Fourteen dry eye volunteers placed one to two drops of 0.15% AF2975 (N,N-dimethyl-2-phenylethylamine HCl) in one eye and the vehicle in their other eye four times a day for 21 days. AF2975 is a sigma agonist known to stimulate the release of tear proteins after instillation in rabbit eyes and was tested for its ability to stabilize protein film extracted from dry eye volunteers. After day 7 and again after day 21, Schirmer test strips were inserted in each eye for 5 minutes, measured for wetting, and stored at -20 degrees C for protein analysis. A volume of 600 microliters was used to extract total protein. A portion of the extract was analyzed for total protein. The remainder was used to measure surface tension, to determine in vitro break up time (in vitro BUT) in a newly designed apparatus, and to further analyze for tear lipocalin, formerly known as presystemic tear albumin. Statistically significant differences were obtained between the drug treated eye and the vehicle treated eye for measurements determined for days 7 and 21. Tear extracts from the drug treated eye showed statistically significant decreases in surface tension and increases in in vitro BUT. Extracts from the drug treated eye also showed statistically significant increases in protein content and tear lipocalin. The results suggest that AF2975 may be able to stabilize the tear film by increasing the concentration of proteins in human tears.

3H-DTG (1.3-di(2-[5-3H]tolyl)guanidine) or 3H-haloperidol was added to areceptors (25 nM) in the presence of 25 nM splperone and incubated with increasing concentrations of bromhexine derivatives (phenylalkylamines; 10~9 to 10~2 M) in membrane homogenate suspensions. IC50 values for two derivatives ranged from 3.2 to 8.8 nM for both radioligands. A preferred derivative, 7A (N, N'-dimethy1-2-phenylethylamine) , yielded an IC50 of 7.8 nM for 3H-haloperidol but showed much less affinity in displacing 3H-DTG (IC50=900 nM). Applying the technic of Bromberg [Exp. Eye Res. , 40:313-320, 1985] , in vitro protein secretion rates were measured following stimulation of either lacrimal gland slices or isolated, intact lacrimocytes with the compounds, in vitro protein secretion rates exhibit a dose-response relationship with increases in protein release up to a concentration of 10~8 to 10"4 M for various derivatives of bromhexine and ÎO-4 M for carbachol. By means of Schirmer strips, tear fluid was collected over a five minute period at 10 and 60 minutes post-dosing following the topical application (50/il) to the right eye of New Zealand white rabbits (n=20-24) of 7A at various concentrations. Incubation of lacrimocytes with 7A alone (10"'' M) , with haloperidol (lO"4 M) alone or in combination show that 7A is acting as an agonist to stimulate protein release, whereas haloperidol is acting as an antagonist to inhibit release. In vivo protein secretion rates also show a doseresponse curve (at both 10 and 60 minutes post-dosing) for 7A that reach a statistically significant maximum in the dosed eye at a concentration of 0.15% w/v. Analysis of protein extracts using size exclusion HPLC shows an increase in secretory proteins, particularly tear-specific prealbumin.

This study was undertaken to gain an understanding of the significance of tear proteins in stabilizing the tear film. Either a sigma agonist, N,N-dimethyl-2-phenylethylamine HCl (AF2975), or a sigma antagonist, haloperidol, was administered to rabbit eyes in order to increase or decrease protein secretion, respectively. At 0, 10 and 60 minutes after instillation, tear proteins were extracted from Schirmer strips and measured for total protein. A portion of the extract was used for separating five major protein fractions using size-exclusion HPLC. Total protein extract or individual protein fractions were measured for surface tension by the horizontal capillary method and for in vitro break up time (in vitro BUT), a newly designed procedure. A statistically significant decrease was measured for surface tension and a concomitant increase was measured for in vitro BUT for the total protein samples at 10 and 60 minutes after instillation of AF2975 compared to the vehicle treated eye. The results for haloperidol yielded an increase in surface tension and an decrease in in vitro BUT. When the tear proteins were separated into five major fractions, only the 23 minute protein fraction was found to decrease surface tension and increase in vitro BUT following AF2975 administration. Haloperidol, a sigma antagonist, showed an exact opposite effect for the total protein and the 23 minute protein fraction.

No abstract

In an effort to develop a long-term topically active tear stimulant, it was important to determine if tolerance developed following the repeated instillation of N,N-Dimethyl-2-phenylethylamine hydrochloride (AF2975) to the albino rabbit eye. New Zealand white rabbits were administered AF2975 (0.15%) twice a day (9 am and 4:30 pm) for 10 days. The right eye received the drug solution (50 microliters) and the left eye received an equal volume of the vehicle. Prior to dosing and at the end of first and last dose (10 and 60 minutes post-dosing), protein secretion was measured with the use of Schirmer tear test strips placed under the lower lid of each eye for five minutes. The strips absorbed tears from which protein was extracted. Eyes treated with AF2975 showed a statistically significant % increase in protein release compared to baseline values. Control eyes did not show statistically significant increases over baseline. A comparison of % changes from baseline in protein secretion rates after the first and last dose showed no significant differences in either treated or control eyes at 10 and 60 minutes postdosing. These results indicate that tolerance does not occur for protein secretion of topically administered AF2975 (0.15%) following a twice a day dosing schedule for 10 days to the rabbit eye.