Effects of timolol on Ca2+ handling and viability in human prostate cancer cells.

Timolol is a medication used widely to treat glaucoma. Regarding Ca2+ signaling, timolol was shown to modulate Ca2+-related physiology in various cell types, however, the effect of timolol on Ca2+ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100-1000 µM induced [Ca2+]i rises. The Ca2+ signal in Ca2+-containing medium was reduced by removal of extracellular Ca2+ by approximately 75%. Timolol (1000 µM) induced Mn2+ influx suggesting of Ca2+ entry. Timolol-induced Ca2+ entry was partially inhibited by three inhibitors of store-operated Ca2+ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished timolol-evoked [Ca2+]i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca2+]i rises. Timolol at concentrations between 200 and 600 µM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca2+]i rises by evoking Ca2+release from the endoplasmic reticulum in a PLC-dependent manner, and Ca2+ influx via PKC-regulated store-operated Ca2+ entry. Timolol also caused cell death that was not linked to preceding [Ca2+]i rises.

Gelatinized core liposomes: A new Trojan horse for the development of a novel timolol maleate glaucoma medication.

Glaucoma treatment with ocular medications requires overcoming the corneal barrier to drug penetration. Liposomes have a great corneal penetration ability and affinity while suffering from poor stability and low entrapment of hydrophilic drugs accompanied by rapid drug release. This work aims to develop a new, effective and stable glaucoma medication with sustained drug release properties; Timolol maleate gelatinized core liposomes. A full factorial design was utilized to study the effects of three formulation variables on drug loading and vesicle particle size. Vesicles were prepared by the thin-film hydration method, and characterized for in-vitro drug release and stability. Intra-ocular pressure (IOP) reduction was evaluated in-vivo on glaucomatous rabbit's eyes. The safety profile was assessed using histopathological examinations. Gelatin significantly increased the drug entrapment percentage reaching 50% with a particle size of 38.81 µm. Sustained drug release was recorded compared to a marketed product and to a conventional liposomal formulation. The prepared vesicles caused the highest reduction in IOP accompanied by safe histological findings. This work provided a new, safe and effective ocular glaucoma medication; Timolol maleate gelatinized core liposomes, solving the main problems of ocular liposomal formulations of hydrophilic drugs, suitable for the pharmaceutical industry and comprising abundant and relatively cheap components.

Proposal of degradation pathway with toxicity prediction for hydrolytic and photolytic degradation products of timolol.

Timolol (TIM) is a potent ß-adrenergic blocker, useful in treatment of ocular hypertension or open-angle glaucoma. Development and validation of stability indicating LCMS assay method for TIM was accomplished coherent with ICH guideline. Successful chromatographic separation of TIM with its four degradation products was attained by using gradient elution mode on reverse phase column using ammonium acetate buffer, pH 4.6 as mobile phase A and organic solvent as the mobile phase B. Chromatographic conditions were set such as 1.0 mL min-1 flow rate, 20 µL injection volume, 30 °C column temperature and 320 nm detection wavelength. Four major degradation products obtained from hydrolysis and photolysis, were identified and characterized with the combination of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS/MS) and accurate mass measurements. Degradation pathways were identified based on a comparison of the fragmentation pattern of the [M+H]+ ions of TIM and its degradation products. The method validation was performed as per ICH guideline Q2 (R1).

Pure Glaucoma Drugs Are Toxic to Immortalized Human Corneal Epithelial Cells, but They Do Not Destabilize Lipid Membranes.

PURPOSE: Most pure glaucoma drugs (pGDs) are hydrophobic substances intended to reduce elevated intraocular pressure. The aims of our study were to determine the toxicity of pGDs (brimonidine tartrate, brinzolamide, latanoprost, timolol maleate, and pilocarpine hydrochloride) on ocular surface cells and to establish whether their toxicity is subsequent to cellular membrane destabilization. METHODS: The toxicity of clinically efficient doses of pGDs was measured at different time points in a cell culture of human corneal epithelial cells using a redox indicator. pGD interaction with the plasma membrane was analyzed using a hemolysis assay and liposome electrokinetic chromatography. The capacity of pGDs to induce endoplasmic reticulum stress was investigated by immunoblotting. RESULTS: The toxicity assay showed that all pGDs decrease the viability of the epithelial cells to variable degrees. Early toxicity was measured for 4% pilocarpine and 0.15% brimonidine with 60% cell death at 4 hours, whereas 2% pilocarpine and 0.005% latanoprost showed almost 100% toxicity but only after 16 hours. The hemolysis assay and liposome electrokinetic chromatography experiments suggested that interaction between pGDs and lipid membranes is weak and cannot explain cell death through lysis. Immunoblotting revealed that the drugs activate endoplasmic reticulum stress and, with the exception of pilocarpine, have the capacity to induce apoptosis through upregulation of C/EBP homologous protein. CONCLUSIONS: Our study indicates that all studied pGDs decrease the viability of the corneal epithelial cells, but none of the tested compounds were able to destabilize cellular membranes. The pGDs seem to be internalized and can induce apoptosis through C/EBP homologous protein recruitment.

Influence of Pilocarpine and Timolol on Human Meibomian Gland Epithelial Cells.

PURPOSE: Investigators have discovered that topical antiglaucoma drugs may induce meibomian gland dysfunction. This response may contribute to the dry eye disease commonly found in patients with glaucoma taking such medications. We hypothesize that drug action involves a direct effect on human meibomian gland epithelial cells (HMGECs). To test this hypothesis, we examined the influence of the antiglaucoma drugs, pilocarpine and timolol, on the morphology, survival, proliferation, and differentiation of HMGECs. METHODS: Immortalized (I) HMGECs (n = 2-3 wells/treatment/experiment) were cultured with multiple concentrations of pilocarpine or timolol for up to 7 days. Experiments included positive controls for proliferation (epidermal growth factor and bovine pituitary extract) and differentiation (azithromycin). Cells were enumerated using a hemocytometer and evaluated for morphology, neutral lipid staining, and lysosome accumulation. RESULTS: Our results demonstrate that pilocarpine and timolol cause a dose-dependent decrease in the survival of IHMGECs. The clinically used concentrations are toxic and lead to cell atrophy, poor adherence, or death. By contrast, drug levels that are known to accumulate within the conjunctiva, adjacent to the meibomian glands, do not influence IHMGEC survival. These latter concentrations also have no effect on IHMGEC proliferation or differentiation, and they do not interfere with the ability of azithromycin to stimulate cellular neutral lipid and lysosome accumulation. This dose of pilocarpine, though, did suppress the epidermal growth factor+bovine pituitary extract-induced proliferation of IHMGECs. CONCLUSIONS: Our results support our hypothesis and demonstrate that these antiglaucoma drugs, pilocarpine and timolol, have direct effects on HMGECs that may influence their morphology, survival, and proliferative capacity.

Ocular Cubosome Drug Delivery System for Timolol Maleate: Preparation, Characterization, Cytotoxicity, Ex Vivo, and In Vivo Evaluation.

Glaucoma is an ocular disease featuring increased intraocular pressure (IOP) and its primary treatment strategy is to lower IOP by medication. Current ocular drug delivery in treating glaucoma is confronting a variety of challenges, such as low corneal permeability and bioavailability due to the unique anatomical structure of the human eye. To tackle these challenges, a cubosome drug delivery system for glaucoma treatment was constructed for timolol maleate (TM) in this study. The TM cubosomes (liquid crystalline nanoparticles) were prepared using glycerol monooleate and poloxamer 407 via high-pressure homogenization. These constructed nanoparticles appeared spherical using transmission electron microscopy and had an average particle size of 142 nm, zeta potential of -6.27 mV, and over 85% encapsulation efficiency. Moreover, using polarized light microscopy and small-angle X-ray scattering (SAXS), it was shown that the TM cubosomes have cubic liquid crystalline D-type (Pn3m) structure, which provides good physicochemical stability and high encapsulation efficiency. Ex vivo corneal permeability experiments showed that the total amount of TM cubosomes penetrated was higher than the commercially available eye drops. In addition, in vivo studies revealed that TM cubosomes reduced the IOP in rabbits from 27.8∼39.7 to 21.4∼32.6 mmHg after 1-week administration and had a longer retention time and better lower-IOP effect than the commercial TM eye drops. Furthermore, neither cytotoxicity nor histological impairment in the rabbit corneas was observed. This study suggests that cubosomes are capable of increasing the corneal permeability and bioavailability of TM and have great potential for ocular disease treatment.

Comparison of Cytotoxic Effects on Rabbit Corneal Endothelium between Preservative-free and Preservative-containing Dorzolamide/timolol.

PURPOSE: To evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium. METHODS: This study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy. RESULTS: Corneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal. CONCLUSIONS: The main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.

Toxicity evaluation of antiglaucoma drugs using stratified human cultivated corneal epithelial sheets.

PURPOSE: To investigate the toxicity profiles of seven antiglaucoma topical eye drops and benzalkonium chloride (BAC) using stratified cultivated human corneal epithelial cell sheets (HCES) in a serum-free culture system. METHODS: A range of prostaglandin analogies and preservatives, including BAC, sofZia (SZ), sodium benzoate (SB), and polyquaternium-1 (PQ) were tested. The barrier function and cell viability were examined by a carboxyfluorescein permeability assay and WST-1 assay. Histological evaluation of the HCES was also performed after application of each solution. RESULTS: The carboxyfluorescein permeability assay had a higher sensitivity for the detection of toxicity of test solutions than the WST-1 assay or histological examination. Latanoprost BAC, latanoprost/timolol BAC, and 0.02% or higher concentration of BAC were the most toxic, followed by latanoprost SB, latanoprost preservative-free, BAC 0.002%, and travoprost/ latanoprost PQ. Travoprost SZ and tafluprost BAC (preserved with 0.001% BAC) was the least toxic in our experimental conditions. CONCLUSIONS: The carboxyfluorescein permeability assay using HCES in a serum-free system was the most useful for the quantification of toxicity of ophthalmic solutions. Among the regimens examined, a BAC concentration of 0.001% or lower or non-BAC preservative sofZia was suggested to be the least toxic to the ocular surface.

In vitro comparative toxicology of polyquad-preserved and benzalkonium chloride-preserved travoprost/timolol fixed combination and latanoprost/timolol fixed combination.

PURPOSE: To compare, in vitro, the cytotoxicity profile of a new formulation of travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution preserved with polyquaternium-1 0.001% (travoprost/timolol PQ) instead of benzalkonium chloride (BAK) with (1) commercially available travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution (travoprost/timolol BAK), (2) commercially available latanoprost 0.005%/timolol 0.5% fixed combination ophthalmic solution (latanoprost/timolol BAK), and (3) their associated BAK concentrations. METHODS: Compounds tested on Wong-Kilbourne-derived human conjunctival epithelial cells: (1) phosphate-buffered saline, (2) polyquaternium-1 0.001% (Polyquad(®), PQ), (3) travoprost/timolol PQ, (4) travoprost/timolol BAK with 0.015% BAK (DuoTrav(®)), (5) BAK 0.015%, (6) latanoprost/timolol BAK with 0.020% BAK (Xalacom(®)), and (7) BAK 0.020%. Toxicological assays were used to assess cell viability [neutral red (NR), Alamar blue (AB)], apoptosis (YO-PRO-1, Hoechst 33342), and oxidative stress (H(2)DCF-DA, hydroethidine). The apoptosis and oxidative stress assays were each reported according to cell viability as observed with NR and AB (totaling 10 analyses per treatment). RESULTS: The NR and AB assays demonstrated that cells incubated with travoprost/timolol PQ had significantly better viability than cells incubated with latanoprost/timolol BAK, travoprost/timolol BAK, BAK 0.015%, and BAK 0.020% (P<0.0001 for all). As assessed with YO-PRO-1 and Hoechst 33342 relative to cell viability determined with NR or AB, travoprost/timolol PQ produced significantly less apoptosis than travoprost/timolol BAK and latanoprost/timolol BAK and their respective BAK concentrations alone (P<0.0001 for all). Also, travoprost/timolol BAK induced less apoptosis than latanoprost/timolol BAK (P<0.0001). As assessed with H(2)DCF-DA as a ratio to NR or AB, all of the compounds without BAK (phosphate-buffered saline, PQ 0.001%, and travoprost/timolol PQ) and travoprost/timolol BAK produced significantly less reactive oxygen species than latanoprost/timolol BAK (P<0.0001 for all). As assessed with hydroethidine as a ratio to NR or AB, travoprost/timolol PQ produced significantly fewer superoxide anions than latanoprost/timolol BAK (P<0.0001). In contrast, release of superoxide anions (hydroethidine method) after incubation with travoprost/timolol BAK was not significantly different from incubation with latanoprost/timolol BAK or travoprost/timolol PQ. CONCLUSION: Travoprost/timolol PQ may be better for ocular surface health than either BAK preserved formulations of latanoprost/timolol or travoprost/timolol.

Ion- and pH-activated novel in-situ gel system for sustained ocular drug delivery.

Poor bioavailability (<1%) of drugs from conventional eye drops is mainly due to the various precorneal loss factors which include rapid tear turnover, systemic drug absorption through naso-lachrymal duct, transient residence time of the drug solution in the cul-de-sac and the relative impermeability of the drugs to corneal epithelial membrane. The present study describes the formulation and evaluation of chitosan and gellan gum based novel in-situ gel system activated by dual physiological mechanisms. Chitosan (a pH-sensitive polymer) in combination with gellan gum (an ion-activated polymer) were used as gelling agent. Timolol maleate, the drug which is frequently used for glaucoma therapy was used as model drug to check the efficacy of the formulation. The developed formulation was characterized for various in vitro parameters, for example, clarity, gelation pH, isotonicity, sterility, viscosity, transcorneal permeation profile, and ocular irritation. Ocular retention was studied by gamma scintigraphy and a significant increase in retention time was observed. The formulation was also found to be nonirritant and well tolerable. The developed system can be a viable alternative to conventional eye drops for the treatment of various ocular diseases and is suitable for clinical application.

Very-high-frequency ultrasound corneal imaging as a new tool for early diagnosis of ocular surface toxicity in rabbits treated with a preserved glaucoma drug.

AIM: To evaluate very-high-frequency (VHF) ultrasound imaging as a new method to detect and quantify early corneal epithelium changes induced by chronic exposure to a benzalkonium-chloride-containing antiglaucoma drug. METHODS: Timolol preserved with 0.01% benzalkonium chloride solution was applied b.i.d. in 1 eye of 10 rabbits for 56 days. Unpreserved timolol solution was used as control. Ocular surface changes were assessed weekly combining clinical examinations, in vivo 60-MHz ultrasound imaging and ex vivo histological analysis. RESULTS: VHF ultrasound imaging allowed quantitative measurement of corneal epithelium thickness and qualitative imaging of toxic epithelial damage. It revealed significantly decreased epithelial thickness in vivo as early as the 21st day of treatment (40.75 +/- 1.72 microm at D0 vs. 39 +/- 2 at D21, vs. 31.9 +/- 2.98 at D56; p = 0.017 and p = 0.005, respectively). The first clinical changes appeared from the 42nd day of treatment (conjunctival redness, conjunctival staining and corneal staining; D56 compared to D0: p = 0.005, 0.01 and 0.004, respectively) and then correlated with VHF ultrasound data. Epithelial thickness measured with VHF ultrasound was correlated with histological epithelial pachymetry (p < 0.001) and with the corneal damage score assessed with scanning electron microscopy (p = 0.038). CONCLUSION: VHF ultrasound imaging provided an early in vivo diagnosis of corneal epithelium pathology induced by chronic exposure to a preserved glaucoma drug, before the first clinical evidence of ocular toxicity. It could be a new reproducible method to detect the toxicity of glaucoma medication so that therapy can then be adapted.

Drug modification of angiogenesis in a rat cornea model.

PURPOSE: To evaluate the influence of some widely used antiglaucoma agents on angiogenesis in a novel rat cornea model. METHODS: Angiogenesis was induced in 32 rats by slow-release polymer pellets containing basic fibroblast growth factor (bFGF) placed in a corneal micropocket. Angiogenesis was later measured and compared in groups of rats given one of four antiglaucoma drug therapies and one control group. The drugs were commercially available preparations of prostaglandins, beta-blockers, alpha-2 agonists, and carbonic anhydrase inhibitors given for 7 days in a manner similar to that used in humans. Growth was measured by calculating the maximum linear vessel growth divided by pellet-limbus distance. RESULTS: Biomicroscopic observation disclosed that all tested animals showed an induction of neovascular reactions in their corneal stroma. The growth index results for the control, latanoprost, dorzolamide, brimonidine, and timolol malate groups were 1.65 +/- 0.16, 1.98 +/- 0.18, 1.85 +/- 0.19, 2.03 +/- 0.38, and 1.65 +/- 0.14, respectively, confirming the hypothesis that topically delivered antiglaucoma drugs modify the normal angiogenic response. Of them, the prostaglandins showed the most prominent angiogenic stimulatory effect (P = 0.03). CONCLUSIONS: This modified micropocket assay of corneal angiogenesis in rats demonstrated the stimulatory effect of several widely used topically delivered antiglaucoma medications on the angiogenic process. The results indicate that the selection of drugs for treating different ophthalmic diseases should take into account their influence on angiogenic processes.

Cellular cytotoxicity of antiglaucoma drugs in cultured corneal endothelial cells.

In this study, the various antiglaucoma drugs including betaxolol, timolol, levobunolol, carteolol, brimonidine, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine were used to investigate the effects of cellular cytotoxicity in cultured bovine corneal endothelial cells. After exposure to the drugs in three dilutions, 1/100, 1/1,000, and 1/10,000, for 100 minutes, cells were estimated based on the release assay of lactate dehydrogenase (LDH) enzyme. It was found that cellular LDH was significantly released in the medium only at 1/100th dilution of betaxolol, brimonidine, dorzolamide, dipivefrin, latanoprost and unoprostone to 130%, 123%, 145%, 157%, 128% and 237%, respectively, compared with controls upon exposure to drugs for 100 minutes. Moreover, benzalkonium chloride preservative at the concentrations ranging from 0.001 to 0.00001 mg/mL did not affect cellular LDH release in bovine corneal endothelial cells. These results indicate that high concentrations of antiglaucoma drugs may induce cytotoxicity in corneal endothelial cells.

[In vivo assessment of corneal epithelial toxicity of timolol with benzalkonium chloride using very-high-frequency ultrasound imaging]. / Evaluation in vivo par échographie à très haute fréquence des modifications épithéliales cornéennes induites par un bêta-bloquant avec 0,01% de chlorure de benzalkonium.

PURPOSE: To assess in vivo the corneal epithelial damage caused by a topical toxic medication using a 60-MHz ultrasound device. MATERIAL: and methods: A solution of timolol with 0.01% benzalkonium chloride (BAC) was applied twice a day in the test eyes of ten rabbits, and a BAC-free solution of timolol in the control eyes, for 56 days. We used a 60-MHz ultrasound device to evaluate the epithelial damage in BAC-exposed eyes, compared to control eyes. The clinical and ultrasound examinations were performed every week, and the histological analysis at the end of the experiment. RESULTS: The clinical findings were conjunctival redness, corneal staining and instability of the tear film. In vivo VHF ultrasound revealed a thinning of the epithelium of test eyes (from 40.9+/-1,6 microm at D0 to 31.8+/-3.4 microm at D56; p=0.0006 for D0 vs D56), while the epithelium of control eyes remained unchanged. Ultrasound epithelial thickness was correlated with corneal staining (at D34 and D56; p=0.0025 and 0.0377, respectively) and histological epithelial pachymetry (p=0.0176 for control and 0.0505 for tested epithelium). Moreover, we report qualitative VHF ultrasound imaging of early epithelial damage. CONCLUSION: This new device could be very useful in ocular toxicity evaluation as a reproducible and reliable tool for multicentric clinical research.

[A comparative study of the ocular tolerance of 3 timolol-based preparations: the influence of preservatives on ocular tolerance]. / Etude comparative de la tolérance oculaire de 3 spécialités à base de timolol: influence du conservateur sur la tolérance oculaire.

PURPOSE: Ophthalmic preparations can cause toxic ocular reactions, often associated with the use of preservatives. The aim of this study was to compare the ocular tolerance of three ophthalmic preparations based on timolol: a preservative free ophthalmic preparation (Timabak) and two other commercially available preserved preparations (Timoptol) and Timoptol LP). METHODS: The effect of repeatedly instilling eye drops for 28 days on rabbit eyes was assessed in vivo by mean of a confocal laser scanning ophthalmoscope. The corneal microlesions were selectively marked by fluorescein. RESULTS AND CONCLUSION: The overall results show the good ocular tolerance of the three tested products. However, a closer comparison between the products brought out differences in the extent of lesions among the tested products depending on their composition. Indeed the preservative free eye drops appeared better tolerated than the two preserved preparations.

[Toxicity of preserved and unpreserved beta-blocker eyedrops in an in vitro model of human conjunctival cells]. / Toxicité des collyres bêta-bloquants avec ou sans conservateur dans un modèle in vitro de cellules conjonctivales humaines.

PURPOSE: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in an in vitro model of human conjunctival cells. METHODS: Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation, mitochondrial mass and activity, free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. In addition, cell size and the expression of an apoptotic marker Apo2.7 were studied by flow cytometry. RESULTS: Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small, significantly less important decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-). 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol -BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation, decrease in mitochondrial membrane potential and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Also reactive oxygen species (ROS) production was significantly more important after cell exposure to timolol-BAC(+). CONCLUSION: In our model of conjunctival cells in vitro, timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for ROS production and for cell viability variations. Oxidative stress could also play a role in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

Comparison of the effects of preserved and unpreserved formulations of timolol on the ocular surface of albino rabbits.

Thirty-six albino rabbits, randomly divided into six groups, were used to study their ocular tolerance to (a) 0.25 and 0.50% Timoptol preserved with 0.01% benzalkonium chloride, (b) 0.25 and 0.50% Timoptol-LP, a gel-forming solution preserved with 0. 012% benzododecinium bromide, and (c) 0.25 and 0.50% Timabak unpreserved in the ABAK eyedrops dispenser. All eyedrops were applied in the right eye for 60 days. A clinical follow-up with slitlamp examination and break-up time evaluation was performed for 2 months. At the end of the experimentation, the animals were sacrificed and their eyes enucleated for histological analyses of the conjunctiva and cornea. There was no significant difference in the clinical examination between each group, except for the break-up time evaluation between Timoptol and Timabak at each concentration which was better with the unpreserved timolol. Histological results showed a significant difference in the corneal stroma edema between preserved and unpreserved timolol. This study confirms that using unpreserved timolol may be beneficial for the long-term treatment of glaucomatous patients as it increases tear film stability and decreases epithelial permeability and stromal aggression of the cornea.

Relationship between amount of beta-blockers permeating through the stratum corneum and skin irritation after application of beta-blocker adhesive patches to guinea pig skin.

We evaluated the relationship between the cumulative amounts of 5 kinds of beta-blockers (alprenolol, oxprenolol, timolol, acebutolol and atenolol) permeating through the stratum corneum and a* values obtained by measuring the formation of erythema, a skin irritation reaction, with a chromameter after transdermal application of adhesive patches containing 2 beta-blocker to the skin of guinea pigs. The cumulative amount of beta-blocker released from each adhesive patch to the skin increased with the increase in application time. The contents of alprenolol, oxprenolol and timolol in the stratum corneum and in the stripped skin increased markedly up to 4 h after application and thereafter were maintained at high levels up to 24 h. The contents of acebutolol and atenolol, on the other hand, increased up to 24 h, but these values were low. a* values of all adhesive patches 24 h after application were higher than those before application. The correlation coefficients between the cumulative amounts of alprenolol, oxprenolol, timolol, acebutolol or atenolol permeating through the stratum corneum and (delta a* -delta a*Placebo) values were 0.739, 0.717, 0.722, 0.551 and 0.633, respectively. The correlation coefficient calculated by averaging the cumulative amounts of 6 kinds of beta-blockers permeating through the stratum corneum [including propranolol which was reported previously (Kobayashi I., et al., Biol. Pharm. Bull., 19, 839-844 (1996))] was 0.731, higher than the correlation coefficient between contents of these beta-blockers in the stripped skin and (delta a* -delta a*Placebo) values (r = 0.552). This suggests that there was a high correlation between the cumulative amounts of beta-blockers permeating through the stratum corneum and (delta a* -delta a*Placebo) values.

[The action of amphiphilic drugs on human fibroblasts in vitro. Their possible use in antiviral and antitumor chemotherapy]. / Observations sur l'action de quelques drogues amphiphiles sur les fibroblastes humains in vitro. Applications possibles en chimiothérapie antivirale et antitumorale.

The LDH cytotoxicity test is able to emphasize the stabilisation of human embryo fibroblast membranes, in vitro, by two amphiphilic drugs: the metomidate and the thymolol. The membrane stabilisation is emphasized by the reduced LDH externalization as compared with the untreated cells. The use of the LDH test for the selection of some natural complexes or synthetic drugs with membrane stabilising and potential antiviral activity is proposed. The inhibition of LDH by metomidate was recorded, as decreasing of the enzyme activity with increasing metomidate concentration. This fact explains the known lactic acid accumulation, under metomidate treatment of human subjects. The use of metomidate in classical cancer treatment potentiation is proposed instead of lactate externalization inhibitors, already used for this aim.

Microemulsions as topical delivery vehicles: ocular administration of timolol.

The topical administration of timolol as an ion-pair with octanoate was achieved by use of an oil-in-water microemulsion containing lecithin as a surfactant. The microemulsion, a solution of the ion-pair and a solution of timolol alone were instilled in the conjunctival sac of rabbits. A rapid method for the separation and determination of timolol in aqueous humour by HPTLC was used. The bioavailability of timolol from the microemulsion and the ion-pair solution was higher than that obtained from timolol alone. The areas under the curve for timolol in aqueous humour after administration of the microemulsion and the ion-pair solution were 3.5 and 4.2 times higher, respectively, than that observed after the administration of timolol alone.