Comprehensive Pharmacokinetic Evaluation of High Melanin Binder Levofloxacin in Rabbits Shows Potential of Topical Eye Drops for Posterior Segment Treatment

Abstract

Purpose: The purpose of this work was to understand the impact of melanin binding on ocular pharmacokinetics after administration of a high-binder model drug via different administration routes.

Methods: We applied levofloxacin to pigmented and albino rabbits as eye drops (single and multiple), as well as by intravitreal and intravenous injections. Ocular tissues and plasma were analyzed for levofloxacin concentrations with liquid chromatography–mass spectrometry (LC-MS/MS), and pharmacokinetic parameters were calculated.

Results: The data show enrichment of levofloxacin and weeks-long retention in pigmented tissues. Upon intravitreal injection, the area under the curve (AUC) values in pigmented tissues were about 9 to 15 times higher than the respective values in the albino rabbits, but this difference expanded to 255- to 951-fold following topical eye drop administration. Multiple dosing of eye drops led to substantial accumulation of levofloxacin in the pigmented tissues: AUC values were 3 to 12 times higher than after intravitreal injection. The AUCs were much lower after single topical or intravenous drug administrations. High drug levels (0.1–35 µM) were always observed in the neural retinas of pigmented eyes; the highest exposure was seen after intravitreal administration followed by multiple doses of topical drops. Single topical instillation and intravenous injections to the albino rabbits resulted in vitreal bioavailability values of 0.009% and 0.003%, respectively.

Conclusions: Melanin binding can be used to achieve targeted drug delivery and extended retention in pigmented ocular tissues. The results from topical multiple dosing experiments suggest that eye drop treatment may yield drug exposures and responses comparable to intravitreal delivery, even in the retinal pigment epithelium and choroid.

Anatomical and physiological barriers of the eye impose significant limitations on drug delivery in ophthalmic diseases. Globally, hundreds of millions of individuals are affected by ocular diseases, a number that is steadily increasing within both working and aging populations.¹ For example, it is anticipated that the number of people with age-related macular degeneration (AMD) will rise to 288 million by 2040.² To date, many common retinal diseases, such as the dry form of AMD and glaucomatous retinopathy, still lack effective drug treatments. Currently, disorders of the posterior eye segment are mainly treated invasively with intravitreally administered solutions of proteins that inhibit vascular endothelial growth factor (VEGF) or with corticosteroid injections that are based on controlled-release implants (e.g., Ozurdex) or suspensions.^3,4 The injectable solutions of anti-VEGF proteins are clinically useful at 4-week up to 16-week dosing intervals due to their half-lives in the eye being in the range of a few days to a week and the feasibility of administering high doses.⁵ However, as small molecules typically have vitreal half-lives of less than 10 hours,^6,7 they are not feasible for use as intravitreally injected solutions. Thus, novel strategies are needed to enable delivery of small molecule drugs to the posterior eye segment.^8,9 

Even though anterior segment diseases, such as elevated intraocular pressure and infections, are commonly treated with daily or multiple daily doses of small-molecule drugs administered as topical eye drops, this mode of administration has proven to be ineffective in treating posterior segment diseases. Topical administration of a small-molecule drug generally delivers around 0.1% to 5% of the dose to the aqueous humor (AH),^10–12 with bioavailability to the retina and choroid being roughly two orders of magnitude lower.^13,14 As a result, there have been clinical studies of topically applied drugs that have failed due to inadequate efficacy.¹⁴ Successful drug treatment of the retina and choroid may only be viable for highly potent drugs active at concentrations below 30 nM.¹⁵ Of note, a cyclodextrin nanoparticle–based formulation that can deliver dexamethasone to the posterior eye segment in animal models¹⁶ is currently being investigated in phase 3 clinical trials for treatment of diabetic macular edema (www.clinicaltrials.gov, NCT05343156). 

Oral administration is the most common and patient friendly method for drug delivery. However, systemic delivery has not been successful in chronic ocular treatment due to the nonspecific drug distribution in the body, which necessitates high doses. Nevertheless, adjunctive use of systemic antibiotics (in combination with either intravitreal injection or topical administration) in bacterial endophthalmitis is commonly employed in clinical practice due to the severity of the disease.^17,18 Blood–ocular barriers limit the access of therapeutics into the eye.¹⁹ For example, blood–retinal barriers, such as the retinal pigment epithelium (RPE) and retinal capillaries, restrict the retinal access of protein-bound drugs from the blood circulation.²⁰ Therefore, the use of oral medications for ophthalmic treatment is often associated with adverse off-target effects and inadequate efficacy.²¹ Targeted delivery into the retina offers the potential to change this paradigm. 

In principle, drug delivery to the site of action in the retina and choroid could be achieved using targeted drug delivery technologies, such as nanomedicines that utilize biological transport pathways.²² Another strategy for small molecules involves binding to endogenous melanin, which does not require specific transport pathways or advanced formulation technology. Melanin is an insoluble macromolecule located in the melanosomes of the RPE and the uveal tract (iris, ciliary body, choroid) of the eye. We have previously screened melanin binding of approximately 3400 small-molecule drugs and observed that 56% of the studied compounds did bind to melanin, obtaining a >95% bound fraction under in vitro study conditions.²³ The scientific literature demonstrates the impact of melanin binding on pharmacokinetic (PK) and pharmacodynamic (PD) properties of several ophthalmic drugs^15,24–29 without associated toxicity.^15,30–32 The mydriatic response half-life of atropine was extended from 43.5 hours in albino rabbits to over 96 hours in pigmented rabbits following the topical administration of atropine eye drops.³³ Similarly, the duration of miotic action of pilocarpine was longer in pigmented rabbits than in albino rabbits, even though the peak response was higher in albino rabbits at a low dose of pilocarpine.²⁹ Based on experimental data and pharmacokinetic simulations of melanin binding, we can conclude that high melanin binding of a drug would lead to its disposition and prolonged retention in the pigmented tissues.¹⁵ The extended retention of drugs in pigmented cells results from a complex interplay between the binding affinity and capacity to melanin, permeation in the melanosomal and plasma membranes of the pigmented cells, and local elimination factors.^15,34 The extended retention in the pigmented tissues may result in lower and/or prolonged drug response, depending on the pharmacokinetics and potency of the drug.²⁹ Drug activity at low unbound concentrations favors the extension of drug action. 

Melanin binding has a significant impact on ocular drug distribution and elimination,³⁵ but the ocular pharmacokinetics of melanin-binding drugs has not been systematically investigated following different routes of drug administration. Several studies have examined the ocular and systemic PK of melanin-binding molecules.^36–38 In the present study, we evaluated the PK of a highly melanin-binding drug in both albino and pigmented rabbits, employing several administration routes (i.e., intravitreal, topical, and systemic), along with single-dose and multiple-dose regimens for topical instillations. To our knowledge, this is the first time such an extensive PK evaluation on various ocular tissues over a long period has been conducted. Levofloxacin was selected as the model compound, as it is highly bound to melanin.^23,39 Previously, we demonstrated substantial enrichment and prolonged retention of levofloxacin in pigmented rat eyes compared to their albino counterparts following a single intravenous injection.⁴⁰ In this study, we explored targeted delivery in the rabbit eye offering better translational values to human eyes in comparison to rodents (such as rats and mouse). Ocular retention of levofloxacin was investigated in the pigmented eye tissues following intravitreal, topical, and systemic drug administrations. The results suggest that it may be possible to use a non-invasive topical route of administration to deliver highly melanin-binding drugs to the posterior eye segment. 

Materials and Methods

Chemicals

Levofloxacin was obtained through the Compound Depository Group at F. Hoffmann-La Roche (Basel, Switzerland). Water, acetonitrile, methanol, and formic acid were supplied by Merck (Darmstadt, Germany). All chemicals were of high purity or reagent grade. 

Animal Experiments

Animal experimentation was approved by the local veterinary authorities and was conducted at the facilities of F. Hoffmann-La Roche in strict accordance with the Swiss federal regulations on animal care and laboratory use and in adherence to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The ARVO Statement for the Use of Animals in Ophthalmic and Vision Research was followed. 

PK Studies in Rabbits

Female New Zealand White rabbits (NZW rabbits, with body weights in the range of 2.6–5.1 kg) were purchased from Charles River Laboratories (Saint-Germain-Nuelles, France), and female pigmented Dutch Belted rabbits (DB rabbits, 1.2–2.8 kg) were purchased from Envigo Global Services (Indianapolis, IN, USA). The rabbits were pair housed in open cages (type 4541P Tecniplast boxes; Tecniplast, West Chester, PA, USA) and maintained on a 12:12-hour light:dark cycle, with constant temperature (18°C–22°C) and humidity (40%–80%). Each cage was provided with unrestricted access to municipal water and sterilized food (#3409; Kliba Nafag, Kaiseraugst, Switzerland). All cages were supplied with environmental enrichments, which complied with best-practice animal welfare standards, and were rotated weekly. The rabbits were acclimated for at least 1 week before the start of the study. 

Study Design

The rabbits were allocated into four groups (n = 3 NZW rabbits or n = 4 DB rabbits per group) according to three different routes of administration: intravenous (IV) bolus injection, intravitreal (IVT) bolus injection, and topical eye drop instillation. Levofloxacin was formulated as a solution (0.6% w/v) in phosphate-buffered saline (pH 7) and administered as a single dose at dose levels presented in Table 1 and dosing volumes of 0.5 mL/kg (IV) and 50 µL (IVT and topical). In addition, an ophthalmic solution of 2% (w/v) was used in a topical multiple-dose study in pigmented DB rabbits (n = 10) following a twice-a-day (BID) dosing regimen, in the morning and in the evening, over a period of 32 consecutive days. 

Table 1.

 

Dosing Regimens of Levofloxacin for the IVT, Topical, and IV Pharmacokinetic Studies. After a Single Dose, the Tissues Were Collected From Two Eyes at Each Time Point Per Dosing Regimen. Four Eyes Were Collected at Each Time Point After Starting Multiple Dosing of Eye Drops. For Plasma Samples, the Numbers of Pigmented and Albino Rabbits Were 3 to 4 and 1 to 3, Respectively

 

Drug Administration

IV dosing was performed as a slow bolus injection into the marginal ear vein of a restrained rabbit. The site of puncture of the vein with a butterfly needle was shaved, locally anesthetized (EMLA Cream 2.5%; Aspen Pharma, Bridgewater, NJ, USA), and cleaned with 70% alcohol. Local anesthetics eye drops (tetracaine 1% SDU Faure, tetracaine hydrochloride; Novartis, Basel, Switzerland) were administered before IVT injection. The IVT dosing was performed under anesthesia using Dorbene (medetomidine, 0.10–0.25 mg/kg subcutaneous; Zoetis, Parsippany, NJ, USA) and Alfaxan (alfaxalone, 0.5–6 mg/kg, IV; Zoetis), and an aseptic technique. The conjunctiva was flushed with a 0.5% to 2% povidone iodine solution before the injection was done through the sclera and pars plana, directed posterior to the lens and into the mid-vitreous. Post-procedure, Alzane (atipamezole, intramuscularly; Zoetis) was used to antagonize medetomidine. For topical administration, the levofloxacin solution was dropped slowly into the middle of the restrained rabbit's eye using an Eppendorf pipette. After dosing, the eyes were kept open for approximately 20 seconds to prevent spillage. 

Tissue Sampling

Blood samples were collected at predefined time points (Table 1) from each rabbit into K3EDTA-coated polypropylene tubes followed by plasma separation by centrifugation at 2000g for 10 minutes at 4°C. The samples were stored at –20°C for further processing and analysis. Ocular tissues were collected within 20 minutes post-euthanasia. Prior to ocular tissue collections, the animals were euthanized by an overdose of Esconarkon (pentobarbital; Streuli Pharma, Uznach, Switzerland), diluted to a final concentration of 150 mg/mL and administered intravenously at a dose of 150 mg/kg, followed by opening of the abdominal and thoracic wall and exsanguination from the vena cava. The ocular fluids and tissues including AH, vitreous humor (VH), ciliary body, iris, neural retina, and choroid–RPE were collected from one rabbit of each strain (single-dose studies) or two pigmented rabbits (multiple-dose study) per scheduled terminal time point into Precellys homogenization tubes containing steel beads (CK14; Bertin Technologies, Montigny le Bretonneux, France). In brief, the ocular tissue dissection involved the following procedures: The eyelid was removed using forceps followed by a 360° conjunctival incision made 2 to 3 mm posterior to the limbus, then dissection of the conjunctiva and Tenon's capsule from the globe was carried out. The extraocular muscles were cut, the optic nerve was clamped and cut, and the globe removed. The eyeball was held in place with forceps and cleaned of any residual tissues. AH was collected slowly using a single-use insulin syringe inserted, bevel up, into the anterior chamber of the globe. The globe was opened by making a cut 3 to 4 mm behind the limbus, and the anterior and posterior parts of the eyeball were then separated with straight scissors to expose the inner tissues for collection. The lens, ciliary body, and iris were carefully removed from the cornea and separated with a pair of toothless forceps, and scissors were used to cut the peripheral cornea to remove any residual tissues. VH (liquid and jellied part) was collected using a single-use syringe, and an absorbent stick was used to remove any remaining VH before collecting the retina. The sclera was cut sagittal to the visual streak using straight scissors, and the it was held in place with forceps to collect the retina by gently pushing it toward the visual streak with a spatula. The choroid–RPE was collected by scraping it off from the sclera with a scalpel blade. Additionally, the sclera was cleaned using an absorbent stick to remove any possible choroid residue, and a check was made to ensure that all surrounding tissue had been removed. All tissues, with the exception of the cornea, lens, and sclera, were stored at –20°C for further processing and analysis. 

Tissue Preparation

The collected samples were diluted with blank rabbit plasma at a ratio of 1:4 and homogenized using the Precellys 24 instrument (Bertin Technologies) at 6500 rpm for 3 × 10 seconds followed by centrifugation at 4000 rpm for 2 minutes. Next, protein in the resulting tissue and plasma mixture was precipitated by adding methanol solution containing bosentan (20 ng/mL) as the analytical internal standard (10× sample volume). The plates were stirred and then centrifuged at 5600 rpm for 10 minutes at 4°C, followed by the addition of 0.1% formic acid water solution (10× sample volume) and a second centrifugation step. 

Liquid Chromatography–Tandem Mass Spectrometry Analysis

Bioanalysis was performed using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) applying a previously described method.⁴⁰ In brief, a Shimadzu high-performance liquid chromatography (HPLC) controller featuring two Shimadzu LC pumps (Shimadzu Scientific Instruments, Columbia, MD, USA) was coupled to a hybrid triple quadrupole QTRAP 5500 mass spectrometer (SCIEX, Framingham, MA, USA) equipped with an electrospray ionization Turbo source, which operated in the positive ion mode. Sample volumes of 1 µL were injected into the system, and the chromatographic separation was achieved on an YMC-Triart C18 Narrowbore HPLC column (3-µm particle size, 50 × 2.0 mm; YMC Co., Kyoto, Japan) at 70°C using an aqueous mobile phase A (0.5% formic acid in water) and an organic mobile phase B (acetonitrile) at a flow rate of 500 µL/min. The following LC gradient was applied: 0.0 to 0.26 minutes, 95% solvent A; 0.26 to 0.90 minutes, from 95% solvent A to 95% solvent B; 0.90 to 1.23 minutes, 95% solvent B; and 1.23 to 1.30 minutes, from 95% solvent B to 95% solvent A to re-equilibrate the system. Analyst 1.6.2 software (SCIEX) was employed for both LC–MS/MS data acquisition and subsequent analysis. Quality-control samples were analyzed prior to each analysis, and blank samples were injected before analyzing samples from each distinct tissue to prevent carryover. 

The lower limit of quantification (LLOQ) was 0.5 ng/mL for plasma and 2.5 ng/mL for the remaining tissues. For ocular tissue samples, the limit of detection (LOD) was set at 20% of the LLOQ (i.e., 0.5 ng/mL). The LOD was incorporated as PK data, and data below the LOD was considered unquantifiable.^41–43 This method seems appropriate for the use of concentrations below limits of quantification that are still above LOD and can benefit the performance of PK analysis (recommended by several authors^41–43). 

Pharmacokinetic Analysis

Non-compartmental analysis (NCA) was performed using Phoenix WinNonlin 8.3. software (Certara, Radnor, PA, USA), utilizing the linear-up log-down calculation method. Average drug concentration–time profiles of plasma and ocular tissues were used for all four PK studies, except in the case of plasma data after IV injection, wherein individual profiles were analyzed. The PK parameters included the following: AUC from zero to the last quantified sampling point time (AUC_0-tlast), AUC from zero to infinity (AUC_0-∞), terminal half-life (t_1/2), and mean residence time (MRT) based on MRT_0-∞. The last three parameters were reported when the terminal phase of the concentration–time profile extended over a minimum of two terminal half-lives. Additionally, the primary pharmacokinetic parameters, clearance (CL) and volume of distribution (V_ss) in VH and plasma after IVT and IV injections, respectively, were also estimated. In these cases, IV bolus administration was selected for the NCA, whereas for the rest of the analysis the NCA was based on extravascular administration. For the multiple-dose topical study, the concentration–time profile after the last administered dose was analyzed, in which 768 hours represented the time of the last eye drop installation. 

The bioavailability of levofloxacin in VH after topical eye drops and IV injections was calculated using Equations 1 and 2, respectively:  

\begin{eqnarray} &&{\rm{Vitreal\ bioavailabilit}}{{{\rm{y}}}_{{\rm{Topical }}}}{\rm{ }}\left( \% \right)\nonumber\\ &&\qquad = 100 \times \frac{{\left( {\frac{{{\rm{Topical\ AU}}{{{\rm{C}}}_{0 - \infty }}{\rm{\ }}}}{{{\rm{Topical\ dose}}}}} \right)}}{{\left( {\frac{{{\rm{Intravitreal\ AU}}{{{\rm{C}}}_{0 - \infty }}{\rm{ }}}}{{{\rm{Intravitreal\ dose}}}}} \right)}} \end{eqnarray}

(1)

 

\begin{eqnarray} &&{\rm{Vitreal\ bioavailabilit}}{{{\rm{y}}}_{{\rm{Intravenous }}}}\left( \% \right)\nonumber\\ &&\qquad = 100\times \frac{{\left( {\frac{{{\rm{Intravenous\ AU}}{{{\rm{C}}}_{0 - \infty }}{\rm{ }}}}{{{\rm{Intravenous\ dose}}}}} \right)}}{{\left( {\frac{{{\rm{Intravitreal\ AU}}{{{\rm{C}}}_{0 - \infty }}{\rm{ }}}}{{{\rm{Intravitreal\ dose}}}}} \right)}} \end{eqnarray}

(2)

 

Results

The concentration–time profiles of levofloxacin, for all examined routes of administration, differed between pigmented and albino rabbits. The pigmented rabbits exhibited significant levofloxacin enrichment in melanin-containing tissues, such as iris, ciliary body, and choroid–RPE (Figs. 1A–1C). In those tissues, the MRT of levofloxacin was much longer in pigmented rabbits (weeks) compared to that in albino animals (hours or a few days), as shown in Table 2. The time curves of levofloxacin concentrations in ocular tissues are presented in Supplementary Figures S1 and S2. 

Figure 1.

 

(A–C) Bar plots of the AUC_0-∞ values of levofloxacin in plasma and six ocular tissues after IVT injection (A), IV injection (B), and topical single dose (C) in both pigmented (gray bar) and albino (white bar) rabbits. (D) The estimations for multiple topical dosing to pigmented rabbits. *AUC_0-tlast is depicted when AUC_0-∞ could not be determined.

Table 2.

 

Pharmacokinetic Parameters Estimated by NCA Analysis for the Three Administration Routes in Pigmented and Albino Rabbits

 

Intravitreal Administration

Supplementary Figures S1A and S2A show the concentration–time profiles of IVT levofloxacin concentrations in pigmented and albino rabbits, respectively. Most levofloxacin was eliminated within 24 hours in albino rabbits (Supplementary Fig. S2), but in pigmented tissues levofloxacin was present up to 42 days (Supplementary Fig. S1). The primary PK parameters for levofloxacin after IVT injection are presented in Table 3 for albino rabbits; however, the lack of early data points in pigmented rabbits prevented determination of these parameters after IVT injection. 

Table 3.

 

NCA Pharmacokinetic Parameters of Levofloxacin in Plasma and VH Following IV and IVT Administration in Pigmented and Albino Rabbits

 

In albino rabbits, the neural retina and VH showed the highest drug exposure after IVT injections, whereas AH had the lowest AUC_0-∞ (Fig. 1, Supplementary Fig. S2A). In pigmented rabbits, high levofloxacin exposure was seen in the choroid–RPE, ciliary body, and iris, with AUC values 9 to 15 times higher than in the respective tissues of albino rabbits (Fig. 1A). Furthermore, the AUC values of levofloxacin in VH, neural retina, and AH were 3 to 11 times higher in the pigmented rabbits compared to the albino rabbits. The terminal t_1/2 values and MRTs were longer in the melanin-containing tissues of pigmented rabbits (∼1 week) as compared to the respective tissues in albino rabbits (3.6–8.6 hours) (Table 2). 

Topical Administration: Single- and Multiple-Dose Regimens

The concentration–time profiles of levofloxacin after a single topical dose (Supplementary Fig. S1C) and multiple dosing (Supplementary Fig. S1D) showed drug accumulation in the melanin-containing tissues of the pigmented rabbits. In albino rabbits, the highest drug exposure after a single eye drop instillation was seen in the AH (Supplementary Fig. S2C). Concentrations of levofloxacin in the AH and VH were reliably measured in pigmented rabbits only at the first time point; therefore, no pharmacokinetic parameters were determined for these tissues (Table 2). 

After a single eye drop instillation, the AUC_0-∞ values of the pigmented iris, ciliary body, and choroid–RPE were at least 2 to 3 orders of magnitude higher than in their albino counterparts; the choroid–RPE showing the greatest difference (Fig. 1C). The longest MRTs and t_1/2 values were estimated to be in the pigmented ciliary body, being 21.1 and 15.9 days, respectively (Table 2). These parameters, however, could not be determined for the choroid–RPE as they did not meet the inclusion requirements. Levofloxacin concentrations in the neural retina at 1 hour after dosing were similar in albino and pigmented rabbits (Supplementary Figs. S1C, S2C), but the drug exposure in the pigmented rabbits was extended, being 44 times higher than in the albino eyes (Fig. 1C, Supplementary Fig. S2C). The bioavailability of levofloxacin in the VH of albino rabbits was 0.009%. 

Administration of levofloxacin eye drops twice daily for 32 days to pigmented rabbits, resulted in the highest drug concentrations in the melanin-containing tissues, followed by AH, neural retina, and VH in decreasing order. Sustained concentrations of levofloxacin were observed in the pigmented tissues, but at lower levels also in the neural retina and VH for 43 days after the last topical dose (Supplementary Fig. S1D). The AUC ranking of the tissues in pigmented rabbits was the same as after a single levofloxacin dose, but the AUC values after multiple dosing were 6 to 18 times higher (in pigmented tissues) and 7 times higher (in neural retina) than after a single dose (Table 2, Fig. 1D). These results clearly demonstrate remarkable accumulation of levofloxacin in the pigmented tissues during multiple dosing using a BID regimen. Interestingly, this multiple dosing regimen also resulted in long drug retention in the neural retina (MRT of 22 days), in addition to the pigmented tissues (MRT of 12–20 days) (Table 2). 

Intravenous Administration

Levofloxacin concentrations in plasma and ocular tissues after a 1-mg/kg IV injection are presented in Supplementary Fig. S1B (pigmented rabbits) and Supplementary Fig. S2B (albino rabbits). Rapid drug elimination from plasma and ocular tissues was observed during 24 hours in albino rabbits. In contrast, pigmented rabbits showed drug concentrations in the melanin-containing tissues that were at least an order of magnitude higher than in albino tissues. Drug levels were sustained for up to 42 days in the pigmented eye tissues, even though the plasma levels were not quantifiable at 24 hours. Among the melanin-free tissues, the neural retina of pigmented rabbits showed a peak concentration at 6 hours and quantifiable levels for at least 7 days. 

Systemic pharmacokinetic parameters of IV levofloxacin demonstrate rapid elimination from plasma (Table 3). The secondary PK parameters of levofloxacin could only be determined in the VH, neural retina, and plasma of the albino rabbit, whereas in the pigmented rabbit these parameters could be determined in melanin-containing tissues, neural retina, and plasma (Table 2). 

The substantial enrichment of intravenously injected levofloxacin in the pigmented tissues is reflected by the AUC values (Fig. 1B). We observed an order of magnitude increase in levofloxacin exposure in the neural retina of pigmented rabbits, as compared to albino rabbits. High AUC values in the pigmented tissues were attributed to higher drug distribution to these tissues and prolonged tissue retention (with MRT values of 16–20 days) (Table 2). Plasma exposure was three times lower in the pigmented rabbits than in the albino rabbits. Moreover, vitreal bioavailability after IV injection was 0.003% in albino rabbits (data not available for pigmented animals). 

Comparison Between the Routes of Administration

The topical multiple dosing resulted in the highest enrichment of levofloxacin (i.e., AUC values) in the melanin-containing tissues (Fig. 2). It is interesting to note that repeated topical administration resulted in a 12-fold higher iridial AUC compared to a single IVT injection. Furthermore, compared to the IVT injection, the multiple dosing topically resulted in 3- and 5-fold AUC values in the choroid–RPE and ciliary body, respectively. Interestingly, the AUC of the choroid–RPE was only slightly smaller than that of ciliary body (Table 2). The highest exposure of levofloxacin in the neural retina was observed after IVT injection followed by the topical multiple dose regimen with 37 times lower AUC (Table 2). 

Figure 2.

 

The AUC_0-∞ values of levofloxacin in ocular tissues after a single topical eye drop dose of 0.3 mg and multiple topical doses of 1 mg twice per day for 32 days (corresponding to the last drop instillation), an IVT dose of 0.05 mg, and an IV injection of 1 mg/kg in pigmented rabbits. *AUC_0-tlast is depicted when AUC_0-∞ could not be determined.

Figure 2 illustrates drug exposure to ocular tissues in pigmented rabbits for all modes of administration. Topical multiple dosing exhibited the highest enrichment in melanin-containing tissues, even 1 order of magnitude higher than IVT injection. For example, iridial exposure was 12-fold higher than after IVT injection, whereas choroid–RPE and ciliary body exposures were 3- and 5-fold higher, respectively. As expected, melanin-free tissues received the highest tissue exposure after the IVT injection. When comparing the concentration patterns in the melanin-containing tissues of pigmented rabbits following four different dosing regimens (Supplementary Fig. S3), we noticed significant variations between the IVT injection and the rest of the administered routes. For example, choroid–RPE concentrations were on average an order of magnitude larger in the topical multiple-dose study than with IVT injection but provided similar VH concentrations in the terminal phase. This resulted in a smaller choroid–RPE/VH concentration ratio after IVT injection (see Supplementary Table S1). Also, the choroid–RPE/retina ratio was even smaller in all studied groups. 

Discussion

In this study, we demonstrated the pharmacokinetic impact of melanin binding after administering a high-binder compound (levofloxacin) intravitreally, topically, and intravenously to rabbits. Overall, melanin binding leads to significant drug accumulation and retention in pigmented rabbit tissues, such as iris, ciliary body, RPE, and choroid, but sustained levofloxacin concentrations were also seen in the non-pigmented neighboring tissues (i.e., neural retina, AH, and VH) in the pigmented rabbit eyes. In addition to the drug depots in the pigmented tissues, the unbound drug levels in the tissues are critically important for pharmacological responses. To our knowledge, this is the first study to quantitatively compare tissue-level drug exposure in albino and pigmented rabbits following drug administration via various routes. Such data are important for understanding melanin-based targeted drug delivery. 

IVT injection is the current method of choice in retinal drug treatment because it provides relatively direct access to the retina with limited systemic drug exposure. However, small molecules, capable of posterior drug elimination, have short half-lives of a few hours in the vitreous.^6,7 In principle, melanin binding might lead to prolonged drug retention in the pigmented tissues, enabling extended dosing intervals. IVT injection yielded the highest drug exposure in neural retina and VH. The data suggest that melanin binding not only enhanced ocular drug exposure in the pigmented tissues but also contributed to the extended levels in the adjacent tissues. For example, levofloxacin levels in the vitreous and neural retina were equal in the albino rabbits, but in the pigmented rabbits levofloxacin levels in the neural retina were several times higher than in the vitreous, suggesting drug release from the pigmented choroid–RPE to the neural retina. In short, the extended MRTs in pigmented rabbit tissues suggest that high melanin binder drugs have potential for prolonged pharmacological activity after IVT injections. 

Systemic administration is not commonly used for chronic eye treatment because high doses are required to overcome blood–ocular barriers and provide adequate drug levels. In fact, the vitreal bioavailability of IV levofloxacin in albino rabbits was only 0.003%, demonstrating that normally only a negligible fraction of the systemic dose enters the eye. The situation is different in the systemic antibiotic treatment of endophthalmitis with levofloxacin, as the blood–ocular barriers are compromised in this disease state. Sometimes serious systemic side effects are associated with systemic eye medications, such as acetazolamide tablets in glaucoma therapy.^44,45 However, targeted drug delivery to the eye might alter this situation. Indeed, significant distribution and retention of 11 intravenously administered drugs to the pigmented rat eyes were seen, as AUC and MRT values in the pigmented eyes were 175 times and 65 times higher than in their albino counterparts, respectively.⁴⁰ Compared to the single dose, multiple doses of per oral levofloxacin resulted in a 6- to 9-fold accumulation index of the drug in the pigmented rat iris–ciliary body and choroid–sclera.³⁷ Boyer et al.³⁸ also investigated this aspect in albino and pigmented rabbits after single and multiple oral drug doses. Drug accumulation was evident in pigmented rabbits, but not in albino rabbits. In our rabbit study, the highest AUC values were observed in the pigmented choroid–RPE, ciliary body, iris, and neural retina in decreasing order. This may indicate that systemic dosing favors accumulation in posterior ocular tissues. From the patients’ perspective, oral drug administration would be convenient in chronic disease treatment. Targeted delivery and extended drug retention in the pigmented posterior segment tissues should enable treatments with smaller drug doses and reduce the risks of adverse effects. 

Topical delivery of eye drops is the most common way for ophthalmic drug delivery to the anterior eye segment. Eye drops are administered once or multiple times daily to treat glaucoma and ocular inflammations, leading to low patient compliance (∼50 % in glaucoma treatment).^46,47 However, topical dosing is adequate for treating only anterior eye segment tissues (e.g., ciliary body, iris) that are reached with eye drop treatment, as shown in this study. Our data revealed the exact and very low bioavailability (0.009%) of any topical drug to the VH. Interestingly, significant levofloxacin AUCs were achieved with a single eye drop in both anterior (iris, ciliary body) and posterior (choroid–RPE) pigmented tissues, as well as in the neural retina. This is due to slow drug clearance from melanin-containing tissues. Melanin binding may be a useful tool in the development of long-acting drugs with reduced dosing frequency and improved patient compliance, especially among elderly patients who struggle with eye drop administration. 

As expected, based on principles of drug accumulation, multiple topical dosing of levofloxacin with a long half-life in pigmented tissues resulted in remarkable drug accumulation in those tissues (e.g., ciliary body, choroid–RPE). Compared to the single eye drop, dose-normalized AUCs after multiple levofloxacin eye drops revealed a sixfold increase of AUCs in choroid–RPE, even though true steady-state levels may not have yet been reached within 32 days (i.e., steady-state levels are reached in five half-lives, meaning 40–80 days in the pigmented tissues with half-lives of 8–16 days) (Table 2). Furthermore, about twofold greater dose-normalized exposure of levofloxacin was seen in the neural retina with multiple dosing, which reflects the high levels in the choroid–RPE. 

The topical multiple-dose experiments resulted in the highest drug exposures, being 3 to 12 times greater than those observed after IVT injection in the pigmented tissues (AUC ranking: iris > ciliary body > choroid–RPE). Interestingly, IVT injection showed higher exposure to the posterior tissues (choroid–RPE > ciliary body > iris), which is in line with drug elimination via the blood–retina barrier to the blood circulation. Compared to the topical multiple dosing, the AUC values of IVT levofloxacin in the neural retina and VH were 37 and 107 times higher, respectively. Overall, it seems that multiple topical dosing can result in choroid–RPE drug levels that are comparable to those achieved with IVT injection, demonstrating the potential of topical melanin-binding drugs to target choroid–RPE in a non-invasive manner. It should be noted that the IVT dose (0.05 mg) was lower than the daily topical dose (2 mg). In general, higher drug doses can be safely delivered topically than intravitreally, where the injected drug has more direct access to the ocular tissues (e.g., retina, lens, ciliary body). 

Multiple topical dosing resulted in adequate neural retina levels of the model drug levofloxacin (∼20–300 nM), indicating the real possibility of pharmacological efficacy of potent melanin-binding drugs in the neural retina. We measured total drug concentrations in the eye tissues, but the concentration of free drug determines the pharmacological action. Unfortunately, free drug levels in the pigmented tissues have not been determined in vivo. Sometimes the vitreal concentration is used as a substitute for unbound concentrations in the vitreous. Even though drugs may bind to the components of the VH (generally < 70%), there is some binding of small molecule drugs.⁵¹ However, we can assume that vitreal concentrations would represent the low end of possible free concentrations in the neural retina of pigmented eye. After multiple topical dosing, the AUC of levofloxacin in the vitreous was about 2 orders of magnitude lower than in the choroid–RPE, a smaller difference than in the in vitro binding equilibrium studies (high binder drugs show 3 to 4 orders of magnitude difference between bound and free states).^15,34,52 Overall, chronic eye drop administration of high melanin-binding drugs holds promise for treating the retina, RPE, and choroid non-invasively, without the need for frequent IVT injections. The best chance of success will be achieved with potent compounds that exert their effects at low nanomolar concentrations of unbound drug (<100 nM but preferably <10 nM). 

Clinical Implications

This study was performed using rabbits as the preclinical animal model. Rabbits are superior to rats and mouse in translating ocular PK studies to the human eye after local ocular administration.^53,54 Rabbit eye dimensions and melanin content are more comparable but the retina vasculature is different from that of the human eye.⁵⁵ For example, the melanin concentrations in the choroid–RPE in DB rabbits (37 µg/mg tissue) and humans (24 µg/mg tissue) are close to each other, unlike the monkey choroid–RPE at 213 µg/mg tissue.^52,56,57 However, it is important to note that the neurosensory retina in rabbits features a visual streak, an elongated region with a high density of photoreceptors and ganglion cells, which differs from the more centralized fovea in humans. The fovea is a small, central pit densely packed with cone photoreceptors, responsible for sharp central vision. Despite these differences, the main findings of the study in the context of studying melanin binding–related pharmacokinetics are translatable to humans. 

Levofloxacin is a broad-spectrum antibiotic effective against Gram-negative and Gram-positive bacteria commonly found in endophthalmitis. The minimum inhibitory concentration (MIC) against various bacteria ranges from 500 to 2000 ng/mL.¹⁸ A modest systemic dose of 1 mg/kg levofloxacin (veterinary IV doses are 5–29 mg/kg^48–50) resulted in MIC levels in the pigmented tissues. Although levofloxacin levels in ocular tissues were mainly below the MIC, IVT injection resulted in higher levels in both pigmented tissues and VH. Multiple topical doses delivered MIC levels only to the pigmented tissues and AH. It should be noted that these are total levels, and only a fraction of levofloxacin is in its active unbound form; therefore, it is not possible to draw conclusions on the impact of melanin binding on the clinical efficacy of levofloxacin as an antibiotic. For clinical activity, unbound concentrations should remain at MIC levels or higher. This study was not designed to assess the antibiotic activity of levofloxacin; rather, levofloxacin was used as a model substance to represent the pharmacokinetic profiles of high melanin-binding drugs. 

Overall, IVT injections are the most commonly used route of drug administration for the treatment of posterior segment eye diseases. In the United States alone, more than 7 million injections are given each year, and this number is increasing annually.⁵⁸ The risk of ocular complications of IVT injections ranges from 0.019% to 1.6%,⁵⁹ but frequent IVT injections, even for the rest of the patient's life, may pose complications (e.g., injection pain, intraocular hemorrhage, retinal detachment, even endophthalmitis).⁶⁰ In the present study, we have shown that small-molecule drugs may reach the target tissues in the back of the eye after topical and systemic delivery. In both cases, multiple dosing should result in major accumulation in the pigmented tissues, offering possibilities for non-invasive home treatment of retinal and choroidal pathologies. This is an important option for patient compliance and convenience, as most posterior eye diseases are chronic and require long-term therapy. Many retinal diseases would certainly benefit from early intervention that is not compatible with IVT injections. 

Extending the proposed pharmacological administration to a posterior segment disease model is outside the scope of this PK-focused paper, but future studies could explore this approach further. Such studies would provide valuable insights into the therapeutic efficacy of melanin-binding drugs in treating conditions such as age-related macular degeneration, diabetic retinopathy, and other retinal diseases. Demonstrating the effective potential of melanin binding in these models would strengthen the clinical relevance of our findings and support the development of new treatment strategies for posterior segment diseases. Based on our results, topical administration is appealing in many ways, as it provides targeted delivery and extended drug retention in important anterior and posterior target tissues. For example, in glaucoma, prolonged retention could yield longer dosing intervals and increased patient compliance by reducing the dosing frequency from multiple daily instillations to even once weekly administration. Moreover, topical administration with reduced frequency would minimize the risk of adverse effects. 

Conclusions

In conclusion, melanin binding of levofloxacin was extensive following topical, IV and IVT administrations. Depending on the drug potency, target tissue, and route of administration, melanin binding may be useful in targeted posterior segment drug delivery and in extending the duration of pharmacological responses in anterior and posterior tissues. Melanin binding seems to be a promising development criterion for discovery of drugs with targeted delivery and accumulation to the pigmented tissues. Such developments might replace repeated IVT injections and introduce non-invasive eye drop or tablet treatments for posterior segment pathologies. Overall, our study demonstrates the versatile potential of melanin binding in ocular drug delivery. 

Acknowledgments

The authors thank Pawel Dzygiel, Régine Hérissé, Frédéric Avenel, Anthony Vandjour, Peter Schrag, and Thomas Thelly for their technical assistance. Sina Bahrpeyma acknowledges the Doctoral School of the University of Eastern Finland and Finnish Cultural Foundation. The eyeball anatomy in the graphical abstract was created using BioRender (https://biorender.com/). 

Supported by F. Hoffmann-La Roche; by a grant from the Academy of Finland (357470 to AU); and by strategic funding of the University of Eastern Finland, Finland (EMdA). 

Disclosure: S. Bahrpeyma, None; P. Jakubiak, F. Hoffmann-La Roche/Genentech (E, F); R. Alvarez-Sánchez, None (Former employee of F. Hoffmann-La Roche); A. Caruso, F. Hoffmann-La Roche (E, F); M. Leuthardt, F. Hoffmann-La Roche (E, F); C. Senn, F. Hoffmann-La Roche (E, F); E.M. del Amo, Allen & Overy Shearman Sterling LLP (C), Anidal Pharma (C); A. Urtti, Active Biotech (C), Pharming (C), Allen & Overy Shearman Sterling LLP (C), Ocular Therapeutics (C), UNITHER (C), ReBio Technologies (F), Bayer (F), Roche (F)