Intravitreal treatment in patients with exudative age-related macular degeneration and visual acuity ≤ 0.05.

BACKGROUND: To investigate intravitreal treatment efficiencies in patients suffering from exudative ARMD with a BCVA ≤ 0.05. METHODS: Retrospective analysis: Analysis parameters were lesion type, BCVA at baseline and at follow-up, the intravitreal drug used, and its application frequency. Patients were divided into: 1) following injections of bevacizumab, triamcinolone, their combination, or ranibizumab regardless of their lesion subtype, 2) or by lesion subtype. Statistical tests were performed using Wilcoxon signed-rank tests, Kruskal-Wallis tests and multivariable logistic regressions. RESULTS: Seventy four eyes of 74 patients were analyzed. Follow-up was at 12.0 to 15.7 weeks. Median difference of BCVA (logMAR) between baseline and follow-up was 0.000 (-0.030, 0.175) in classic (p = 0.105), 0.000 (-1.15, 0.20) in occult (p = 0.005), -0.200 (-1.20, 0.60) in cases with subretinal fluid (p = 0.207), 0.000 (-0.60, 0.30) in pigment epithelial detachment (p = 0.813), and 0.050 (-0.40, 0.70) in Junius Kuhnt maculopathy (p = 0.344). BCVA increased ≥ 0.2 logMAR in 4 (24 %) classic, 9 (47 %) occult, 6 (33 %) pigment epithelial detachment, 6 (55 %) subretinal fluid, in 29 (39 %) eyes regardless of the lesion type, and reached a BCVA ≥ 0.05 in 7 (9 %) of those with a baseline BCVA <0.05. CONCLUSIONS: Results indicate that in patients with ARMD and a BCVA lower 0.05, intravitreal treatment may improve visual acuity, most probably in cases with occult lesions.

Three-dimensional quantitative structure-activity relationship analyses of substrates of the human proton-coupled amino acid transporter 1 (hPAT1).

The proton-coupled amino acid transporter hPAT1 has recently gained much interest due to its ability to transport small drugs thereby allowing their oral administration. A three-dimensional quantitative structure-activity relationship (3D QSAR) study has been performed on its natural and synthetic substrates employing comparative molecular similarity indices analysis (CoMSIA) to investigate the structural requirements for substrates and to derive a predictive model that may be used for the design of new prodrugs. The cross-validated CoMSIA models have been derived from a training set of 40 compounds and the predictive ability of the resulting models has been evaluated against a test set of 10 compounds. Despite the relatively narrow range of binding affinities (K(i) values) reliable statistical models with good predictive power have been obtained. The best CoMSIA model in terms of a proper balance of all statistical terms and the overall contribution of individual properties has been obtained by considering steric, hydrophobic, hydrogen bond donor and acceptor descriptors (q(cv)(2)=0.683, r(2)=0.958 and r(PRED)(2)=0.666). The 3D QSAR model provides insight in the interactions between substrates and hPAT1 on the molecular level and allows the prediction of affinity constants of new compounds. A pharmacophore model has been generated from the training set by means of the MOE (molecular operating environment) program. This model has been used as a query for virtual screening to retrieve potential new substrates from the small-molecule, 'lead-like' databases of MOE. The affinities of the compounds were predicted and 11 compounds were identified as possible high-affinity substrates. Two selected compounds strongly inhibited the hPAT mediated l-[(3)H]proline uptake into Caco-2 cells constitutively expressing the transport protein.

Recognition of 2-aminothiazole-4-acetic acid derivatives by the peptide transporters PEPT1 and PEPT2.

The H(+)/peptide cotransporters PEPT1 and PEPT2 have gained considerable interest in pharmaceutical sciences as routes for drug delivery. It is, therefore, of interest to develop uncommon artificial substrates for the two carriers. This study was initiated to investigate the binding affinity of 2-aminothiazole-4-acetic acid (ATAA) conjugates with amino acids to PEPT1 and PEPT2. The 2-aminothiazole-4-acetic acid derivatives have been synthesised and tested for their affinity to PEPT1 and PEPT2. The K(i) values were compared with in silico predicted values from CoMSIA models. C-terminal ATAA-Xaa conjugates proved to be low to medium inhibitors of the [(14)C]Gly-Sar uptake at both carrier systems whereas N-terminal Xaa-ATAA conjugates exhibited medium to high affinity. A promising candidate for further functionalisation is Val-ATAA which shows extraordinary high affinity to PEPT1.

Structural requirements for the substrates of the H+/peptide cotransporter PEPT2 determined by three-dimensional quantitative structure-activity relationship analysis.

The renal type H(+)/peptide cotransporter PEPT2 has a substantial influence on the in vivo disposition of dipeptides and tripeptides as well as peptide-like drugs within the body, particularly in kidney, lung, and the brain. The comparative molecular similarity indices analysis (CoMSIA) method was applied to identify those regions in the substrate structures that are responsible for recognition and for differences in affinity. We have developed a comprehensive 3D quantitative structure-activity relationship (3D-QSAR) model based on 83 compounds that is able to explain and predict the binding affinities of new PEPT2 substrates. This 3D-QSAR model possesses a high predictive power (q(2) = 0.755; r(2) = 0.893). An additional 3D-QSAR model based on the same compounds was generated and correlated with affinity data of the intestinal H(+)/peptide cotransporter PEPT1. By comparing the CoMSIA contour plots, differences in selectivity between the intestinal and the renal type peptide carrier become evident.

Secretory phospholipase A2 from Arabidopsis thaliana: insights into the three-dimensional structure and the amino acids involved in catalysis.

A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been shown to be a true secretory phospholipase A2 that requires Ca2+ ions in the millimolar range and belongs to group XIB. On the basis of the three-dimensional structures of secretory phospholipase A2 forms (sPLA2s) from bee venom and bovine pancreas, a homology model was generated. Analysis of this model and alignments of different plant sPLA2s showed that the common His-Asp dyad of animal sPLA2s does not exist in plant sPLA2s. In place of the aspartate residue of the dyad, the plant enzymes of group XIA contain a histidine residue, and the enzymes of group XIB contain a serine or an asparagine residue. Mutagenesis of amino acids supposed to be involved in catalysis has shown that His62, the calcium-coordinating Asp63, and the above-mentioned Ser79 residue are essential for activity.

Three-dimensional quantitative structure-activity relationship analyses of beta-lactam antibiotics and tripeptides as substrates of the mammalian H+/peptide cotransporter PEPT1.

The utilization of the membrane transport protein PEPT1 as a drug delivery system is a promising strategy to enhance the oral bioavailability of drugs. Since very little is known about the substrate binding site of PEPT1, computational methods are a meaningful tool to gain a more detailed insight into the structural requirements for substrates. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using the comparative molecular similarity indices analysis (CoMSIA) method were performed on a training set of 98 compounds. Affinity constants of beta-lactam antibiotics and tripeptides were determined at Caco-2 cells. A statistically reliable model of high predictive power was obtained (q(2) = 0.828, r(2) = 0.937). The results derived from CoMSIA were graphically interpreted using different field contribution maps. We identified those regions which are crucial for the interaction between peptidomimetics and PEPT1. The new 3D-QSAR model was used to design a new druglike compound mimicking a dipeptide. The predicted K(i) value was confirmed experimentally.

Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells.

The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H(+)/peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [(14)C]glycylsarcosine with K(i) values of 0.19 +/- 0.01 mm and 0.07 +/- 0.01 mm for PEPT1 and PEPT2, respectively. Saturation kinetic studies revealed that in both cell types alafosfalin affected only the affinity constant (K(t)) but not the maximal velocity (V(max)) of glycylsarcosine (Gly-Sar) uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters: apical uptake and transepithelial apical to basolateral flux of [(14)C]Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin (3 mm) by 73%. In SKPT cells, uptake of [(14)C]Gly-Sar but not flux was inhibited by 61%. We found no evidence for an inhibition of the basolateral to apical uptake or flux of [(14)C]Gly-Sar by alafosfalin. Alafosfalin (3 mm) did not affect the apical to basolateral [(14)C]mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in Snapwells trade mark, alafosfalin increased the short-circuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H(+)/peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H(+)-symport, explaining its oral availability. The results also demonstrate that dipeptides where the C-terminal carboxyl group is substituted by a phosphonic function represent high-affinity substrates for mammalian H(+)/peptide cotransporters.

Three-dimensional quantitative structure-activity relationship analyses of peptide substrates of the mammalian H+/peptide cotransporter PEPT1.

The utilization of the carrier protein PEPT1 for the absorption of peptidomimetic drug molecules is a promising strategy for oral drug administration and increasing bioavailability. In the absence of structural information on the binding mode of substrates to PEPT1, a computational study was conducted to explore the structural requirements for substrates and to derive a predictive model that may be used for the design of novel orally active drugs. A comparative molecular field analysis (CoMFA) and a comparative molecular similarity indices analysis (CoMSIA) were performed on a series of 79 dipeptide-type substrates for which affinity data had been collected in a single test system under the same conditions. These studies produced models with conventional r(2) and cross-validated coefficient (q(2)) values of 0.901 and 0.642 for CoMFA and 0.913 and 0.776 for CoMSIA. The models were validated by an external test set of 19 dipeptides and dipeptide derivatives. CoMSIA contour maps were used to identify the recognition elements that are relevant for the binding of PEPT1 substrates. The 3D QSAR models provide an insight in the interactions between substrates and PEPT1 on the molecular level and allow the prediction of affinity constants of new compounds.