Different concentrations of betaxolol switch cell fate between necroptosis, apoptosis, and senescence in human corneal stromal cells.

Betaxolol is commonly used to manage glaucoma in clinical practice. However, its long-term use may damage the cornea. Thus, the cytotoxicity and mechanisms of betaxolol in human corneal stromal cells (HCSCs) warrant further study. In this study, we used in vitro HCSCs and in vivo rabbit corneal models to investigate betaxolol cytotoxic effects and mechanism of action. At near-clinical concentrations (0.28% and 0.14%), betaxolol inhibited caspase-8 activity, activated receptor-interacting protein kinase (RIPK)1, RIPK3, and mixed-spectrum kinase-like domain (MLKL), and phosphorylated MLKL to induce necroptosis in HCSCs. Similarly, moderate concentrations of betaxolol (0.07%-0.0175%) activated caspase-8 to trigger the exogenous apoptotic pathway. Through the intrinsic apoptotic pathway, betaxolol upregulated the expression of Bcl-2 family apoptotic proteins Bax and Bad and downregulated that of anti-apoptotic proteins Bcl-2 and Bcl-xL. This subsequently disrupted the mitochondrial membrane potential and cytoplasmic transfer of cytochrome c and apoptosis-inducing factor, activated caspase-9, and induced apoptosis in HCSCs. Furthermore, continuous treatment with low betaxolol concentrations (0.00875%) for three generations of HCSCs prevented apoptosis by promoting the expression of Bcl-xL and suppressing that of Bax. However, its toxic effects initiated cellular senescence by increasing reactive oxygen species, leading to the disruption of energy metabolism and DNA damage. Finally, clinical concentrations of betaxolol had a pro-apoptotic effect on rabbit corneal stromal cells in vivo. These results suggest that betaxolol induces cytotoxicity in a concentration-dependent manner in HCSCs, and that caspase-8 and Bcl-2 family proteins may be critical switches in the conversion of different HCSC death mechanisms.

An in vitro approach to investigate ocular metabolism of a topical, selective ß1-adrenergic blocking agent, betaxolol.

1. Topical glaucoma treatments have often been limited by poor absorption and bioavailability. Betaxolol, a selective ß1-blocker, has been well studied for its pharmacokinetics and disposition. Limited ocular, betaxolol metabolism data is available despite a growing number of novel ocular treatments. 2. In vitro ocular fractions indicated the formation of an active metabolite, across rat, rabbit and human, which was only observed historically in the liver. 3. Ocular metabolic profiles of preclinical toxicology species, rat and rabbit, were not predictive of human in vitro ocular data. M1 was specific to human and only captured by the liver data. 4. Liver S9 over predicted the extent of ocular metabolism compared to ocular fractions. Rabbit liver S9 fractions demonstrated extensive glucuronidation and higher parent turn-over in 1 h as compared to other matrices. 5. This research assesses in vitro species and organ differences across preclinical species and human. The complex data set highlights the need for an in vitro ocular system to explore poorly documented ocular metabolism.

Binding of betaxolol, metoprolol and oligonucleotides to synthetic and bovine ocular melanin, and prediction of drug binding to melanin in human choroid-retinal pigment epithelium.

PURPOSE: To characterize the binding of betaxolol, metoprolol and oligonucleotides to synthetic and bovine ocular melanin, and to predict the binding to melanin in human choroid-retinal pigment epithelium (RPE). MATERIALS AND METHODS: The shape, size and specific surface area of synthetic melanin and isolated melanin granules from bovine choroid-retinal pigment epithelium (RPE) were characterized by SEM, laser diffractometry and BET. The binding of betaxolol, metoprolol, fluorescein isothiocyanate (FITC)-labeled phosphodiesther oligonucleotides and 6-carboxyfluorescein (6-CF) to melanin was determined. The binding of beta-blockers to melanin in human choroid-RPE was estimated based on binding parameters and the melanin content in human choroid-RPE. RESULTS: Bovine melanin granules were round or oval with a mean diameter of ca. 1 mum. Synthetic granules were slightly smaller and irregular and had a two times higher specific surface area than bovine melanin. Synthetic melanin bound more betaxolol and metoprolol than bovine melanin and both melanin types showed a high affinity and a low affinity binding sites. The human choroid-RPE was predicted to contain 3-19 times more melanin bound drug than unbound drug at typical therapeutic concentrations (1-1,000 ng/ml). FITC-labeled oligonucleotides and 6-CF did not bind to melanin. CONCLUSIONS: The binding of lipophilic drugs to biological melanin differs from that of synthetic melanin. Lipophilic beta-blockers are expected to bind significantly to melanin in human choroid-RPE: only a small fraction of the drug being in active free form. In contrast, phosphodiesther oligonucleotides do not seem to bind to melanin.

Assessment of systemic adverse reactions induced by ophthalmic beta-adrenergic receptor antagonists.

To assess quantitatively the risks of ophthalmic beta-blocking agents for cardiovascular and respiratory adverse reactions, we analyzed the binding kinetics of beta-blocking agents to the beta-1 and beta-2 adrenoceptors. The relationship between the occupancies for beta-1 and beta-2 adrenoceptors and the effects on the exercise pulse rate or the forced expiratory volume in one second (FEV1) after topical administration of carteolol, befunolol, timolol and betaxolol was analyzed using a ternary complex model. The beta-1 and beta-2 receptor occupancies after ophthalmic administration were calculated to be quite high as well as those after oral administration. The maximum occupancies for beta-1 and beta-2 receptors after ordinary ophthalmic administration were 52% and 88% for carteolol, 52% and 61% for befunolol, 62% and 82% for timolol, and 44% and 3% for betaxolol, respectively. Concave relationships were obtained between a decrease in exercise pulse rate and the beta-1 receptor occupancy and between a decrease in FEV1 and beta-2 receptor occupancy, respectively. Nasolacrimal occlusion was estimated to decrease the exercise pulse rate and FEV1 by 65% and 50%, respectively. The beta-1 and beta-2 adrenoceptor occupancies were proved to be the most appropriate indicators for cardiac and pulmonary adverse reactions evoked by ophthalmic beta-blocking agents.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Betaxolol, a beta1-adrenoceptor antagonist, has an affinity for L-type Ca2+ channels.

The effect of betaxolol on the specific binding of [3H]diltiazem and [3H]nitrendipine to rat cortical membranes was examined. Betaxolol inhibited specific [3H]diltiazem and [3H]nitrendipine binding with IC50 values of 19.7 and 46.3 microM, respectively. The effect of betaxolol on L-type Ca2+ channels showed little stereospecificity, since similar inhibitions of radioligand binding were observed with both racemic betaxolol and L-betaxolol. The dissociation kinetics of [3H]diltiazem were unaffected by 30 microM betaxolol, whereas it increased the [3H]nitrendipine dissociation rate, thus suggesting that betaxolol directly interacts with the benzothiazepine binding site and allosterically modulates the dihydropyridine binding site. Carteolol, propranolol and timolol were also found to inhibit both specific [3H]diltiazem and [3H]nitrendipine binding to rat cortical membranes, but with less potency than betaxolol. The ability of betaxolol to interact with L-type Ca2+ channels may have a role in its therapeutic effects in the management of systemic hypertension and in reducing neuronal death as occurring in glaucoma.

Ocular-specific chemical delivery systems of betaxolol for safe local treatment of glaucoma.

Novel ketomethoxime (BMO) and oxime (BO) analogs of betaxolol (B) were prepared through the oxidation of betaxolol, followed by quenching of the ketone with the appropriate oxyamine. The Z isomers were kinetically favored and thermodynamically more stable. Isomerization to reach an equilibrium mixture of Z/E was observed for all pure isomers in buffers. Equilibration is much faster, however in biological fluids. Ocular administration of any of the oxime derivatives, delivers betaxolol specifically to the eye tissues, with the highest concentration in the iris ciliary body. Both BMO and BO, when applied topically, showed marked reduction of intraocular pressure (IOP) in normotensive rabbits. No effect on isoproterenol-induced tachycardia in rabbits and rats were observed, even after iv. administration. Very mild eye irritation, which was less than that of betaxolol hydrochloride, was observed particularly with BMO maleate, which is an excellent candidate for safe treatment of glaucoma.

A fatal case of betaxolol poisoning.

We report the first case in the literature of fatal betaxolol (Kerlone) self-poisoning. After a single-step liquid-liquid alkaline extraction, betaxolol was identified by a high-performance liquid chromatographic-diode array detection screening procedure and then quantitated in cardiac blood, heart, brain, muscle, spleen, stomach contents, duodenum contents, liver, and kidney. The blood betaxolol concentration was 36 mg/L.

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors.

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.

(-)-pindolol and (+/-)-tertatolol affect rat hippocampal 5-HT levels through mechanisms involving not only 5-HT1A, but also 5-HT1B receptors.

The present work examined, using in vivo microdialysis, the effects of 0.16-10 mg/kg of the beta-adrenoceptor antagonists, (-)-pindolol and (+/-)-tertatolol, which have additional 5-HT1A receptor antagonist properties, on extracellular 5-HT levels in the ventral hippocampus of chloral hydrate-anaesthetized rats. These effects were compared with those observed when (-)-pindolol and (+/-)-tertatolol were given together with the 5-HT1A agonist 8-OH-DPAT (0.31 mg/kg i.p.). When given alone, (-)-pindolol and (+/-)-tertatolol increased 5-HT levels not only after systemic administration (at 2.5 and 10 mg/kg s.c.), but also when perfused locally through the dialysis probe (at a concentration of 10 microM). At doses equal to or lower than those that increased 5-HT when given alone, (-)-pindolol and (+/-)-tertatolol inhibited the decrease of extracellular 5-HT levels induced by 8-OH-DPAT. At higher doses, however, (-)-pindolol and (+/-)-tertatolol were less able to reverse these effects of 8-OD-DPAT. The selective beta 1-adrenoceptor antagonist, (+/-)-betaxolol, did not alter 5-HT levels, either when given alone or when given together with 8-OD-DPAT. Although the antagonism of the 8-OH-DPAT-induced decrease of 5-HT levels by (-)-pindolol and (+/-)-tertatolol is likely to be related to their 5-HT1A antagonist properties, their ability to increase extracellular 5-HT levels when given alone may involve interactions with 5-HT1B receptors at hippocampal 5-HT terminals.

Autoradiographic characterization of beta-adrenergic receptor subtype in the canine conduction system.

It has been hypothesized, based on physiological evidence, that there is a greater proportion of beta 2-adrenergic receptors on the myocytes of the conduction system when compared with the working myocardium. The purpose of these studies was to examine beta-adrenergic receptor subtype in the conduction system of the dog by using the technique of coverslip autoradiography. Scintillation studies of [125I]pindolol binding to ventricular sections demonstrated that binding was saturable (dissociation constant of 116 pM), had the correct order of potency for a beta-receptor, and was stereoselective. Both betaxolol (beta 1-selective) and ICI-118,551 (beta 2-selective) competition curves fit a two-site model in nonlinear curve-fitting analyses (78% beta 1-receptors). Autoradiographic studies determined that the myocytes of the sinoatrial node had approximately twice as many autoradiographic grains as the surrounding atrial myocytes. The myocytes of the atrioventricular bundle had a number of grains similar to the number in surrounding septal myocytes. Autoradiographic inhibition curves with betaxolol or ICI-118,551 demonstrated that both the sinoatrial node and the atrioventricular bundle had inhibition profiles similar to the surrounding myocytes (predominantly beta 1) but unlike the inhibition profiles of arterioles (predominantly beta 2). Calculations using the dissociation constants derived from the nonlinear curve-fitting analysis and the percent specific binding in the presence of 4 x 10(-7) M betaxolol or ICI-118,551 determined that the proportion of beta 1- to beta 2-receptors was the same (70-80% beta 1) when comparing the sinoatrial node and the surrounding atrial myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Formulation influence on conjunctival penetration of four beta blockers in the pigmented rabbit: a comparison with corneal penetration.

The objective of this study was to compare the influence of pH, tonicity, benzalkonium chloride, and EDTA on the conjunctival and corneal penetration of four beta blockers--atenolol, timolol, levobunolol, and betaxolol. Drug penetration was evaluated using the isolated pigmented rabbit conjunctiva and cornea in the modified Ussing chamber. The conjunctiva was more permeable than the cornea to all four beta blockers. Formulation changes caused larger changes in corneal than in conjunctival drug penetration, especially for the hydrophilic beta blockers, atenolol and timolol. Raising the solution pH to 8.4 caused the largest increase in corneal penetration for all drugs except atenolol. This increase was greater than that obtained by removing the corneal epithelium. The same formulation also increased conjunctival drug penetration, although to a lesser extent. In the case of timolol, the formulation changes evaluated brought about similar changes in its ocular and systemic absorption with good in vitro-in vivo correlations. The above findings indicate that in making formulation changes to maximize corneal drug penetration, it is necessary to evaluate possible changes in conjunctival drug penetration, hence systemic absorption. Moreover, because the conjunctiva plays an active role in the noncorneal route of ocular drug absorption, the relative contribution of the noncorneal to the corneal routes to ocular drug absorption may also be altered by formulation changes.

Determination of betaxolol and its metabolites in blood and urine by high-performance liquid chromatography with fluorimetric detection.

Analytical methods for the determination of betaxolol and two of its metabolites in blood and urine are described. Betaxolol, alpha-hydroxybetaxolol, and the acid metabolite were extracted, with over 65% efficiency, from biological samples by liquid-liquid extraction methods. Analysis was performed using reversed-phase high-performance liquid chromatography with fluorimetric detection. N,N-Dimethyloctylamine (0.005 M) was used to improve the chromatography of betaxolol and alpha-hydroxybetaxolol, while acetic acid (1%) was used for the acid metabolite. An excitation wavelength of 200 nm was found to produce the best detector response. Linear standard curves were obtained for all three compounds with detection limits (signal-to-noise ratio = 3) varying between 1 and 10 ng/ml. The coefficients of variation of the determination for all three compounds in blood and urine varied between 3.0 and 8.7%. The metabolism of betaxolol was studied in twelve healthy male subjects. The amounts (mean +/- S.D.) of betaxolol, alpha-hydroxybetaxolol and the acid metabolite renally excreted in the first 48 h after intravenous administration of 10 mg of betaxolol hydrochloride are 17.1 +/- 6.2, 0.4 +/- 0.1 and 14.5 +/- 3.7%, respectively, of the administered dose.

The distribution of beta-adrenoceptors in human cervix.

The distribution of beta-adrenoceptors in sections of human cervix taken at the proliferative phase of the menstrual cycle was studied using light-microscopic autoradiography. The radioligand [125I]iodocyanopindolol (125ICYP) was used to identify specific binding sites. Moderate density of labelling by 125ICYP was seen over smooth muscle and blood vessels. The most intense labelling, however, was seen over glands and surface columnar epithelium. The association of beta-adrenoceptors with glands and surface columnar epithelium suggests a possible adrenergic regulation of secretory function in the cervix.