Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-02-26
2002-10-22
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S029000, C435S034000, C435S975000, C435S283100
Reexamination Certificate
active
06468735
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to assays and kits using porcine carotid arteries for screening compounds to identify modulators of angiogenesis. In particular, an assay for rapidly screening compounds that inhibit angiogenesis is provided.
Angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. The control of angiogenesis is a highly regulated system of angiogenic stimulators and inhibitors. Thus, angiogenesis is a critical component of the body's normal physiology, especially during wound healing.
In addition, the control of angiogenesis has been found to be altered in certain disease states, and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis. It also has a detrimental aspect, for example, when blood vessels multiply and enhance growth and metastasis of tumors. Aberrant angiogenesis is also associated with numerous disorders, including rheumatoid arthritis, where blood vessels invade the joint and destroy cartilage, and numerous ophthalmologic pathologies, such as diabetic retinopathies in which new capillaries invade the vitreous, bleed and cause blindness, and macular degeneration, prostate cancer and Kaposi's carcinoma. Angiogenesis is essential to tumor development and growth. Prevention of angiogenesis can inhibit solid tumor growth.
Compounds that have anti-angiogenic activity can be used, for example, as anti-tumor agents and for the treatment of ophthalmic disorders, particularly involving the retina and vitreous humor, and for hyperproliferative dermatological disorders, such as psoriasis, that have an angiogenic component. Thus, compounds that enhance angiogenesis and compounds that inhibit angiogenesis are being sought.
This has led to a search for specific inhibitors of endothelial cell growth. As a result, there is an interest in measuring proliferation of endothelial cells under inhibitory and stimulatory conditions as screens for discovery of inhibitors (or alternatively stimulators) of angiogenesis.
Direct assessment of cell numbers, either microscopically or by particle counter is time consuming and not amenable for high throughput screening. Consequently, direct assessment has been replaced by indirect methods, such as by packed cell volume, by chemical determination of a cellular component, for example, protein or deoxyribonucleic acid, or by uptake of a chromogenic dye such as neutral red.
These methods can be laborious when handling large numbers of cultures, and also inaccurate at low cell densities. For high throughput screening protocols it is necessary to rapidly and accurately measure low cell densities and/or relatively small changes in cell number over a large range of cell densities. Presently available protocols to not provide a means to do this. Thus, there is a need for convenient, rapid and reproducible assays for identifying agents that modulate angiogenesis.
Therefore it is an object herein to provide a method for identifying compounds that modulate angiogenesis. In particular, it is an object herein to provide a method of screening for inhibitors of angiogenesis.
SUMMARY OF THE INVENTION
The present invention relates to assays and kits using porcine carotid arteries for screening compounds to identify modulators of angiogenesis. In particular, an assay for rapidly screening compounds that inhibit angiogenesis is provided.
DETAILED DESCRIPTION OF THE INVENTION
A first embodiment of the present invention is illustrated by a method of analyzing the angiogenesis modulating effect of a compound, comprising the steps of:
(a) incubating a sample comprised of a fragment of porcine carotid artery with the compound;
(b) generating images of the sample; and
(c) quantitating the images to determine the extent of angiogenesis.
In a second embodiment, the incubation of step (a) is done in a humidified atmosphere of about 5% carbon dioxide at about 30° C. to about 40° C. for about 1 to 4 weeks.
In yet another embodiment, the incubation of step (a) is done in a humidified atmosphere of about 5% carbon dioxide at about 37° C. for about 2 to 3 weeks.
The process wherein the incubation of step (a) is done using Matrigel™ as the matrix is yet one more embodiment.
Also encompassed by the present invention is the method described above wherein the images generated in step (b) are digital.
In still another embodiment, the digital images are quantitated in step (c) using image analysis software.
Another embodiment is the method described above wherein the digital images are quantitated in step (c) using image analysis software and the vessel body is subtracted out of the pixel calculation.
A preferred embodiment is a method of analyzing the angiogenesis modulating effect of a compound, comprising the steps of:
(a) incubating a sample comprised of a fragment of porcine carotid artery with the compound in a humidified atmosphere of about 5% carbon dioxide at about 37° C. for about 2 to 3 weeks;
(b) generating digital images of the sample; and
(c) quantitating the digital images to determine the extent of angiogenesis by using image analysis software.
A subembodiment of the present invention is the method described above wherein the angiogenesis modulating effect is inhibition of angiogenesis.
Another embodiment is a method of analyzing the angiogenesis inhibiting effect of a compound, comprising the steps of:
(a) incubating a sample comprised of a fragment of porcine arotid artery with the compound in a humidified atmosphere of about 5% carbon dioxide at about 37° C. for about 2 to 3 weeks in a Matrigel™ matrix;
(b) generating digital images of the sample; and
(c) quantitating the digital images to determine the extent of angiogenesis by using image analysis software.
Also encompassed by the instant claims is a kit for analyzing the angiogenesis modulating effect of a compound, comprising a fragment of porcine carotid artery in a medium with sufficient nutrients to allow growth of new vascular tissue.
In the claims, “porcine carotid artery” refers to blood vessels from both adult and fetal animals.
The study of angiogenesis as a therapeutic target requires a reliable, physiologically relevant, and technically straightforward assay. An ex vivo assay bridges the gap between cell-based assays, which may not realistically represent the complex process of vessel sprouting, and in vivo assays, which are time consuming and expensive. Porcine carotid arteries provide an ideal tissue source for angiogenesis inhibitor screens due to their availability, physiological relevance and large size. The present assay has numerous advantages over the rat aortic ring assay (Nicosia, R. F., et al,
In Vitro.
18:538-549). Among these advantages is that it reduces the number of animals used, increases reproducibility (decreased animal-to-animal variability), decreases costs by eliminating need to house animals since tissue source is commercially available, and increases productivity.
Angiogenesis is a complex biological process which is the result of a variety of cellular interactions. Tissue fragments growing in a three-dimensional matrix provide a model system for the study of angiogenic processes with a complete source of relevant cell types. Evaluation of angiogenic compounds is a difficult process and often requires a variety of assays to determine the potential of a given compound as a therapeutic agent. An assay that is often used to evaluate angiogenesis is the aortic ring assay as mentioned above. Although this assay addresses the complexity of cell-type interactions, it has numerous disadvantages including animal to animal variability.
By purchasing blood vessels from an abattoir, many issues associated with the use of research animals are avoided. Not only is the number of animals used per assay reduced, but need to house research animals is also eliminated. This creates both social and economic savings. Due to the close approximation of porcine to human vasculature, adult porcine caroti
Brown Dianne
Daniel Mark R.
Leary Louise N.
Merck & Co. , Inc.
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