Ocular Pharmacokinetic Effects of Drug Binding to Melanin Pigment and the Vitreous Humor

Diseases of the posterior segment of the eye, such as age-related macular degeneration and diabetic retinopathy, are the leading causes of blindness in the developed world. Drug delivery to the posterior eye tissues, including the retina and choroid, is accomplished by frequent intravitreal injections, which are expensive and burdensome to the health care providers and patients, and may cause harmful side effects. Therefore, sustained and less invasive delivery strategies are vitally needed to improve the treatment. Melanin pigment is found at high concentrations in ocular tissues, and many clinical drugs bind to it. Targeting the pigment to obtain sustained drug action in ocular tissues could be a feasible approach for improving ocular drug therapy. Therefore, this thesis project aimed to study the effects of pigment binding on pharmacokinetics in the eye with in vitro, in vivo and in silico methods. In addition, we investigated drug binding to the vitreous humor, as it may participate in modulating ocular pharmacokinetics and has been studied only scarcely. Melanin is a negatively charged, hydrophobic polymer, and it is expected to bind all basic and lipophilic drugs to some extent. Melanin is located in intracellular melanosomes, which are cell organelles surrounded by a lipid membrane, and are expected to have an acidic intraluminal pH. Due to the location of melanin, cellular and physiological factors act in concert with the binding to the melanin polymer, to impact ocular pharmacokinetics of melanin-binding drugs. We investigated pigment binding and related cellular and physiological factors with seven small molecule drug or drug-like compounds: chloroquine, timolol, nadolol, propranolol, methotrexate, 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) and diclofenac. A pH-dependent binding (pH 5.0 vs. 7.4) to isolated porcine ocular melanin was observed, mainly with the acidic compounds that are less negatively charged and bind better at pH 5.0. Therefore, pH plays an important role in drug-melanin binding in the case of acidic drugs, but is less important in the case of basic drugs. The binding parameters, maximum binding capacity and affinity, were reliably calculated with the Sips binding isotherm instead of the commonly used Langmuir isotherms. The Sips isotherm is in line with the heterogeneous nature of the melanin surface to where the drugs bind, and, therefore, is better suitable for parameter analysis. Cellular uptake and intracellular binding in pigmented retinal pigment epithelial cells were shown to correlate with melanin binding, but other factors, such as lipophilicity of the drug, also need to be taken into account. Pharmacokinetic simulations of the retention of drugs in pigmented posterior segment tissues demonstrated that low drug permeability in the plasma and melanosomal membranes and the entrapment of positively charged drugs in the melanosome increases melanin binding related retention in these tissues. In addition, both the intracellular binding experiments with pigmented cells and the pharmacokinetic simulations showed that only a small fraction (~0.01%) of the highest binding drug of this study, chloroquine, is in the free form inside the cells. The free form of the drug elicits the drug action (beneficial or harmful), and it is, therefore, important to differentiate between the free and total drug inside the cells when assessing drug response. Furthermore, we demonstrated the distribution and retention of 123I-chloroquine in the eyes of pigmented but not albino rats after intravenous administration, establishing the use of single photon emission computed tomography/computed tomography imaging in monitoring melanin binding related kinetics in vivo. All in all, in the variety of experiments performed, melanin binding was shown to have a major impact on ocular pharmacokinetics in pigmented tissues. In addition to investigating melanin binding, drug binding to the vitreous humor was studied with a cassette of 35 clinical small molecule drugs. The binding was rather low and vitreal binding was concluded to have only a modest effect on ocular pharmacokinetics. In conclusion, this thesis project generated important information of the extensive pharmacokinetic impact of drug-melanin binding in ocular tissues. In addition, we demonstrated the moderate pharmacokinetic impact of vitreal drug binding, which is not comparable to melanin binding. The computational models developed on melanin binding and its pharmacokinetic implications can be used in drug discovery and development. The in vitro methods can also be implemented to the industry scale drug development process. The results obtained support the feasibility of using melanin targeting to attain sustained action in pigmented ocular tissues, but more research into the approach is needed before it can be employed in practice.Silmän takaosan sairaudet, kuten silmänpohjan ikärappeuma ja diabeettinen retinopatia, ovat yleisimpiä sokeuden aiheuttajia teollisuusmaissa. Lääkkeenanto ja lääkeaineen pääsy silmän takaosan kudoksiin on kuitenkin hankalaa, ja annostelu joudutaan toteuttamaan kuukausittain annettavilla lasiaisen sisäisillä injektioilla. Nämä injektiot voivat olla epämiellyttäviä ja pelottavia potilaille, voivat aiheuttaa vakavia haittavaikutuksia ja kuormittavat terveydenhuoltoa. Siksi tarvitaankin pitkävaikutteisia, kohdennettuja lääkehoitoja helpottamaan silmän takaosan sairauksien hoitoa. Silmän etu- ja takaosassa on paljon melaniiniksi kutsuttua pigmenttiä. Melaniini sitoo useita lääkeaineita, ja sitoutuessaan melaniiniin lääkeainetta kertyy silmän pigmentoituneisiin kudoksiin. Melaniinisitoutumista voitaisiinkin mahdollisesti hyödyntää pidentämään lääkeaineen vaikutusta silmässä ja kohdentamaan hoitoa silmän pigmentoituneisiin kudoksiin. Tämän väitöstutkimuksen tavoitteena oli tutkia melaniinisitoutumisen vaikutusta lääkeaineiden kulkeutumiseen silmässä. Lisäksi tutkimme lääkeaineiden sitoutumista silmän lasiaiseen, sillä lasiaissitoutumisen vaikutus lääkeaineen vaiheisiin silmässä tunnetaan huonosti. Melaniini sijaitsee solujen sisällä melanosomeissa; soluelimissä, joita peittää lipidikalvo. Lääkeaineen tulee läpäistä sekä solu- että melanosomikalvo ennen kuin se pääsee sitoutumaan melaniiniin. Solutason tekijöiden, kuten solukalvon läpäisevyyden, vaikutus sitoutumiseen elimistössä tunnetaan huonosti. Tutkimmekin lääkeaineiden melaniinisitoutumisen ja solutason tekijöiden yhteisvaikutusta lääkeaineiden kulkeutumiseen silmässä. Tulosten perusteella voimme todeta, että melaniinisitoutuminen voi pidentää lääkeaineen viipymistä silmässä huomattavasti. Solukalvon läpäisevyys ja lääkeaineen sitoutuminen muihin solun osiin kuin melaniiniin vaikuttavat myös huomattavasti solutason sitoutumiseen. Huomioitavaa on, että suurin osa pigmentoituneissa kudoksissa olevasta melaniiniin sitoutuvasta lääkeaineesta on sitoutuneena, ja lääkeaineen tulee olla riittävän tehokas, jotta vapaana oleva lääkeainepitoisuus riittää vaikutuksen aikaansaamiseen. Lääkeaineiden sitoutuminen silmän lasiaiseen ei ole verrattavissa melaniinisitoutumiseen, eikä sillä ole suurta vaikutusta lääkeaineiden kulkeutumiseen silmässä. Tämä väitöskirjatyö tuotti tärkeää tietoa lääkeaineiden melaniinisitoutumisen vaikutuksesta niiden kulkuun silmässä. Melaniinisitoutuminen määrittää lääkeaineiden kertymistä silmään ja viipymistä silmän pigmentoituneissa kudoksissa, jotka ovat lääkehoidon kohteita monille silmän takaosan sairauksille. Väitöskirjatyössä luotuja menetelmiä ja tietokonemalleja voidaan käyttää lääkekehityksessä helpottamaan melaniinisitoutumisen tutkimista. Melaniinisitoutumisen hyödyntäminen silmälääkkeiden vaikutuksen kohdentamisessa ja pidentämisessä näyttää lupaavalta, mutta lisätutkimuksia tarvitaan ennen kuin tämä lähestymistapa saadaan toteutettua käytännössä

Ocular melanin binding of drugs : in vitro binding studies combined to a pharmacokinetic model

Certain drugs accumulate into pigmented tissues due to their binding to melanin, a macromolecule inside pigmented cells. Melanin can affect the drug's pharmacokinetics by acting as a drug reservoir. Binding can also cause toxic effects by accumulating compounds to pigmented cells. This thesis focuses on ocular melanin. The literature review covers the most common methods used in the study of ocular melanin binding and concentrates on in vitro methods and the analysis and usability of the results in pharmacokinetic modeling. The aim of the experimental part was to study melanin binding of a set of compounds in vitro with melanin isolated from the retinal pigment epithelium (RPE) and choroid of porcine eyes and with primary porcine RPE cells and then construct a pharmacokinetic model of melanin binding with STELLA® software and simulate it with the in vitro results. The compounds chosen for the study; nadolol, timolol, chloroquine, methotrexate, carboxydichlorofluorescein (CDCF) and dexamethasone, are small molecules with diverse physicochemical properties (octanol/water partitioning coefficient (logP), pKa, acid/base status). Some are also efflux substrates. The in vitro binding with melanin was studied at pH 7.4 and in addition at pH 5 for the acidic compounds, since the pH inside melanosomes where melanin is located is acidic. Porcine RPE cells were used to study the amount of uptake and rate of elimination of the set of compounds. The effect of efflux was also evaluated with a general efflux inhibitor probenecid. All the basic compounds bound to melanin in vitro. The acidic compounds did not seem to bind at pH 7.4 but bound at pH 5. Chloroquine, as expected, had the highest binding. In the cell studies, the uptake of chloroquine was significant, at least partly due to melanin binding. The other compounds were taken into the cells to a much smaller extent. The efflux inhibitor did not seem to affect the results. The results of the binding study were used in the models constructed of melanin binding and cellular pharmacokinetics. The constructed model was a very simple one not taking into account many factors affecting cellular pharmacokinetics. The results of both the in vitro studies and the model give a good idea of the importance of melanin binding in ocular drug delivery. The model can be used in the future as a base for more comprehensive models of the effect of melanin binding on ocular pharmacokinetics.Lääkeaineet voivat kerääntyä pigmentoituneisiin kudoksiin sitoutumalla solujen sisältämään melaniinipigmenttiin. Melaniini vaikuttaa lääkeaineen farmakokinetiikkaan toimimalla varastona, josta lääkeaine annostelun päättymisen jälkeen vapautuu. Sitoutuminen voi myös aiheuttaa haittoja suuren paikallisen pitoisuuden vuoksi. Tämä tutkimus käsittelee silmän melaniinia. Kirjallisuuskatsauksessa tutustutaan tavallisimpiin melaniinisitoutumisen tutkimusmenetelmiin ja painotetaan in vitro sitoutumiskokeita ja tulosten sopivuutta farmakokineettiseen mallinnukseen. Kokeellisen tutkimuksen tavoitteena oli tutkia muutaman yhdisteen sitoutumista sian silmän verkkokalvon pigmenttiepiteelistä (RPE) ja suonikalvosta eristettyyn melaniiniin sekä lääkeaineiden kinetiikkaa sian RPE:n primaarisoluilla ja yhdistää tulokset STELLA® -ohjelmalla rakennettuun kineettiseen malliin melaniinisitoutumisesta. Tutkittavaksi valittiin yhdisteitä, joilla on erilaiset fysikokemialliset ominaisuudet (oktanoli/vesi -jakautumiskerroin (logP), pKa, happo/emäs). Osa oli myös efluksitransporttereiden substraatteja. Kaikkien yhdisteiden melaniinisitoutuminen tutkittiin pH:ssa 7,4 ja happamilla molekyyleillä lisäksi pH:ssa 5, sillä melanosomit eli melaniinia sisältävät soluelimet ovat sisällöltään happamia. Solukokeissa mitattiin soluun menevää ainemäärää ja eliminaatiota soluista. Efluksitransporttereiden vaikutusta tutkittiin yleisellä inhibiittorilla (probenesidi). Kaikki emäksiset yhdisteet sitoutuivat melaniiniin. Happamat yhdisteet eivät sitoutuneet pH:ssa 7,4, mutta sitoutuivat pH:ssa 5. Klorokiini sitoutui odotetusti parhaiten. Solukokeissa se oli myös ainut yhdiste, jota meni merkittävästi solun sisään. Tämä johtui osittain melaniinisitoutumisesta. Efluksi-inhibiittori ei näyttänyt vaikuttavan tuloksiin. Sitoutumiskokeen tuloksia käytettiin rakennetussa farmakokineettisessä mallissa. Mallit rakennettiin sekä melaniinisitoutumisesta että solutason kinetiikasta. Solumalli oli erittäin yksinkertainen, mutta sitä voidaan hyödyntää rakennettaessa kattavampia malleja melaniinisitoutumisen vaikutuksesta lääkeaineen farmakokinetiikkaan silmässä. Tutkimus kokonaisuudessaan osoitti melaniinisitoutumisen tärkeyden lääkeaineiden annostelussa silmään

Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells

Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016–0.73 and in non-pigmented cells 0.017–1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, Fic, were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3–4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity.Peer reviewe

Pharmacokinetic Simulations of Intravitreal Biologicals : Aspects of Drug Delivery to the Posterior and Anterior Segments

Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Peer reviewe

Binding of Small Molecule Drugs to Porcine Vitreous Humor

Pharmacokinetics in the posterior eye segment has therapeutic implications due to the importance of retinal diseases in ophthalmology. In principle, drug binding to the components of the vitreous, such as proteins, collagen, or glycosaminoglycans, could prolong ocular drug retention and modify levels of pharmacologically active free drug in the posterior eye segment. Since drug binding in the vitreous has been investigated only sparsely, we studied vitreal drug binding of 35 clinical small molecule drugs. Isolated homogenized porcine vitreous and the drugs were placed in a two compartment dialysis system that was used to separate the bound and unbound drug. Free drug concentrations and binding percentages were quantitated using LC-MS/MS. Drug binding levels varied between 21 and 74% in the fresh vitreous and 0 and 64% in the frozen vitreous. The vitreal binding percentages did not correlate with those in plasma. Our data-based pharmacokinetic simulations suggest that vitreal binding of small molecule drugs has only a modest influence on the AUC of free drug or drug half-life in the vitreous. Therefore, it is likely that vitreal binding is not a major reason for interindividual variability in ocular drug responses or drug-drug interactions.Peer reviewe

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology

Microscale Thermophoresis as a Screening Tool to Predict Melanin Binding of Drugs

Interactions between drugs and melanin pigment may have major impacts on pharmacokinetics. Therefore, melanin binding can modify the efficacy and toxicity of medications in ophthalmic and other disease of pigmented tissues, such as melanoma. As melanin is present in many pigmented tissues in the human body, investigation of pigment binding is relevant in drug discovery and development. Conventionally, melanin binding assays have been performed using an equilibrium binding study followed by chemical analytics, such as LC/MS. This approach is laborious, relatively slow, and limited to facilities with high performance quantitation instrumentation. We present here a screening of melanin binding with label-free microscale thermophoresis (MST) that utilizes the natural autofluorescence of melanin. We determined equilibrium dissociation constants (Kd) of 11 model compounds with melanin nanoparticles. MST categorized the compounds into extreme (chloroquine, penicillin G), high (papaverine, levofloxacin, terazosin), intermediate (timolol, nadolol, quinidine, propranolol), and low melanin binders (atropine, methotrexate, diclofenac) and displayed good correlation with binding parameter values obtained with the conventional binding study and LC/MS analytics. Further, correlation was seen between predicted melanin binding in human retinal pigment epithelium and choroid (RPE-choroid) and Kd values obtained with MST. This method represents a useful and fast approach for classification of compounds regarding melanin binding. Thus, the method can be utilized in various fields, including drug discovery, pharmacokinetics, and toxicology

Pharmacokinetic aspects of retinal drug delivery

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory.Peer reviewe

Quantification of Drugs in Distinctly Separated Ocular Substructures of Albino and Pigmented Rats

The rat is a commonly used species in ocular drug research. Detailed methods of separating rat ocular tissues have not been described in literature. To understand the intraocular drug distribution, we developed a robust method for the separation of individual anterior and posterior substructures of pigmented Brown Norway (BN) and albino Wistar Han (WH) rat eyes, followed by quantification of drug concentration in these substructures. A short formalin incubation, which did not interfere with drug quantification, enabled the preservation of individual tissue sections while minimizing cross-tissue contamination, as demonstrated by histological analysis. Following oral administration, we applied the tissue separation method, in order to determine the ocular concentrations of dexamethasone and levofloxacin, as well as two in-house molecules BI 113823 and BI 1026706, compounds differing in their melanin binding. The inter-individual variability in tissue partitioning coefficients (Kp) was low, demonstrating the reproducibility of the separation method. Kp values of individual tissues varied up to 100-fold in WH and up to 46,000-fold in BN rats highlighting the importance of measuring concentration directly from the ocular tissue of interest. Additionally, clear differences were observed in the BN rat tissue partitioning compared to the WH rat. Overall, the developed method enables a reliable determination of small molecule drug concentrations in ocular tissues to support ocular drug research and development