Claims for Patent: 4,806,464

Summary for Patent: 4,806,464

Title:	Nuclear binding assay for steroid receptor functionality in cancerous cells
Abstract:	A method for rapidly determining the presence of functional cellular steroid receptors by assaying a tissue sample for nuclear steroid or antisteroid binding is disclosed which comprises treating the tissue with collagenase, incubating the isolated cells with a labelled steroid or a labelled antisteroid capable of complexing said receptors and measuring the bound nuclear radioactivity and the DNA of the isolated cellular nuclei.
Inventor(s):	Spelsberg; Thomas C. (Rochester, MN)
Assignee:	Mayo Foundation for Medical Education and Research (Rochester, MN)
Application Number:	07/013,569
Patent Claims:	1. A method for rapidly assaying a tissue sample for nuclear antisteroid binding comprising: (a) fragmenting said tissue sample; (b) digesting said fragmented tissue with collagenase; (c) isolating the cells from said digested tissue; (d) incubating said cells with an amount of a radiolabelled antisteroid capable of complexing with and saturating the steroid receptors in said cells; (e) isolating the cellular nuclei; (f) measuring the bound radioactivity and the total DNA of said nuclei; and (g) comparing the amount of bound radioactivity and the total DNA to determine a measure of the total nuclear bound steroid receptors. 2. The method of claim 1 wherein the tissue sample is carcinoma tissue. 3. The method of claim 2 wherein the carcinoma is selected from the group consisting of breast, endrometrium, testicular, lung, myeloma and prostate. 4. The method of claim 1 wherein about 1-1000 mg of tissue is fragmented. 5. The method of claim 4 wherein the tissue is contacted with about 0.5-15 units of collagenase per mg of tissue. 6. The method of claim 1 wherein the isolated cells are incubated in a medium which is about 5-50 nM in radiolabelled antisteroid. 7. The method of claim 6 wherein the radiolabelled antisteroid is a radiolabelled antiestrogen. 8. The method of claim 1 wherein the nuclei are isolated by layering a homogenate of said incubated cells onto a cold, neutral, aqueous medium comprising sucrose, glycerol, octoxynol-9, tris(hydroxymethyl)amino-methane hydrochloride and KCl and centrifuging said layered medium to obtain a pellet of purified nuclei. 9. The method of claim 8 further comprising dispersing said pellet in a neutral solution comprising tris(hydroxymethyl)amino-methane hydrochloride and glycerol, and collecting the dispersed nuclei by filtration. 10. The method of claim 1 wherein the DNA of said nuclei is measured by a diphenylamine assay. 11. A method for rapidly assaying a cancerous tissue sample for nuclear antisteroid binding comprising: (a) obtaining a sample of cancerous tissue by biopsy; (b) fragmenting said tissue sample; (c) disrupting said fragmented tissue sample in contact with about 1-5 units of collagenase per mg of tissue; (d) isolating the cells from said tissue sample; (e) incubating said cells with an amount of a radiolabelled antisteroid capable of complexing with and saturating the steroid receptors in said cells; (f) homogenizing said incubated cells; (g) layering said homogenate onto a cold, neutral aqueous medium comprising sucrose, glycerol, octoxynol-9, tris(hydroxymethyl)amino-methane hydrochloride and KCl; (h) centrifuging said layered medium to obtain a pellet of purified nuclei; (i) measuring the bound radioactivity and the total DNA of said nuclei; and (j) comparing the amount of bound radioactivity and the total DNA to determine a measure of the total nuclear bound steroid receptors. 12. The method of claim 11 wherein about 1-25 mg of tissue are obtained by biopsy. 13. The method of claim 11 wherein about 1-5 mg of tissue are obtained by biopsy. 14. The method of claim 11 wherein said cells are incubated in medium comprising about 5-50 nM of the radiolabelled antisteroid. 15. The method of claim 14 wherein the cells are incubated for about 0.5-1.5 hr at about 18-37.degree. C. 16. The method of claim 14 wherein step (h) further comprises dispersing the pellet of purified nuclei in a neutral aqueous solution comprising tris(hydroxymethyl)amino-methane hydrochloride and glycerol and collecting them. 17. A method for rapidly determining the presence of functional cellular steroid receptors by assaying a tissue sample for nuclear steroid binding comprising: (a) fragmenting said tissue sample into pieces containing intact cells; (b) digesting said fragmented tissue with collagenase; (c) isolating the cells from said digested tissue; (d) incubating said cells with an amount of a radiolabelled steroid or a radiolabelled antisteroid capable of complexing with and saturating said receptors; (e) isolating the cellular nuclei; (f) extracting the bound radioactivity from said nuclei and measuring the bound radioactivity; (g) measuring the total DNA of said nuclei; and (h) comparing the amount of bound radioactivity and the total DNA to determine a measure of the total nuclear bound steroid receptors. 18. The method of claim 17 wherein the tissue sample is carcinoma tissue. 19. The method of claim 18 wherein the carcinoma is selected from the group consisting of breast, endrometrium, testicular, lung, myeloma and prostate. 20. The method of claim 17 wherein said 1-1000 mg of tissue is fragmented. 21. The method of claim 17 wherein the tissue is contacted with about 0.5-15 units of collagenase per mg of tissue. 22. The method of claim 17 wherein the isolated cells are incubated in a medium which is about 5-50 mM in radiolabelled steroid or radiolabelled antisteroid. 23. The method of claim 22 wherein the radiolabelled steroid is [.sup.3 H]-progesterone or [.sup.3 H]-estradiol. 24. The method of claim 17 wherein the nuclei are isolated by layering a homogenate of said incubated cells onto a cold, neutral, aqueous medium comprising sucrose, glycerol, octoxynol-9, tris(hydroxymethyl)amino-methane hydrochloride and KCl and centrifuging said layered medium to obtain a pellet of purified nuclei. 25. The method of claim 24 further comprising dispersing said pellet in a neutral solution comprising tris(hydroxymethyl)amino-methane hydrochloride and glycerol, and collecting the dispersed nuclei by filtration. 26. The method of claim 17 wherein, in step (g), the nuclei are hydrolyzed with perchloric acid and reacted with diphenylamine. 27. The method of claim 17 wherein the DNA of said nuclei is measured by a method comprising treating said nuclei with a protease and ribonuclease, mixing the treated nuclei with ethidium bromide and measuring the fluorescence of said nuclei. 28. The method of claim 17 wherein the bound radioactivity is extracted from said nuclei with alcohol. 29. The method of claim 18 wherein the alcohol is ethanol. 30. A method for rapidly determining the presence of functional steroid receptors in cancerous cells comprising assaying a tissue sample for nuclear steroid binding comprising: (a) obtaining a tissue sample by biopsy; (b) fragmenting said tissue sample into pieces containing intact cells; (c) disrupting said fragmented tissue sample in contact with about 1-5 units of colagenase per mg of tissue; (d) isolating the cells from said tissue sample; (e) incubating said cells with an amount of a radiolabelled steroid or a radiolabelled antisteroid capable of complexing with and saturating said receptors; (f) homogenizing said incubated cells; (g) isolating the cellular nuclei; (h) extracting the bound radioactivity from said nuclei; (i) measuring the total DNA of said extracted nuclei; and (j) comparing the amount of bound radioactivity and the total DNA to determine a measure of the total nuclear bound steroid receptors. 31. The method of claim 30 wherein about 1-25 mg of tissue are obtained by biopsy. 32. The method of claim 31 wherein about 1-5 mg of tissue are obtained by biopsy. 33. The method of claim 30 wherein said cells are incubated in medium comprising about 10-30 nM [.sup.3 H]-progesterone or [.sup.3 H]-estradiol. 34. The method of claim 30 wherein the cells are incubated for about 0.5-1.5 hr at about 18.degree.-37.degree. C. 35. The method of claim 13 wherein, in step (i), the nuclei are hydrolyzed with perchloric acid and reacted with diphenylamine. 36. The method of claim 30 wherein the bound radioactivity is extracted with absolute ethanol. 37. The method of claim 36 wherein said absolute ethanol comprises sodium acetate.

Details for Patent 4,806,464

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29