Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.

The leukocyte integrin very late antigen-4 (alpha(4)beta(1), CD49d/CD29) is an adhesion receptor that plays an important role in allergic inflammation and contributes to antigen-induced late responses (LAR) and airway hyperresponsiveness (AHR). In this study, we show that single doses of a new small-molecule, tight-binding inhibitor of alpha(4), BIO-1211, whether given by aerosol or intravenously, either before or 1.5 h after antigen challenge blocks allergen- induced LAR and post-antigen-induced AHR in allergic sheep. Multiple treatments with doses of BIO-1211 that were ineffective when given singly, were protective. BIO-1211 also provided dose-dependent inhibition of the early airway response (EAR) to antigen. In conjunction with the functional protection against the antigen-induced LAR and AHR, sheep treated with BIO-1211 before challenge showed significantly reduced: (1) numbers of eosinophils in bronchoalveolar lavage (BAL), (2) BAL levels of the inflammatory marker tissue kallikrein, and (3) numbers of inflammatory cells (lymphocytes, eosinophils, metachromatic staining cells, and neutrophils) in bronchial biopsies obtained after challenge when compared with corresponding biopsies after vehicle treatment. More importantly, we show for the first time that an inhibitor of alpha(4) was able to reverse post-antigen-induced AHR, thereby decreasing the time of recovery from the normal period of > 9 d to 3 d. Our results show that effective inhibition of antigen-induced airway responses can be achieved with single doses of a potent small-molecule inhibitor of alpha(4) and that such agents may be used therapeutically, as well as prophylactically, to alleviate allergen- induced inflammatory events. These data provide further support and extend the evidence for the role of alpha(4) integrins in the pathophysiologic events that follow airway antigen challenge.

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A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.

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We have used chemical cross-linking to identify sequences in integrin alpha4beta1 that are involved in its interactions with ligands. A recently described leucine-aspartic acid-valine (LDV)-based small molecule inhibitor of alpha4beta1 (BIO-1494), that contained a single reactive amino group for targeting the cross-linking, was used for these studies. The specificity of the interaction was defined by (i) the ability to block the interaction with a competitive inhibitor lacking the reactive group, (ii) the absolute requirement of divalent cations for cross-linking, and (iii) the lack of cross-linking to the functionally related integrin alpha4beta7. With ANB-NOS as the cross-linker, only the beta1 chain was labeled with BIO-1494, while with the more flexible cross-linker DSS both the alpha4 and beta1 chains were modified. Similar results were obtained when cross-linking was performed on K562 cells expressing alpha4beta1 but not on K562 cells expressing alpha2beta1. The site of cross-linking on the beta1 chain was localized by CNBr peptide mapping within residues 130-146, a region that contains the putative metal binding site DXSXS and for which analogous data had been generated with RGD binding to integrin alphaIIbbeta3. The striking similarity between the data we generated for an LDV ligand and published data for the RGD family supports the notion of a common ligand binding pocket formed by both integrin chains. The cross-linking strategy developed here should serve as a useful tool for studying alpha4beta1 function.

We aimed to evaluate its safety, pharmacokinetics (PK) and pharmacodynamics (PD).Methods: Thirty-six subjects received single subcutaneous injection of ropeginterferon alfa-2b at doses ranging from 24 to 270 μg, and 12 subjects received pegylated IFN alfa-2a subcutaneously at 180 μg. Primary endpoints were safety/PK profiles of ropeginterferon alfa-2b, while secondary endpoints were to compare PK/PD parameters with pegylated IFN alfa-2a.Results: Adverse events in ropeginterferon alfa-2b and pegylated IFN alfa-2a groups were similar, and most of them were mild or moderate. Mean C max increased from 1.78 to 24.84 ng/mL along with the dose escalations in ropeginterferon alfa-2b groups and was 12.95 ng/mL for pegylated IFN alfa-2a. At 180 μg, ropeginterferon alfa-2b showed statistically significant C max geometric mean ratio (1.76; P = .0275).Mean T max ranged from 74.52 to 115.69 h for ropeginterferon alfa-2b groups, and was 84.25 h for pegylated IFN alfa-2a. Mean AUC 0-t increased from 372.3 to 6258 ng•h/mL with the dose escalations in the ropeginterferon alfa-2b groups, while for pegylated IFN alfa-2a it was found to be 2706 ng•h/mL in pegylated IFN alfa-2a.For neopterin and 2 0 ,5 0 -oligoadenylate synthase, mean E max , T max and AUC 0-t of ropeginterferon alfa-2b were similar to those of pegylated IFNα-2a at 180 μg. Conclusion:Ropeginterferon alfa-2b up to 270 μg was safe and well tolerated. The PK/PD parameters of ropeginterferon alfa-2b showed increase in dose-response. Ropeginterferon alfa-2b had higher drug exposures and showed similar safety profile when compared to pegylated IFN alfa-2a at the same dose level.