Patent Application: US-201113698730-A

Abstract:
a method of treating endometriosis in a person by reducing a cytokine mcp - 1 related activity level of an endometriosis implant in said person by 50 % or more while not affecting a corresponding tnf - α related activity level by more than comprises providing a pharmaceutical composition comprising a local anaesthetic , in particular lidocaine hydrochloride , and a pharmaceutically acceptable carrier , administering the composition intraperitoneally to said person , thereby substantially reducing the recruitment of macrophages by mcp - 1 released from the endometriosis implant . also disclosed is a corresponding pharmaceutical composition .

Description:
preparing cultures of endometriosis cells . during laparotomy , human endometriosis tissue biopsies from endometriosis cysts were obtained from four women during laparotomy . the tissue samples were handled aseptically , transported to the laboratory and frozen in fluid nitrogen to − 150 ° c . the biopsies were later verified histologically as endometriosis tissue . the sampling procedure was approved by the ethical committee . the frozen endometriosis tissues were thawed in water at + 37 ° c . and then transferred to test tubes . two tissue biopsy samples were taken from each endometrioma and prepared as follows : the endometriosis tissue covering the inside of endometriosis cysts was detached from encapsulating fibrous tissue by gentle scraping with a knife . the tissue was then exposed for 0 . 2 % collagenase type iii ( worthington biochemical corporation freehold , n . j ., usa ) for 2 h . the cell suspension was then centrifuged at 1500 rpm for 10 min . the cells were re - suspended in rpmi 1640 cell culture medium with l - glutamine and phenol red ( invitrogen ltd ), 20 % foetal bovine serum ( invitrogen ltd ), sodium pyruvate , mem 100 mm ( invitrogen ltd ), non - essential amino acids ( invitrogen ltd ), heparin 90 μg / ml ( sigma - aldrich fine chemicals st louis , mo ., usa ), penicillin - streptomycin with 10 . 000 units / ml penicillin g sodium and 10 . 000 μg / ml streptomycin sulphate in 0 . 85 % saline ( invitrogen ltd ), hepes buffer solution 1m ( invitrogen ltd ) and seeded in t - 75 cm 2 cell culture flasks ( nunc , roskilde , denmark ) pre - coated with 0 . 2 % gelatin ( sigma - aldrich ). the culture medium was changed the following day and thereafter once a week . when confluent monolayers were obtained after 3 weeks , the cultures were tryptinized cultures from passages 4 and 6 were frozen in fluid nitrogen to − 150 ° c . and later used for experiments . incubations of endometriosis cells and lidocaine . the lidocaine treatments of the cells were performed in duplicates on the endometriosis cells that were used for experiments . the thawed cells were re - suspended in rpmi 1640 cell culture medium with l - glutamine and phenol red ( 21875 - 042 invitrogen , stockholm , sweden invitrogen ltd ) including 15 % foetal bovine serum ( cat . nr . 10270 - 106 invitrogen , stockholm , sweden invitrogen ltd ), 0 . 8 % sodium pyruvate , 1 % hepes ( cat . nr . 15630 - 056 , invitrogen , stockholm , sweden ), 0 . 8 % ecgs ( cat . no . 354006 , becton - dickinson ), 0 . 8 % mem non - essential amino acids ( cat . no . 11140 - 035 invitrogen , stockholm , sweden invitrogen ltd ), heparin 90 μg / m1 ( cat . no . h3149 sigma - aldrich fine chemicals st . louis , mo ., usa ), penicillin - streptomycin with 10 . 000 units / ml penicillin g sodium and 10 . 000 μg / m1 streptomycin sulphate in 0 . 85 % saline ( cat . no . 151140 - 122 , invitrogen , stockholm , sweden ), and seeded in t - 25 cm 2 cell culture flasks ( nunc , roskilde , denmark ) pre - coated with 0 . 2 % gelatin ( sigma - aldrich ) and maintained in a 5 % co 2 / 95 % humidified air atmosphere at 37 ° c . the cells were incubated with added lidocaine hydrochlorideat a final concentration of 1 mg / ml . the lidocaine treatment had duration of 24 and 48 hours respectively . in the control cultures there was no addition of lidocaine to the incubation medium . after incubation the culture media was collected and snap frozen − 70 ° c . the production of mcp - 1 , tnf - α and il - 6β was measured according to the manufacturer &# 39 ; s using an enzyme - linked immunosorbent assay ( elisa , quantikine , r & amp ; d systems , minneapolis , minn . 55413 , usa ). in vitro results . the elisa analyses showed a reduced cytokine production from the endometriosis cells during incubation with lidocaine . a substantial reduction of mcp - 1 was noted whereas the production of tnf - α was unaffected . the concentration of il - 1β in the cell medium was at a low level both before and after incubation with lidocaine ( table 1 ). treatment of endometriosis by selectively reducing release of mcp - 1 by endometric cells while not substantially effecting the release of tnf - a from the same cells . to a female subject diagnosed with inflammation caused by endometriosis is administered intraperitoneally a single dose of 10 ml of saline comprising 1 mg / ml of lidocaine . hydrochloride . 1 . hao m , shi y , dong m . measurements of interleukin - 6 , interleukin - 8 and transforming growth factor - beta 1 levels in peritoneal fluid of patients with endometriosis . zhonghua fu chan ke za zhi . 2000 ; 35 ( 6 ): 329 - 31 . 2 . agic a , xu h , finas d , banz c , diedrich k , hornung d . is endometriosis associated with systemic subclinical inflammation ? 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