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.

The penetration and distribution of topical atropine in animal ocular tissues.

PURPOSE: To conduct a multi-tissue investigation on the penetration and distribution of topical atropine in myopia treatment, and determine if atropine is detectable in the untreated contralateral eye after uniocular instillation. METHODS: Nine mature New Zealand white rabbits were evenly divided into three groups. Each group was killed at 5, 24 and 72 hr, respectively, following uniocular instillation of 0.05 ml of 1% atropine. Tissues were sampled after enucleation: conjunctiva, sclera, cornea, iris, ciliary body, lens, retina, aqueous, and vitreous humors. The assay for atropine was performed using liquid chromatography-mass spectrometry (LC-MS), and molecular tissue distribution was illustrated using matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) via an independent experiment on murine eyes. RESULTS: At 5 hr, the highest (mean ± SEM) concentration of atropine was detected in the conjunctiva (19.05 ± 5.57 ng/mg, p < 0.05) with a concentration gradient established anteriorly to posteriorly, as supported by MALDI-IMS. At 24 hr, preferential binding of atropine to posterior ocular tissues occurred, demonstrating a reversal of the initial concentration gradient. Atropine has good ocular bioavailability with concentrations of two magnitudes higher than its binding affinity in most tissues at 3 days. Crossing-over of atropine to the untreated eye occurred within 5 hr post-administration. CONCLUSION: Both transcorneal and transconjunctival-scleral routes are key in atropine absorption. Posterior ocular tissues could be important sites of action by atropine in myopic reduction. In uniocular atropine trials, cross-over effects on the placebo eye should be adjusted to enhance results reliability. Combining the use of LC-MS and MALDI-IMS can be a viable approach in the study of the ocular pharmacokinetics of atropine.

Objective evaluation of the systemic effects of topical application of 1% atropine sulfate ophthalmic solution in healthy horses.

OBJECTIVE To determine the safety of topical administration of 1% atropine ophthalmic solution in healthy horses by objectively measuring gastrointestinal transit time. DESIGN Randomized, masked, controlled crossover study. ANIMALS 6 adult geldings. PROCEDURES Horses were randomly assigned (3/group) to first receive topical treatment of the left eye with 1% atropine or artificial tears solution; the right eye was left untreated. After 24 hours of treatment every 6 hours, 200 nontoxic beads were administered to each horse via nasogastric intubation and treatment frequency was decreased to every 12 hours for 4 more days. Pupillary light reflexes (PLRs), mydriasis, heart rate, fecal bead passage, abdominal girth measurements, auscultable gut sounds, fecal weight, and clinical signs of abdominal pain were monitored. Following a 4-week washout period, horses received the opposite treatment in the left eye and measurements were repeated. Serum atropine concentration (reflecting systemic absorption) was measured with an ELISA at various points after initial atropine administration. RESULTS No horse had subjective or objective evidence of colic or ileus at any monitoring point. Complete mydriasis of the left eye with absence of the PLR was identified in 5 horses within 6 hours and in all 6 horses within 12 hours after initial atropine administration. One horse had mydriasis with an absent PLR in the untreated eye by day 5 of atropine treatment. At no point was atropine detected in serum samples of any horse. CONCLUSIONS AND CLINICAL RELEVANCE Topical atropine application at clinically appropriate doses induced no evidence of ileus in healthy horses.

Efficacy of an eye drop mixture for pupillary dilatation: A randomized comparative study / Eficacia de la mezcla de gotas oculares para la dilatación de la pupila: estudio aleatorizado y comparativo

Purpose: Pupillary dilatation with three types of eye drops is used regularly in the clinic; however, a mixture of these drops in a single bottle may be more beneficial in reducing workloads and resources. This study compared the efficacy in pupillary dilatation between two protocols of dilating drop instillation. Methods: This prospective, randomized, comparative study included 30 eligible Thai patients. The patients randomly received preoperative pupillary dilatations by either the conventional protocol (1% tropicamide (T), 10% phenylephrine (P) and 0.1% diclofenac (D) in three separate bottles) or the fixed combination (TPD) protocol which had the three types of eye drops mixed in a single bottle in a ratio of 4:3:3. The chi-square test and independent t-test were used to analyze the data. Results: The conventional protocol group and TPD protocol group each had 15 patients. Sixty minutes after the initial instillation, all patients in the TPD protocol and 13 patients (86.7%) in conventional protocol achieved at least 6mm in the shortest diameter. The mydriatic rate between protocols showed no difference. In patients who received the TPD protocol, the systemic effects on the mean arterial blood pressure and pulse rate decreased over time. Conclusion: The mixture of tropicamide, phenylephrine and diclofenac had a comparable efficacy for a pupillary dilatation to the conventional dilating drops in separate bottles. The systemic complications on blood pressure and arterial pulse of the TPD mixture were less than the conventional protocol (AU)

Objetivo: La dilatación de la pupila con tres tipos de gotas oculares se utiliza normalmente en la práctica clínica; sin embargo, la mezcla de dichas gotas en un único envase puede resultar más beneficiosa a la hora de reducir las cargas de trabajo y los recursos. Este estudio comparó la eficacia entre dos protocolos de dilatación de pupilas. Métodos: Este estudio prospectivo, aleatorizado y comparativo incluyó a 30 pacientes tailandeses elegibles. A dichos pacientes se les dilató aleatoria y preoperatoriamente la pupila utilizando el protocolo convencional (1% tropicamida (T), 10% fenilefrina (P) y 0,1% diclofenaco (D) en tres envases separados), o el protocolo de combinación fija (TPD), que contenía los tres tipos de gotas oculares mezclados en un único envase, a un ratio de 4:3:3. Se utilizaron las pruebas de χ2 y la prueba independiente t para analizar los datos. Resultados: Tanto el grupo de protocolo convencional como el grupo TPD incluyeron a 15 pacientes. A los sesenta minutos de la instilación inicial, todos los pacientes del protocolo TPD y 13 pacientes (86,7%) del protocolo convencional lograron un mínimo de 6mm en el diámetro menor. La tasa midriática entre ambos protocolos no reflejó diferencia alguna. En los pacientes del protocolo TPD, los efectos sistémicos sobre la presión sanguínea media y el índice de pulso disminuyeron con el tiempo. Conclusión: La mezcla de tropicamida, fenilefrina y diclofenaco mostró una eficacia comparable a la de las gotas para dilatación de pupilas suministradas en envases separados. Las complicaciones sistémicas sobre la presión sanguínea y la presión arterial de la mezcla de TPD fueron menores a las del protocolo convencional (AU)

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.

Systemic Absorption of Cyclopentolate and Adverse Events After Retinopathy of Prematurity Exams.

PURPOSE: Preterm infants undergoing Retinopathy of Prematurity Eye Exams (ROPEE) may experience adverse events, possibly from systemic absorption of cyclopentolate. The purpose of this study was to analyze the association between adverse events and drug levels found in neonates undergoing ROPEE. MATERIALS AND METHODS: 25 infants were randomized into two groups during routine ROP screening: 5 infants for blood collection before mydriatic drops and 20 for blood collection 1 h after eye drops. Blood was collected onto dried blood spot cards, extracted, and analyzed for cyclopentolate and phenylephrine using liquid chromatography and mass spectrometry. Relationships between drug levels and adverse events were assessed. RESULTS: Cyclopentolate (range 6-53 ng/ml) was observed in 15 of 18 infants, while phenylephrine was not detected. Levels of cyclopentolate were significantly higher in infants who were on oxygen (p = 0.01). There was a significant association between cyclopentolate levels and gastric residuals in tube-fed infants not receiving oxygen (p = 0.01). CONCLUSIONS: Cyclopentolate levels varied among preterm infants after ROPEE. Cyclopentolate was positively associated with increased gastric residuals. Underlying medical conditions requiring oxygen administration may affect absorption and metabolism of cyclopentolate. There is a need to predict infants at risk for high blood levels of cyclopentolate in order to prevent or treat adverse events after ROPEE.

Atropine and Roscovitine Release from Model Silicone Hydrogels.

PURPOSE: Drug delivery to the anterior eye has a low compliance and results in significant drug losses. In pediatric patients, eye diseases such as myopia and retinoblastoma can potentially be treated pharmacologically, but the risk associated with high drug concentrations coupled with the need for regular dosing limits their effectiveness. The current study examined the feasibility of atropine and roscovitine delivery from model silicone hydrogel materials which could potentially be used to treat myopia and retinoblastoma, respectively. METHODS: Model silicone hydrogel materials that comprised TRIS and DMA were prepared with the drug incorporated during synthesis. Various materials properties, with and without incorporated drug, were investigated including water uptake, water contact angle, and light transmission. Drug release was evaluated under sink conditions into phosphate buffered saline. RESULTS: The results demonstrate that up to 2 wt% of the drugs can be incorporated into model silicone hydrogel materials without adversely affecting critical materials properties such as water uptake, light transmission, and surface hydrophilicity. Equilibrium water content ranged from 15 to 32% and transmission exceeded 89% for materials with at least 70% DMA. Extended release exceeding 14 days was possible with both drugs, with the total amount of drug released from the materials ranging from 16% to over 76%. Although a burst effect was noted, this was thought to be due to surface-bound drug, and therefore storage in an appropriate packaging solution could be used to overcome this if desired. CONCLUSIONS: Silicone hydrogel materials have the potential to deliver drugs for over 2 weeks without compromising lens properties. This could potentially overcome the need for regular drop instillation and allow for the maintenance of drug concentration in the tear film over the period of wear. This represents a potential option for treating a host of ophthalmic disorders in children including myopia and retinoblastoma.

Effects of MDMA alone and after pretreatment with reboxetine, duloxetine, clonidine, carvedilol, and doxazosin on pupillary light reflex.

RATIONALE: Pupillometry can be used to characterize autonomic drug effects. OBJECTIVE: This study was conducted to determine the autonomic effects of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), administered alone and after pretreatment with reboxetine, duloxetine, clonidine, carvedilol, and doxazosin, on pupillary function. METHODS: Infrared pupillometry was performed in five placebo-controlled randomized studies. Each study included 16 healthy subjects (eight men, eight women) who received placebo-MDMA (125 mg), placebo-placebo, pretreatment-placebo, or pretreatment-MDMA using a crossover design. RESULTS: MDMA produced mydriasis, prolonged the latency, reduced the response to light, and shortened the recovery time. The impaired reflex response was associated with subjective, cardiostimulant, and hyperthermic drug effects and returned to normal within 6 h after MDMA administration when plasma MDMA levels were still high. Mydriasis was associated with changes in plasma MDMA concentration over time and longer-lasting. Both reboxetine and duloxetine interacted with the effects of MDMA on pupillary function. Clonidine did not significantly reduce the mydriatic effects of MDMA, although it produced miosis when administered alone. Carvedilol and doxazosin did not alter the effects of MDMA on pupillary function. CONCLUSIONS: The MDMA-induced prolongation of the latency to and reduction of light-induced miosis indicate indirect central parasympathetic inhibition, and the faster recovery time reflects an increased sympathomimetic action. Both norepinephrine and serotonin mediate the effects of MDMA on pupillary function. Although mydriasis is lasting and mirrors the plasma concentration-time curve of MDMA, the impairment in the reaction to light is associated with the subjective and other autonomic effects of MDMA and exhibits acute tolerance.

Effects of unilateral topical atropine on binocular pupil responses and eye growth in mice.

PURPOSE: Studies on drugs selected to target myopia development often use the vehicle-treated fellow eye as a control. However, it is not clear how much of the drug reaches the fellow eye, rendering it a potentially invalid control. Therefore, in this study, pupil responses were used to probe the effects of atropine in both eyes in mice, after unilateral topical application. In a second experiment, interocular differences in refractive development and axial eye growth were studied while atropine was applied daily to one eye. METHODS: In 20 C57BL/6 (B6) wildtype mice, a single drop of 1% atropine solution was instilled into one eye. Mice were gently restrained by holding their necks while video image processing software detected the pupil and measured its diameter at a sampling rate of 30 Hz. A bright green LED, attached to the photoretinoscope of the video camera, was flashed. Pupil responses were quantified daily over a period of 2 weeks. In another group of 24 mice, one drop of 1% atropine was applied daily for 28 days. Axial length was measured pre- and post-treatment, using low coherence interferometry (the Zeiss AC-Master). Refractive development was measured by infrared photorefraction. RESULTS: Similar to previous findings with the same device, untreated eyes displayed a pupil constriction of 24.84+/-1.73% upon stimulation with the green LED. A single drop of 1% atropine caused complete suppression with no significant recovery over the whole observation period of two weeks. The responses in the fellow eye were temporarily reduced to about 75% and then recovered towards baseline. After daily atropine application, there was significant reduction in axial length of the eyes, relative to the saline-treated fellow eyes (3.234+/-0.186 versus 3.378+/-0.176 mm, n=24, p<0.01, paired t-test) and the refractions became more hyperopic/less myopic (+13.46+/-2.15 D versus +10.06+/-2.02 D, n=24, p<0.01). CONCLUSIONS: In line with previous findings, one drop of atropine solution caused a long lasting suppression of pupil responses in the mouse eye. New data show that the transfer to the fellow eye was limited, making interocular comparisons feasible. It is also new that topical atropine reduced axial eye growth even when mice had largely normal vision.

Systemic adverse effects of topical ocular treatments.

Some eyedrops, gels or ointments may cause adverse effects as serious as those observed with systemic therapies. Because of their relatively poor penetration into eye tissue, ophthalmic drugs usually contain high concentrations of their active ingredient. Asking patients about these drugs to prevent interactions is useful when prescribing a new systemic treatment. Conversely, it is advisable to ask about ophthalmic drugs during the etiological investigation of possible iatrogenic effects.

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%.

Design, pharmacokinetic, and pharmacodynamic evaluation of a new class of soft anticholinergics.

PURPOSE: To design and evaluate a new class of soft anticholinergics with subtype selectivity. METHODS: A new class of soft anticholinergics was designed based on the "inactive metabolite" approach. Four compounds were synthesized. The potency and soft nature of the compounds were evaluated by receptor binding, cardiac, and mydriatic studies. Stability and pharmacokinetic studies were also performed on these newly synthesized soft anticholinergics. RESULTS: Receptor binding studies of the soft anticholinergics on cloned muscarinic receptors indicated pKi values in the range of 7.5 to 8.9. Two compounds, 9a and 13a, of the series showed muscarinic subtype receptor selectivity (M3/M2). In mydriatic studies, 13a and 13b showed shorter duration of action in the treated eyes than tropicamide. In the control eyes, significant dilation of pupils was found only in rabbits treated with atropine and tropicamide, indicating that the soft anticholinergics lack systemic effects because of their facile hydrolytic deactivation. Consistent with their soft nature, this new class of soft anticholinergics displayed much shorter cardiovascular effects in the carbachol-induced bradycardia (10 to 15 min) in rats than atropine (> 60 min). Stability and pharmacokinetic studies suggested that the new soft anticholinergics were rapidly eliminated from plasma (systemic circulation) after i.v. administration. CONCLUSIONS: A new class of anticholinergics was designed and synthesized, and the PK/PD evaluation confirmed they were potent "soft" anticholinergics; two of them showed muscarinic receptor subtype selectivity (M3/M2).

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.

Intraoperative pulmonary oedema in a child following systemic absorption of phenylephrine eyedrops.

Ophthalmic surgeons often apply phenylephrine topically to effect pupillary dilatation. We describe a paediatric patient in whom cardiac arrhythmias, severe hypertension and pulmonary oedema occurred following intraoperative ocular phenylephrine administration. We believe that systemic absorption of the drug was responsible and discuss ways in which this might be reduced when ocular phenylephrine is used in this context.

Design, pharmacokinetic, and pharmacodynamic evaluation of soft anticholinergics based on tropyl alpha-phenylcyclopentylacetate.

Four new soft anticholinergic agents based on tropyl alpha-phenylcyclopentylacetate, 15a, 15b, 18a, and 18b, were designed and synthesized. Receptor binding studies on the cloned human muscarinic receptors indicated that the new soft anticholinergic agents possessed moderate potency as pKi ranged from 6.7 to 7.6. Mydriatic studies in rabbit eyes revealed that the duration of the action of the new soft anticholinergics (8.5-11.0 h) were shorter than that of atropine (about 24 h) under pharmacodynamic equivalent dose, and one of them, 18a, showed even shorter than that of tropicamide. In addition, after unilateral administration, significant dilation of pupil in the control eyes was observed with tropicamide and atropine but not with soft drugs, suggesting the systemic activity of soft drugs was minimal. With their soft nature, the new soft anticholinergics displayed much shorter protective effect against carbachol-induced bradycardia (about 30 min) than atropine (at least 60 min) in rats. In vitro and in vivo pharmacokinetic studies demonstrated that the soft anticholinergics were rapidly hydrolyzed into the corresponding inactive metabolites once they were introduced into the systemic circulation.