Claims for Patent: 5,360,720

Summary for Patent: 5,360,720

Title:	Method of preparing human conjunctival mast cells for mast cell stabilization assays
Abstract:	By allowing the mast cell enriched population of human conjunctival tissue cells to incubate for a minimum of about forty (40) hours post enzymatic digestion, the treatment window between spontaneous histamine release and anti-human IgE stimulated histamine release is increased. Culturing the human conjunctival tissue mast cells decreases the spontaneous release of histamine and increases the anti-IgE stimulated histamine release, greater than ten fold over spontaneous, at time points over forty (40) hours. This treatment window is sufficient to detect a compound\'s stabilizing or anti-allergic activity.
Inventor(s):	Miller; Steven T. (Arlington, TX), Yanni; John M. (Burleson, TX)
Assignee:	Alcon Laboratories, Inc. (Fort Worth, TX)
Application Number:	08/134,148
Patent Claims:	1. A method of preparing human conjunctival mast cells for use in mast cell stabilization assays and evaluation of antiallergic drugs comprising the steps of: (a) enzymatically digesting human conjunctival tissue; (b) washing the tissue of step (a) with a buffer over a filter so that the filtrate is collected; (c) enriching the filtrate of step (b) for mast cells; (d) forming a mast cell preparation by resuspending the enriched cells in a buffer; (e) allowing the mast cell preparation to equilibrate at about 30.degree.-37.degree. C. in a supplemented tissue culture medium comprising serum and non-stable essential amino acids for a minimum of about 40 hours; (f) harvesting the mast cells from the culture medium; and (g) determining viability and number of mast cells. 2. The method of claim 1 wherein the filtrate is enriched in stpe (C) by centrifugation over a Percoll.RTM. cushion of about 1.058 g/L. 3. The method of claim 1 wherein the buffer is Tyrode's buffer containing 0.1% gelatin. 4. The method of claim 1 wherein the culture medium is supplemented with serum, non-stable essential amino acids, an antibiotic, a fungicide, and a biological buffer. 5. The method of claim 4 wherein the culture medium is RPMI 1640 supplemented with fetal bovine serum (20%), L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 .mu.g/ml), amphotericin B (2.5 .mu.g/ml), and HEPES (10 mM). 6. The method of claim 1 wherein the enzymatic digestion is accomplished with a mixture of collagenase (Type IV) and hyaluronidase (Type I-S). 7. The method of claim 1 wherein the enzymatic digestion in step (a) is accomplished in two stages. 8. The method of claim 7 wherein the amount of enzyme per gram of tissue in the first stage is about 200 U each of collagenase (Type IV) and hyaluronidase (Type I-S), and the amount of enzyme per gram of tissue in the second stage is about 2000 U each of collagenase (Type IV) and hyaluronidase (Type I-S). 9. The method of claim 1 wherein the mast cell preparation is allowed to equilibrate for a minimum of about 60 hours. 10. A method of preparing human conjunctival mast cells for use in mast cell stabilization or anti-allergic activity assays comprising the steps of: (a) enzymatically digesting human conjunctival tissue using about 200 U each of collagenase (Type IV) and hyaluronidase (Type I-S) per gram of tissue; (b) washing the human conjunctival tissue of step (a) with Tyrode's buffer containing 0.1% gelatin over a filter so that some intact tissue remains on top of the filter and the filtrate is collected; (c) further enzymatically digesting the intact human conjunctival tissue of step (b) using about 2000 U each of collagenase (Type IV) and hyaluronidase (Type I-S); (d) washing the intact human conjunctival tissue of step (c) with Tyrode's buffer containing 0.1% gelatin over a filter so that the filtrate is collected; (e) centrifuging the filtrate of steps (b) and (d) over a Percoll.RTM. cushion of about 1.058 g/L to form pelleted cells; (f) forming a mast cell preparation by resuspending the pelleted cells in Tyrode's buffer containing about 0.1% gelatin; (g) allowing the mast cell preparation to equilibrate at about 30.degree.-37.degree. C. in a culture medium of RPMI 1640 supplemented with fetal bovine serum (20%), L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (100 .mu.g/ml), amphotericin B (2.5 .mu.g/ml), and HEPES (10 mM) for a minimum of about 60 hours; (h) harvesting the mast cells from the culture medium; and (i) determining viability and number of mast cells.

Details for Patent 5,360,720

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	05/05/2004		2039-02-26
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	12/02/2004		2039-02-26
Amphastar Pharmaceuticals, Inc.	AMPHADASE	hyaluronidase	Injection	021665	10/26/2004		2039-02-26
Akorn, Inc.	HYDASE	hyaluronidase	Injection	021716	10/25/2005		2039-02-26
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-02-26