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 This review is based on the literature and unpublished data from the authors' laboratory.Peer reviewe

Regenerative potential of corneal endothelium from patients with fuchs endothelial corneal dystrophy

La dystrophie cornéenne endothéliale de Fuchs (FECD, pour l’abréviation du terme anglais « Fuchs endothelial corneal dystrophy ») est une maladie de l'endothélium cornéen. Sa pathogenèse est mal connue. Aucun traitement médical n’est efficace. Le seul traitement existant est chirurgical et consiste dans le remplacement de l’endothélium pathologique par un endothélium sain provenant de cornées de la Banque des yeux. Le traitement chirurgical, en revanche, comporte 10% de rejet immunologique. Des modèles expérimentaux sont donc nécessaires afin de mieux comprendre cette maladie ainsi que pour le développement de traitements alternatifs. Le but général de cette thèse est de développer un modèle expérimental de la FECD en utilisant le génie tissulaire. Ceci a été réalisé en trois étapes. 1) Tout d'abord, l'endothélium cornéen a été reconstruit par génie tissulaire en utilisant des cellules endothéliales en culture, provenant de patients atteints de FECD. Ce modèle a ensuite été caractérisé in vitro. Brièvement, les cellules endothéliales cornéennes FECD ont été isolées à partir de membranes de Descemet prélevées lors de greffes de cornée. Les cellules au deuxième ou troisième passages ont ensuite été ensemencées sur une cornée humaine préalablement décellularisée. Suivant 2 semaines de culture, les endothélia cornéens reconstruits FECD (n = 6) ont été évalués à l'aide d'histologie, de microscopie électronique à transmission et d’immunomarquages de différentes protéines. Les endothélia cornéens reconstruits FECD ont formé une monocouche de cellules polygonales bien adhérées à la membrane de Descemet. Les immunomarquages ont démontré la présence des protéines importantes pour la fonctionnalité de l’endothélium cornéen telles que Na+-K+/ATPase α1 et Na+/HCO3-, ainsi qu’une expression faible et uniforme de la protéine clusterine. 2) Deux techniques chirurgicales (DSAEK ; pour « Descemet stripping automated endothelial keratoplasty » et la kératoplastie pénétrante) ont été comparées pour la transplantation cornéenne dans le modèle animal félin. Les paramètres comparés incluaient les défis chirurgicaux et les résultats cliniques. La technique « DSAEK » a été difficile à effectuer dans le modèle félin. Une formation rapide de fibrine a été observée dans tous les cas DSAEK (n = 5). 3) Finalement, la fonctionnalité in vivo des endothélia cornéens reconstruits FECD a été évaluée (n = 7). Les évaluations in vivo comprenaient la transparence, la pachymétrie et la tomographie par cohérence optique. Les évaluations post-mortem incluaient la morphométrie des cellules endothéliales, la microscopie électronique à transmission et des immunomarquage de protéines liées à la fonctionnalité. Après la transplantation, la pachymétrie a progressivement diminué et la transparence a progressivement augmenté. Sept jours après la transplantation, 6 des 7 greffes étaient claires. La microscopie électronique à transmission a montré la présence de matériel fibrillaire sous-endothélial dans toutes les greffes d’endothelia reconstruits FECD. Les endothélia reconstruits exprimaient aussi des protéines Na+-K+/ATPase et Na+/HCO3-. En résumé, cette thèse démontre que les cellules endothéliales de la cornée à un stade avancé FECD peuvent être utilisées pour reconstruire un endothélium cornéen par génie tissulaire. La kératoplastie pénétrante a été démontrée comme étant la procédure la plus appropriée pour transplanter ces tissus reconstruits dans l’œil du modèle animal félin. La restauration de l'épaisseur cornéenne et de la transparence démontrent que les greffons reconstruits FECD sont fonctionnels in vivo. Ces nouveaux modèles FECD démontrent une réhabilitation des cellules FECD, permettant d’utiliser le génie tissulaire pour reconstruire des endothelia fonctionnels à partir de cellules dystrophiques. Les applications potentielles sont nombreuses, y compris des études physiopathologiques et pharmacologiques.Fuchs endothelial corneal dystrophy (FECD) is a primary disease of the corneal endothelium. Its pathogenesis is poorly understood. No medical treatment is effective. Surgical treatment (the only available treatment) carries 10% of immunogenic rejection. Experimental models are needed in order to better understand the disease and to investigate potential autologous treatments (to prevent immunogenic rejection). The overall goal of this thesis is to develop an experimental model for FECD using tissue engineering. This was achieved in three steps. 1) An in vitro tissue-engineered FECD model was created and characterized. Briefly, Descemet’s membranes from patients with late-stage FECD undergoing Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on a previously decellularized human cornea. After 2 weeks in culture, TE-FECD corneas (n=6) were assessed using histology, transmission electron microscopy (TEM) and immunofluorescence labeling of various proteins. TE-FECD endothelium yielded a monolayer of polygonal cells well adhered to Descemet’s membrane. The TE-FECD corneal endothelium expressed the function-related proteins Na+-K+/ATPase α1 and Na+/HCO3-. Clusterin expression was faint and uniform. 2) In order to determine the best surgical procedure to transplant the TE-FECD corneas in the feline model, a DSAEK procedure was evaluated and compared to penetrating keratoplasty technique. DSAEK assessments included surgical challenges and clinical outcomes. DSAEK technique was challenging to perform in the feline model. Rapid fibrin formation was observed in all DSAEK cases (n=5). 3) The in vivo functionality of the TE-FECD corneas was assessed. TE-FECD corneas were grafted in the feline model (n=7) using penetrating keratoplasty procedure and observed for seven days. In vivo assessments included transparency, pachymetry, optical coherence tomography, endothelial cell morphometry, TEM and immunostaining of function-related proteins. After transplantation, pachymetry gradually decreased and transparency gradually increased. Seven days after transplantation, 6 out of 7 grafts were clear. Post-mortem TEM showed subendothelial loose fibrillar material deposition in all TE-FECD grafts. The TE grafted endothelium expressed Na+-K+/ATPase and Na+/HCO3-. This thesis demonstrates that endothelial cells from late-stage FECD corneas can be used to engineer a corneal endothelium. Compared to DSEAK, penetrating keratoplasty is a more appropriate procedure for corneal transplantation in the feline model, since the DSAEK procedure in the feline model presently yields inconsistent clinical results. Restoration of corneal thickness and transparency demonstrates that the TE-FECD grafts are functional in vivo. This novel FECD living model suggests a potential role of tissue engineering for FECD cell rehabilitation. Potential applications are numerous, including pathophysiological and pharmacological studies

Cutting-Edge Topics in Dry Eye Disease

This text presents cutting-edge topics on and the diagnostic and therapeutic approaches for tear film abnormities, including dry eye disease and Meibomian gland dysfunction. A new management algorithm for Meibomian gland dysfunction, therapeutic eye drops and intense pulsed lasers for dry eye disease, and topical and systemic agents for neuropathic ocular pain are proposed in this issue

Ocular and systemic pharmacokinetic models for drug discovery and development

Drug discovery and development is a long process: it takes usually 12 to 15 years before a drug candidate reaches the market. The pharmacokinetics of the drug is an important aspect of drug discovery and development, because the drug must reach its target site and exert the therapeutic response. The pharmacokinetic parameters of new compounds should be investigated early in drug discovery. Pharmacokinetic predictions can be made with Quantitative Structure-Property Relationships (QSPR) which are computational models that correlate chemical features with pharmacokinetic properties. The correlations are based on in vivo or in vitro pharmacokinetic data and molecular descriptors. QSPR models can be used to predict the pharmacokinetic parameters even before any actual drug synthesis and can be exploited to guide drug discovery. Pharmacokinetic models can also simulate concentration profiles of drugs during the drug discovery and development process. It was decided to develop QSPR models of pharmacokinetic parameters of drugs to be delivered by the systemic or ocular routes. A combination of Principal Component Analysis and Partial Least Square multivariate statistical methods was used to obtain QSPR equations for volume of drug distribution and fraction of unbound drug in plasma. Parallel modelling of these parameters resulted in acceptable R2 (0.58 - 0.77) and Q2 values (0.55 - 0.58). These models are based on a large set of structurally unrelated compounds, they are open and they have a defined applicability domain. Charge and lipophilicity related descriptors were the relevant ones which influenced the volume of distribution and free fraction of drug in plasma. Pharmacokinetics is an important factor in the development of ocular medications, because the ocular drug targets are difficult to reach, particularly in the posterior tissues such as retina and choroid. Therefore, drugs need to be injected intravitreally in the treatment of retina and choroid diseases (e.g. in exudative age-related macular degeneration) and thus prediction of intravitreal pharmacokinetics would be especially advantageous in ocular drug discovery and development. The first comprehensive collection of intravitreal volume of distribution and clearance values of compounds was collated based on extensive rabbit eye data from the literature. Moreover, predictive QSPR models for intravitreal clearance and half-life were created which had R2 and Q2 values of 0.62 0.84 for clearance and 0.61 - 0.80 for half-life. LogD7.4 and hydrogen bonding capacity defined the intravitreal clearance and half-life of compounds with a molecular weight below 1500 Da. The intravitreal volumes of drug distribution lay within a narrow range (80% within 1.18 - 2.28 ml). The QSPR models for intravitreal clearance and the typical values for intravitreal volumes of distribution were implemented in pharmacokinetic simulation models; the simulated profiles based on the real and predicted pharmacokinetic parameter values were similar. Thus, a combination of QSPR and pharmacokinetic models can be used in drug discovery and development to aid in the design of drugs and drug delivery systems. A comprehensive comparison of intravitreal pharmacokinetic data between rabbit and human was carried out to clarify the translational value of the rabbit model. The analysis revealed that the rabbit can be considered as a clinically predictive animal model for intravitreal pharmacokinetics of small molecules (18 Da - 1500 Da) and macromolecules (7.1 kDa - 149 kDa). There was a correlation between the intravitreal clearance values in human patients and healthy rabbits; they showed similar, but not identical, absolute values. The intravitreal pharmacokinetics of small molecules is mainly governed by permeability-limited clearance across blood-ocular barriers and occurs via the posterior route, whereas large molecules are cleared mostly via the anterior route. Although the literature contains some claims about the significance of the viscosity of the vitreous, it seems that this is not a major factor in drug elimination from the eye. In conclusion, new in silico tools were generated for systemic and ocular pharmacokinetics and drug delivery. These models can be exploited in industrial drug discovery and will hopefully speed up the development of new medications.Silmätaudeissa lääkehoitoa vaikeuttaa se, että lääkehoitoa on vaikea saattaa perille silmänpohjaan verkkokalvon soluihin, joissa näkövammaisuuteen ja sokeutumiseen johtavat muutokset tavallisesti tapahtuvat. Näin ollen lääkkeitä joudutaan antamaan silmän sisään toistuvina injektioina esimerkiksi verkkokalvon ikärappeuman hoidossa. Lääkkeiden kulkeutumisen ymmärtäminen ja ennustaminen tietokoneella auttaa pitkävaikutteisten injektioiden ja vaihtoehtoisten lääkkeen antotapojen kehittämistä. Väitöskirjassa kehitettiin tällaisia tietokonemalleja pohjautuen julkaistuihin tutkimuksiin

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

An assessment of the differentiation potential of epithelial stem cells in adult endometrium

Background: We are at the beginning of a new era where regenerative medicine is really starting to take hold. An appreciation of adult stem cells and their role in tissue maintenance have heralded novel insight into endometrial remodelling. The idea that human endometrium could be a plentiful source of adult stem/progenitor cells (ASPCs) has captured the imagination of many stem cell biologists. Not only that, they are also hypothesised to be involved in endometrial proliferative diseases, such as endometriosis. However, work remains preliminary and is stunted by a lack of specific endometrial stem cell markers. Recently, stage specific embryonic antigen -1 (SSEA-1) was proposed as a candidate epithelial ASPC marker in adult endometrium. In order to validate this, stem cell assays are required. These assays include measurements of clonogenicity, prolonged self-renewal and differentiation potential. Aim: To assess the differentiation potential of SSEA-1+ cells in vitro. Methods: All endometrial culture samples were collected from pre-menopausal women undergoing surgery for benign disease at Liverpool Women’s Hospital (LWH). Epithelial cells were cultured in 3D MatrigelTM to produce gland-like structures. Gland-like structures were characterised via immuno-histochemistry, to validate their resemblance to glands in endometrial tissue in vivo. To assess multi-potency, magnetic cell sorted (MACS) SSEA-1 enriched and depleted cell fractions were cultured in adipogenic and osteogenic inducing media. In order to assess pluripotency, SSEA-1 enriched and depleted fractions were cultured in neurogenic media. Results: SSEA-1 enriched cell fractions had a greater propensity to produce gland-like structures in 3D culture than SSEA-1 depleted cell fractions. Gland-like structures best resembled glands in post-menopausal (PM) endometrium. Non-gland like structures also formed in 3D culture from endometriosis samples, which stained exclusively and intensely for CK5/6. SSEA-1 enriched and depleted fractions were unable to differentiate into other cell types of mesodermal lineage. However, both enriched and depleted fractions were able to produce ectodermal derived neural-like PGP9.5 positive cells. Discussion: This work confirms SSEA-1 is a reliable epithelial ASPC marker in adult endometrium. SSEA-1+ cells possess an increased ability to produce gland-like structures, reminiscent of in vivo endometrial glands. This therefore confirms unipotency. The presence of CK5/6 in non-gland-like structures from endometriosis samples suggests a potential role in endometriosis aetiology, and warrants further work. SSEA-1 enriched and depleted cells were unable to undergo multi-lineage differentiation, but were capable of producing neural-like cells. As both cell populations were unable to demonstrate multipotency, it unlikely SSEA-1 enriched/depleted cells are pluripotent. More likely, a neural progenitor may exist in adult endometrium, one that has been included in both cell populations. Given the close proximity of endometrial glands to the placenta, the commitment of epithelial stem cells could be an ancillary mechanism to prevent maternal microchimersim in offspring, which is associated with a number of pathologies

The genetic predictors of severe outcome in patients with anterior uveitis

Uveitis is a generic term for a wide variety of different types of intraocular inflammation with different clinical phenotypes and visual outcomes. The explanation for why some patients develop chronic anterior disease whereas others do not is unknown. It seems likely that host factors such as the cytokine milieu of the aqueous humor may be an important factor in determining outcome. In turn, their secretion is genetically determined and cytokine gene polymorphisms have been associated with high or low level production whatever the stimulus. Purpose: The aim of this study was to identify key cytokine and chemokine polymorphisms associated with disease susceptibility, clinical phenotype, and development of visually significant complications in patients with anterior uveitis. Methods: PCR amplification was used to genotype a number of biallelic SNPs in several cytokine genes. This genetic data was then compared between patients and healthy controls, and within the patient group itself for association with clinical disease outcomes. Results: Our results show that a significant difference in the frequency of TNF-857T allele in patients with idiopathic anterior uveitis. We found a significant association between TNF-308 allele G and patients with anterior uveitis who were HLA-B27 positive. Patients with HLA-B27 associated anterior uveitis who developed visually threatening complications were more likely to carry the TNFRSF1A-201T or TNFRSF1A-1135T alleles. In addition, the frequency of IL- 1ra allele T was found to be significantly associated with chronicity of the disease. The frequency of MCP-1 (-2076T) allele was found to be significantly higher in healthy individuals when compared to patients with acute idiopathic anterior uveitis. Conclusions: These results suggest that genetic variations in proinflammatory mediators may influence the susceptibility and severity of the inflammatory response in eyes of patients with anterior uveitis. This knowledge may be useful in identifying prognosis and responsiveness to anti-TNF blockade in patients with anterior uveitis