An UPLC-MS/MS Method for Simultaneous Determination of Tropicamide and Phenylephrine in Rabbit Ocular Tissues and Plasma.

Purpose: Mixed eye drops containing 0.5% tropicamide and 0.5% phenylephrine are commercially available for cycloplegic refraction. Determining the pharmacokinetics (PK) and distribution of tropicamide and phenylephrine simultaneously in ocular tissues is an important but challenging issue. Herein, we developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of tropicamide and phenylephrine concentrations in rabbit ocular tissues and plasma. Methods: The two analytes were extracted with ethyl acetate using etofesalamide as an internal standard and separated using a chromatographic C8 column with isocratic elution. Mass spectrometry analysis was performed with positive electrospray ionization and data were acquired in a multiple reaction monitoring mode. Results: We validated this method over a concentration range of 5-1,600 ng/mL for tropicamide and 1-320 ng/mL for phenylephrine in ocular tissues, as well as 0.5-64 ng/mL for both compounds in plasma. Inter- and intraday precisions in all samples were both <12.9% and the accuracy was within 92.1%-108.4%. The highest concentration of tropicamide was found in aqueous humor (Cmax: 29430 ng/g), while was in cornea for phenylephrine (Cmax: 3465 ng/g). All the ocular tissues concentrations were much higher than those of blood. Conclusion: This UPLC-MS/MS method allowed us to determine the PK and distribution of tropicamide and phenylephrine in rabbit ocular tissue, which may be helpful in the future development and application of mydriatic agents.

Mydriatics release from solid and semi-solid ophthalmic formulations using different in vitro methods.

The aim of the present paper was the development of semi-solid (hydrogels) and solid (film) ophthalmic formulations for the controlled release of two mydriatics: phenylephrine and tropicamide. The formulations - based on polyvinylalcohol and hyaluronic acid - were characterized, and release studies were performed with three different in vitro set-ups, i.e. Franz-type diffusion cell, vial method and inclined plane; for comparison, a solution and a commercial insert, both clinically used to induce mydriasis, were evaluated. Both gels and film allowed for a controlled release of drugs, appearing a useful alternative for mydriatics administration. However, the release kinetic was significantly influenced by the method used, highlighting the need for optimization and standardization of in vitro models for the evaluation of drug release from ophthalmic dosage forms.

[The distribution of tropicamide in the body of warm-blooded animals after its intragastric administration].

The objective of the present study was to elucidate the peculiar features of the distribution of N-ethyl-3-hydroxy-2-phenyl-N-(piridin-4-yl-methyl)propanamide (tropicamide) in the body of warm-blooded animals (rats) after its intragastric administration. The following methods were employed in the study: TLC, chromogenic reactions, electronic spectrophotometry, and GC-MS. The results of the quantitative analysis of N-ethyl-3-hydroxy-2-phenyl-N-(piridin-4-yl-methyl)propanamide in different organs of warm-blooded animals (rats) were compared within 20 minutes, 2.5 and 6 hours after its single intragastric administration at a dose equivalent to 1.5 LD50 of the toxic substance. It was shown that at all time intervals the maximum amount of tropicamide was present in the tissues of the stomach. Small intestines, their contents, brain, lungs, and spleen.

Pharmacodynamics of a new ophthalmic mydriatic insert in healthy volunteers: potential alternative as drug delivery system prior to cataract surgery.

Cataract surgery requires a satisfactory degree of mydriasis throughout the entire operation. A phase I, open-labelled, randomised, cross-over trial was conducted in 18 healthy volunteers to compare mydriasis obtained with subsequent administration of phenylephrine 10% and tropicamide 0.5% eyedrops or a new insoluble-matrix retropalpebral ophthalmic insert containing 5.38 mg phenylephrine and 0.28 mg tropicamide. Phenylephrine serum concentrations were measured over 6 hr following each treatment administration. Secondary end-points included cardiovascular, general and local tolerance and quantification of bacterial colonisation of the conjunctiva and the cultured insert, respectively. When normalized to the pupil diameter after conventional treatment, the diameter achieved with the insert was 1.13 (95% confidence interval, 0.94-1.48, P=0.38). Moreover, standard eye drops provided faster effective mydriasis than the insert, starting 30 min. as compared to 90 min. upon treatment administration (P<0.01, repeated-measures ANOVA). Phenylephrine concentrations remained almost undetectable for both treatments and no change in heart rate or blood pressure were observed throughout the study. Only three superficial punctuate keratitis were diagnosed with the insert and two with the eye drops. No significant bacterial contamination of conjunctiva swab and cultured insert was observed. The new insoluble-matrix retropalpebral ophthalmic mydriatic insert produced similar but delayed effective and prolonged mydriasis as compared to the standard delivery system. In addition to its potential usefulness in patients undergoing cataract surgery, such new ophthalmic delivery system may be an advantage in children who need to undergo fundus photography due to the single administration and excellent tolerance as well.

Pupil response to tropicamide following laser in situ keratomileusis.

PURPOSE: To investigate the effect of corneal thinning after laser in situ keratomileusis (LASIK) on the corneal penetration of topical eye medication. SETTING: Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA. METHODS: Laser in situ keratomileusis surgery was performed in 19 eyes of 10 patients enrolled in this prospective study. Measurements were made before surgery and 3 months postoperatively. After instillation of tropicamide 1%, the change in pupil size over time was measured with a Colvard pupillometer. Central corneal thickness (CCT) was measured with ultrasonic pachymetry before and 3 months after LASIK. The corneal epithelial condition was also examined by fluorescein dye staining. RESULTS: The mean CCT decreased significantly from 564 microm +/- 33 (SD) before LASIK surgery to 514 +/- 48 microm 3 months postoperatively (P<.0001). Pupil diameter 10, 15, and 20 minutes after tropicamide 1% instillation was significantly larger 3 months after surgery than preoperatively (P=.0083, P=.0043, and P=.0144, respectively). The mean time to reach a pupil diameter of 6.0 mm decreased significantly from 14.4 +/- 4.3 minutes in preoperative eyes to 11.5 +/- 2.3 minutes in postoperative eyes (P=.0281). Mild punctate corneal epithelial staining (fewer than 5 spots) were observed in 4 eyes at the 3-month postoperative examination. CONCLUSIONS: Pupil dilation after tropicamide 1% instillation was significantly faster after LASIK surgery. Corneal thinning that resulted from LASIK enhanced corneal penetration of tropicamide 1%.

Insulin and tropicamide accumulate in the contralateral, untreated eye of rats following ipsilateral topical administration by a mechanism that does not involve systemic uptake.

BACKGROUND: This study was designed to determine: (1) whether the accumulation of insulin in the contralateral retina and aqueous humor following ipsilateral topical insulin administration was due to systemic uptake and (2) whether tropicamide, applied to one eye, could induce dilation in the contralateral eye by a mechanism that did not involve systemic uptake. METHODS: Insulin eye drops were applied to the left eye of intact and decapitated rats, and their retinas and aqueous humors were then removed and their insulin levels quantified. In a separate experiment live animals received 0.1% tropicamide in their left eye and had their pupillary dilation response in both eyes measured at different time points. RESULTS: Administration of insulin to the left eye of decapitated rats resulted in its significant accumulation not only in the left retina and aqueous humor, but also in the retina and aqueous humor of the right eye. Similar aqueous humor results were obtained when live animals were used. Tropicamide drops induced marked pupillary dilation in treated eyes; the pupils of the contralateral, untreated eyes also dilated significantly, but less than did the treated pupils. The pupils of rats injected with tropicamide intravenously showed negligible dilation. CONCLUSIONS: These results showed that insulin accumulated in the retina and aqueous humor of contralateral, untreated eyes following topical application, by a mechanism that did not appear to involve systemic uptake. Similarly, tropicamide provoked a dilation response in the unheated eye by a mechanism that similarly did not appear to involve uptake from the blood.

Does photorefractive keratectomy alter the effect of topical medications?

INTRODUCTION: The influence of photorefractive keratectomy (PRK) on drug penetration into the eye is not yet established, as this procedure may alter the barrier function of the cornea as well as the blood ocular barrier. Pupillary response to topical tropicamide depends on its penetration into the anterior chamber. The purpose of this study was to examine the influence of PRK on pupillary response to topical tropicamide, and hence to evaluate whether PRK alters drug penetration into the eye. METHODS: Pupillary diameter was measured using infrared pupillometer at 0, 15 and 30 min after instillation of 0.1% tropicamide to both eyes of 28 patients 4 weeks after PRK in one eye. The untreated eye served as control. RESULTS: Pupillary diameter before and 30 min after dilation was 7.4 +/- 1.2 and 8.0 +/- 1.0 mm in the treated eyes and 7.5 +/- 1.2 and 8.1 +/- 1.0 mm in the untreated eyes, respectively (p = 0.45). Percentage of pupillary dilation (delta pupillary diameter/pupillary diameter at 0 min x 100) was also similar in treated and untreated eyes for the entire study group (12.5 and 10.5%, respectively, p = 0.17). A trend for greater dilation was seen in patients with myopia above 6 diopters (13.7 and 10.5% in treated and untreated eyes, respectively, p = 0.11) and cornea thinner than 540 microm (14.2 and 10.1% in treated and untreated eyes, respectively, p = 0.1). DISCUSSION: Our results confirm animal and human studies that found restoration of the ocular drug barrier at 4 weeks following PRK. The trend for greater penetration in patients with high myopia and thin corneas warrants further study on a larger cohort of this subgroup.

Probing anterior segment kinetics with focally applied mydriatics.

The purpose of this study was to examine the effects of convectional flow and anterior segment configuration on drug kinetics. Mydriatics were applied focally at the limbus in order to produce sector dilation of the pupil. Subjects were tested with either tropicamide or phenylephrine, applied at the superior, temporal, inferior, or nasal limbus (or as a conventional drop). Changes in pupil form were analyzed by means of photography, digitization, and circular Fourier series representation. Both tropicamide and phenylephrine were found to produce sector dilation; however, phenylephrine was approximately twice as effective. Applications at the superior limbus were significantly less effective than applications at the inferior limbus. The results are interpreted in terms of anterior segment convectional flow, which is believed to play a substantial role in pharmacokinetics of the anterior segment.

Enhancement of the mydriatic response to tropicamide by bioadhesive polymers.

The aim of this work was to evaluate how the addition of mucoadhesive polymers to aqueous solutions affects the ocular response of tropicamide (0.2%; w/v). The polymer solutions tested were carboxymethylcellulose sodium salt (CMC-Na; 1%; w/v), hyaluronic acid sodium salt (HA-Na; 0.1%; w/v) and polyacrylic acid (PAA; 0.2%; w/v). Polymeric solutions were compared to a nonviscous formulation (AS). In vitro mucoadhesion measurements were expressed as a percentage of the adhesion force mucin-mucin, considering this one as 100% mucoadhesion. The values ofmucoadhesion obtained were 172%, 127%, 103% and 87.6% for formulations with CMC, PAA, HA and AS, respectively. The mydriatic response of tropicamide was determined in adult male New Zealand rabbits, weighing 1.7-2 Kg, by pupil diameter measurements at different times after instillation. The area under the mydriatic response-time curve (AUC 0-6 hr) was interpreted as an indication of the bioavailability of tropicamide in each vehicle. The AUC 0-6 hr was related to the in vitro mucoadhesion for each formulation. Tropicamide solutions with CMC-Na and PAA resulted in mucoadhesion and AUC 0-6 hr values approximately 1.9 and 1.4 times higher than AS. Although the solution with HA-Na was less mucoadhesive than PAA, the hyaluronic acid solution resulted in a higher AUC mydriasis/time value. Formulations with HA-Na and PAA presented values of surface tension close to that observed in the lacrimal fluid, with the Imax (maximum pupil diameter) being higher than for CMC-Na and AS. Greater than 90% of the mydriatic effect disappeared 4.5 hr after instillation for PAA and AS. Nevertheless, the mydriatic effect remained up to 5.5 hr for HA-Na and CMC-Na. HA-Na solution enhanced the bioavailability oftropicamide, presenting a value of mucoadhesion similar to the reference mucin-mucin.

Preparation and evaluation of liposomal formulations of tropicamide for ocular delivery.

Tropicamide, a mydriatic, cycloplegic drug was entrapped in liposomes. Liposomes were investigated by laser counting studies, transmission electron microscopy and differential scanning calorimetry for characterization. The precorneal clearance of liposomes was compared with solution by gamma-scintigraphy in the rabbit. The neutral liposomes failed to demonstrate significant enhancement in precorneal retention in comparison with aqueous solution. The potential of liposomes as an ophthalmic drug delivery system was investigated by comparing pupil dilatory effect of tropicamide by topical instillation, in the rabbit eye, of the solution and various drug-loaded liposomal forms, i.e. neutral liposomes, positively charged liposomes and neutral liposomes dispersed in 0.25% (w/v) polycarbophil gel. The positively charged liposomal formulation and liposomes dispersed in polycarbophil gel were found to be more effective than neutral liposomal dispersion when data were statistically treated at the 5% level of significance.

Ocular pharmacokinetics and convectional flow: evidence from spatio-temporal analysis of mydriasis.

Pupil shape and placement were studied during pharmacologic dilation, using a photographic technique. Dilation onset was highly asymmetric; typically, the inferior or inferior-nasal side of the pupil moved maximally, while the opposite side barely moved. Pupil shape tended to elongate at a large angle to the direction of maximum shift. Late in dilation, pupils returned to more circular shape and pupil centers returned to near their original position. Results when a subject's head was inverted during and immediately after drug administration were essentially unchanged. However, results when a subject's head was rolled on one side (and kept there) after administration were markedly different; the dilation pattern shifted roughly in accord with head tilt. It is proposed that anterior chamber convectional flow is responsible for the asymmetry. Supportive evidence was obtained from experiments in which cooled or heated gel-packs were applied to the lids. Analysis indicates that convectional flow makes a very substantial contribution to anterior segment pharmacokinetics and is also likely to be of general importance in anterior segment transport.

Systemic absorption and anticholinergic activity of topically applied tropicamide.

We studied the plasma levels and systemic anticholinergic activity of tropicamide after ocular administration in eight women. Two 40 microliters drops of 0.5% tropicamide were instilled into the lower cul-de-sac of one eye of the subjects and concentrations and respective muscarinic receptor occupancy of tropicamide in plasma were monitored using radioligand binding techniques. Tropicamide was rapidly absorbed systemically with the mean peak concentration in plasma being 2.8 +/- 1.7 ng/ml (mean +/- SD) at five minutes after instillation. Tropicamide disappeared rapidly from the systemic circulation: drug concentration in plasma was 0.46 +/- 0.51 ng/ml (mean +/- SD) at 60 minutes and below 240 pg/ml at 120 minutes after instillation. Tropicamide bound to muscarinic receptors of rat brain with an apparent equilibrium binding constant (Ki-value in plasma) 220 +/- 25 nM (mean +/- SD, n = 3). Tropicamide occupied maximally 8% of muscarinic receptors in plasma after ocular application. The low affinity of tropicamide for muscarinic receptors and its negligible receptor occupancy in plasma can explain the low incidence of systemic side-effects of tropicamide eyedrops.

Evaluation of mucoadhesive polymers in ocular drug delivery. II. Polymer-coated vesicles.

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P less than 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.

Effects of pilocarpine and tropicamide on blood-aqueous barrier permeability in man.

The time courses of changes in the effects of topical pilocarpine and tropicamide on the index of the blood-aqueous barrier permeability to plasma protein (Pin) were determined in normal volunteers. Before and after drug instillation in one eye, protein concentration in the anterior chamber (Ca) was determined from aqueous flare intensity with a laser flare-cell meter and from aqueous flow by fluorophotometry. The Pin was calculated from the Ca, plasma protein concentration, and aqueous flow. One percent pilocarpine produced a maximum increase of 21 +/- 10% in the Ca (mean +/- SEM, n = 10), no significant change in the aqueous flow (n = 5), and a maximum increase of 29 +/- 10% in the Pin (n = 10). Three percent pilocarpine produced a maximum increase of 55 +/- 11% in the Ca (n = 8), a maximum increase of 34 +/- 13% in the aqueous flow (n = 5), and a maximum increase of 74 +/- 18% in the Pin (n = 8). Tropicamide (0.4%) produced a maximum decrease of 17 +/- 7% in the Ca (n = 8), a maximum decrease of 15 +/- 11% in the aqueous flow (n = 8), and a maximum decrease of 24 +/- 13% in the Pin (n = 8). The results indicated that pilocarpine increased the blood-aqueous barrier permeability to plasma protein in a dose-dependent manner and that tropicamide reduced it.