Rabbit as an animal model for intravitreal pharmacokinetics : Clinical predictability and quality of the published data

Corrigendum: Experimental Eye Research 169 (2018) 60 doi: 10.1016/j.exer.2018.01.006 WOS: 000430158400008Intravitreal administration is the method of choice in drug delivery to the retina and/or choroid. Rabbit is the most commonly used animal species in intravitreal pharmacokinetics, but it has been criticized as being a poor model of human eye. The critique is based on some anatomical differences, properties of the vitreous humor, and observed differences in drug concentrations in the anterior chamber after intravitreal injections. We have systematically analyzed all published information on intravitreal pharmacokinetics in the rabbit and human eye. The analysis revealed major problems in the design of the pharmacokinetic studies. In this review we provide advice for study design. Overall, the pharmacokinetic parameters (clearance, volume of distribution, half-life) in the human and rabbit eye have good correlation and comparable absolute values. Therefore, reliable rabbit-to-man translation of intravitreal pharmacokinetics should be feasible. The relevant anatomical and physiological parameters in rabbit and man show only small differences. Furthermore, the claimed discrepancy between drug concentrations in the human and rabbit aqueous humor is not supported by the data analysis. Based on the available and properly conducted pharmacokinetic studies, the differences in the vitreous structure in rabbits and human patients do not lead to significant pharmacokinetic differences. This review is the first step towards inter-species translation of intravitreal pharmacokinetics. More information is still needed to dissect the roles of drug delivery systems, disease states, age and ocular manipulation on the intravitreal pharmacokinetics in rabbit and man. Anyway, the published data and the derived pharmacokinetic parameters indicate that the rabbit is a useful animal model in intravitreal pharmacokinetics. (C) 2015 The Authors. Published by Elsevier Ltd.Peer reviewe

Impact of molecular methods in the analysis of the invasiveness of Streptococcus pneumoniae

[eng] This doctoral thesis proves the necessity of applying molecular techniques in direct sample for a more accurate study of the Invasive Disease Potential (IDP) of Streptococcus pneumoniae and for its epidemiological surveillance. These methods allow a better detection and serotype identification of the pneumococcal population in both Invasive Pneumococcal Disease (IPD) and nasopharyngeal carriers. However, to better understand the behavior of the different serotypes a complete analysis of the clonal types of pneumococci (invasive and carriers) should be also performed. In the first two studies the IDP of pneumococcal serotypes found in our pediatric population was estimated. In the first study, traditional methods based only in culture were used for both the detection of Streptococcus pneumoniae and the identification of the capsular type; while in the second study, the same estimation was performed but this time adding molecular techniques in direct sample for both the detection and serotyping. When comparing the results of both studies, the addition of molecular techniques in direct sample doubled the number of pneumococci identified causing IPD, and in the nasopharynx of healthy children. The great increase in the pneumococcus detection revealed a different distribution of the serotypes circulating in the population of the area, when compared with the data that had been obtained only by culture, which was translated in a huge impact in the IDP of these serotypes. In the carrier’s population an important proportion in the increase of serotypes that were able to be identified in carriers was due to the detection of multiple colonies in one sample, with up to four different serotypes distinguished in the same sample. The analysis of the carriers’ population also revealed a rate of co-colonization of 26.4% in the children population of our area. The better estimation of the ranking order of the most common serotypes in IPD and in the nasopharynx also allowed a more accurate analysis of the effects of PCV7 in the study time period. The results from the third study provided additional information about the invasive disease potential of serotypes causing IPD in our area due to the clonal study of invasive strains. In addition, it provided information about the evolution of serotypes causing disease after the introduction of PCV13 in 2010, in both children and adults. Although the most common serotypes found in the study were still PCV13 serotypes (with a special concern for the increase of the ST156 serotype 14), probably due to the low vaccine coverage in our area, a significant decrease was observed. In contrast, a significant increase in the proportion of non-PCV13 serotypes was revealed. Worth mentioning, is the increase experienced by serotype 12F (in high association with ST989) and, when analyzed by age groups, the increase of serotype 24F in children less than 2 years old (the major clone being the multi drug resistant ST230). But most importantly was the data obtained in the serotypes analysis of their clonal composition. Despite the capsule is the most important factor in virulence, strains expressing the same serotype but belonging to different clonal types has been found to present different invasiveness. Certain clonal types may present some characteristics that are likely to be advantageous for invasiveness, like antibiotic resistance. The study of the genetic population of the serotypes offers a more complete knowledge of the pneumococcal invasiveness, explains temporal trends and differences in geographical areas, and provides useful information for the prediction of which serotypes could be replacing the ones included in the vaccines. For a better interpretation of the pneumococcal behavior, the analysis of the clonal types associated with a serotype should be performed along with the capsular identification.[cat] Aquesta tesis doctoral demostra la necessitat d’aplicar tècniques moleculars en mostra directa per a poder realitzar un estudi més acurat del potencial invasiu de Streptococcus pneumoniae, i per a la seva vigilància epidemiològica. Aquests mètodes permeten una millor detecció i un millor serotipatge de la població pneumocòccica, tant en la malaltia pneumocòccica invasiva (MPI) com en els portadors nasofaringis. Tot i això, per realment entendre millor el comportament dels diferents serotips també s’hauria de realitzar un anàlisi complet dels tipus clonals que presenten els pneumococs (tant en malaltia com en portadors). Als dos primers estudis es va calcular el potencial invasiu dels serotips pneumocòccics trobats en la població pediàtrica de Catalunya (Espanya). Al primer estudi es van utilitzar mètodes tradicionals basats en el cultiu tant per la detecció de Streptococcus pneumoniae com per la identificació del tipus capsular; mentre que en el segon estudi, es va realitzar el mateix anàlisi però en aquest cas afegint les tècniques moleculars en mostra directa tant per la detecció com pel serotipatge. Al comparar els resultats d’ambdós estudis, l’addició de les tècniques moleculars en mostra directa va duplicar el número de pneumococs identificats causant MPI i presents a la nasofaringe de nens sans. Aquesta millora en la detecció del pneumococ també es va traduir en un gran impacte en el càlcul del potencial invasiu d’aquest serotips. En la població de portadors la capacitat de detectar múltiples colònies en una mateixa mostra va tenir un paper rellevant en l’increment de serotips que es van poder identificar en el segon estudi. Els resultats del tercer estudi van aportar informació addicional sobre el potencial invasiu dels serotips causant MPI a la nostra àrea gràcies a l’estudi clonal de les soques invasives. A més, van aportar informació sobre l’evolució dels serotips causant malaltia després de la introducció de la PCV13 al 2010, tant en nens com en adults. L’anàlisi dels tipus clonals sent expressats pels serotips pneumocòccics podria explicar perquè alguns tipus capsulars que es troben normalment colonitzant asimptomàticament han experimentat un canvi en la seva invasivitat convertint-se en les principals causes de la MPI

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

Superior fruit total soluble solid content of red x pink F1 hybrids over nearly-isogenic parental lines

Comunicación realizada en el congresoPink-coloured tomatoes are reputed as good flavoured and are very popular in local markets. Pink tomatoes show a transparent, colourless fruit cuticle in contrast to the naringenin-chalcone flavonoid-rich, orange-yellow cuticle of normal, red tomatoes due to the y (colourless epidermis) recessive mutation in MYB12 gene located in chromosome 1. In order to investigate the basis of the alleged higher quality of pink vs. red tomatoes and, especially, our previous observations of good flavour in hybrids between red and pink tomato varieties, two red x pink F1 crosses between pairs of tomato near-isogenic lines were obtained, namely ‘Ailsa Craig’ wt/wt x ‘Ailsa Craig’ y/y and ‘Ponderosa Red’ wt/wt x ‘Ponderosa Red’ y/y, being the latter a pink tomato line in which y allele was introgressed from its closely related ‘Ponderosa Pink’ cultivar. The parental lines and the two F1 hybrids were grown to harvest in a greenhouse and total soluble solids (°Brix) and titratable acidity (TA) were measured in red ripe fruits. While no differences for °Brix were observed between the parents of each cross, significantly higher °Brix was measured in the two red x pink F1’s compared to their corresponding parental lines. No clear differences between parental lines and F1 crosses were shown for TA. The experiment was repeated but including also the pink x red, reciprocal F1 crosses. The overdominance for °Brix but not for TA was confirmed in the red x pink hybrids while the pink x red hybrids produced °Brix values similar to those of their parents.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye

On the surface of the eye, both the cornea and conjunctiva are restricting ocular absorption of topically applied drugs, but barrier contributions of these two membranes have not been systemically compared. Herein, we studied permeability of 32 small molecular drug compounds across an isolated porcine cornea and built a quantitative structure-property relationship (QSPR) model for the permeability. Corneal drug permeability (data obtained for 25 drug molecules) showed a 52-fold range in permeability (0.09-4.70x10(-6) cm/s) and the most important molecular descriptors in predicting the permeability were hydrogen bond donor, polar surface area and halogen ratio. Corneal permeability values were compared to their conjunctival drug permeability values. Ocular drug bioavailability and systemic absorption via conjunctiva were predicted for this drug set with pharmacokinetic calculations. Drug bioavailability in the aqueous humour was simulated to be <5% and trans-conjunctival systemic absorption was 34-79% of the dose. Loss of drug across the conjunctiva to the blood circulation restricts significantly ocular drug bioavailability and, therefore, ocular absorption does not increase proportionally with the increasing corneal drug permeability.Peer reviewe

Mechanisms of cellular retention of melanin bound drugs : Experiments and computational modeling

Melanin binding of drugs is known to increase drug concentrations and retention in pigmented eye tissues. Even though the correlation between melanin binding in vitro and exposure to pigmented eye in vivo has been shown, there is a discrepancy between rapid drug release from melanin particles in vitro and the long in vivo retention in the pigmented tissues. We investigated mechanisms and kinetics of pigment-related drug retention experimentally using isolated melanin particles from porcine retinal pigment epithelium and choroid, isolated porcine eye melanosomes, and re-pigmented ARPE-19 cells in a dynamic flow system. The experimental studies were supplemented with kinetic simulations. Affinity and capacity of levofloxacin, terazosin, papaverine, and timolol binding to melanin revealed Kd values of asymptotic to 50-150 mu M and B-max asymptotic to 40-112 nmol.mg(-1). The drugs were released from melanin in < 1 h (timolol) or in 6-12 h (other drugs). The drugs were released slower from the melanosomes than from melanin; the experimental differences ranged from 1.2-fold (papaverine) to 7.4-fold (timolol). Kinetic simulations supported the role of the melanosomal membrane in slowing down the release of melanin binders. In release studies from the pigmented ARPE-19 cells, drugs were released from the cellular melanin to the extra -cellular space in asymptotic to 1 day (timolol) and asymptotic to 11 days (levofloxacin), i.e., much slower than the release from melanin or melanosomes. Simulations of drug release from pigmented cells in the flow system matched the experimental data and enabled further sensitivity analyses. The simulations demonstrated a significant prolongation of drug retention in the cells as a function of decreasing drug permeability in the melanosomal membranes and increasing melanin content in the cells. Overall, we report the impact of cellular factors in prolonging drug retention and release from melanin-containing cells. These data and simulations will facilitate the design of melanin binding drugs with prolonged ocular actions.Peer reviewe

Role of retinal pigment epithelium permeability in drug transfer between posterior eye segment and systemic blood circulation

Retinal pigment epithelium (RPE) is a major part of blood-retinal barrier that affects drug elimination from the vitreous to the blood and drug distribution from blood circulation into the eye. Even though drug clearance from the vitreous has been well studied, the role of RPE in the process has not been quantified. The aim of this work was to study the role of RPE clearance (CLRpE) as part of drug elimination from the vitreous and ocular drug distribution from the systemic blood circulation. We determined the bidirectional permeability of eight small molecular weight drugs and bevacizumab antibody across isolated bovine RPE-choroid. Permeability of small molecules was 10(-6) -10(-5)cm/s showing 13-15 fold range of outward and inward permeation, while permeability of bevacizumab was lower by 2-3 orders of magnitude. Most small molecular weight drugs showed comparable outward (vitreous-to-choroid) and inward (choroid-to-vitreous) permeability across the RPEchoroid, except ciprofloxacin and ketorolac that had an over 6 and 14-fold higher outward than inward permeability, respectively, possibly indicating active transport, Six of seven tested small molecular weight drugs had outward CLRPE values that were comparable with their intravitreal clearance (CLIvr) values (0.84-2.6 fold difference). On the contrary, bevacizumab had an outward CLRPE that was only 3.5% of the CLIvt, proving that its main route of elimination (after intravitreal injection) is not RPE permeation. Experimental values were used in pharmacokinetic simulations to assess the role of the RPE in drug transfer from the systemic blood circulation to the vitreous (CLBv). We conclude that for small molecular weight drugs the RPE is an important route in drug transfer between the vitreal cavity and blood, whereas it effectively hinders the movement of bevacizumab from the vitreous to the systemic circulation.Peer reviewe

Evaluación del II Plan autonómico de investigación, desarrollo y transferencia de conocimientos de Aragón (II PAID)

Requisitos del sistema: válido con distintas versiones de los principales navegadores; Adobe Acrobat Reader.Forma de acceso: interne

Liposomal sunitinib for ocular drug delivery : A potential treatment for choroidal neovascularization

Choroidal neovascularization (CNV) is a prevalent vision-threatening vascular disorder in aging population. CNV is associated with several diseases in the posterior segment of the eye such as age-related macular degeneration (AMD). In this study we developed sunitinib-loaded liposomes to block the neovascularization signalling pathway through inhibition of tyrosine kinase of vascular endothelial growth factor receptors (VEGFRs). Liposomal sunitinib formulations were prepared by thin film hydration method and studied for their encapsulation efficiency (EE), loading capacity (LC) and drug release profile in buffer andvitreous. Our finding showed that the liposomes (mean size 104 nm) could effectively entrap sunitinib (EE approximate to 95%) at relatively high loading capacity (LC approximate to 5%) and release sunitinib over at least 3 days. Intravitreal sunitinib-loaded liposomes revealed inhibitory effect on established neovascularization in laser-induced CNV mouse model while the intravitreal injection of sunitinib solubilized with cyclodextrin was inefficient in management of neovascularization. Accordingly, liposomal sunitinib is a promising drug delivery system that should be further studied to inhibit the CNV related to AMD.Peer reviewe