) 0.6 0.2 (n =2) 1.1 0.1 (n =2) 0.3 0.4 (n =2) 1.2 0.1 (n =3) 0.9 0.1 (n =2) 1.5 0.1 (n =3) 0.8 0.1 (n =2) 0.3 0.2 (n =2) 0.2 0.3 (n =2) 0.6 0.1 (n =2) 0.5 0.1 (n =2) 0.3 0.1 (n =2) 0.6 0.1 (n =2) 0.8 0.1 (n =3) 0.9 0.1 (n =2) 1.1 0.1 (n =3) 0.8 0.1 (n =2) 0.6 0.2 (n =2) 0.2 0.3 (n =2) 0.5 0.1 (n =2) 0.3 0.1 (n =2) 0.4 0.1 (n =2) 0.2 0.1 (n =2) 1.3 0.1 (n =2) 0.9 0.1 (n =2) 0.6 0.1 (n =2) 0.2 0.1 (n =2) 0.8 0.1 (n =3)

15b 10 28 1.2 0.1 (n =2) 15c 10 29 1.3 0.1 (n =2) 15d 10 26 1.5 0.1 (n =3) 15e 10 28 1.2 0.1 (n =2) 15f 10 33 0.4 0.2 (n =2) 15g 10 33 0.4 0.2 (n =2) 15h 10 29 0.6 0.1 (n =2) 15i 10 29 1.2 0.1 (n =2) 15j 10 33 0.4 0.2 (n =2) 15k 10 33 0.3 0.3 (n =2) 15l 10 30 0.7 0.1 (n =2) 15m 10 26 1.4 0.1 (n =2) 15n 10 29 0.5 0.1 (n =2) 15o 10 30 0.5 0.1 (n =2) 15p 10 26 0.6 0.1 (n =2) 21a 10 27 0.8 0.1 (n =2) 21b 10 25 0.8 0.1 (n =2) 21c 10 28 0.5 0.2 (n =2) 21d 10 26 1.1 0.1 (n =2) 21e 10 24 1.0 0.1 (n =2) 21f 10 27 0.8 0.1 (n =2) 21g 10 17 1.3 0.1 (n =3) 23a 10 24 1.0 0.1 (n =2) 23b 10 22 0.9 0.1 (n =2) 23c 10 25 0.6 0.1 (n =2) 23d 10 23 1.8 0.1 (n =3) 23e 10 22 1.6 0.1 (n =2) 23f 10 24 1.3 0.1 (n =2) Agarose pellet 0.1 0.1 (n =10) -1,4-galactan 50 2.5 1.4 0.1 (n =10) sulphate (LuPS S5) b 0 = no or weak effect, 1 = medium effect, 2 = strong effect

Figure 5.1 A 7 day old chicken egg (Blood vessels are shown through a dissecting microscope).

Figure 6.2 Figure showing capillary free zone for compound 23d. Inner circle representing approximate size of sample disc, and outer circle representing approximate capillary free zone.

Introduction

MTT Cell Proliferation Assay Kit provides a simple method for determination of cell number using standard micro-plate absorbance readers. Determination of cell growth rates is widely used in the testing of drug action, cytotoxic agents and screening other biologically active compounds. Several methods can be used for such determinations, but indirect approaches using fluorescent or chromogenic indicators provide the most rapid and large scale assays. Among such procedures, the MTT assay developed by Mossman 1983 is still among one of the most versatile and popular assays. The MTT assay involves the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) to an insoluble formazan. (Liu et al. 1997, Berridge et al. 1993, Vistica et al. 1991) The formazan is then solubilized, and the concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to approximately 106 cells per well.

MTT Cell Proliferation Assay Kit provides enough material to perform 1000 individual tests using standard 96-well microplates. Following the protocol described below, a complete assay requires an overnight incubation. However, with a slight modification, the whole procedure can be performed in five hours (not including cell preparation time). For additional information concerning the numerous variations and modifications of the MTT assay, we recommend that you consult the citations provided. (Garn et al. 1994, Carmichael et al. 1987, Twentyman et al. 1987, Tada et al. 1986)

Materials
Kit Contents

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MW = 414, Component A), 10 vials, each containing 5 mg SDS sodium dodecyl sulfate (MW = 288, Component B), 10 vials, each containing 1 gm Storage and Handling Upon receipt the kit should be stored at 4C protected from light. Stored properly, the kit components should remain stable for 12 months. Materials Required but Not Provided Phosphate-buffered saline (PBS), sterile HCl, 0.01 M solution dimethylsulfoxide (DMSO) optional

Experimental Protocol
Reagent Preparation 1. Prepare a 12 mM MTT stock solution by adding 1 mL of sterile PBS to one 5 mg vial of MTT (Component A). Mix by vortexing or sonication until dissolved. Occasionally there may be some particulate material that will not dissolve; this can be removed by filtration or centrifugation. Each 5 mg vial of MTT provides sufficient reagent for 100 tests, using 10 L of the stock solution per well. Once prepared, the MTT solution can be stored for four weeks at 4C protected from light.

2. Add 10 mL of 0.01 M HCl to one tube containing 1 gm of SDS (Component B). Mix the solution gently by inversion or sonication until the SDS dissolves. Once prepared, the solution should be used promptly. Each tube makes sufficient solution for 100 tests, using 100 L per well. Culturing Cells The culture conditions used to grow the cells can affect the results and must be taken into consideration when analyzing the data. The age of the cultures, number of passages and details of the growth medium can all be important factors. Natural variation in the requirements and growth rates of different cell lines make it difficult to provide precise guidelines for preparing your cells. In general, cells seeded at densities between 5000-10,000 cells per well should reach optimal population densities within 48-72 hours. Note that the presence of phenol red in the final assay samples can seriously affect results. We strongly recommend that the cells be cultured in medium free of phenol red, if possible. Alternatively, the final incubation with the MTT can be performed after exchanging the cells into medium free of phenol red. Labeling Cells

1. For adherent cells, remove the medium and replace it with 100 L of fresh culture medium. For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as possible and replace it with 100 L of fresh medium.

2. Add 10 L of the 12 mM MTT stock solution (prepared in step 1.1) to each well. Include a negative control of 10 L of the MTT stock solution added to 100 L of medium alone.

3. Incubate at 37C for 4 hours. At high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.

4. Add 100 L of the SDS-HCl solution (prepared in step 1.2) to each well and mix thoroughly using the pipette. 5. Incubate the microplate at 37C for 4 hours in a humidified chamber. Longer incubations will decrease the sensitivity of the assay. (Niks et al. 1990)

6. Mix each sample again using a pipette and read absorbance at 570 nm.

Rapid Protocol Option To shorten the time of the assay it is possible to use DMSO (not provided) as a solubilizing agent to dissolve the formazan.6 1. After labeling the cells with MTT, as described above, remove all but 25 L of medium from the wells. For non-adherent cells it may be necessary to first centrifuge the plates to sediment the cells. 2. Add 50 L of DMSO to each well and mix thoroughly with the pipette. 3. Incubate at 37C for 10 minutes. 4. Mix each sample again and read absorbance at 540 nm not 570 nm, as above.

References