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.

[Biomedical research in France: what has changed since 2004]. / La recherche biomédicale en France: ce qui a changé depuis 2004.

The public health law passed in 2004 is the implementation into French law of the European Clinical Trial directive 2001/20/EC on the implementation of good clinical practice in conducting clinical trials on medicinal products for human use. This law goes further than the European directive since it reconsiders all the components related to all fields of biomedical research. It has (i) standardized communication with the authorities (French Drug Agency/French Health Authority and the Ethics Committee); (ii) reinforced protection to participants in clinical trials and modified the information delivered to participants and how their informed consent is obtained; (iii) added risk-benefit assessment; and (iv) increased transparency for the public.

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).

[Improvement of dry eye symptoms with polyunsaturated fatty acids]. / Amélioration de la symptomatologie chez des patients atteints de sécheresse oculaire et traités oralement par des acides gras polyinsaturés.

INTRODUCTION: Polyunsaturated fatty acids (PUFAs) are involved in inflammatory pathways via prostaglandins. Conjunctival inflammation is a hallmark of all dry eye syndromes. We investigated the role of dietary n-6 and n-3 fatty acids in patients suffering from ocular dryness. PATIENTS AND METHODS: Seventy-one patients presenting with mild to moderate dry eye syndromes were randomly assigned to Nutrilarm or placebo capsules, twice a day for 6 months. The Schirmer test, BUT, fluorescein staining, and lissamin green stainings were performed at inclusion and after 1, 3, and 6 months. Furthermore, a questionnaire related to the dry eye symptoms and global discomfort was provided at every visit. RESULTS: The Schirmer test, BUT, fluorescein staining, and lissamin green stainings were improved with treatment when compared to placebo but the difference was not statistically significant. The efficacy evaluated by the patients and the investigator were nearly significant (p=0.052 and p=0.054, respectively). For some signs, such as reflex tearing and conjunctival hyperemia, the improvement reached the threshold of significance (p=0.047 and p=0.045, respectively). The same results were found with skin quality and emotional condition, which were improved (61% with treatment versus 36% with placebo). CONCLUSION: This double-masked pilot study shows that PUFAs seem to be an interesting tool to alleviate the symptoms related to dry eye syndrome. These results should be confirmed using a larger study population.

Comparison of carteolol plasmatic levels after repeated instillations of long-acting and regular formulations of carteolol 2% in glaucoma patients.

BACKGROUND: A new long-acting (LA) formulation of carteolol 2% instilled once daily has been shown to provide a therapeutic effect similar to that of the regular formulation of carteolol 2% instilled twice daily. This study was designed to test whether the new formulation reduces the systemic delivery of carteolol. METHODS: In this double-masked, randomised, intra-subject comparative study, 23 patients with bilateral primary open-angle glaucoma or bilateral ocular hypertension received sequentially, according to the randomised order of administration, each of the 2 following treatments: carteolol 2% LA once daily for 2 months and carteolol 2% regular twice daily for 2 months. Treatments were instilled in both eyes throughout the study period. At the end of each period of treatment, blood samples were taken immediately before the last morning instillation (residual time), then 30 min, 1 h, 2 h and 4 h after this instillation in order to measure the carteolol plasma concentrations. RESULTS: The mean values of maximal plasma concentration (C(max)), residual level and area under the curve obtained following carteolol 2% LA treatment were significantly lower than the values obtained after carteolol 2% regular treatment (mean+/-SD): C(max) (ng/ml): 1.72+/-0.85 versus 3.64+/-3.65; residual level (ng/ml): 0.70+/-0.58 versus 1.80+/-0.84; area under the curve (ng/mlxh): 5.50+/-2.66 versus 10.27+/-5.46. Regarding safety, two drug-related, non-serious adverse events were reported in the LA group: one case of moderate, superficial, punctate keratitis and one case of "bitter taste in the throat." Both treatments appeared to be well tolerated. CONCLUSIONS: The data from this study showed that the systemic delivery of carteolol is lower for the once-daily LA formulation than for the regular twice-daily formulation. Consequently, long-acting carteolol eye-drops should reduce the risk of beta-blocking systemic side effects.

[Comparison of once-daily nonpreserved timolol and timolol maleate gel-forming solution associated with latanoprost]. / Comparaison du timolol sans conservateur et du timolol à délivrance prolongée donnés une fois par jour en association à du latanoprost.

AIM: To compare the efficacy and safety of a single daily instillation of nonpreserved timolol to a timolol maleate gel-forming solution in patients with chronic glaucoma or ocular hypertension already treated with latanoprost. PATIENTS AND METHODS: A randomized, prospective, multicenter, open, parallel-group clinical trial was undertaken with 73 patients with chronic glaucoma treated with latanoprost and a timolol maleate gel-forming solution. In 36 patients, the previous regimen was substituted by nonpreserved timolol given instead of timolol maleate gel for 3 months. The changes in intraocular pressure (IOP) were recorded as well as local and systemic tolerance and patient compliance. RESULTS: At 3 months, both regimens were found equivalent in maintaining IOP control between D0 and D84. The difference with baseline was -0.08 +/- 2.22 mmHg and -0.38 +/- 2.41 mmHg in the nonpreserved timolol group and in the timolol maleate gel-forming solution group, respectively (CI 95% [-0.79; 1.38]). After 84 days of treatment, blurred vision (5.9%) and eyelid deposits (5.9%) were reduced in the preservative-free timolol group compared to the other group (respectively, 33.3% and 24.2%). These differences were statistically significant for both signs (blurred vision: p < 0.0001 and for eyelid deposits: p = 0.03). CONCLUSION: This short-term study has demonstrated the equivalence of nonpreserved timolol to timolol maleate gel-forming solution in terms of IOP control. Moreover, the local tolerance of nonpreserved timolol was better.

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.

Topical ocular instillation of nitric oxide synthase inhibitors and intraocular pressure in rabbits.

PURPOSE: It has been reported that intravenous injection of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl esther (L-NAME) causes a rapid decrease of intraocular pressure (IOP) in rabbits. This study investigates the effect of topical ocular application of different NOS inhibitors on a raise of IOP induced by an acute water intake (rabbit water loading model). METHODS: Forty New Zealand (albino) male rabbits received randomly (within thirty minutes) in their right eye three 50 microliters installations of either 0.9% NaCl, 0.5% timolol maleate (beta-adrenoreceptor antagonist), 0.5% 7-nitroindazole (7-NI; NOS inhibitor), 0.5% L-NAME, or 0.5% 2-amino-5,6-dihydro-6-methyl-1,3,-thiazine (AMT; NOS inhibitor) before an oral water gavage (60 ml/kg). IOP was measured (TonoPen) before and after topical instillation (time 0), and then 15, 30, 60, 90, and 120 minutes after water intake. RESULTS: In right eyes, the area under the curve (AUC) of the IOP difference versus time (arbitrary units) was 527 +/- 284 for NaCl, 255 +/- 178 for timolol, 466 +/- 242 for 7-NI, 604 +/- 195 for L-NAME, and 394 +/- 202 for AMT. Values of AUC were only significantly lower (p < 0.05) in the timolol-treated group. In left eyes, no significant difference (p > 0.05) could be observed in values of AUC between groups. CONCLUSIONS: Under the present experimental conditions (including concentration and bioavailability of the drugs used), topical application of the NOS inhibitors 7-NI, L-NAME, and AMT does not prevent an IOP increase induced by water intake in rabbits.

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.

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.

Inflammation measurement and immunocharacterization of cell proliferation in an experimental model of proliferative vitreoretinopathy.

An experimental model of proliferative vitreoretinopathy was developed in the rabbit eye by injecting a solution of human platelet-rich plasma. In this model we evaluated the progression with time of intraocular inflammation and the rate and origin of cell proliferation. A sterile solution adjusted to 107 platelets was injected into the right eye of a total of 46 pigmented and 14 albino rabbits. Animals were sequentially sacrificed at days 7, 14, 21 and 1 month after injection. Clinical evaluation of vitreoretinal proliferation, using a classification in six grades, and of anterior segment inflammation assessed by a Laser Flare Meter, were done for 1 month after injection, before histopathological analysis. Eighty percent of eyes developed tractional retinal detachment in 1 month. Histopathology showed intense cell migration and proliferation in the area of the ciliary body, as early as the seventh day, then further increasing rapidly. Infiltrates were composed of cytokeratin- and vimentin-expressing cells. Abnormal expression of vimentin was also found in ciliary and retinal epithelia and in M¿ller cells. Inflammation measured by the Laser Flare Meter was maximal at day 11 and then reached a plateau at significantly higher levels than controls. Albino rabbits showed significantly lower grades of proliferation, as compared to pigmented rabbits. This study thus clarified some characteristics of experimental vitreoretinal proliferations that that proved similar to those in human diseases, such as the involvement of ciliary body and retinal pigment epithelium, the existence of inflammatory reactions preceding cell proliferation and strong changes in intermediate filaments. This may provide a simple and valuable model for antiproliferative assays and shed some light on the pathogenesis of intraocular proliferative disorders.

Effects of different formulations of mitoxantrone (solutions, nanospheres, liposomes) on glaucoma surgery in rabbits.

Pharmacological blockade of fibroblastic proliferation after glaucoma filtration surgery by using antimitotic agents in different formulations (liposomes, nanospheres) is of great clinical interest. However, only limited comparative data are available on the effect of encapsulated drugs on intraocular pressure (IOP) after filtering surgery. Therefore we have studied the effect on IOP of liposomes, nanospheres and a solution of mitoxantrone (MTO), a well-known antimitotic, in rabbits before and after sclerectomy. MTO in solution form, as well as in a liposome formulation, improved the outcome of the surgery by reducing IOP when administered subconjunctivally just following surgery. This effect was similar to mitomycin-C application. In contrast, neither prior administration nor subconjunctival nanosphere injections induced a reduction in IOP. Liposome administration demonstrated no delayed action or promoting effect but reduced the occurrence of corneal opacity observed in groups treated with MTO in solution.

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).

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.

Allergic conjunctivitis and uveitis models: reappraisal with some marketed drugs.

The purpose of this study was to assess the activity of some marketed products in ocular non-immune and immune type I hypersensitivity reactions, and during intra-ocular type III hypersensitivity. In order to compare these activities, we improved and validated three different models of ocular allergic reaction already known for their ability to reproduce allergic conjunctivitis or uveitis. Allergic conjunctivitis was induced by ocular immediate hypersensitivity after instillation of compound 48/80 in the rat, or an active anaphylaxis reaction with ovalbumin immunisation and challenge in the guinea pig. Uveitis was induced by a reverse passive anaphylaxis reaction using intra-vitreal rabbit anti-bovine IgG anti-serum sensitisation and intravenous bovine gamma-globulin challenge in the rabbit. Clinical scores and blood-tissue permeability indices were studied. Using the same schedule of ocular instillation, the effects of Livostin (levocabastine 0.05%), Almide (lodoxamide 0.1%), Opticrom (sodium cromoglycate 2%), Ocufen (flurbiprofen 0.03%), Acular (ketorolac 0.5%) and 0.3% chlorpheniramine maleate were compared to positive and negative controls. We demonstrated the potent activity of chlorpheniramine maleate 0.3% and Livostin in both allergic conjunctivitis models. Significant activity was also evidenced with Almide, which was only active in the non-immune allergy model, while Opticrom was definitely not active in these models. In the uveitis model, Acular and Ocufen are active and potent drugs, while Livostin and Almide were not active. These results are discussed with respect to the models used and the mediators involved.

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.

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.

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.