title: Conjunctival proinflammatory and proapoptotic effects of latanoprost and preserved and unpreserved timolol: an ex vivo and in vitro study.
abstract: OBJECTIVE
  To compare the toxicity of latanoprost and preserved and unpreserved timolol on conjunctival cells. Expression of inflammatory markers and MUC5AC-related mucin production were evaluated by impression cytology in a case-control ex vivo study. The proapoptotic effect of the same drugs was also evaluated in vitro in a conjunctival cell line and compared with that of benzalkonium chloride (BAC).
  
  
  METHODS
  Impression cytology (IC) specimens were obtained from a series of normal subjects and from patients with glaucoma treated for at least 1 year with latanoprost eye drops or preserved or unpreserved timolol. All groups were comparable in age and duration of treatment. Expression of HLA-DR, intercellular adhesion molecule (ICAM)-1, and mucin was evaluated in a masked manner by flow cytometry. For the in vitro study, a human conjunctiva-derived cell line was treated with 0.02% BAC-containing latanoprost or timolol, unpreserved timolol, or 0.02% BAC alone for 15 minutes, followed or not by 4 or 24 hours of cell recovery in normal medium. Cell viability and chromatin condensation were evaluated using microplate cold light cytofluorometry with the neutral red and the Hoechst 33342 tests, respectively. The Hoechst-neutral red ratio was defined for the apoptosis assay, and cytoskeleton changes were assessed by confocal microscopy.
  
  
  RESULTS
  No difference was found between normal eyes and those receiving unpreserved timolol. Preserved latanoprost and timolol significantly increased the inflammatory marker expression and decreased MUC5AC expression, but to a significantly higher extent in the preserved timolol group compared with latanoprost. In vitro, 0.02% BAC-containing timolol and latanoprost triggered conjunctival cell apoptosis-however, to a significantly lesser extent than did 0.02% BAC alone. Unpreserved timolol did not cause any cell toxicity.
  
  
  CONCLUSIONS
  These ex vivo and in vitro studies demonstrate that BAC-containing latanoprost and timolol exhibit higher proinflammatory and proapoptotic effects on conjunctival cells than does unpreserved timolol. Latanoprost caused less toxicity, however, than preserved timolol, and both drugs were less toxic than BAC alone. These results suggest a potential protective effect of the prostaglandin analogue and to a lesser extent of timolol against the toxicity of BAC in conjunctival cells.
authors.affiliation: Department of Ophthalmology, University Hospital of Tours, Tours, France.
authors.forename: Pierre-Jean
authors.initials: PJ
authors.lastname: Pisella
authors.forename: Caroline
authors.initials: C
authors.lastname: Debbasch
authors.forename: Pascale
authors.initials: P
authors.lastname: Hamard
authors.forename: Catherine
authors.initials: C
authors.lastname: Creuzot-Garcher
authors.forename: Patrice
authors.initials: P
authors.lastname: Rat
authors.forename: Françoise
authors.initials: F
authors.lastname: Brignole
authors.forename: Christophe
authors.initials: C
authors.lastname: Baudouin
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000959:Antihypertensive Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001548:Benzalkonium Compounds / Q000494:pharmacology; D016022:Case-Control Studies; D002460:Cell Line; D002470:Cell Survival; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D005902:Glaucoma, Open-Angle / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D000077338:Latanoprost; D008297:Male; D008875:Middle Aged; D055271:Mucin 5AC; D009077:Mucins / Q000378:metabolism; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D011461:Prostaglandins F, Synthetic / Q000494:pharmacology*; D013999:Timolol / Q000494:pharmacology*
chemical_list: D000959:Antihypertensive Agents; D001548:Benzalkonium Compounds; D006684:HLA-DR Antigens; C524678:MUC5AC protein, human; C524680:Muc5ac protein, rat; D055271:Mucin 5AC; D009077:Mucins; D011310:Preservatives, Pharmaceutical; D011461:Prostaglandins F, Synthetic; D018799:Intercellular Adhesion Molecule-1; D000077338:Latanoprost; D013999:Timolol