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Pharmacokinetics and Tissue Distribution of Pilocarpine After Application to Eyelid Skin of Rats
Journal of Pharmaceutical Sciences xxx (2019) 1-7
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Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Pharmacokinetics and Tissue Distribution of Pilocarpine After Application to Eyelid Skin of Rats Gerard Lee See 1, 2, Ayano Sagesaka 1, Hiroaki Todo 1, 3, Konstanty Wierzba 1, 3, Kenji Sugibayashi 1, 3, * 1 2 3
Graduate School of Pharmaceutical Sciences, Josai University, Saitama, Japan Department of Pharmacy, School of Health Care Professions, University of San Carlos, Cebu, Philippines Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 February 2019 Revised 23 March 2019 Accepted 10 April 2019
Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance in the management of chronic ocular diseases. Topical application onto the lower eyelid skin, as an alternative to eye drops, is seen to be a valuable strategy in the treatment of chronic eye diseases. To elucidate the critical value of delivering drugs in solution onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted, and the results were verified using a direct pharmacodynamic study in rats. The mean residence time of pilocarpine after topical eyelid application to the eyelid skin, conjunctiva, eyeball, and plasma were 14.9, 8.50, 6.29, and 8.11 h, respectively. Conjunctiva and eyeball concentrations of pilocarpine at 8 h were 80-fold and 8-fold higher after topical eyelid application, respectively, than those for eye drops. Pupillary constriction was sustained over 8 h after topical eyelid application. Topical eyelid skin application exhibited a localized drug absorption and specific drug accumulation in the ocular tissues. Hence, it is rational to prepare topical formulations directed onto the eyelid skin, which is suitable for drugs required for long-term treatment. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: ophthalmic drug delivery skin drug targeting pharmacokinetics residence time
Introduction Ocular diseases such as glaucoma, conjunctivitis, and eye infections require immediate management and often entail frequent eye drops instillation, and chronic conditions may involve extended treatment. While instillation of eye drops is regarded as a simple task, recent studies have revealed that patients often have difficulty instilling eye drops, a challenge that leads to poor adherence1-3 and a consequent risk of treatment failure. Instilled drugs show low bioavailability because they are rapidly eliminated from the lacrimal fluid by drainage of extra solution, induced lacrimation, and tear turnover. Moreover, frequent administration of eye drops leads to substantial systemic absorption, which may result in undesirable side effects.4,5 The eyelid skin provides an alternative route to deliver drugs into the eyes. We previously established that dermal penetration by
Conflict of interest: The authors declare no known conflict of interest. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.04.012. * Correspondence to: Kenji Sugibayashi (Telephone: þ81 49 271 8137). E-mail address: [email protected] (K. Sugibayashi).
drugs depends on the skin site. The thinnest skin layer, eyelid skin, has been shown to be more permeable to some drugs than the skin localized on the abdominal surface.6 With the fewer stratum corneum layers in human eyelid skin (8 ± 2), greater skin permeation was observed.7 Therefore, it prompted us to explore the possibility of drug delivery to the eyes by topical application to the eyelids. Topical application of drugs onto the lower eyelid skin is noninvasive, and it has sustained delivery features maintaining a constant drug concentration beneath the application site for a longer duration.8-10 Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance, especially for the management of chronic ocular diseases. The direct and sustained delivery of drugs into the eyes is seen to be a practical strategy in the management of these diseases. Furthermore, the eyelid skin is proximate to the conjunctiva, which could function as a reservoir for ocular drugs. In this case, the drug may not be eliminated rapidly because it is localized in the vasculature, allowing sustained delivery of the drug into the eyeball from the eyelid skin. Of note, the conjunctiva is 2 to 30 times more permeable to drugs than the cornea,11 and it has a direct relation with the eyeball, the target site in most chronic ocular diseases.
https://doi.org/10.1016/j.xphs.2019.04.012 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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We selected pilocarpine as a model drug in this investigation. Pilocarpine hydrochloride, a nonselective muscarinic receptor agonist, has long been used in the treatment of glaucoma by increasing trabecular outflow, leading to reduced intraocular pressure. In ciliary smooth muscle cells, pilocarpine binds to and activates muscarinic M3 receptors and stimulates the contraction of the longitudinal ciliary muscle manifested as miosis.12 Current delivery systems, eye drops and ocular inserts, are available for the transport of pilocarpine into the eyes, but they are linked with disadvantages. Pilocarpine drops deliver high dose of drugs, which initially cause side effects such as headache, dimmed vision due to miosis, and myopia.13 There have been no investigations whether topical application onto the lower eyelid skin may be useful for the treatment of chronic eye diseases. The only evidence is in our previous work wherein the eyelid skin was found to be more permeable to drugs.6 To further elucidate the value of delivering drugs onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted. Our pharmacokinetic results affirmed our hypothesis, and these results were verified using a direct pharmacodynamic study of pilocarpine in rats. Materials and Methods Materials and Experimental Animals Pilocarpine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Male hairless rats (WBM/ILA-Ht, 8 weeks of age, body weight of 210 ± 2 g) were obtained from the Life Science Research Center, Josai University (Sakado, Saitama, Japan) or Ishikawa Experimental Animal Laboratories (Fukaya, Saitama, Japan). The animals were housed in appropriate cages with free access to food and water. They were kept in a controlled environment (25 C, 42% humidity,12 h light and dark cycle). Rats were anesthetized with 3 types of anesthesia (0.375 mg/kg medetomidine, 2.5 mg/kg butorphanol, and 2 mg/kg midazolam) by an intraperitoneal route before experiments. The animal studies were conducted in accordance with the Association for Research in Vision and Ophthalmology statement for use of animals as well as approved by the Institutional Animal Care and Use Committee of Josai University with the approval number JU 18003. All efforts were made to reduce the number of animals used in this study. Preparation of Solutions for Administration Pilocarpine hydrochloride (1%) solution was prepared by dissolving the drug in a sufficient volume of phosphate-buffered saline pH 7.4 to reach the desired concentration.
point) and immediately placed in heparin-coated plastic tubes. The blood samples were centrifuged (15,000 rpm and 4 C for 10 min), and the supernatant plasma was transferred into a separate tube and stored at 30 C until analysis. Furthermore, for the tissue distribution studies, rats from each group (n ¼ 4) were ethically sacrificed by overdose of pentobarbital sodium (100 mg/kg) intraperitoneally for each time point after drug administration. Tissue samples (lower eyelid skin, conjunctiva, and eyeball) were collected, rinsed 3 times with phosphate-buffered saline, pat-dried, and weighed. The collected samples were reduced in size using scissors, and 0.5 mL of acetonitrile was added before homogenization at 12,000 rpm and 4 C for 5 min using a homogenizer (Polytron PT 1200 E; Kinematica AG, Littau, Lucerne, Switzerland). The sample homogenate was then centrifuged at 15,000 rpm and 4 C for 5 min. The pilocarpine concentration in the resulting supernatant was determined by liquid chromatographyetandem mass spectrometry (LC-MS/MS). Abdominal Skin Application Studies A Teflon tube, similar to those used for eyelids, was attached to the abdomen of rats and then loaded with pilocarpine solution. After 4 h, the rats were ethically sacrificed, and tissue samples from the same sites (lower eyelid skin, conjunctiva, and eyeball) were collected and processed as in the abovementioned section. A similar method for the detection of pilocarpine was used. Quantification of Pilocarpine Using LC-MS/MS Supernatant (50 mL) from the plasma or tissue homogenates was mixed with an equal amount of acetonitrile and centrifuged at 4 C for 5 min. Ten microliters of the resulting supernatant was injected into an LC/MS/MS system for quantification of pilocarpine, as described previously.8 The LC/MS/MS system consisted of a system controller (CBM-20A; Shimadzu Corporation), pump (LC-20AD; Shimadzu Corporation), an auto-sampler (SIL-20ACHT; Shimadzu Corporation), a column oven (CTO-20A; Shimadzu Corporation), detector (4000QTRAP; AB Sciex LLC, Tokyo, Japan), and an analysis software program (Analyst® version 1.4.2; Shimadzu Corporation). The column was Shodex ODP2HPG-2A 2.0 mm 10 mm (Showa Denko Inc., Tokyo, Japan), which was kept at 40 C. The mobile phase was a mixture of acetonitrile and 0.2% trifluoroacetic acid containing 40 mM ammonium acetate (85:15), and the flow rate was 0.2 mL/min. Mass spectrometric quantification was carried out in the multiple reaction monitoring mode, monitoring transition ions of m/z 209.1 to m/z 95.1, with collision energy of 36 eV in the positive ion mode.14 Pharmacodynamic Studies
Pharmacokinetics and Tissue Distribution Studies Sixty rats were randomly divided into 3 groups. Three methods of applicationdtopical eyelid, eye drops, and intravenousdwere performed separately on each of the 3 groups. For topical eyelid application, a Teflon™ tube (internal diameter 0.48 cm and height 2 cm) was glued onto the lower eyelid skin using cyanoacrylate bond (Aron Alpha Konishi Company Ltd., Osaka, Japan) and then loaded with 100 mL of pilocarpine solution (equivalent to 4.7 mg/kg dose) using a micropipette. For eye drops, a practical application volume of 10 mL of pilocarpine solution (equivalent to 0.47 mg/kg) was instilled directly into the eyeball using a micropipette. For intravenous administration, pilocarpine solution was injected into the tail vein. The dose of pilocarpine (4 mg/kg) was administered to allow comparison of pharmacokinetic data. A 10-mg/kg dose was additionally used for the intravenous route. At predetermined time points (0.25, 2, 4, 6, and 8 h) after drug administration, approximately 200 mL of blood samples were collected from the jugular vein (n ¼ 4 per time
Direct pharmacodynamic studies on the pupil size were conducted. Anesthetized rats (n ¼ 6) were divided into 2 groups. The first group was administered 10 mL of pilocarpine solution by eye drops, whereas the other group was administered the same solution in the form of a tube glued directly onto the lower eyelid skin of hairless rats. The changes in pupil size were observed and measured using a digital microscope (VHX-5000; Keyence Company Ltd., Osaka, Japan). The area of the pupil was calculated using a program VH-M100 XY Measurement system (VHX-5000; Keyence Company Ltd., Osaka, Japan). The measurement of pupil size was performed in room light conditions using the same observation parameters throughout the study. Pharmacokinetic Calculation and Statistical Analysis The estimation of pharmacokinetic parameters was carried out using a 2-compartmental open model and a noncompartmental
G.L. See et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
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Figure 1. Disposition of pilocarpine after topical eyelid (a), eye drops (b), and intravenous administration (c). Eyelid skin ( ); conjunctiva ( ); eyeball ( ); plasma ( ). Each value represents the mean ± standard deviation (n ¼ 4) (*p < 0.05).
analysis for intravenous and topical application, respectively. Primary pharmacokinetic parameters for intravenous administration (A, B, a, and b) were obtained from a graphical analysis of the concentration-time curve and then used for the calculation of the respective secondary parameters. The following equation was used in the calculation: Cp ¼ A*Exp (a*t) þ B*Exp(b*t), where A and B represent the intercepts of 2 exponential functions, a and b are the hybrid rate constants for the distribution and elimination phases, respectively, and t is the time after intravenous administration. The following pharmacokinetic parameters were calculated: half-life time (T0.5a, T0.5b) for the distribution and elimination phases, distribution rate constants (k21, k12, and k10) from one tissue compartment to another tissue compartment, overall elimination rate constant, volume of distribution (Vc and VT) of the central compartment and peripheral compartment, and total clearance. The area under the plasma concentration-time curve (AUC) was calculated using a linear trapezoidal rule with extrapolation to infinity.15 Pharmacokinetic parameters estimated using noncompartmental analysis were as follows: area under plasma concentration-time curve (AUC0-t) and extrapolated to infinity (AUC0-∞), area under first moment curve (AUMC0-t) and extrapolated to infinity (AUMC0-∞), terminal elimination rate constant (lz), mean residence time (MRT), apparent volume of distribution at steady state (Vdss),
elimination rate constant (Kel), and its half-life (t0.5).16 The tissue-toplasma partition coefficients were calculated as the ratio of the respective AUC0-t values. The MRTs estimated after intravenous administration were used for calculation of the mean absorption of tissue penetration time (MAT), which is described by the following equation: MAT ¼ MRTtopical or eye drops e MRTiv. Time courses of pilocarpine in tissues and in blood plasma were also subjected to a noncompartmental pharmacokinetic analysis. Experimental data were tested for statistical significance (p < 0.05) using one-way ANOVA and Tukey’s honestly significant difference post hoc analysis. All data were expressed as mean with standard deviation. Results Pharmacokinetics Study The time course of the plasma and tissue concentrations of pilocarpine after different routes of administration are shown in Figure 1 and Table 1. Noncompartmental pharmacokinetic parameters for plasma and tissues are presented in Table 2. Plasma concentration-time profiles after intravenous administration at 10 mg/kg and 4 mg/kg are presented in Figure 2, and the corresponding pharmacokinetic parameters are presented in
Table 1 Plasma and Tissue Concentrations of Pilocarpine After Intravenous, Topical Eyelid, and Eye Drop Administration to Hairless Rats Application Site and Dose
Time (h)
Plasma, Average (ng/mL)
Eyelid Skin, Average (ng/g)
Conjunctiva, Average (ng/g)
Eyeball, Average (ng/g)
IV injection, 4 mg/kg
0.25 2 4 6 8 0.25 2 4 6 8 0.25 2 4 6 8
773 ± 132 287 ± 98 113 ± 31 66 ± 27 29 ± 16 4 ± 0.1a,b 7 ± 2.3a,b 10 ± 2.8a,b 11 ± 4.2a,b 7 ± 0.6a,b 84 ± 37 126 ± 51 83 ± 58 37 ± 34 21 ± 16
1871 ± 52 215 ± 68 134 ± 10 90 ± 15 42 ± 10 13,657 ± 5427a 204,916 ± 81,243a 296,747 ± 91,994a 444,215 ± 49,771a 374,539 ± 28,864a Not determined
1154 ± 66 82 ± 18 139 ± 59 75 ± 14 41 ± 6 892 ± 620a,b 8112 ± 2051a,b 32,822 ± 7643a,b 19,455 ± 7842a,b 16,241 ± 4352a,b 16,107 ± 4323 5755 ± 2825 1595 ± 554 600 ± 442 206 ± 101
2156 ± 120 225 ± 12 218 ± 32 157 ± 38 68 ± 17 82 ± 56a,b 336 ± 68a,b 1747 ± 216a,b 913 ± 206a,b 547 ± 145a,b 2926 ± 1606 619 ± 231 275 ± 175 78 ± 40 66 ± 23
Topical eyelid application, 4.7 mg/kg
Eye drops, 0.47 mg/kg
Each value represents a mean ± SD (n ¼ 4). IV, intravenous; SD, standard deviation. a Significant difference (p < 0.05) between topical eyelid application and IV injection. b Significant difference (p < 0.05) between topical eyelid application and eye drops.
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Table 2 Mean Noncompartmental Pharmacokinetic Parameters of Pilocarpine in Rats After IV Injection, Topical Eyelid, and Eye Drop Administration Drug Application
Organ
AUC(0-t) (ng.h/mL)
AUC(0-∞) (ng.h/mL)
AUMC(0-t) (ng.h2/mL)
AUMC(0-∞) (ng.h2/mL)
lz (h1)
MRT (h)
MAT (h)
Vdss (mL/kg)
Kel (h1)
T0.5 (h)
IV injection
Plasma Eyelid skin Conjunctiva Eyeball Plasma
1842 2762 1778 3396 62
1925 2907 1910 3626 102
3189 3755 2941 5554 290
4093 5410 4423 8187 829
0.10 0.29 0.31 0.29 0.18
2.13 1.86 2.32 2.26 8.11
N/A N/A N/A N/A 5.98
4852 2561 4418 2491 347,691
0.47 0.54 0.43 0.44 0.12
1.47 1.29 1.61 1.56 5.62
Eyelid skin Conjunctiva Eyeball Plasma Eyelid skin Conjunctiva Eyeball
2,163,838 6,554,340 132,962 225,298 6370 8256 660 721 Not determined 31,494 31,898 4858 5025
11,315,618 650,294 30,311 1829
97,906,949 1,913,950 51,899 2494
0.09 0.18 0.29 0.34
14.9 8.50 6.29 3.46
13.0 6.18 4.03 1.33
11 172 3438 2667
0.07 0.12 0.16 0.29
10.4 5.89 4.36 2.40
47,213 6715
51,222 8463
0.51 0.40
1.61 1.68
0.71 0.58
23 158
0.62 0.59
1.11 1.17
Topical eyelid application
Eye drops
AUC, area under the plasma concentration-time curve; AUMC, area under first moment curve; IV, intravenous; MAT, mean absorption of tissue penetration time; MRT, mean residence time.
Table 3. Pilocarpine concentration declined with 2 distinct elimination phases (Fig. 2). Pharmacokinetic parameters for both doses were similar, and the only significant and obvious difference was related to the AUC values, which were dose-dependent and indicated a linear pattern of pharmacokinetics (Table 3). Plasma and tissue concentrations of pilocarpine after intravenous administration peaked at 0.25 h, and the values declined over time (Table 1). In Table 2, the AUC values for 0-8 h ranged from 1.78 103 ng*h/mL to 3.40 103 ng*h/mL for the conjunctiva and eyeball, respectively. The values of MRT for plasma and examined tissues ranged between 1.86 h and 2.32 h for skin and conjunctiva. The other parameters such as half-life time and elimination rate constant related to the process of drug eliminations were similar for both plasma and tissues (Table 2). In Figure 1c, results showed no significant difference between plasma and tissue concentration of pilocarpine after intravenous administration, showing an almost homogenous distribution of pilocarpine to the investigated tissues (Fig. 1c).
respectively (Table 2). On the other hand, the corresponding values of 2.32 h and 2.26 h were calculated for intravenous injection of pilocarpine. After topical eyelid application, the concentration of pilocarpine in plasma at 2 h was 7 ng/mL and 126 ng/mL for eye drops, which corresponded to 18-fold lower plasma concentration at 2 h than with eye drops (Figs. 1a and 1b). This suggests localization of the drug within the ocular region, whereas very limited amounts were detected in the systemic circulation. Low concentrations of pilocarpine in plasma and very high concentrations in the conjunctiva and eyeballs demonstrated a possibility of strong ocular activity and low toxicity to other organs. A significant difference in the eyeball concentration can be observed after the different methods of application (Fig. 3). The MRT of pilocarpine in the eyeball was found to be 6 h, which corresponded
Tissue Distribution Study The disposition of pilocarpine in the ocular system of hairless rats after topical eyelid, eye drops, and intravenous administration are shown in Figure 1, and the mean concentration of pilocarpine is presented in Table 1. For eye drops, the conjunctiva and eyeball concentrations peaked at 15 min and decreased rapidly thereafter in a first-order fashion (Fig. 1b). Conversely, eyelid application (Fig. 1a) peaked at 4 h, with pilocarpine concentrations ranging from 1.74 103 ng/g to 3.28 104 ng/g in the eyeball and conjunctiva. This indicated a slow penetration of pilocarpine into the target tissues, which was followed by a slow elimination from the conjunctiva and eyeball (Fig. 1a). However, the eyelid skin still contained very high concentrations of pilocarpine, which was capable of being a reservoir for an extended period. The disposition of pilocarpine after topical eyelid application exhibited a distinctive and sustained profile over 8 h (Fig. 1a). However, both eye drops and intravenous administration showed a similar trend, decreasing pilocarpine concentrations in a short time. Conjunctival and eyeball concentrations of pilocarpine at 8 h were 1.62 104 ng/g and 547 ng/g, respectively, which corresponded to 80-fold and 8-fold higher concentrations after topical eyelid application, respectively, than with eye drops (Table 1, Fig. 1a). Pilocarpine delivered by topical eyelid application was present in the conjunctiva and eyeball at high concentrations until 8 h. In contrast, eye drop instillation was accompanied by a rapid elimination of pilocarpine from the eyeballs, the target tissues. The MRTs in the conjunctiva and eyeball were 1.61 h and 1.68 h,
Figure 2. Pharmacokinetics of pilocarpine in hairless rats after intravenous administration of 4 mg/kg ( ) or 10 mg/kg ( ). Each value represents the mean ± standard deviation (n ¼ 4). Circles represent actual experimental data while lines represent the predicted time course of pilocarpine concentrations.
G.L. See et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
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Table 3 Pharmacokinetic Parameters for a 2-Compartment Open Model After Intravenous Injection of Pilocarpine Parameters
A (ng/mL) B (ng/mL) Cp0 (ng/mL) a (h1) b (h1) T0.5,b (h) K21 (h1) K12 (h1) K10 (h1) Vc (mL/kg) VT (mL/kg) AUC (ng.h/mL) CL (mL/h/kg)
Dose 4 mg/kg
10 mg/kg
930 ± 150 237 ± 147 1166 ± 483 2.06 ± 0.8 0.34 ± 0.04 2.01 ± 0.38 0.69 ± 0.1 0.69 ± 0.4 1.02 ± 0.03 3430 ± 1080 3397 ± 1124 1139 ± 2032 3512 ± 763
1395 ± 591 947 ± 227 2341 ± 692 1.66 ± 1.2 0.36 ± 0.06 1.89 ± 0.27 0.89 ± 0.39 0.45 ± 0.57 0.68 ± 0.25 4271 ± 1544 2173 ± 1890 3426 ± 1183 2919 ± 1162
Each value represents a mean ± SD (n ¼ 4). AUC, area under the plasma concentration-time curve; CL, total clearance; SD, standard deviation.
to a 3- and 4-fold higher MRTafter topical eyelid application than after intravenous and eye drops administration, respectively (Table 2). For topical eyelid application, the AUC (0-∞) was found to be 8.26 103 ng*h/mL (Table 2), which corresponded to be 2-fold higher than for eye drops and intravenous administration. MRT of pilocarpine and its tissue-to-plasma concentration ratio were shown to be positively correlated, wherein a higher tissue-toplasma concentration existed with a longer MRT (Fig. 4). Figure 4 was obtained by plotting tissue-to-plasma concentration ratio against plasma MRT obtained from each application route. Eyelid skin application, intravenous, and eye drops have MRTs of 8.50 h, 2.32 h, and 1.61 h, respectively, in the conjunctiva. In addition, eyelid skin application, intravenous, and eye drops have MRTs of 6.29 h, 2.26 h, and 1.68 h, respectively, in the eyeball (Table 2). Eyelid skin application was shown to have the highest MRT; likewise, a higher drug concentration was also present in the
Figure 4. The relationship of mean residence time with tissue-to-plasma concentration ratio of pilocarpine in the eyeball ( ) and the conjunctiva ( ).
conjunctiva and eyeball (Fig. 4). The observed MRTs after topical application exceeded those for intravenous injection, 8.50 h and 6.29 h versus 2.32 h and 2.26 h for the conjunctiva and eyeball, respectively. The difference in MRT between intravenous injection and topical application ranges from 6.18 h to 4.03 h, which demonstrated MAT, the mean time of absorption into the conjunctiva and eyeball (Table 2). Abdominal Skin Application Studies Pilocarpine reached the eyeball 4 h after administration at different anatomical sites (supporting info I). Eyeball concentrations of pilocarpine after topical eyelid application was 1.75 103 ng/g, which corresponded to a 6- to 8-fold higher concentration than for eye drops and intravenous administration. Eyelid skin proved to be the most effective site of application for pilocarpine, followed by eye drops and intravenous administration. Abdominal skin as a site of application appeared to be the least efficient. Pharmacodynamics Study The measurement of time-dependent changes in the pupil size in hairless rats after eye drops and topical eyelid skin application is presented in Figure 5. The mean decrease in the size of the pupils 68 h after eye drops administration was 316 mm, 152 mm, and 95 mm, whereas for topical eyelid application, it was 1.59 103 mm, 1.74 103 mm, and 1.63 103 mm, respectively (Fig. 5). A significantly greater decrease in pupil size after topical eyelid installation of pilocarpine was observed when compared with eye drops over 8 h. The decrease in pupil size peaked 1 h after eye drops administration and declined gradually, returning back to the baseline size. As opposed to that of eye drops, the constriction of the pupils was sustained over 8 h after topical eyelid application.
Figure 3. Distribution of pilocarpine in the eyes of hairless rats after administration of 4 mg/kg by intravenous ( ), 0.47 mg/kg by eye drops ( ), and 4 mg/kg by topical administration onto the eyelid skin ( ). Each value represents the mean ± standard deviation (n ¼ 4). Significant difference (*p < 0.05) between the eyelid skin and intravenous or eye drops means of administration.
Discussion Although many medications exist to treat ocular conditions, the challenge remains to deliver them effectively and over prolonged
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Figure 5. Constriction of the pupil diameter induced by pilocarpine administration of 0.47 mg/kg by eye drops ( ) and 0.47 mg/kg by topical eyelid ( ) administration. Each value represents the mean ± standard deviation (n ¼ 4) (*p < 0.05).
periods of time with minimal side effects.17 In this study, the disposition of pilocarpine in ocular tissues (eyelid skin, conjunctiva, and eyeball) was determined after topical eyelid application, eye drops, and intravenous administration. Plasma concentrations were also determined to understand the extent of systemic distribution of pilocarpine. In our previous studies,6,8 we established that in vitro permeability of many drugs through the eyelid skin was much higher than that in the case of abdominal skin. Furthermore, topical application onto the eyelid skin enhanced the penetration of a drug to the eyeball. We then explored further the possibility of extending the action of the drugs and its distribution to ocular tissues using both pharmacokinetic and pharmacodynamic approaches. Among the 3 methods of application, topical eyelid application proved to be superior in delivering pilocarpine into the eyes for an extended period (Fig. 1). It was evident that the MRT of pilocarpine in the ocular system was significantly longer than that for eye drops and the intravenous route (Table 2). A longer residence time corresponded to a slower absorption rate and consequently a slower elimination process, which allowed the drug to accumulate in the specific ocular tissues for a longer period of time and exhibit its therapeutic efficacy. Thus, high drug concentrations were detected in the conjunctiva and eyeball after topical eyelid application. Notably, pilocarpine plasma concentrations were lower after topical application onto the eyelid skin (Fig. 1), suggesting ocular absorption and specific accumulation of the drug in the conjunctiva driven by a high-concentration gradient in the eyelid skin and then diffusing into the eyeball. Very high concentrations of the drug in the application site, eyelid skin, remained for the whole observation time. However, the concentration started to decline in the conjunctiva and eyeball from 4 h after drug instillation. A possible depletion of drug from the application tube may have occurred because our previous in vitro study showed that about 5%-10% of the applied dose permeated over 4 h.6 Hence, a longer half-life time and MRT after eyelid skin administration in comparison to eye drop instillation are good indicators for a longer duration of therapeutic response, possibly resulting in a lower dosing frequency. When drugs were delivered as eye drops (Fig. 1b), a very different situation was observed. Drugs penetrate the eye by absorption across the cornea from the precorneal tear film. The kinetic behavior of drugs in the tears has a direct bearing on the efficiency of drug absorption by the eye.18 Our study demonstrated
that drugs rapidly entered the eyeball following eye drops and exerted a pharmacologic effect, but this lasted for a short duration due to the short MRT and particularly fast elimination rate from the application site (Table 2). Eye drops typically remain on the ocular surface for a very short period of time before being washed away by the tears into the nasolacrimal duct, a gateway for the systemic circulation. Moreover, high plasma drug concentration, as shown in this study, after eye drops indicated nonspecific accumulation of the drug in the nontarget organs. This phenomenon emphasizes the possible occurrence of unwanted side effects associated with pilocarpine when given as eye drops.19 Furthermore, in the case of intravenous route (Fig. 1c), although pilocarpine in the systemic circulation reached the ocular tissues, high doses were essential, which may lead to unwanted side effects. Thus, the ocular kinetics of pilocarpine after eye drops application is similar to that after intravenous dose. There is extensive evidence indicating differences in the permeability of skin to chemical substances depending on the application site. Based on the previous experiments, it was shown that the skin permeability of pilocarpine was 2.6-fold higher in the case of eyelid skin than for the abdominal skin of hairless rats.6 To prove the results obtained from in vitro experiments in in vivo conditions, topical application on the abdominal skin was performed. The results of this experiments confirmed the trend observed in vitro, where skin permeability for pilocarpine was low for abdominal skin (supporting information I), whereas the eyelid skin was permeable to a greater extent. The eyelid skin, through the conjunctiva, behaves like a reservoir wherein a drug absorbed after topical application remains in the specific target organ and exerts a specific pharmacologic effect. The higher tissue-to-plasma concentration indicates that drug accumulation takes place in the specific target organ after topical application (Fig. 4). Direct pharmacologic observation was conducted to further confirm the results of the pharmacokinetic data. Pupil size reduction, miosis, is attributed to the pharmacologic effect of pilocarpine. The measurement of pupil size was performed in room light conditions because Kronschl€ ager et al.20 reported no apparent difference in pupil diameter between darkness and room light condition in anesthetized topically treated animals. Our findings showed that pupil size reduction increases over time after topical eyelid application, indicating a prolonged pharmacologic effect of pilocarpine (Fig. 5). This further confirmed localized drug action with sustained release features. In the present study, the determination of drug disposition in the specific eye region (i.e., anterior or posterior segment) has not been investigated. Correspondingly, the possibility of selective drug targeting to these regions was not fully elucidated. However, the possible mechanism for the distribution of pilocarpine into the eyeball after topical eyelid skin delivery was that topical eyelid application allows direct partitioning of pilocarpine into the eyelid skin, followed by permeation into the bulbar conjunctiva by passive diffusion. The conjunctival permeability coefficient of pilocarpine was reported to be 103 cm/h.21 From the conjunctiva, pilocarpine may directly transfer to cornea through the sclera, a site more permeable to hydrophilic drugs such as pilocarpine.22 Therefore, the development of topical formulations directed onto the eyelid skin is rational and suitable for drugs required for long-term treatment. Slow absorption and the possible application of a larger dose of a drug provide the ideal circumstances for the treatment of ophthalmic diseases requiring chronic drug application. Many viral infections may serve as examples where there is the necessity to deliver a drug at a rate providing a constant and effective concentration and maintain it for a long period. The proposed new type of topical application will improve treatment effectiveness by delivering a drug during long sleeping time, potentially resulting in much better adherence by patients.
G.L. See et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
Conclusion Topical eyelid skin application was shown to have the highest MRT and a higher drug concentration in the conjunctiva and eyeball, suggesting localized drug absorption and specific drug accumulation in the ocular tissues. Consequently, a longer duration of therapeutic response may permit less-frequent dosing. Hence, it is rational to prepare the topical formulations directed to the eyelid skin, which is suitable for drugs used in long-term treatment. Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References 1. Sayner R, Carpenter DM, Robin AL, et al. How glaucoma patient characteristics, self-efficacy and patient-provider communication are associated with eye drop technique. Int J Pharm Pract. 2016;24:78-85. 2. Schwartz GF, Hollander DA, Williams JM. Evaluation of eye drop administration technique in patients with glaucoma or ocular hypertension. Curr Med Res Opin. 2013;11:1515-1522. 3. Lavik E, Kuehn MH, Kwon YH. Novel drug delivery systems for glaucoma. Eye. 2011;25:578-586. 4. Ustundag-Okur N, Gokce EH, Bozbiyik DI, Egrilmez S, Ozer O, Ertan G. Preparation of in vitro-in vivo evaluation of ofloxacin loaded ophthalmic nano structured lipid carriers modified with chitosan oligosaccharide lactate for the treatment of bacterial keratitis. Eur J Pharm Sci. 2014;63:204-215. 5. Ramsay E, del Amo EM, Toropainen E, Tengvall-Unadike U, Ranta VP, Ruponen M. Corneal and conjunctival drug permeability: systematic comparison and pharmacokinetic impact in the eye. Eur J Pharm Sci. 2018;119:83-89. 6. See G, Sagesaka A, Sugasawa S, Todo H, Sugibayashi K. Eyelid skin as a potential site for drug delivery to conjunctiva and ocular tissues. Int J Pharm. 2017;533: 198-205. 7. Ya-Xian Z, Suetake T, Tagami H. Number of cell layers of the stratum corneum in normal skin e relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291(10):555-559.
7
8. See GL, Sagesaka A, Torizuka H, Todo H, Sugibayashi K. Iontophoresis-aided drug delivery into the eyeball via eyelid skin. J Drug Deliv Sci Technol. 2018;47: 380-385. 9. Kimura C, Tojo K. Development of a stick-type transdermal eyelid delivery system of ketotifen fumarate for ophthalmic diseases. Chem Pharm Bull. 2007;55(7):1002-1005. 10. Isowaki A, Ohtori A, Matsuo Y, Tojo K. Drug delivery to the eye with a transdermal therapeutic system. Biol Pharm Bull. 2003;26(1):69-72. 11. Davies NM. Biopharmaceutical considerations in topical ocular drug delivery. Clin Exp Pharmacol Physiol. 2000;27:558-562. 12. Skaat A, Rosman MS, Chien JL, et al. Effect of pilocarpine hydrochloride on the Schlemm canal in healthy eyes and eyes with open-angle glaucoma. JAMA Ophthalmol. 2016;134:976-981. 13. Smith SE, Smith SA, Friedmann AI, Chaston J. Comparison of the pupillary, refractive, and hypotensive effects of Ocusert-40 and pilocarpine eyedrops in the treatment of chronic simple glaucoma. Br J Ophthalmol. 1979;63:228-232. 14. Van de Merbel NC, Tinke AP, Oosterhuis B, Jonkman JHG, Bohle JF. Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection. J Chromatogr B. 1998;708:103-112. 15. Rosenbaum S. Pharmacokinetics of an intravenous bolus injection in a twocompartment model. In: Basic Pharmacokinetic and Pharmacodynamics: An Integrated Textbook and Computer Simulations. New Jersey: John Wiley & Sons Inc; 2011:158-181. 16. Gabrielsson J, Weiner D. Non-compartmental analysis. In: Reisfeld B, Mayeno AN, eds. Computational Toxicology: Volume I, Methods in Molecular Biology. New Jersey: Humana Press; 2012:377-389. 17. Rupenthal D. Drug-device combination approaches for delivery to the eye. Curr Opin Pharmacol. 2017;36:44-51. 18. Mishima S. Clinical pharmacokinetics of the eye. Prector lecture. Invest Ophthalmol Vis Sci. 1981;21:504-541. 19. Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev. 2006;58:1131-1135. €derberg P. Topically applied 20. Kronschl€ ager M, Yu Z, Talebizadeh N, Meyer LM, So caffeine induces miosis in ketamine/xylazine anesthetized rat. Exp Eye Res. 2014;127:179-183. 21. Conrad JM, Robinson JR. Mechanisms of anterior segment absorption of pilocarpine following subconjunctival injection in albino rabbits. J Pharm Sci. 1980;69:875-884. 22. Al-Kinani AA, Zidan G, Elsaid Nm Seyfoddin A, Alani AWG, Alany RG. Ophthalmic gels: past, present and future. Adv Drug Deliv Rev. 2018;126:113126.
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Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Pharmacokinetics and Tissue Distribution of Pilocarpine After Application to Eyelid Skin of Rats Gerard Lee See 1, 2, Ayano Sagesaka 1, Hiroaki Todo 1, 3, Konstanty Wierzba 1, 3, Kenji Sugibayashi 1, 3, * 1 2 3
Graduate School of Pharmaceutical Sciences, Josai University, Saitama, Japan Department of Pharmacy, School of Health Care Professions, University of San Carlos, Cebu, Philippines Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Saitama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 February 2019 Revised 23 March 2019 Accepted 10 April 2019
Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance in the management of chronic ocular diseases. Topical application onto the lower eyelid skin, as an alternative to eye drops, is seen to be a valuable strategy in the treatment of chronic eye diseases. To elucidate the critical value of delivering drugs in solution onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted, and the results were verified using a direct pharmacodynamic study in rats. The mean residence time of pilocarpine after topical eyelid application to the eyelid skin, conjunctiva, eyeball, and plasma were 14.9, 8.50, 6.29, and 8.11 h, respectively. Conjunctiva and eyeball concentrations of pilocarpine at 8 h were 80-fold and 8-fold higher after topical eyelid application, respectively, than those for eye drops. Pupillary constriction was sustained over 8 h after topical eyelid application. Topical eyelid skin application exhibited a localized drug absorption and specific drug accumulation in the ocular tissues. Hence, it is rational to prepare topical formulations directed onto the eyelid skin, which is suitable for drugs required for long-term treatment. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: ophthalmic drug delivery skin drug targeting pharmacokinetics residence time
Introduction Ocular diseases such as glaucoma, conjunctivitis, and eye infections require immediate management and often entail frequent eye drops instillation, and chronic conditions may involve extended treatment. While instillation of eye drops is regarded as a simple task, recent studies have revealed that patients often have difficulty instilling eye drops, a challenge that leads to poor adherence1-3 and a consequent risk of treatment failure. Instilled drugs show low bioavailability because they are rapidly eliminated from the lacrimal fluid by drainage of extra solution, induced lacrimation, and tear turnover. Moreover, frequent administration of eye drops leads to substantial systemic absorption, which may result in undesirable side effects.4,5 The eyelid skin provides an alternative route to deliver drugs into the eyes. We previously established that dermal penetration by
Conflict of interest: The authors declare no known conflict of interest. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.04.012. * Correspondence to: Kenji Sugibayashi (Telephone: þ81 49 271 8137). E-mail address: [email protected] (K. Sugibayashi).
drugs depends on the skin site. The thinnest skin layer, eyelid skin, has been shown to be more permeable to some drugs than the skin localized on the abdominal surface.6 With the fewer stratum corneum layers in human eyelid skin (8 ± 2), greater skin permeation was observed.7 Therefore, it prompted us to explore the possibility of drug delivery to the eyes by topical application to the eyelids. Topical application of drugs onto the lower eyelid skin is noninvasive, and it has sustained delivery features maintaining a constant drug concentration beneath the application site for a longer duration.8-10 Extending the delivery of drugs into the eyes while reducing systemic bioavailability is of utmost importance, especially for the management of chronic ocular diseases. The direct and sustained delivery of drugs into the eyes is seen to be a practical strategy in the management of these diseases. Furthermore, the eyelid skin is proximate to the conjunctiva, which could function as a reservoir for ocular drugs. In this case, the drug may not be eliminated rapidly because it is localized in the vasculature, allowing sustained delivery of the drug into the eyeball from the eyelid skin. Of note, the conjunctiva is 2 to 30 times more permeable to drugs than the cornea,11 and it has a direct relation with the eyeball, the target site in most chronic ocular diseases.
https://doi.org/10.1016/j.xphs.2019.04.012 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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We selected pilocarpine as a model drug in this investigation. Pilocarpine hydrochloride, a nonselective muscarinic receptor agonist, has long been used in the treatment of glaucoma by increasing trabecular outflow, leading to reduced intraocular pressure. In ciliary smooth muscle cells, pilocarpine binds to and activates muscarinic M3 receptors and stimulates the contraction of the longitudinal ciliary muscle manifested as miosis.12 Current delivery systems, eye drops and ocular inserts, are available for the transport of pilocarpine into the eyes, but they are linked with disadvantages. Pilocarpine drops deliver high dose of drugs, which initially cause side effects such as headache, dimmed vision due to miosis, and myopia.13 There have been no investigations whether topical application onto the lower eyelid skin may be useful for the treatment of chronic eye diseases. The only evidence is in our previous work wherein the eyelid skin was found to be more permeable to drugs.6 To further elucidate the value of delivering drugs onto the eyeball through the eyelid skin, pharmacokinetic studies of pilocarpine were conducted. Our pharmacokinetic results affirmed our hypothesis, and these results were verified using a direct pharmacodynamic study of pilocarpine in rats. Materials and Methods Materials and Experimental Animals Pilocarpine hydrochloride was purchased from Sigma-Aldrich (St. Louis, MO). Male hairless rats (WBM/ILA-Ht, 8 weeks of age, body weight of 210 ± 2 g) were obtained from the Life Science Research Center, Josai University (Sakado, Saitama, Japan) or Ishikawa Experimental Animal Laboratories (Fukaya, Saitama, Japan). The animals were housed in appropriate cages with free access to food and water. They were kept in a controlled environment (25 C, 42% humidity,12 h light and dark cycle). Rats were anesthetized with 3 types of anesthesia (0.375 mg/kg medetomidine, 2.5 mg/kg butorphanol, and 2 mg/kg midazolam) by an intraperitoneal route before experiments. The animal studies were conducted in accordance with the Association for Research in Vision and Ophthalmology statement for use of animals as well as approved by the Institutional Animal Care and Use Committee of Josai University with the approval number JU 18003. All efforts were made to reduce the number of animals used in this study. Preparation of Solutions for Administration Pilocarpine hydrochloride (1%) solution was prepared by dissolving the drug in a sufficient volume of phosphate-buffered saline pH 7.4 to reach the desired concentration.
point) and immediately placed in heparin-coated plastic tubes. The blood samples were centrifuged (15,000 rpm and 4 C for 10 min), and the supernatant plasma was transferred into a separate tube and stored at 30 C until analysis. Furthermore, for the tissue distribution studies, rats from each group (n ¼ 4) were ethically sacrificed by overdose of pentobarbital sodium (100 mg/kg) intraperitoneally for each time point after drug administration. Tissue samples (lower eyelid skin, conjunctiva, and eyeball) were collected, rinsed 3 times with phosphate-buffered saline, pat-dried, and weighed. The collected samples were reduced in size using scissors, and 0.5 mL of acetonitrile was added before homogenization at 12,000 rpm and 4 C for 5 min using a homogenizer (Polytron PT 1200 E; Kinematica AG, Littau, Lucerne, Switzerland). The sample homogenate was then centrifuged at 15,000 rpm and 4 C for 5 min. The pilocarpine concentration in the resulting supernatant was determined by liquid chromatographyetandem mass spectrometry (LC-MS/MS). Abdominal Skin Application Studies A Teflon tube, similar to those used for eyelids, was attached to the abdomen of rats and then loaded with pilocarpine solution. After 4 h, the rats were ethically sacrificed, and tissue samples from the same sites (lower eyelid skin, conjunctiva, and eyeball) were collected and processed as in the abovementioned section. A similar method for the detection of pilocarpine was used. Quantification of Pilocarpine Using LC-MS/MS Supernatant (50 mL) from the plasma or tissue homogenates was mixed with an equal amount of acetonitrile and centrifuged at 4 C for 5 min. Ten microliters of the resulting supernatant was injected into an LC/MS/MS system for quantification of pilocarpine, as described previously.8 The LC/MS/MS system consisted of a system controller (CBM-20A; Shimadzu Corporation), pump (LC-20AD; Shimadzu Corporation), an auto-sampler (SIL-20ACHT; Shimadzu Corporation), a column oven (CTO-20A; Shimadzu Corporation), detector (4000QTRAP; AB Sciex LLC, Tokyo, Japan), and an analysis software program (Analyst® version 1.4.2; Shimadzu Corporation). The column was Shodex ODP2HPG-2A 2.0 mm 10 mm (Showa Denko Inc., Tokyo, Japan), which was kept at 40 C. The mobile phase was a mixture of acetonitrile and 0.2% trifluoroacetic acid containing 40 mM ammonium acetate (85:15), and the flow rate was 0.2 mL/min. Mass spectrometric quantification was carried out in the multiple reaction monitoring mode, monitoring transition ions of m/z 209.1 to m/z 95.1, with collision energy of 36 eV in the positive ion mode.14 Pharmacodynamic Studies
Pharmacokinetics and Tissue Distribution Studies Sixty rats were randomly divided into 3 groups. Three methods of applicationdtopical eyelid, eye drops, and intravenousdwere performed separately on each of the 3 groups. For topical eyelid application, a Teflon™ tube (internal diameter 0.48 cm and height 2 cm) was glued onto the lower eyelid skin using cyanoacrylate bond (Aron Alpha Konishi Company Ltd., Osaka, Japan) and then loaded with 100 mL of pilocarpine solution (equivalent to 4.7 mg/kg dose) using a micropipette. For eye drops, a practical application volume of 10 mL of pilocarpine solution (equivalent to 0.47 mg/kg) was instilled directly into the eyeball using a micropipette. For intravenous administration, pilocarpine solution was injected into the tail vein. The dose of pilocarpine (4 mg/kg) was administered to allow comparison of pharmacokinetic data. A 10-mg/kg dose was additionally used for the intravenous route. At predetermined time points (0.25, 2, 4, 6, and 8 h) after drug administration, approximately 200 mL of blood samples were collected from the jugular vein (n ¼ 4 per time
Direct pharmacodynamic studies on the pupil size were conducted. Anesthetized rats (n ¼ 6) were divided into 2 groups. The first group was administered 10 mL of pilocarpine solution by eye drops, whereas the other group was administered the same solution in the form of a tube glued directly onto the lower eyelid skin of hairless rats. The changes in pupil size were observed and measured using a digital microscope (VHX-5000; Keyence Company Ltd., Osaka, Japan). The area of the pupil was calculated using a program VH-M100 XY Measurement system (VHX-5000; Keyence Company Ltd., Osaka, Japan). The measurement of pupil size was performed in room light conditions using the same observation parameters throughout the study. Pharmacokinetic Calculation and Statistical Analysis The estimation of pharmacokinetic parameters was carried out using a 2-compartmental open model and a noncompartmental
G.L. See et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
3
Figure 1. Disposition of pilocarpine after topical eyelid (a), eye drops (b), and intravenous administration (c). Eyelid skin ( ); conjunctiva ( ); eyeball ( ); plasma ( ). Each value represents the mean ± standard deviation (n ¼ 4) (*p < 0.05).
analysis for intravenous and topical application, respectively. Primary pharmacokinetic parameters for intravenous administration (A, B, a, and b) were obtained from a graphical analysis of the concentration-time curve and then used for the calculation of the respective secondary parameters. The following equation was used in the calculation: Cp ¼ A*Exp (a*t) þ B*Exp(b*t), where A and B represent the intercepts of 2 exponential functions, a and b are the hybrid rate constants for the distribution and elimination phases, respectively, and t is the time after intravenous administration. The following pharmacokinetic parameters were calculated: half-life time (T0.5a, T0.5b) for the distribution and elimination phases, distribution rate constants (k21, k12, and k10) from one tissue compartment to another tissue compartment, overall elimination rate constant, volume of distribution (Vc and VT) of the central compartment and peripheral compartment, and total clearance. The area under the plasma concentration-time curve (AUC) was calculated using a linear trapezoidal rule with extrapolation to infinity.15 Pharmacokinetic parameters estimated using noncompartmental analysis were as follows: area under plasma concentration-time curve (AUC0-t) and extrapolated to infinity (AUC0-∞), area under first moment curve (AUMC0-t) and extrapolated to infinity (AUMC0-∞), terminal elimination rate constant (lz), mean residence time (MRT), apparent volume of distribution at steady state (Vdss),
elimination rate constant (Kel), and its half-life (t0.5).16 The tissue-toplasma partition coefficients were calculated as the ratio of the respective AUC0-t values. The MRTs estimated after intravenous administration were used for calculation of the mean absorption of tissue penetration time (MAT), which is described by the following equation: MAT ¼ MRTtopical or eye drops e MRTiv. Time courses of pilocarpine in tissues and in blood plasma were also subjected to a noncompartmental pharmacokinetic analysis. Experimental data were tested for statistical significance (p < 0.05) using one-way ANOVA and Tukey’s honestly significant difference post hoc analysis. All data were expressed as mean with standard deviation. Results Pharmacokinetics Study The time course of the plasma and tissue concentrations of pilocarpine after different routes of administration are shown in Figure 1 and Table 1. Noncompartmental pharmacokinetic parameters for plasma and tissues are presented in Table 2. Plasma concentration-time profiles after intravenous administration at 10 mg/kg and 4 mg/kg are presented in Figure 2, and the corresponding pharmacokinetic parameters are presented in
Table 1 Plasma and Tissue Concentrations of Pilocarpine After Intravenous, Topical Eyelid, and Eye Drop Administration to Hairless Rats Application Site and Dose
Time (h)
Plasma, Average (ng/mL)
Eyelid Skin, Average (ng/g)
Conjunctiva, Average (ng/g)
Eyeball, Average (ng/g)
IV injection, 4 mg/kg
0.25 2 4 6 8 0.25 2 4 6 8 0.25 2 4 6 8
773 ± 132 287 ± 98 113 ± 31 66 ± 27 29 ± 16 4 ± 0.1a,b 7 ± 2.3a,b 10 ± 2.8a,b 11 ± 4.2a,b 7 ± 0.6a,b 84 ± 37 126 ± 51 83 ± 58 37 ± 34 21 ± 16
1871 ± 52 215 ± 68 134 ± 10 90 ± 15 42 ± 10 13,657 ± 5427a 204,916 ± 81,243a 296,747 ± 91,994a 444,215 ± 49,771a 374,539 ± 28,864a Not determined
1154 ± 66 82 ± 18 139 ± 59 75 ± 14 41 ± 6 892 ± 620a,b 8112 ± 2051a,b 32,822 ± 7643a,b 19,455 ± 7842a,b 16,241 ± 4352a,b 16,107 ± 4323 5755 ± 2825 1595 ± 554 600 ± 442 206 ± 101
2156 ± 120 225 ± 12 218 ± 32 157 ± 38 68 ± 17 82 ± 56a,b 336 ± 68a,b 1747 ± 216a,b 913 ± 206a,b 547 ± 145a,b 2926 ± 1606 619 ± 231 275 ± 175 78 ± 40 66 ± 23
Topical eyelid application, 4.7 mg/kg
Eye drops, 0.47 mg/kg
Each value represents a mean ± SD (n ¼ 4). IV, intravenous; SD, standard deviation. a Significant difference (p < 0.05) between topical eyelid application and IV injection. b Significant difference (p < 0.05) between topical eyelid application and eye drops.
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Table 2 Mean Noncompartmental Pharmacokinetic Parameters of Pilocarpine in Rats After IV Injection, Topical Eyelid, and Eye Drop Administration Drug Application
Organ
AUC(0-t) (ng.h/mL)
AUC(0-∞) (ng.h/mL)
AUMC(0-t) (ng.h2/mL)
AUMC(0-∞) (ng.h2/mL)
lz (h1)
MRT (h)
MAT (h)
Vdss (mL/kg)
Kel (h1)
T0.5 (h)
IV injection
Plasma Eyelid skin Conjunctiva Eyeball Plasma
1842 2762 1778 3396 62
1925 2907 1910 3626 102
3189 3755 2941 5554 290
4093 5410 4423 8187 829
0.10 0.29 0.31 0.29 0.18
2.13 1.86 2.32 2.26 8.11
N/A N/A N/A N/A 5.98
4852 2561 4418 2491 347,691
0.47 0.54 0.43 0.44 0.12
1.47 1.29 1.61 1.56 5.62
Eyelid skin Conjunctiva Eyeball Plasma Eyelid skin Conjunctiva Eyeball
2,163,838 6,554,340 132,962 225,298 6370 8256 660 721 Not determined 31,494 31,898 4858 5025
11,315,618 650,294 30,311 1829
97,906,949 1,913,950 51,899 2494
0.09 0.18 0.29 0.34
14.9 8.50 6.29 3.46
13.0 6.18 4.03 1.33
11 172 3438 2667
0.07 0.12 0.16 0.29
10.4 5.89 4.36 2.40
47,213 6715
51,222 8463
0.51 0.40
1.61 1.68
0.71 0.58
23 158
0.62 0.59
1.11 1.17
Topical eyelid application
Eye drops
AUC, area under the plasma concentration-time curve; AUMC, area under first moment curve; IV, intravenous; MAT, mean absorption of tissue penetration time; MRT, mean residence time.
Table 3. Pilocarpine concentration declined with 2 distinct elimination phases (Fig. 2). Pharmacokinetic parameters for both doses were similar, and the only significant and obvious difference was related to the AUC values, which were dose-dependent and indicated a linear pattern of pharmacokinetics (Table 3). Plasma and tissue concentrations of pilocarpine after intravenous administration peaked at 0.25 h, and the values declined over time (Table 1). In Table 2, the AUC values for 0-8 h ranged from 1.78 103 ng*h/mL to 3.40 103 ng*h/mL for the conjunctiva and eyeball, respectively. The values of MRT for plasma and examined tissues ranged between 1.86 h and 2.32 h for skin and conjunctiva. The other parameters such as half-life time and elimination rate constant related to the process of drug eliminations were similar for both plasma and tissues (Table 2). In Figure 1c, results showed no significant difference between plasma and tissue concentration of pilocarpine after intravenous administration, showing an almost homogenous distribution of pilocarpine to the investigated tissues (Fig. 1c).
respectively (Table 2). On the other hand, the corresponding values of 2.32 h and 2.26 h were calculated for intravenous injection of pilocarpine. After topical eyelid application, the concentration of pilocarpine in plasma at 2 h was 7 ng/mL and 126 ng/mL for eye drops, which corresponded to 18-fold lower plasma concentration at 2 h than with eye drops (Figs. 1a and 1b). This suggests localization of the drug within the ocular region, whereas very limited amounts were detected in the systemic circulation. Low concentrations of pilocarpine in plasma and very high concentrations in the conjunctiva and eyeballs demonstrated a possibility of strong ocular activity and low toxicity to other organs. A significant difference in the eyeball concentration can be observed after the different methods of application (Fig. 3). The MRT of pilocarpine in the eyeball was found to be 6 h, which corresponded
Tissue Distribution Study The disposition of pilocarpine in the ocular system of hairless rats after topical eyelid, eye drops, and intravenous administration are shown in Figure 1, and the mean concentration of pilocarpine is presented in Table 1. For eye drops, the conjunctiva and eyeball concentrations peaked at 15 min and decreased rapidly thereafter in a first-order fashion (Fig. 1b). Conversely, eyelid application (Fig. 1a) peaked at 4 h, with pilocarpine concentrations ranging from 1.74 103 ng/g to 3.28 104 ng/g in the eyeball and conjunctiva. This indicated a slow penetration of pilocarpine into the target tissues, which was followed by a slow elimination from the conjunctiva and eyeball (Fig. 1a). However, the eyelid skin still contained very high concentrations of pilocarpine, which was capable of being a reservoir for an extended period. The disposition of pilocarpine after topical eyelid application exhibited a distinctive and sustained profile over 8 h (Fig. 1a). However, both eye drops and intravenous administration showed a similar trend, decreasing pilocarpine concentrations in a short time. Conjunctival and eyeball concentrations of pilocarpine at 8 h were 1.62 104 ng/g and 547 ng/g, respectively, which corresponded to 80-fold and 8-fold higher concentrations after topical eyelid application, respectively, than with eye drops (Table 1, Fig. 1a). Pilocarpine delivered by topical eyelid application was present in the conjunctiva and eyeball at high concentrations until 8 h. In contrast, eye drop instillation was accompanied by a rapid elimination of pilocarpine from the eyeballs, the target tissues. The MRTs in the conjunctiva and eyeball were 1.61 h and 1.68 h,
Figure 2. Pharmacokinetics of pilocarpine in hairless rats after intravenous administration of 4 mg/kg ( ) or 10 mg/kg ( ). Each value represents the mean ± standard deviation (n ¼ 4). Circles represent actual experimental data while lines represent the predicted time course of pilocarpine concentrations.
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Table 3 Pharmacokinetic Parameters for a 2-Compartment Open Model After Intravenous Injection of Pilocarpine Parameters
A (ng/mL) B (ng/mL) Cp0 (ng/mL) a (h1) b (h1) T0.5,b (h) K21 (h1) K12 (h1) K10 (h1) Vc (mL/kg) VT (mL/kg) AUC (ng.h/mL) CL (mL/h/kg)
Dose 4 mg/kg
10 mg/kg
930 ± 150 237 ± 147 1166 ± 483 2.06 ± 0.8 0.34 ± 0.04 2.01 ± 0.38 0.69 ± 0.1 0.69 ± 0.4 1.02 ± 0.03 3430 ± 1080 3397 ± 1124 1139 ± 2032 3512 ± 763
1395 ± 591 947 ± 227 2341 ± 692 1.66 ± 1.2 0.36 ± 0.06 1.89 ± 0.27 0.89 ± 0.39 0.45 ± 0.57 0.68 ± 0.25 4271 ± 1544 2173 ± 1890 3426 ± 1183 2919 ± 1162
Each value represents a mean ± SD (n ¼ 4). AUC, area under the plasma concentration-time curve; CL, total clearance; SD, standard deviation.
to a 3- and 4-fold higher MRTafter topical eyelid application than after intravenous and eye drops administration, respectively (Table 2). For topical eyelid application, the AUC (0-∞) was found to be 8.26 103 ng*h/mL (Table 2), which corresponded to be 2-fold higher than for eye drops and intravenous administration. MRT of pilocarpine and its tissue-to-plasma concentration ratio were shown to be positively correlated, wherein a higher tissue-toplasma concentration existed with a longer MRT (Fig. 4). Figure 4 was obtained by plotting tissue-to-plasma concentration ratio against plasma MRT obtained from each application route. Eyelid skin application, intravenous, and eye drops have MRTs of 8.50 h, 2.32 h, and 1.61 h, respectively, in the conjunctiva. In addition, eyelid skin application, intravenous, and eye drops have MRTs of 6.29 h, 2.26 h, and 1.68 h, respectively, in the eyeball (Table 2). Eyelid skin application was shown to have the highest MRT; likewise, a higher drug concentration was also present in the
Figure 4. The relationship of mean residence time with tissue-to-plasma concentration ratio of pilocarpine in the eyeball ( ) and the conjunctiva ( ).
conjunctiva and eyeball (Fig. 4). The observed MRTs after topical application exceeded those for intravenous injection, 8.50 h and 6.29 h versus 2.32 h and 2.26 h for the conjunctiva and eyeball, respectively. The difference in MRT between intravenous injection and topical application ranges from 6.18 h to 4.03 h, which demonstrated MAT, the mean time of absorption into the conjunctiva and eyeball (Table 2). Abdominal Skin Application Studies Pilocarpine reached the eyeball 4 h after administration at different anatomical sites (supporting info I). Eyeball concentrations of pilocarpine after topical eyelid application was 1.75 103 ng/g, which corresponded to a 6- to 8-fold higher concentration than for eye drops and intravenous administration. Eyelid skin proved to be the most effective site of application for pilocarpine, followed by eye drops and intravenous administration. Abdominal skin as a site of application appeared to be the least efficient. Pharmacodynamics Study The measurement of time-dependent changes in the pupil size in hairless rats after eye drops and topical eyelid skin application is presented in Figure 5. The mean decrease in the size of the pupils 68 h after eye drops administration was 316 mm, 152 mm, and 95 mm, whereas for topical eyelid application, it was 1.59 103 mm, 1.74 103 mm, and 1.63 103 mm, respectively (Fig. 5). A significantly greater decrease in pupil size after topical eyelid installation of pilocarpine was observed when compared with eye drops over 8 h. The decrease in pupil size peaked 1 h after eye drops administration and declined gradually, returning back to the baseline size. As opposed to that of eye drops, the constriction of the pupils was sustained over 8 h after topical eyelid application.
Figure 3. Distribution of pilocarpine in the eyes of hairless rats after administration of 4 mg/kg by intravenous ( ), 0.47 mg/kg by eye drops ( ), and 4 mg/kg by topical administration onto the eyelid skin ( ). Each value represents the mean ± standard deviation (n ¼ 4). Significant difference (*p < 0.05) between the eyelid skin and intravenous or eye drops means of administration.
Discussion Although many medications exist to treat ocular conditions, the challenge remains to deliver them effectively and over prolonged
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G.L. See et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
Figure 5. Constriction of the pupil diameter induced by pilocarpine administration of 0.47 mg/kg by eye drops ( ) and 0.47 mg/kg by topical eyelid ( ) administration. Each value represents the mean ± standard deviation (n ¼ 4) (*p < 0.05).
periods of time with minimal side effects.17 In this study, the disposition of pilocarpine in ocular tissues (eyelid skin, conjunctiva, and eyeball) was determined after topical eyelid application, eye drops, and intravenous administration. Plasma concentrations were also determined to understand the extent of systemic distribution of pilocarpine. In our previous studies,6,8 we established that in vitro permeability of many drugs through the eyelid skin was much higher than that in the case of abdominal skin. Furthermore, topical application onto the eyelid skin enhanced the penetration of a drug to the eyeball. We then explored further the possibility of extending the action of the drugs and its distribution to ocular tissues using both pharmacokinetic and pharmacodynamic approaches. Among the 3 methods of application, topical eyelid application proved to be superior in delivering pilocarpine into the eyes for an extended period (Fig. 1). It was evident that the MRT of pilocarpine in the ocular system was significantly longer than that for eye drops and the intravenous route (Table 2). A longer residence time corresponded to a slower absorption rate and consequently a slower elimination process, which allowed the drug to accumulate in the specific ocular tissues for a longer period of time and exhibit its therapeutic efficacy. Thus, high drug concentrations were detected in the conjunctiva and eyeball after topical eyelid application. Notably, pilocarpine plasma concentrations were lower after topical application onto the eyelid skin (Fig. 1), suggesting ocular absorption and specific accumulation of the drug in the conjunctiva driven by a high-concentration gradient in the eyelid skin and then diffusing into the eyeball. Very high concentrations of the drug in the application site, eyelid skin, remained for the whole observation time. However, the concentration started to decline in the conjunctiva and eyeball from 4 h after drug instillation. A possible depletion of drug from the application tube may have occurred because our previous in vitro study showed that about 5%-10% of the applied dose permeated over 4 h.6 Hence, a longer half-life time and MRT after eyelid skin administration in comparison to eye drop instillation are good indicators for a longer duration of therapeutic response, possibly resulting in a lower dosing frequency. When drugs were delivered as eye drops (Fig. 1b), a very different situation was observed. Drugs penetrate the eye by absorption across the cornea from the precorneal tear film. The kinetic behavior of drugs in the tears has a direct bearing on the efficiency of drug absorption by the eye.18 Our study demonstrated
that drugs rapidly entered the eyeball following eye drops and exerted a pharmacologic effect, but this lasted for a short duration due to the short MRT and particularly fast elimination rate from the application site (Table 2). Eye drops typically remain on the ocular surface for a very short period of time before being washed away by the tears into the nasolacrimal duct, a gateway for the systemic circulation. Moreover, high plasma drug concentration, as shown in this study, after eye drops indicated nonspecific accumulation of the drug in the nontarget organs. This phenomenon emphasizes the possible occurrence of unwanted side effects associated with pilocarpine when given as eye drops.19 Furthermore, in the case of intravenous route (Fig. 1c), although pilocarpine in the systemic circulation reached the ocular tissues, high doses were essential, which may lead to unwanted side effects. Thus, the ocular kinetics of pilocarpine after eye drops application is similar to that after intravenous dose. There is extensive evidence indicating differences in the permeability of skin to chemical substances depending on the application site. Based on the previous experiments, it was shown that the skin permeability of pilocarpine was 2.6-fold higher in the case of eyelid skin than for the abdominal skin of hairless rats.6 To prove the results obtained from in vitro experiments in in vivo conditions, topical application on the abdominal skin was performed. The results of this experiments confirmed the trend observed in vitro, where skin permeability for pilocarpine was low for abdominal skin (supporting information I), whereas the eyelid skin was permeable to a greater extent. The eyelid skin, through the conjunctiva, behaves like a reservoir wherein a drug absorbed after topical application remains in the specific target organ and exerts a specific pharmacologic effect. The higher tissue-to-plasma concentration indicates that drug accumulation takes place in the specific target organ after topical application (Fig. 4). Direct pharmacologic observation was conducted to further confirm the results of the pharmacokinetic data. Pupil size reduction, miosis, is attributed to the pharmacologic effect of pilocarpine. The measurement of pupil size was performed in room light conditions because Kronschl€ ager et al.20 reported no apparent difference in pupil diameter between darkness and room light condition in anesthetized topically treated animals. Our findings showed that pupil size reduction increases over time after topical eyelid application, indicating a prolonged pharmacologic effect of pilocarpine (Fig. 5). This further confirmed localized drug action with sustained release features. In the present study, the determination of drug disposition in the specific eye region (i.e., anterior or posterior segment) has not been investigated. Correspondingly, the possibility of selective drug targeting to these regions was not fully elucidated. However, the possible mechanism for the distribution of pilocarpine into the eyeball after topical eyelid skin delivery was that topical eyelid application allows direct partitioning of pilocarpine into the eyelid skin, followed by permeation into the bulbar conjunctiva by passive diffusion. The conjunctival permeability coefficient of pilocarpine was reported to be 103 cm/h.21 From the conjunctiva, pilocarpine may directly transfer to cornea through the sclera, a site more permeable to hydrophilic drugs such as pilocarpine.22 Therefore, the development of topical formulations directed onto the eyelid skin is rational and suitable for drugs required for long-term treatment. Slow absorption and the possible application of a larger dose of a drug provide the ideal circumstances for the treatment of ophthalmic diseases requiring chronic drug application. Many viral infections may serve as examples where there is the necessity to deliver a drug at a rate providing a constant and effective concentration and maintain it for a long period. The proposed new type of topical application will improve treatment effectiveness by delivering a drug during long sleeping time, potentially resulting in much better adherence by patients.
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Conclusion Topical eyelid skin application was shown to have the highest MRT and a higher drug concentration in the conjunctiva and eyeball, suggesting localized drug absorption and specific drug accumulation in the ocular tissues. Consequently, a longer duration of therapeutic response may permit less-frequent dosing. Hence, it is rational to prepare the topical formulations directed to the eyelid skin, which is suitable for drugs used in long-term treatment. Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References 1. Sayner R, Carpenter DM, Robin AL, et al. How glaucoma patient characteristics, self-efficacy and patient-provider communication are associated with eye drop technique. Int J Pharm Pract. 2016;24:78-85. 2. Schwartz GF, Hollander DA, Williams JM. Evaluation of eye drop administration technique in patients with glaucoma or ocular hypertension. Curr Med Res Opin. 2013;11:1515-1522. 3. Lavik E, Kuehn MH, Kwon YH. Novel drug delivery systems for glaucoma. Eye. 2011;25:578-586. 4. Ustundag-Okur N, Gokce EH, Bozbiyik DI, Egrilmez S, Ozer O, Ertan G. Preparation of in vitro-in vivo evaluation of ofloxacin loaded ophthalmic nano structured lipid carriers modified with chitosan oligosaccharide lactate for the treatment of bacterial keratitis. Eur J Pharm Sci. 2014;63:204-215. 5. Ramsay E, del Amo EM, Toropainen E, Tengvall-Unadike U, Ranta VP, Ruponen M. Corneal and conjunctival drug permeability: systematic comparison and pharmacokinetic impact in the eye. Eur J Pharm Sci. 2018;119:83-89. 6. See G, Sagesaka A, Sugasawa S, Todo H, Sugibayashi K. Eyelid skin as a potential site for drug delivery to conjunctiva and ocular tissues. Int J Pharm. 2017;533: 198-205. 7. Ya-Xian Z, Suetake T, Tagami H. Number of cell layers of the stratum corneum in normal skin e relationship to the anatomical location on the body, age, sex and physical parameters. Arch Dermatol Res. 1999;291(10):555-559.
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8. See GL, Sagesaka A, Torizuka H, Todo H, Sugibayashi K. Iontophoresis-aided drug delivery into the eyeball via eyelid skin. J Drug Deliv Sci Technol. 2018;47: 380-385. 9. Kimura C, Tojo K. Development of a stick-type transdermal eyelid delivery system of ketotifen fumarate for ophthalmic diseases. Chem Pharm Bull. 2007;55(7):1002-1005. 10. Isowaki A, Ohtori A, Matsuo Y, Tojo K. Drug delivery to the eye with a transdermal therapeutic system. Biol Pharm Bull. 2003;26(1):69-72. 11. Davies NM. Biopharmaceutical considerations in topical ocular drug delivery. Clin Exp Pharmacol Physiol. 2000;27:558-562. 12. Skaat A, Rosman MS, Chien JL, et al. Effect of pilocarpine hydrochloride on the Schlemm canal in healthy eyes and eyes with open-angle glaucoma. JAMA Ophthalmol. 2016;134:976-981. 13. Smith SE, Smith SA, Friedmann AI, Chaston J. Comparison of the pupillary, refractive, and hypotensive effects of Ocusert-40 and pilocarpine eyedrops in the treatment of chronic simple glaucoma. Br J Ophthalmol. 1979;63:228-232. 14. Van de Merbel NC, Tinke AP, Oosterhuis B, Jonkman JHG, Bohle JF. Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection. J Chromatogr B. 1998;708:103-112. 15. Rosenbaum S. Pharmacokinetics of an intravenous bolus injection in a twocompartment model. In: Basic Pharmacokinetic and Pharmacodynamics: An Integrated Textbook and Computer Simulations. New Jersey: John Wiley & Sons Inc; 2011:158-181. 16. Gabrielsson J, Weiner D. Non-compartmental analysis. In: Reisfeld B, Mayeno AN, eds. Computational Toxicology: Volume I, Methods in Molecular Biology. New Jersey: Humana Press; 2012:377-389. 17. Rupenthal D. Drug-device combination approaches for delivery to the eye. Curr Opin Pharmacol. 2017;36:44-51. 18. Mishima S. Clinical pharmacokinetics of the eye. Prector lecture. Invest Ophthalmol Vis Sci. 1981;21:504-541. 19. Urtti A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv Drug Deliv Rev. 2006;58:1131-1135. €derberg P. Topically applied 20. Kronschl€ ager M, Yu Z, Talebizadeh N, Meyer LM, So caffeine induces miosis in ketamine/xylazine anesthetized rat. Exp Eye Res. 2014;127:179-183. 21. Conrad JM, Robinson JR. Mechanisms of anterior segment absorption of pilocarpine following subconjunctival injection in albino rabbits. J Pharm Sci. 1980;69:875-884. 22. Al-Kinani AA, Zidan G, Elsaid Nm Seyfoddin A, Alani AWG, Alany RG. Ophthalmic gels: past, present and future. Adv Drug Deliv Rev. 2018;126:113126.