Tear concentrations of azithromycin following topical administration of a single dose of azithromycin 0.5%, 1.0%, and 1.5% eyedrops (T1225) in healthy volunteers.

PURPOSE: To evaluate azithromycin tear concentrations after one drop of T1225 0.5%, 1.0%, and 1.5% eyedrops. METHODS: In this randomized, double-masked study, 91 healthy volunteers received one drop into each eye of T1225 0.5% (n=23), T1225 1.0% (n=38), or T1225 1.5% (n=38). Azithromycin tear concentrations were measured by HPLC-MS at seven time points for 24 hours. Tolerability was evaluated. RESULTS: T1225 1.0% and 1.5% had similar pharmacokinetic profiles. After a post-instillation peak (167 to 178 mg/L after 10 minutes), mean concentrations remained above 7 mg/L for 24 hours (except for T1225 1% at H24). A delayed increase of the azithromycin mean tear concentration might be explained by the known late azithromycin release from tissues after storage in cells. Areas under inhibitory curve (AUICs) of T1225 1.0% and 1.5% were higher than AUICs of T1225 0.5% and ranged between 47 and 90. The three T1225 concentrations were safe for the ocular surface. CONCLUSIONS: Once daily instillation of T1225 1.0% and 1.5% was shown to reach an AUIC markedly above the required threshold for an antibacterial activity against Gram-positive bacteria (25-35). These results suggest that a BID instillation is more likely to ensure antimicrobial activity against Gram-negative bacteria (threshold >100).

Long-term tolerance of preservative-free eye drops containing macrogol hydroxystearate as an excipient.

PURPOSE: This in vivo animal study was conducted to assess the tolerability of macrogloglycerol hydrostearate 40 (MGH 40), commonly used as a solubilizing excipient in prostaglandin F2α analogue eye drops without benzalkonium chloride. METHODS: Twenty-eight (14 males and 14 females) New Zealand white albino rabbits in good health and with no signs of ocular irritation were randomly assigned to receive 25 µL instillations of a solution containing 10% MGH 40 in the right eye 3 times daily for either 3 or 6 months. Ocular examinations of the conjunctiva, cornea and iris (using an ophthalmoscope and slit-lamp), corneal sensitivity, and intraocular pressure were assessed in both the right (treated) and left (untreated) eyes throughout the study. General characteristics, hematology and serum biochemistry parameters were also assessed throughout the study and necropsy examinations were performed at study completion. RESULTS: There were no treatment-related effects on the cornea, conjunctiva, iris, or intraocular pressure. Transient findings were generally seen in the untreated as well as the treated eye. Similarly, there were no treatment-related findings in either the hematology or serum biochemistry data or at necropsy. There were no differences based on gender. CONCLUSIONS: Long-term administration of a 10% MGH 40-containing formulation three times per day in a standard in vivo animal model was well tolerated and had no ocular or other effect.

Autoradiographic localization of beta-adrenergic receptors in rabbit eye.

Using an in vitro autoradiography, beta-adrenergic receptors were localized in the rabbit eye. Autoradiograms were generated by apposition of isotope-sensitive film to slide-mounted eye sections, labelled with [125I](-)Iodocyanopindolol. A high density of silver grains was obtained in conjunctival, corneal and ciliary process epithelium. Binding sites were also present in corneal endothelium, iris epithelium, lens epithelium, choroid and extraocular muscles. In some areas, retina was also labelled. Studies with beta-adrenergic compounds showed that the majority of beta-adrenergic receptors, detectable by autoradiography, were of the beta2 type in the rabbit eye.

Localization and characterization of substance P binding sites in rat and rabbit eyes.

Specific and high-affinity binding sites for Substance P (SP) were found in eyes from albino rabbits and rats using an in vitro autoradiographic method with 125I-Bolton Hunter SP (BHSP). autoradiograms were generated by apposing 10-20 microns-thick cryostat eye sections to 3H-Hyperfilm or liquid emulsion and quantified by means of image-analysis procedures. Kinetic studies showed that equilibrium was reached after a 75-min incubation at room temperature. In rat retina, specific binding corresponding to approximately 90% of total binding, was reversible, of high affinity (dissociation constant [Kd], 0.13 +/- 0.02 nM). Half-time for dissociation of 125I-BHSP was about 15 min. Unlabeled SP and the two neurokinins (NK) A and B competed in a concentration-dependent manner for retinal sites labeled by 125I-BHSP with the following order of potencies: SP greater than NKA greater than NKB, in agreement with a pharmacologic profile of a SP receptor site. In both species, specific binding was found in the iris sphincter muscle, choroid, and retina. In rats, detectable amounts of SP-binding sites were also expressed in the corneal epithelium and iridial stroma. Quantitative analysis of the autoradiograms revealed that the highest densities of 125I-BHSP binding sites were localized in the iris sphincter muscle in rabbits and the inner retina in rats.

Monoclonal antibodies to human amnion recognize different components of the rabbit eye.

The epithelium of the eye originates from embryonic ectoderm, whereas the amnion is an extra-embryonic membrane that bears close relationship with many ectodermal tissues. Shared antigens have been identified between human amnion and cornea using rabbit antisera to human amnion. Three monoclonal antibodies to human amnion, GB4, GB9, and GB11 were studied by immunofluorescence on the anterior segment of the rabbit eye. GB4 recognized the epithelium of the conjunctiva and the subcapsular epithelium of the lens. GB9 reacted only with the central four fifths of the corneal epithelium; the peripheral epithelium near the limbus was not reactive. GB11 detected the pigmented epithelial cells in ciliary processes.

Retinal dopaminergic receptor affinity and ocular pharmacokinetic profile of piribedil.

Binding studies on retinal dopamine receptors have revealed the existence of both D1 and D2 receptors. Human retina micro-autoradiographs confirm the distribution of dopaminergic receptors in the plexiform layers. Piribedil, a dopaminergic agonist, is able to bind to D2 receptors, while its metabolite (S584) preferentially displaces D1-specific radioligands. These results demonstrate that piribedil has a dopamine-like pharmacological profile including direct interaction with receptors. When instilled into the rabbit eye, piribedil penetrates rapidly and accumulates in the pigmented epithelia--the iris ciliary body and chorioretina--before being rapidly cleared. Macro-autoradiographs confirm this distribution and show the levels to be compatible with the affinity of piribedil for retinal dopaminergic receptors.

Autoradiographic localization of D1 and D2 dopamine binding sites in the human retina.

The localization of dopamine binding sites was studied by in vitro autoradiography in the normal human retina using [125I]SCH 23982 for D1 receptor labelling and [125I]iodosulpride for D2 receptors. Results demonstrated that both types of binding sites were present in human retina. Binding of [125I]SCH 23982 to D1 dopamine receptor was blocked by 1 microM SKF 38393, SCH 23390 (D1 specific compounds) whereas bromocriptine and domperidone (D2 specific compounds) were inactive at the same concentration. On the contrary, binding of [125I]iodosulpride to D2 dopamine receptor was inhibited only by D2 drugs. Precise cellular distribution was given by microautoradiographic techniques and showed that binding sites were exclusively localized to the plexiform layers.

Neurotransmitters and intraocular pressure.

The role of the ocular autonomic nervous system in IOP regulation has been well established. Pharmacological and autohistoradiographic studies confirmed the high density of beta 2 and alpha 2 receptors on ciliary processes and iris epithelium. Their respective pharmacological activation or blockade is discussed. The role of other ocular neurotransmitters is also complex, as shown by the paradoxical similar action of dopamine agonists and antagonists on IOP. Concerning the cholinergic system, ocular muscarinic receptors are pharmacologically not well documented. Numerous other neurotransmitters may modulate IOP without necessarily leading to the development of new drugs. Drugs of the future will probably concentrate on dopaminergic agonists, cAMP-stimulators such as forskolin, prostaglandins, and cannabinoids.

Central nervous system control of intraocular pressure.

Normal intraocular pressure (IOP) is the result of an equilibrium between aqueous humor (AH) production, AH outflow and episcleral venous pressure. Most available antiglaucoma agents produce their effects by interacting with autonomic mechanisms (beta-blockers, epinephrine or parasympathomimetics). In contrast, the role of the central nervous system (brain and nerves) in the regulation of IOP remains unclear in view of the complex haemodynamic, metabolic or hormonal changes which occur under experimental conditions. In this paper, we discuss a basic understanding of the anatomic and physiological relationships between central nervous system and IOP and describe how the brain can affect functions in ciliary body and trabeculum meshwork.

Physiological roles of dopamine and neuropeptides in the retina.

The retina is a highly complex nervous tissue that converts light into patterns of electrical action potentials in order to process visual information. To carry out its function as a transducer and processor of visual information, the retina must be structurally and biochemically organized to send a coherent signal to the visual areas of the brain. In recent years, a number of biologically active substances have been demonstrated to be located within neurons in the retina. Most of them are thought to be involved in the modulation of the signal and its transmission to the brain through the optic nerve. The present paper attempts to summarize the immunocytochemical distribution and physiology of some neuronally localized substances in the mammalian retina, namely dopamine and neuropeptides.

Characterization of beta adrenergic receptors in bovine pigmented ciliary processes.

To analyse the molecular mechanism of action of beta-adrenergic compounds in reducing the intraocular pressure, the binding of 125I-iodohydroxybenzylpindolol to bovine pigmented ciliary processes was studied. The binding was found to be highly specific, saturable, reversible and displayed stereospecificity. Only one class of binding sites was detected. Values for KD of 0.18 nM and 0.32 nM were derived from kinetic and equilibrium experiments, respectively. The total number of beta receptors was large : 1.28 pmoles/mg protein. Competitive inhibition of 125I-iodohydroxybenzylpindolol binding by agonists and antagonists revealed that the majority of beta receptors in bovine pigmented ciliary processes were of the beta 2 type. The pharmacological and biochemical characteristics of the binding of adrenergic drugs found in these membranes are consistent with a regulation of aqueous humor production by the beta adrenergic system.

Dopamine receptors in rabbit and rat eye: characterization and localization of DA1 and DA2 binding sites.

In vitro autoradiography is used to characterize and localize DA1 and DA2 receptors in rabbit and rat eyes. The bindings of 125I-SCH 23982 (DA1 antagonist) and of 125I-Iodosulpride (DA2 antagonist) to slide mounted tissue sections take place with characteristics expected of substances that recognize DA1 and DA2 receptors respectively. They are saturable, have high affinity and exhibit an appropriate pharmacology. The regional distribution of these receptors indicates that they are mainly present in retina. Precise cellular localizations are visualized in retinal structures by means of histoautoradiography: DA1 receptors are found in inner plexiform layer, outer plexiform layer and inner nuclear layer and DA2 binding sites are present in the two plexiform layers.

Pharmacological characteristics of beta-adrenergic-sensitive adenylate cyclase in non pigmented and in pigmented cells of bovine ciliary process.

Adenylate cyclase activity was studied on the two epithelial cell types of bovine ciliary process after digestion by trypsin and purification by density gradient centrifugation. cAMP formation after isoproterenol stimulation showed that non pigmented cells were particularly enriched in adenylate cyclase activity. Furthermore pharmacological characteristics of this enzyme indicated that adenylate cyclase-associated beta adrenergic receptors were of beta 2 type in each kind of epithelial cells. These results support the hypothesis that beta adrenergic compounds modulate aqueous humor production through a direct effect on ciliary epithelium and enrichment of beta adrenergic sensitive adenylate cyclase activity in non pigmented epithelial cells suggests that these particular cells may play an important role in aqueous humor secretion.

Effects of a new anti-allergic agent: the magnesium salt of N-acetyl-aspartyl-glutamic acid on experimental allergic inflammation of the rabbit eye.

We evaluated the efficacy of N-Acetyl-Aspartyl-Glutamic acid (magnesium salt) (NAAGA) eye-drops in preventing the conjunctival and uveal allergic inflammation induced by reverse passive Arthus reaction in the rabbit. The permeability of the blood-conjunctival and blood-aqueous barriers was investigated by the leakage of 125I-labelled rabbit serum albumin injected intravenously prior to the challenge with the antigen. Permeability of both barriers was markedly increased 2 hours after the triggering of the allergic reaction; marked edema of the conjunctiva was also observed. Pretreatment with 1, 3, 6% (w/w) NAAGA eye-drops prevented the conjunctival inflammatory changes in a dose related manner and to a lesser degree, the breakdown of the blood-aqueous barrier. The distribution of topically applied 14C-NAAGA in the rabbit eyes was also investigated.

Pharmacokinetic and autoradiographic studies of basic fibroblast growth factor on de-epithelialized and intact rabbit eye.

In a previous work, we showed and compared the wound healing properties of aFGF and bFGF topically administered on totally de-epithelialized rabbit corneas. Pharmacokinetic and autoradiographic studies were then performed to investigate the sites of accumulation of bFGF in ocular structures, both on de-epithelialized and intact rabbit eyes. After one single instillation of 125I-bFGF, all the ocular structures were dissected and the measurement of the radioactivity allowed to establish kinetic curves. The results showed a very important and early fixation of bFGF on denuded cornea (10 minutes) and a posterior distribution of the drug between 10 and 30 minutes. A second accumulation of bFGF in the anterior segment appeared 8 hours after application and then decreased till the 48th hour. These findings were confirmed by the macroautoradiographies and the microautoradiographies pointed out the fixation of bFGF not only at the location of the Bowman membrane, but also on the corneal endothelium. These experiments also demonstrated the systemic diffusion of bFGF into the untreated controlateral eye. The integrity of bFGF in the cornea and other structures was then confirmed by SDS PAGE followed by autoradiography. In the intact eye, bFGF was shown to penetrate in extremely low amounts, illustrating the major role of the corneal barrier. For a therapeutic use bFGF may be recommended as an efficient wound healing agent for epithelial but also endothelial defects. Its eventual unwanted side effects must be kept in mind to perfect an efficient low dose and short term clinical treatment.

Ocular distribution of lomefloxacin 0.3% after a single instillation in the infected eye of pigmented rabbits.

We investigated the distribution of lomefloxacin, a fluoroquinolone, in the infected eye of pigmented rabbits after one instillation of 50 microliters lomefloxacin 0.3% eye drop, and compared tissue concentrations with therapeutic levels. Infection was induced by 6 instillations of Pseudomonas aeruginosa inoculum 6 x 10(5) bacteria) on centrally scratched cornea of 32 pigmented rabbits. Fifty microliters of 14[C] lomefloxacin 0.3% were instilled 24 hours after infection and animals were sacrificed 0.25, 0.5, 1, 2, 4, 8 and 12 hours later. Ocular structures, tear fluid, whole blood and plasma were then sampled, and tissue levels over time, Tmax, Cmax and area under the curve (AUC) were calculated. Lomefloxacin rapidly penetrated into the ocular structures: tear (Tmax 0.25 hours, Cmax 366 micrograms/ml, AUC 360 micrograms/ml x hour), corresponding value were for: eye lids (0.25, 7, 33), conjunctiva (0.25, 11, 7), cornea (0.25, 56, 70), aqueous humor (1, 8, 14), iris ciliary-body (4, 34, 252), lens (1, 0.2, 0.6). Tear levels remained high for many hours and were 34, 48, 1 and 27 micrograms/ml at 1, 2, 4 and 8 hours following a single instillation. Elimination from ocular structures was rapid. Therapeutic levels were achieved after a single instillation (mainly in the external ocular structures) when compared with MIC 90% values (1-10 micrograms/ml).

Alginic acid effect on carteolol ocular pharmacokinetics in the pigmented rabbit.

The effect of alginic acid addition to 1% or 2% carteolol solutions on the ocular penetration of the drug has been evaluated in the pigmented rabbit. During single dose studies, an increase in bioavailability ranging from 40% to 60% was observed in the aqueous humor and in the iris-ciliary body. During repeated dose studies, this increased ocular bioavailability of carteolol in the presence of alginic acid led to an equivalent concentration in the target tissue, although the dosage was only once a day compared with twice a day for the usual carteolol eyedrops. 14C-carteolol distribution studies demonstrated the binding of carteolol in pigmented ocular tissues. Thus, the presence of alginic acid as a new excipient supports a possible decrease in dosage regimen, while retaining sufficient ocular bioavailability to lower intraocular pressure.

Antiproliferative activity of a liposomal delivery system of mitoxantrone on rabbit subconjunctival fibroblasts in an ex-vivo model.

Wound healing is the main cause of the failure of filtering surgery in glaucoma. We developed a liposomal delivery system of mitoxantrone (MITX), an anthracyclin derivative, to allow a single adjuvant administration and to lessen ocular side-effects of the drug. In order to evaluate the antiproliferative activity of liposomal MITX, an ex vivo model consisting in the culture of subconjunctival tissue explants from rabbits pretreated with subconjunctival injections of free or liposomal MITX was used. We found that both forms of MITX decreased the growth rate as well as the explant proliferation surfaces 15 days or 1 month after a single administration of the drug in vivo. A morphometric analysis of the cells showed that the surface of the fibroblasts exposed to both forms of MITX was from 10 to 12 times as important as that of the control cells exposed to the empty liposomes and to the control buffer. A radioautographic study showed that more than 95% of the fibroblasts exposed to both forms of MITX were in the G1 phase of the cell cycle, while the control cell population was equally distributed among the different phases of the cell cycle.

The renin-angiotensin system in the rabbit eye.

We analyzed the components of the renin-angiotensin system (RAS) in ocular tissues of normal rabbit eyes and compared the results with those measured in rabbit eyes with proliferative vitreoretinopathy and ocular hypertension. Proliferative vitreoretinopathy was induced by injection of human platelets into the vitreous humor, and ocular hypertension was induced by injection of alpha-chymotrypsin into the posterior chamber. Angiotensinogen, renin, angiotensin converting enzyme (ACE), angiotensin II (Ang II), and Ang II receptors were assessed using conventional biochemical techniques. The vascularized tissues of normal eyes contained high renin and ACE activities concomitant with low concentration of angiotensinogen and Ang II. In general, in the ocular humors, the opposite was found. The Ang II receptor density was highest in the uveal tract [range 35-190 fmol/mg protein]. The AT1 receptor subtype predominated [> 80%]. The RAS was only minimally different in the two pathological models except that, in ocular hypertension, the renin activity in the uveal tract was reduced [-50%]. Also, the ratio of AT1 to AT2 receptors changed as compared to control, although the total receptor density remained unaltered. In conclusion, we present evidence for the presence of a complete local RAS in the rabbit eye, which is only marginally affected by the two pathological models studied.

Effect of SR121463, a selective non-peptide vasopressin V2 receptor antagonist, in a rabbit model of ocular hypertension.

The activity on intraocular pressure (IOP) of SR121463, a selective non-peptide arginin-vasopressin (AVP) V2 receptor antagonist, was investigated in a rabbit model of ocular hypertension. We first demonstrated that, in vitro, SR121463 displayed high competitive affinity for rabbit vasopressin V2 receptors (Ki = 2.1 +/- 1.2 nM). In vivo, SR121463 was instilled once (at concentrations ranging from 0.1 to 3%), or for 10 days (20 instillations) at 1% concentration, in the eye of ocular hypertensive rabbits (intraocular injection of 0.14 mg alpha-chymotrypsin). SR121463 also was instilled at 1% in the normotensive eye or intravenously injected (100 microg/kg) to ocular hypertensive rabbits. SR121463 was compared to timolol 0.5% or to clonidine 0.25%. Additionally, local and systemic safety aspects were examined. Results showed that SR121463 was locally well-tolerated and had no anesthetic effect. A significant decrease in IOP of the hypertensive eye was observed for concentrations of SR121463 > or =1%. This decrease was comparable to that obtained with reference compounds. A similar activity was found after intravenous administration. No tachyphylaxis was observed after 10 days, and no contralateral or systemic effect was noted. Also, when applied on the normotensive eye or when intravenously injected, SR121463 had no effect on the normotensive eye. These results on IOP and the good local and systemic safety profile, suggest that a potent vasopressin V2 receptor antagonist, SR121463, could be of value for the treatment of glaucoma, through a mechanism of action that remains to be elucidated.