Efficacy and safety of 4 months of sublingual immunotherapy with recombinant Mal d 1 and Bet v 1 in patients with birch pollen-related apple allergy.

BACKGROUND: Birch pollen-related apple allergy is among the most prevalent food allergies in adolescent/adult subjects and mainly results from sensitization to the major birch pollen allergen Bet v 1 and subsequent cross-reaction with the apple protein Mal d 1. However, specific immunotherapy with birch pollen has inconsistent effects on apple allergy. OBJECTIVE: We sought to compare the safety and efficacy of sublingual immunotherapy (SLIT) with 2 formulations containing either rMal d 1 or rBet v 1 on birch pollen-related apple allergy. METHODS: Sixty participants with birch pollen-related apple allergy were randomized to daily sublingual application of placebo (n = 20) or 25 µg of rMal d 1 (n = 20) or rBet v 1 (n = 20) for 16 weeks. Adverse events were regularly recorded. Sublingual challenges with standardized doses of rMal d 1, skin prick tests with recombinant allergens, and measurements of allergen-specific IgE and IgG4 antibodies were performed before and after treatment. RESULTS: Both formulations caused comparable, mainly local adverse events. No systemic reactions occurred. Compared with the placebo and rBet v 1-treated groups, SLIT with rMal d 1 reduced rMal d 1-induced oral symptoms (P = .001 and P = .038) accompanied by longitudinally reduced rMal d 1-specific cutaneous reactions (P = .022) and enhanced IgG4/IgE ratios (P = .012). SLIT with rBet v 1 neither improved the clinical reactivity to rMal d 1 nor enhanced rMal d 1-specific IgG4/IgE ratios. Participants receiving placebo showed no allergen-specific changes. CONCLUSION: Sublingual treatment with a recombinant food allergen was safe and clinically effective, as determined by using standardized challenges. We present a promising approach for the effective treatment of birch pollen-related apple allergy.

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

A pilot study to assess the efficacy of tariquidar to inhibit P-glycoprotein at the human blood-brain barrier with (R)-11C-verapamil and PET.

UNLABELLED: Tariquidar, a potent, nontoxic, third-generation P-glycoprotein (P-gp) inhibitor, is a possible reversal agent for central nervous system drug resistance. In animal studies, tariquidar has been shown to increase the delivery of P-gp substrates into the brain by severalfold. The aim of this study was to measure P-gp function at the human blood-brain barrier (BBB) after tariquidar administration using PET and the model P-gp substrate (R)-(11)C-verapamil. METHODS: Five healthy volunteers underwent paired (R)-(11)C-verapamil PET scans and arterial blood sampling before and at 2 h 50 min after intravenous administration of tariquidar (2 mg/kg of body weight). The inhibition of P-gp on CD56-positive peripheral lymphocytes of each volunteer was determined by means of the (123)Rh efflux assay. Tariquidar concentrations in venous plasma were quantified using liquid chromatography/mass spectrometry. RESULTS: Tariquidar administration resulted in significant increases (Wilcoxon test for paired samples) in the distribution volume (DV, +24% +/- 15%) and influx rate constant (K(1), +49% +/- 36%) of (R)-(11)C-verapamil across the BBB (DV, 0.65 +/- 0.13 and 0.80 +/- 0.07, P = 0.043; K(1), 0.034 +/- 0.009 and 0.049 +/- 0.009, P = 0.043, before and after tariquidar administration, respectively). A strong correlation was observed between the change in brain DV after administration of tariquidar and tariquidar exposure in plasma (r = 0.90, P = 0.037). The mean plasma concentration of tariquidar achieved during the second PET scan (490 +/- 166 ng/mL) corresponded to 100% inhibition of P-gp function in peripheral lymphocytes. CONCLUSION: Tariquidar significantly increased brain penetration of (R)-(11)C-verapamil-derived activity due to increased influx. As opposed to peripheral P-gp function, central P-gp inhibition appeared to be far from complete after the administered tariquidar dose.

Interactions between ABC-transport proteins and the secondary Fusarium metabolites enniatin and beauvericin.

Enniatins (ENN) and beauvericin (BEA) exert cytotoxic properties. Here, we observed that their impact on Ca(2+)-homeostasis can be reversed by exogenous ATP. Thus, we investigated whether membrane-located ATP-binding cassette (ABC) transporters influence ENNs- and BEA-induced cytotoxicity. In short-term exposure assays breast cancer resistance protein (ABCG2)-overexpression weakly but significantly reduced the cytotoxic activity of BEA but not ENNs. In contrast, multidrug resistance-associated protein-1 (ABCC1)- and P-glycoprotein (ABCB1)-overexpression was not protective under identical conditions. ABCG2-mediated resistance against BEA was reversible by ABCG2 modulators. In long-term exposure assays, ABCG2 and ABCB1 significantly protected against ENNs- and to a lesser extent BEA-induced cytotoxicity. Moreover, both fusariotoxins potently inhibited the ABCG2- and ABCB1-mediated efflux of specific fluorescent substrates, with BEA being more effective. Additionally, ATPase and photoaffinity-labelling assays proofed interaction of both substances with ABCG2 and ABCB1. Remarkably, 2 years selection of KB-3-1 cells against both fusariotoxins resulted only in two-fold ENNs but negligible BEA resistance. Interestingly, the selected sublines displayed upregulation of multidrug resistance proteins and crossresistance to other chemotherapeutics. Summarizing, ABCG2 and ABCB1 slightly but significantly protect human cells against ENNs- and BEA-induced cytotoxicity. However, both mycotoxins potently interact with ABCB1 and ABCG2 transport functions suggesting influences on bioavailability of xenobiotics and pharmaceuticals.

Multispecificity of drug transporters: probing inhibitor selectivity for the human drug efflux transporters ABCB1 and ABCG2.

Multidrug resistance transporters P-glycoprotein/ABCB1 and ABCG2 limit the effect of a large number of cytostatic and cytotoxic drugs by energy-dependent efflux. In experimental models, pump inhibitors reestablish sensitivity towards these drugs. Both transporters demonstrate remarkably broad and partly overlapping substrate specificity. Propafenone analogues are inhibitors of a large number of drug efflux pumps including P-glycoprotein and ABCG2 as well as the microbial pumps. Here the two human ABC transporters ABCB1 and ABCG2 have been studied with respect to interaction with this class of compounds. Data indicate that within the same chemical scaffold, selectivity indices span three orders of magnitude. Compounds with the highest selectivity indices contain a non-ionizable nitrogen atom. Qualitative and quantitative pharmacophore models indicate that hydrophobicity, the number of rotatable bonds, and the number of H-bond acceptors are key features for both activity and selectivity.

Catalytic cycle of ATP hydrolysis by P-glycoprotein: evidence for formation of the E.S reaction intermediate with ATP-gamma-S, a nonhydrolyzable analogue of ATP.

Structural and biochemical studies of ATP-binding cassette (ABC) transporters suggest that an ATP-driven dimerization of the nucleotide-binding domains (NBDs) is an important reaction intermediate of the transport cycle. Moreover, an asymmetric occlusion of ATP at one of the two ATP sites of P-glycoprotein (Pgp) may follow the formation of the symmetric dimer. It has also been postulated that ADP drives the dissociation of the dimer. In this study, we show that the E.S conformation of Pgp (previously demonstrated in the E556Q/E1201Q mutant Pgp) can be obtained with the wild-type protein by use of the nonhydrolyzable ATP analogue ATP-gamma-S. ATP-gamma-S is occluded into the Pgp NBDs at 34 degrees C but not at 4 degrees C, whereas ATP is not occluded at either temperature. Using purified Pgp incorporated into proteoliposomes and ATP-gamma-35S, we demonstrate that the occlusion of ATP-gamma-35S has an Eact of 60 kJ/mol and the stoichiometry of ATP-gamma-35S:Pgp is 1:1 (mol/mol). Additionally, in the conserved Walker B mutant (E556Q/E1201Q) of Pgp, we find occlusion of the nucleoside triphosphate but not the nucleoside diphosphate. Furthermore, Pgp in the occluded nucleotide conformation has reduced affinity for transport substrates. These data provide evidence for the ATP-driven dimerization and ADP-driven dissociation of the NBDs, and although two ATP molecules may initiate dimerization, only one is driven to an occluded pre-hydrolysis intermediate state. Thus, in a full-length ABC transporter like Pgp, it is unlikely that there is complete association and disassociation of NBDs and the occluded nucleotide conformation at one of the NBDs provides the power-stroke at the transport-substrate site.

Self-organizing maps for identification of new inhibitors of P-glycoprotein.

Self-organizing maps were trained to separate high- and low-active propafenone-type inhibitors of P-glycoprotein. The trained maps were subsequently used to identify highly active compounds in a virtual screen of the SPECS compound library.

Role of transmembrane domain/transmembrane domain interfaces of P-glycoprotein (ABCB1) in solute transport. Convergent information from photoaffinity labeling, site directed mutagenesis and in silico importance prediction.

Human P-glycoprotein (P-gp, ABCB1) plays an important role in the development of resistance to anticancer therapy. This ABC-transporter (ATP-binding cassette transporter) intercepts drugs at the level of the plasma membrane and effluxes them before they are able to reach their intracellular target structures. Inhibition of P-gp by low molecular weight compounds has been advocated as a concept for resensitization of cells to anticancer agents and several clinical studies in oncological patients have advanced to phase III. Even more importantly, P-glycoprotein also represents an antitarget. Its expression in cells lining the intestinal tract, the canalicular side of hepatocytes, renal tubuli and the blood brain barrier lead to interference with pharmacokinetics of compounds that are recognized as pump substrates. An early prediction of ADMET (Absorption-Distribution-Metabolism-Excretion-Toxicity) properties is important during drug development, since interference of a compound with P-gp might compromise its future development into a drug. Despite considerable efforts, the mechanism by which P-gp binds and transports its solutes remains unclear. Generation of homology models of the protein allowed integration of data obtained by photoaffinity labeling, in silico prediction of functional importance by evolutionary tracing and site directed mutagenesis. An integral view of data indicates that these three lines of evidence converge to indicate two pseudosymmetric P-gp drug binding pockets located at the two transmembrane domain interfaces.

Remodeling the regulation of iron metabolism during erythroid differentiation to ensure efficient heme biosynthesis.

Terminal erythropoiesis is accompanied by extreme demand for iron to ensure proper hemoglobinization. Thus, erythroblasts must modify the "standard" post-transcriptional feedback regulation, balancing expression of ferritin (Fer; iron storage) versus transferrin receptor (TfR1; iron uptake) via specific mRNA binding of iron regulatory proteins (IRPs). Although erythroid differentiation involves high levels of incoming iron, TfR1 mRNA stability must be sustained and Fer mRNA translation must not be activated because iron storage would counteract hemoglobinization. Furthermore, translation of the erythroid-specific form of aminolevulinic acid synthase (ALAS-E) mRNA, catalyzing the first step of heme biosynthesis and regulated similarly as Fer mRNA by IRPs, must be ensured. We addressed these questions using mass cultures of primary murine erythroid progenitors from fetal liver, either undergoing sustained proliferation or highly synchronous differentiation. We indeed observed strong inhibition of Fer mRNA translation and efficient ALAS-E mRNA translation in differentiating erythroblasts. Moreover, in contrast to self-renewing cells, TfR1 stability and IRP mRNA binding were no longer modulated by iron supply. These and additional data stemming from inhibition of heme synthesis with succinylacetone or from iron overload suggest that highly efficient utilization of iron in mitochondrial heme synthesis during normal erythropoiesis alters the regulation of iron metabolism via the IRE/IRP system.

Photoaffinity labeling of P-glycoprotein.

The aim of the present review is to summarize recent progress in identifying substrate binding domains of P-glycoprotein by photoaffinity labeling. Preferred substrate binding regions have been identified using a number of photoaffinity ligands, including anthracyclines, the quinazoline iodoarylazidoprazosine (IAAP), dihydropyridines, taxanes and propafenones. These studies allowed identification of protein regions, which are involved in ligand interaction.

P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach.

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.

A three-dimensional model for the substrate binding domain of the multidrug ATP binding cassette transporter LmrA.

Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the alpha-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.

Intramolecular distribution of hydrophobicity influences pharmacological activity of propafenone-type MDR modulators.

Lipophilicity is one of the major determining physicochemical descriptors for P-glycoprotein (P-gp) inhibitory activity. Recently, Pajeva and Wiese showed that in case of P-gp interaction, lipophilicity may be regarded as space-directed property. In the present study, a series of propafenone-type P-gp inhibitors with systematically varying hydrophobicity distribution within the molecules were synthesised and pharmacologically tested. QSAR studies on the basis of multiple linear regression analysis showed that with increasing lipophilicity of the substituents on the amine moiety, the statistical significance of the indicator variables, denoting the substitution pattern on the central aromatic ring system, also increases. This indicates that the distribution of hydrophobicity within the molecules influences the mode of interaction with P-gp.

A subset of highly effective propafenone-type multidrug resistance modulators lacks effects on cardiac action potential and mechanical twitch parameters of rat papillary muscles.

In this study, we tested a series of 12 previously identified, highly effective propafenone-type multidrug resistance (MDR) modulators for their possible undesirable effects on cardiac tissue. We used rat papillary muscle preparations and quantitatively determined the potency of these substances to block action potential (AP) upstroke velocity (Vmax) and to prolong APD50. Simultaneously, the effects on isometric twitch parameters were evaluated. Concentration-response curves were obtained for all parameters. Within a subset of the compounds, we found a significant rank correlation (r' = 0.87; p < 0.05) between potencies to block Vmax (kiVmax) and to inhibit daunomycin efflux in MDR cells (IC50). Surprisingly, the most lipophilic compounds with additional aromatic side chains completely lacked effects on AP and mechanical twitch parameters, although they are the most effective MDR modulators. Additional structural modifications such as fluoride substitution of the aromatic ring, introduction of arylpiperazine or piperidine side chains, as well as modifying the hydrogen bond acceptor strength of the carbonyl group did not reestablish cardiac side effects. In contrast, when these substances were truncated at the phenylpropiophenone moiety of the propafenone core structure, cardiac effects reoccurred. We conclude that aromatic substituents in the vicinity of the nitrogen atom prevent interaction with ion channels, likely due to steric hindrance, and are thus a prerequisite for eliminating unwanted cardiac effects.

In silico screening with benzofurane- and benzopyrane-type MDR-modulators.

Development of inhibitors of the drug efflux pump P-glycoprotein is a versatile approach to overcome multi drug resistance (MDR) in tumor therapy. In an approach to lower the conformational flexibility of the lead compound propafenone, we synthesized a set of dihydrobenzofuranes and benzopyranones. In the case of the 4 diastereomeric dihydrobenzofuranes, no significant differences in activity regarding the configuration on the side-chains at the dihydrofurane moiety (cis or trans) was observed. This may be due to the high flexibility of the side-chains, which still allow mutually overlap of pharmacophores. The benzopyranones showed a good correlation between lipophilicity and activity with gnerally lower logpotency/logP ratios. This decrease may be due to the rigidization of the molecules. In an in silico screening approach, a set of diverse propafenone-type compounds was used to establish a pharmacophore model, which was used to screen the world drug index. Among the hits retrieved there are several compounds, which were previously described as MDR-modulators. This demonstrates the validity of the model.

Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by Pgp is still incompletely understood. This study introduces a series of propafenone-related photoaffinity ligands, which combine high specificity and selectivity for Pgp with high labeling efficiency. Molecules are intrinsically photoactivatable in the arylcarbonyl group, which represents a pharmacophoric substructure for this group of ligand molecules. A detailed study of the structure-activity relationship for this type of photoligand is presented. In subsequent experiments, these ligands were used to characterize the drug-binding domain of propafenone-type analogs. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry shows that propafenone-type ligands preferentially label fragments assigned to putative transmembrane segments 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a highly charged region of 15 amino acids in the second cytoplasmic loop. This region corresponds to the so-called EAA-like motif, which has been proposed to play a role in the interaction between transmembrane domain and nucleotide binding domain of peroxisomal ATP-binding cassette transporters. In addition, a region in cytoplasmic loop 3 and between TM12 and the N terminus of the Walker A sequence of NBD2 are labeled by the ligands. Therefore, a number of confined protein regions contribute to the drug-binding domain of propafenone-type analogs.

Similarity based SAR (SIBAR) as tool for early ADME profiling.

Estimation of bioavailability and toxicity at the very beginning of the drug development process is one of the big challenges in drug discovery. Most of the processes involved in ADME are driven by rather unspecific interactions between drugs and biological macromolecules. Within the past decade, drug transport pumps such as P-glycoprotein (Pgp) have gained increasing interest in the early ADME profiling process. Due to the high structural diversity of ligands of Pgp, traditional QSAR methods were only successful within analogous series of compounds. We used an approach based on similarity calculations to predict Pgp-inhibitory activity of a series of propafenone analogues. This SIBAR approach is based on selection of a highly diverse reference compound set and calculation of similarity values to these reference compounds. The similarity values (denoted as SIBAR descriptors) are then used for PLS analysis. Our results show, that for a set of 131 propafenone type compounds, models with good predictivity were obtained both in cross validation procedures and with a 31-compound external test set. Thus, these new descriptors might be a versatile tool for generation of predictive ADME models.