Cross-species comparisons of the pharmacokinetics of ibudilast.

To enable clinical development of ibudilast for new indications, its pharmacokinetics were characterized in mice, rats, rabbits, dogs, cynomolgus monkeys, and minipigs. Animal pharmacokinetics were compared with a separate study in healthy volunteers. Following oral dosing, the dose-normalized area under the curve (AUC) (DN-AUC(24h)) in humans is 896 ((ng*h ml(-1))/(mg kg(-1))), and in animals ranges from 0.3 to 87. The variability among species cannot be explained by intrinsic clearance, which in intravenous dosing experiments shows only moderate interspecies variation (13-41 l h(-1) m(-2)). A portal vein rat pharmacokinetics model suggested that differences in first-pass gut clearance may explain some of the interspecies variation in oral bioavailability. Ibudilast shows auto-induction of metabolism in some animals, but not in humans. Plasma protein binding in humans and some animals is greater than or equal to 95%. The primary metabolite 6,7-dihyrdodiol-ibudilast is measurable and has been quantitated in plasma from animals and humans. Finally, biodistribution studies show that ibudilast distributes rapidly, extensively, and reversibly to the central nervous system.

Ligand interactions with E-selectin. Identification of a new binding site for recognition of N-acyl aromatic glucosamine substituents of sialyl Lewis X.

Several N-acylglucosamine derivatives of sialyl Lewis X (1-3) were prepared using a combined chemical enzymatic approach and evaluated as an inhibitor of E-selectin-mediated cellular adhesion. Compounds with aromatic functionality, 1 and 2, were found to be 3-10 times more potent than the N-acetyl derivative (14) in an ELISA E-selectin cell adhesion assay. Conformational analysis with NMR indicated that the sialyl Lewis x domain of 1 retained the conformation of the N-acetyl derivative (14) despite the presence of the N-naphthamido group. The dramatic order of magnitude increase in potency of these monovalent structures can be utilized to design more potent selectin-based cell adhesion inhibitors.

Structure--activity relationships of sialyl Lewis x-containing oligosaccharides. 1. Effect of modifications of the fucose moiety.

Leukocyte adhesion to the vasculature is mediated by E-, P-, and L-selectins. The natural ligands for E- and P-selectins have not been fully characterized but have been shown to contain the tetrasaccharide sialyl Lewis x structure (SLe(x)). To determine the importance of the fucose moiety of SLe(x), various analogs of SLe(x) containing modifications thereof were prepared and tested as inhibitors of E-selectin-mediated cell adhesion. Cellular experiments indicate that replacement of the hydroxyl groups of fucose by hydrogen abrogated E-selectin binding. However, the arabinose analog of fucose (CH3 delta H) inhibited cell adhesion but was 5-fold less potent than native SLe(x). This data suggests that modifications of fucose on SLe(x) are generally deleterious toward E-selectin binding.

Thrombin receptor peptide-mediated leukocyte rolling in rat mesenteric venules: roles of P-selectin and sialyl Lewis X.

Neutrophil adhesion to monolayers of cultured endothelial cells is enhanced, via a P-selectin-mediated mechanism, by a 14-amino acid peptide fragment (TRP-14) of the thrombin receptor. The objective of this study was to determine whether TRP-14 promotes P-selectin-mediated sialyl Lewis X-dependent leukocyte rolling in postcapillary venules. Superfusion of the rat mesentery with TRP-14 for 30 min resulted in the recruitment of rolling leukocytes and a concomitant reduction in leukocyte rolling velocity. Analogues of TRP-14 were largely ineffective in promoting leukocyte-endothelial cell adhesion. Treatment with either a monoclonal antibody directed against rat P-selectin or soluble sialyl Lewis X oligosaccharide (the carbohydrate ligand to P-selectin found on leukocytes) significantly attenuated the TRP-14-induced recruitment of rolling leukocytes. However, no effect was observed with a nonbinding antibody or a control fucose-deficient oligosaccharide. These results indicate that TRP-14 elicits the recruitment of rolling leukocytes in postcapillary venules via a P-selectin-dependent mechanism. The results also support the view that sialyl Lewis X participates in P-selectin-mediated leukocyte-endothelial cell adhesion.

Failure to demonstrate long-lived MHC saturation both in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides.

Peptides that bind with high affinity to class II MHC molecules can inhibit T cell activation both in vitro and in vivo. Thus, they have been suggested as potential therapeutic agents for MHC-associated autoimmune diseases. We have constructed nonnatural peptides with high affinity for certain disease-associated MHC alleles. More specifically, a particular peptide, designated as CY-760.50, was found to have a high binding affinity for DR1, slow dissociation kinetics after binding to MHC, and prolonged stability in human serum. However, when the ability of this peptide to block peptide presentation to an influenza hemagglutinin 307-319 peptide-specific, DR1-restricted T cell clone was examined, it was found that MHC blockade could only be achieved when high concentrations of peptide were present along with Ag in the fluid phase. Thus, pretreatment of APC with MHC class II blocker, followed by removal of unbound blocker, did not result in saturation of MHC molecules, because practically immediate reacquisition of Ag-presenting capacity was observed after removal of fluid phase blocker. The pharmacokinetic behavior and the duration of blocking activity of CY-760.50 were also examined in vivo, taking advantage of the fact that the mouse MHC class II molecule I-Ab also bound CY-760.50 with high affinity. CY-760.50 administered i.v. to C57BL/6 mice was rapidly cleared from the circulation and virtually undetectable in the serum 10 min after injection. This fast clearance rate was paralleled by a similarly short duration of the MHC blockade effect. These in vivo results have implications concerning the biology of peptide-MHC interactions, and suggest that MHC blockade may not be feasible as a therapeutic approach unless effective concentrations of inhibitor can be maintained over extended periods of time in the extracellular fluids.

Sialyl Lewisx-containing oligosaccharide attenuates myocardial reperfusion injury in cats.

Neutrophil (PMN) adhesion to the vascular endothelium is an important mechanism of myocardial reperfusion injury. The adhesion process is initially mediated by selectins (e.g., P- and L-selectin), and monoclonal antibodies directed against these adhesion molecules exert cardioprotective activity in ischemia/reperfusion models. The counterreceptors for these selectins are thought to be carbohydrate-containing moieties. In this connection, we studied the effect of a soluble sialyl Lewisx-containing oligosaccharide (SLex-OS) on PMN-endothelial interactions in a feline model of myocardial ischemia/reperfusion (MI/R). SLex-OS (10 mg/kg), administered 10 min before R, significantly reduced myocardial necrosis compared with its vehicle 270 min after reperfusion (6 +/- 1% vs. 35 +/- 4% of area at risk, P < 0.01). The cardioprotection was confirmed by significantly lower plasma creatine kinase activities in SLex-OS vs. vehicle-treated cats (P < 0.01). Cardiac contractility (dP/dt max) of cats receiving SLex-OS was significantly preserved after 270 min of R (97 +/- 2% vs. 78 +/- 5% of initial, P < 0.01). Furthermore, endothelium-dependent relaxation to acetylcholine in coronary artery rings isolated from MI/R cats treated with SLex-OS was significantly preserved (73 +/- 7% vs. 22 +/- 6% vasorelaxation, P < 0.01). In vitro PMN adherence to coronary vascular endothelium after 270 min of R was significantly attenuated in the SLex-OS-treated group compared with the vehicle group (14 +/- 5 vs. 91 +/- 12 PMN/mm2, P < 0.01). Our results indicate that a SLex-OS is cardioprotective and preserves coronary endothelial function after MI/R, indicating an important role of sialyl Lewisx in PMN accumulation, endothelial dysfunction, and myocardial injury in myocardial ischemia/reperfusion.

Expression and functional significance of alternatively spliced CS1 fibronectin in rheumatoid arthritis microvasculature.

Expression of fibronectin (FN) isoforms containing CS1, a 25-amino acid sequence present within the alternatively spliced IIICS region of FN, has been analyzed in rheumatoid arthritis (RA) synovium. Unexpectedly, CS1-containing FN variants were exclusively found on endothelium but not extracellular matrix (ECM) of RA synovium. Lumenal expression of CS1 on RA endothelial cells, as observed by electron microscopy, correlated with inflammation in RA, since normal synovium expressed little CS1 without appreciable decrease in ECM FN. CS1 expression on human endothelial cells was further shown by FN mRNA analyses. In adhesion assays on frozen RA synovial sections, T lymphoblastoid cells expressing functionally activated alpha 4 beta 1 integrin specifically attached to the intravascular surface of RA endothelium. Binding was abrogated by both anti-alpha 4 integrin and CS1 peptides. Our observations suggest direct involvement of CS1-containing FN in recruitment of alpha 4 beta 1-expressing mononuclear leukocytes in synovitis, and provide basis for therapeutic intervention in RA.

HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions.

In the present study, we describe the definition of a DRB1*0401 (DR4w4)-specific motif. The strategy used entailed a three-step process: 1) screening a large set of unrelated peptide ligands to identify good MHC binders; 2) truncation analysis of several DR4w4 binding peptides of high affinity to identify the crucial core-binding regions; 3) the use of single amino acid substitutions of the DR4w4-binding peptide hemagglutinin (HA) 307-319 to elucidate the specific residues crucial for binding. The DR4w4 motif is characterized by the presence of a hydrophobic or aromatic (F, W, Y, L, I, V, M) anchor residue in position 1, and a second hydroxyl (S, T) or aliphatic (L, I, V, or M) anchor residue in position 6. Furthermore, positive charges (R, K) are not allowed in positions 4, 7, and 9, and negative charges (D, E) are not allowed in position 9. This motif was present in 92% of good (IC50 < or = 100 nM) DR4w4-binding peptides, but less than 25% of the negative (IC50 > 45 microM) binders, indicating that the presence of the motif is necessary, but not sufficient for good DR4w4 binding capacity. The results of the present study are discussed in relation to previous work defining binding motifs and rules for other DR alleles, illustrating how different DR alleles bind variations on a similar structural theme. Finally, using two different peptide ligands [tetanus toxoid 830-843 and HA 307-319] as model systems, it is demonstrated how the fine allelic specificity of the DR binders can be predictably modulated by introducing subtle changes in the primary amino acid sequence.

Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum.

The determination of peptide stability in human serum (HS) or plasma constitutes a powerful screening assay for eliminating unstable peptides from further development. Herein we report on the stability in HS of several major histocompatibility complex (MHC)-binding peptides. Some of these peptides are in development for the novel treatment of selected autoimmune disorders such as rheumatoid arthritis and insulin-dependent diabetes. For most of the l-amino acid peptides studied, the predominant degradation mechanism is exopeptidase-catalyzed cleavage. Peptides that were protected by d-amino acids at both termini were found to be more stable than predicted, based on additivity of single substitutions. In addition, N-acetylglucosamine glycopeptides were significantly stabilized, even when the glycosylation site was several amino acids from the predominant site(s) of cleavage. This indicates that long-range stabilization is possible, and likely due to altered peptide conformation. Finally, the effect of single amino acid substitutions on peptide stability in HS was determined using a model set of poly-Ala peptides which were protected from exopeptidase cleavage, allowing the study of endopeptidase cleavage pathways.

Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration.

Leukocyte extravasation is mediated by multiple interactions of adhesive surface structures with ligands on endothelial cells and matrix components. The functional role of beta 1 (CD29) integrins (or very late antigen [VLA] proteins) in eosinophil migration across polycarbonate filters was examined under several in vitro conditions. Eosinophil migration induced by the chemoattractant C5a or platelet-activating factor was fully inhibited by monoclonal antibody (mAb) 8A2, a recently characterized "activating" CD29 mAb. However, inhibition by mAb 8A2 was observed only under filter conditions that best reflected the in vivo situation, i.e., when the eosinophils migrated over filters preincubated with the extracellular matrix (ECM) protein fibronectin (FN), or when the filters were covered with confluent monolayers of cultured human umbilical vein endothelial cells (HUVEC). When bare untreated filters were used, mAb 8A2 had no effect, whereas the C5a-directed movement was prevented by CD18 mAb. Studies with alpha-subunit (CD49)-specific mAbs indicated that the integrins VLA-4 and -5 mediated migration across FN-preincubated filters, and VLA-2, -4, -5, and -6 were involved in eosinophil migration through filters covered with HUVEC. In contrast with the activating CD29 mAb 8A2, a combination of blocking CD49 mAbs or the nonactivating but blocking CD29 mAb AIIB2 failed to inhibit completely eosinophil migration over FN-preincubated or HUVEC-covered filters. mAb 8A2 stimulated binding to FN but not to HUVEC. Moreover, eosinophil migration over FN-preincubated or HUVEC-covered filters was significantly inhibited by anti-connecting segment 1 (CS-1) mAbs, as well as the soluble CS-1 peptide (unlike migration across bare untreated filters). Thus, inhibition of eosinophil migration by mAb 8A2 depended upon the presence of ECM proteins and not upon the presence of HUVEC per se. In conclusion, "freezing" adhesion receptors of the beta 1 integrin family into their high-avidity binding state by the activating CD29 mAb 8A2 results in a complete inhibition of eosinophil migration under physiological conditions. Hence, activation of beta 1 integrin-mediated cell adhesion may represent a new approach to prevent influx of inflammatory cells.

MHC class II molecules bind indiscriminately self and non-self peptide homologs: effect on the immunogenicity of non-self peptides.

Synthetic peptides spanning the entire sequence of both human and mouse beta 2-microglobulin (beta 2M) have been tested for their capacity to bind to three different mouse (I-Ad, I-Ed, and I-Ak) or human (DR1, DR2, and DR5) class II molecules. The results demonstrate that class II molecules do not discriminate between self and non-self peptides. When the immunogenicity of the human beta 2M peptides was measured by their ability to prime H-2d mice for in vitro T cell proliferation, it was found that peptides incapable of binding class II molecules in vitro were also non-immunogenic in vivo. Interestingly, however, several binders, including the human beta 2M peptide 1-16, the best binder in this series to Iad molecules, were found to be non-immunogenic. Since the corresponding mouse beta 2M peptide 1-16 was also capable of binding to Iad molecules, this suggested that lack of responsiveness to the non-self peptide could arise either from central or peripheral tolerance induced by the self homolog. Alternatively, lack of responsiveness could arise from other mechanisms, such as negative selection by other non-homolog sequences or lack of suitable T cell receptor genes. To discriminate between these possibilities, H-2d mice with disrupted beta 2M genes were immunized with the human beta 2M peptide 1-16. This peptide also failed to prime for T cell responsiveness in beta 2M-negative mice, suggesting that a hole in the T cell repertoire for this antigen was not mediated by negative selection or peripheral tolerance induced by self beta 2M peptides.

Functional consequences of engagement of the T cell receptor by low affinity ligands.

The mechanisms involved in TCR antagonism by Ag analog/MHC have been analyzed. A detailed structure-activity relationship study indicated that modification of any of the major T cell contact residues of the peptide molecule can yield a powerful antagonist. It was also shown that as the analog structure increased in similarity to the Ag, the capacity to antagonize Ag-TCR interaction increased up to the point that the analogs themselves became antigenic. These data strongly suggest an affinity-related mechanism whereby a certain affinity is required for signaling through the TCR, and that below this level there can be sufficient affinity to engage the receptor such that triggering does not occur and antagonism can be detected. Taking advantage of this information, antagonist peptides active down to the 10 nM range were engineered. Thus, this approach demonstrates for the first time a rational approach to designing effective, selective low m.w. compounds with high potential in treatment of allergies and autoimmune diseases.

High affinity for class II molecules as a necessary but not sufficient characteristic of encephalitogenic determinants.

A direct binding assay specific for IAs molecules has been developed and its immunological relevance validated by examining, for a panel of nine different synthetic peptides, the correlation between their capacity to bind purified IAs and to inhibit IAs-restricted antigen presentation. The IAs assay thus developed has then been used to study the IAs binding affinity of a set of overlapping peptides spanning the entire myelin basic protein (MBP). It was found that the encephalitogenic MBP region corresponds to peptides with high MHC binding affinities. Other regions of the MBP that have not been described as being pathogenic in the context of IAs molecules have also been found to be high IAs binders, suggesting that variables other than MHC affinity are also involved in determining the pathogenic potential of self-derived determinants.

MHC interaction and T cell recognition of carbohydrates and glycopeptides.

The T cell independence of complex polysaccharide Ag has suggested the possibility that carbohydrates may be incapable of T cell recognition because of a failure to interact with MHC restriction elements and/or a failure of MHC/carbohydrate complexes to interact with and be recognized by Ag-specific TCR. We have used two approaches to obtain information about T cell recognition of carbohydrate. First, we have determined the capacity of a series of oligosaccharides and glycolipids to bind a murine class II MHC molecule, IAd. No significant binding was observed with the 26 compounds tested, but the limitation to these studies was that there was a relatively limited collection of synthetic carbohydrate and glycolipid structures of limited complexity available for analysis. The second approach involved the study of the effect of glycosylation of a known peptide T cell epitope (OVA 323-339) on MHC binding of the peptide and on T cell recognition. Three patterns of effects were observed: 1) no effect on either binding or T cell recognition. This pattern was observed when the carbohydrate was located at residues removed from the core MHC-binding region. When the carbohydrate was located within the core MHC-binding regions, either 2) glycosylation destroyed both MHC binding and T cell recognition; or 3) glycosylation did not ablate MHC binding or T cell recognition. In this latter instance, there was evidence to indicate that the carbohydrate moiety was an important part of the antigenic determinant recognized by T cells.

Effect of pH on MHC class II-peptide interactions.

The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II-peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II-peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides.

Antigen analog-major histocompatibility complexes act as antagonists of the T cell receptor.

A novel mechanism for inhibition of T cell responses is described. Using the recognition of the influenza hemagglutinin (HA) 307-319 peptide in the context of DR1 class II major histocompatibility complex molecules, we have found that nonstimulatory analogs of the HA peptide preferentially inhibit HA-specific T cells in inhibition of antigen presentation assays. This antigen-specific effect could be generalized to another DR1-restricted peptide, Tetanus toxoid 830-843. Direct binding and cellular experiments indicated that the mechanism responsible was distinct from competition for binding to DR1 molecules. Likewise, negative signaling and induction of T cell tolerance could also be excluded as effector mechanisms. Thus, the most likely mechanism for this effect is engagement of antigen-specific T cell receptors by DR1-peptide analog complexes, which results in antigen-specific competitive blocking of T cell responses by virtue of their capacity to compete with DR1-antigen complexes for binding to the T cell receptor.

On the interaction of promiscuous antigenic peptides with different DR alleles. Identification of common structural motifs.

We have investigated the interaction between DR1 molecules and the two antigenic peptides, tetanus toxoid 830-843 and hemagglutinin 307-319, previously known to bind most DR alleles (degenerate binding) and to be recognized by the same T cell clones in the context of different DR alleles (promiscuous T cell recognition). The DR1 affinity of these two peptides was compared with that of two other different T cell epitopes (pertussis toxin 30-42 and ragweed allergen Ra3 51-65). It was found that degeneracy and promiscuity were associated with high affinity interactions, whereas binding and T cell selectivity were associated with weaker interactions. Thus, the selectivity of DR-peptide interactions, as is commonly observed with the antibody molecule, appears to be inversely correlated to affinity. Several singly substituted analogs of the hemagglutinin 307-319 determinant have also been tested for capacity to bind various DR alleles (DR1, DR2, DR5, and DR7). The results obtained suggest that this determinant may bind the different DR alleles in a similar orientation. Similar conclusions were reached when the interaction between the tetanus toxoid 830-843 determinant and three different DR alleles (DR1, DR2, and DR7) was studied following the same experimental approach. When crucial DR-binding residues of the two peptides were compared, it was found that they were very similar in both chemical nature and spacing in the peptide primary structure, suggesting that the two peptides may bind DR in a very similar orientation. Finally, a putative motif has been derived and shown to be present in a majority of the DR binders tested, but only in a minority of the non-DR binding peptides.

Single amino acid changes in DR and antigen define residues critical for peptide-MHC binding and T cell recognition.

Single amino acid substitutions of Ag and MHC were used to analyze the fine structure of the influenza hemagglutinin (HA)-derived epitope (HA 307-319) recognized in the context of DR7 molecules by a T cell clone. Putative T cell (HA 308, 310, 311, 313, and 316) and DR (HA 309, 312, and 317) contact residues of the Ag were identified by the use of single amino acid-substituted analogs that were tested for their T cell-activating and DR-binding capacities. The peptide-DR7-T cell interaction was further characterized by the use of a panel of 13 site-directed DR7 mutant transfectants analyzed for their capacity to present Ag to T cells, and for their purified mutant DR7 molecules to bind HA 307-319 or its single amino acid-substituted analogs. Eight mutants lost their Ag-presenting function, whereas only one had any decrease in peptide binding. Finally, for three of the mutants it was possible to correct the deleterious effects of mutation by using a particular single amino acid-substituted analog of the peptide molecule. The observed pattern of complementation led to a model that predicts that the Ag assumes an extended conformation, with a turn, in the binding groove, such that the following residues are in close proximity: DR 86-HA 309, DR 71-HA 312, DR 30-HA 314, and 315.