Nonsteroidal selective glucocorticoid modulators: the effect of C-5 alkyl substitution on the transcriptional activation/repression profile of 2,5-dihydro-10-methoxy-2,2,4-trimethyl-1H-[1]benzopyrano[3,4-f]quinolines.

The preparation and characterization of a series of selective glucocorticoid receptor modulators are described. The preliminary structure-activity relationship of nonaromatic C-5 substitution on the tetracyclic quinoline core showed a preference for small lipophilic side chains. Proper substitution at this position maintained the transcriptional repression of proinflammatory transcription factors while diminishing the transcriptional activation activity of the ligand/glucocorticoid receptor complex. The optimal compounds described in this study were the allyl analogue 18 and cyclopentyl analogue 32. These candidates showed slightly less potent, highly efficacious E-selectin repression with significantly reduced levels of glucocorticoid response element activation in reporter gene assays vs prednisolone. Allyl analogue 18 was evaluated in vivo. An oral dose of 18 showed an ED(50) = 1.7 mg/kg as compared to 1.2 mg/kg for prednisolone in the Sephadex-induced pulmonary eosinophilia model and an ED(50) = 15 mg/kg vs 4 mg/kg for prednisolone in the carrageenan-induced paw edema model.

Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with prednisolone-equivalent functional activity.

A novel class of functional ligands for the human glucocorticoid receptor is described. Substituents in the C-10 position of the tetracyclic core are essential for glucocorticoid receptor (GR) selectivity versus other steroid receptors. The C-5 position is derivatized with meta-substituted aromatic groups, resulting in analogues with a high affinity for GR (K(i) = 2.4-9.3 nM) and functional activity comparable to prednisolone in reporter gene assays of glucocorticoid-mediated gene transcription. The biological activity of these novel quinolines was also prednisolone-equivalent in whole cell assays of glucocorticoid function, and compound 13 was similar to prednisolone (po ED(50) = 2.8 mpk for 13 vs ED(50) = 1.2 mpk for prednisolone) in a rodent model of asthma (sephadex-induced eosinophil influx).

Retention of immunosuppressant activity in an ascomycin analogue lacking a hydrogen-bonding interaction with FKBP12.

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Delta(23,24)-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyascomycin was found to be equipotent to the parent compound both in its immunosuppressive potency and in its interaction with the immunophilin, FKBP12. Conversely, Delta(23,24)-dehydroascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency. NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to understand this phenomenon at the molecular level. The NMR structures of the complexes formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex formation.

A macrolactam inhibitor of T helper type 1 and T helper type 2 cytokine biosynthesis for topical treatment of inflammatory skin diseases.

T lymphocytes play a critical part in inflammatory skin diseases but are targeted by available therapies that have only partial efficacy, significant side-effects, or both. Because psoriasis, atopic dermatitis, and allergic contact hypersensitivity are associated with T helper type 1 (Th1), T helper type 2 (Th2), or mixed Th1-Th2 cell subsets and cytokine types, respectively, there is a need for a better broad-based inhibitor. The macrolactam ascomycin analog, ABT-281, was found to inhibit potently T cell function across species and to inhibit expression of multiple cytokines in human peripheral blood leukocytes which have been found in human skin disease cells and tissues. These included immunoregulatory Th1 (interleukin-2 and interferon-gamma) and Th2 (interleukin-4 and interleukin-5) cytokines. ABT-281 was shown to have potent topical activity (ED50 = 0.6% in acetone/olive oil) in a stringent swine model of allergic contact hypersensitivity, but its potency was markedly reduced compared with ascomycin when administered systemically due to more rapid clearance. Topical application of 3% ABT-281 in acetone/olive oil over 25% of the body surface in swine resulted in undetectable blood levels. Compared with a wide potency range of topical corticosteroids in clinical formulations, 0.3% and 1% ABT-281 ointments profoundly inhibited dinitrochlorobenzene-induced contact hypersensitivity in the pig by 78% and 90%, respectively, whereas super-potent steroids such as clobetasol propionate only inhibited in the 50% range and mild to moderate potency steroids such as fluocinolone acetonide were inactive. The potent topical activity of ABT-281 in swine, its superior efficacy, its rapid systemic clearance following uptake into the bloodstream, and its ability to inhibit cytokine biosynthesis of both Th1 and Th2 cell subsets, suggests that it will have a broad therapeutic value in inflammatory skin diseases, including psoriasis, atopic dermatitis, and allergic contact dermatitis.

32-Ascomycinyloxyacetic acid derived immunosuppressants. Independence of immunophilin binding and immunosuppressive potency.

The potent immunosuppressant ascomycin (1b) was selectively alkylated at the C-32 carbinol, thus providing esters and amides of 32-ascomycinyloxyacetic acid (4, AOAA). These compounds present structural variation at the FKBP/calcineurin interface. While the native carboxylic acid 4 shows no activity in vitro, esters and simple amides of 4 exhibit potent immunosuppression in the human MLR assay. Moreover, amides show inhibitory activity in the rat popliteal lymph node hyperplasia assay. Surprisingly, FKBP binding was weakened by several orders of magnitude when secondary hydrophobic aryl amides of 4 were tested, while maintaining potent immunosuppressive efficacy in vitro.

Studies on an immunosuppressive macrolactam, ascomycin: synthesis of a C-33 hydroxyl derivative.

Ascomycin 2, a close analogue of the immunosuppressant FK506 1, was modified to incorporate a hydroxyl group at the C-33 position. This increased the aqueous solubility of ascomycin by a hundred-fold at pH 7.4 and by approximately 300-fold at pH 6.5. Ascomycin 3 also exhibited an excellent immunosuppressive activity in vitro, as tested in a human mixed lymphocyte proliferation (HuMLR) assay, and in vivo using a rat popliteal lymph node (rPLN) hyperplasia assay.

Discovery of ascomycin analogs with potent topical but weak systemic activity for treatment of inflammatory skin diseases.

Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

High-level synthesis of the 12-kDa human FK506-binding protein in Escherichia coli using translational coupling.

The gene encoding the 12-kDa cytosolic form of human FK506-binding protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational coupling to an upstream segment of kdsB under the control of this modified lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was shown to have the authentic N terminus. The level of active re-hFKBP-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.

Structure-activity profiles of macrolactam immunosuppressant FK-506 analogues.

The immunosuppressive agent FK-506 has received much attention due to its efficacy and potency in the areas of transplant rejection and autoimmune disease. Calcineurin, a Ca(2+)-calmodulin activated phosphatase, was recently implicated in the immunosuppressive mechanism of FK-506. In our ongoing search for superior immunosuppressive agents, we have synthesized several analogues of FK-506 and tested their mechanistic and immunosuppressive actions. It was found that C-18 hydroxyl analogues of ascomycin, an analogue of FK-506 also called FR900520, bound tightly to immunophilin FKBP-12, but do not show any immunosuppressive activity in vitro or in vivo despite good bioavailability. Further, they reverse the inhibition of calcineurin caused by FK-506/FKBP-12 complex.

Spoilage of hydrogel contact lenses by lipid deposits. Tear-film potassium depression, fat, protein, and alcohol consumption.

"Jelly-bump" deposits, which are composed mainly of lipids, on high water content, extended-wear hydrogel lenses, are a clinically significant problem of unknown etiology. The authors explored the effect of nutrition on the frequency of deposition of these jelly bumps. Forty-three patients were included in this masked case-control study from five contact lens practices in North America. Thirty patients were lipid-depositor cases and 13 were controls. Tear specimens were drawn and analyzed for potassium by atomic-absorption spectrophotometry. Detailed dietary histories were conducted and diet nutrient composition was computed. Patients with diminished tear-potassium concentrations have an increased risk of jelly-bump deposition. Elevated intake of protein, alcohol, and cholesterol also appeared to be correlated significantly with increased jelly-bump deposition. The authors found that diabetics and the use of diuretics, anticholinergics, sympathomimetics, were associated with tear-film potassium depletion and lipid deposit formation.

Cytolysis of actinomycin D-treated target cells by cell-free supernatants from human monocytes.

The lytic mechanism of human peripheral blood monocytes was studied by using as targets actinomycin D-treated WEHI-164, an NK-insensitive murine fibrosarcoma cell line. Monocytes, but not lymphocytes, lysed WEHI-164 target cells pre-treated with actinomycin D within 6 h in 51Cr-release assays. Because cytolysis could not be inhibited competitively by unlabeled WEHI/D target cells, contact-independent mechanisms of cytolysis were investigated. Cell-free supernatants collected from monocytes cultured for 4-6 h at 37 degrees C lysed target cells as effectively as effector cell preparations of monocytes. Supernatants from lymphocytes cultured in parallel were not cytolytic. Cytolytic activity was not detected in supernatants from preparations of monocytes that were held on ice. However, monocytes produced cytolytic activity whether they were isolated by adherence or remained unseparated in suspensions of mononuclear cells. The cytolytic activity in cell-free supernatants (CFS) from monocytes was unaffected by incubation with protease inhibitors. CFS activity was destroyed by heat. Storage of CFS at 37 degrees, 22 degrees, 4 degrees, or -20 degrees C for 24 h decreased cytolytic activity; however, loss of cytotoxicity was minimized by storage at 4 degrees C. The cytolytic substance detected in 4-h CFS from monocytes appeared to be a protein(s) based on the sensitivity of the cytolytic activity to proteases. Cytolytic activity of CFS eluted from Sephacryl 200 in a single peak with an apparent molecular weight between 25,000 and 45,000 daltons.

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein.

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.

Induction of selective biological responses to chemoattractants in a human monocyte-like cell line.

The availability of monocyte cell lines that can be induced to differentiate in a predictable fashion can provide important tools for the study of the biochemical mechanisms of specific cellular responses. The U937 human monocyte cell line was previously shown to differentiate into chemotactically responsive cells when incubated with supernatants of lectin-stimulated lymphocytes (conditioned medium). Considering the heterogeneous nature of stimulated lymphocyte supernatants, attempts were made to identify well-defined agents that could reproducibly induce U937 cell differentiation. Both dimethyl sulfoxide and dibutyryl cAMP induced expression of receptors for the N-formylated oligopeptide chemoattractants in U937 cells. Unstimulated U937 cells contained no detectable receptors. After cells were exposed to 1 mM dibutyryl cAMP, 1.3% dimethyl sulfoxide, or 5% conditioned medium for 72 h, the average number of oligopeptide chemoattractant receptors per U937 cell was 33,000, 4,000, and 3,400, respectively. Specific binding proteins for the chemoattractants were identified by covalent affinity labeling on the differentiated U937 cells as well as on normal human monocytes. Cells exposed to conditioned medium responded chemotactically, secreted lysosomal enzymes, and formed superoxide anion when incubated with the chemoattractant. Treatment of U937 cells with dibutyryl cAMP resulted in the most reproducible and rapid increase in the number of chemoattractant receptors as well as in chemotactic responsiveness. The receptors on dibutyryl cAMP-treated cells and on dimethyl sulfoxide-treated cells initiated chemotaxis and lysosomal enzyme secretion in response to chemoattractants, but not the formation of superoxide anion. These findings demonstrate that development of the chemotactic and respiratory burst functions during the differentiation of a monocyte-like cell line can occur independently.

Fc receptors of mouse cell lines. I. Distinct proteins mediate the IgG subclass-specific Fc binding activities of macrophages.

To determine the biochemical basis for the two distinct IgG subclass binding specificities on mouse macrophage-like cell lines, Fc-binding proteins were isolated from P388D1 and J774 cells by affinity chromatography using immobilized IgG. Similar IgG subclass-specific Fc gamma-binding proteins were identified on both cell lines. The Fc-binding proteins were characterized by their IgG subclass binding specificity, size, charge, and trypsin sensitivity. Using immobilized IgG1, a protein was isolated with an average m.w. of 65,000 and a pI range of pH 4.7 to 5.8. This protein could also be isolated, although in reduced amounts, with immobilized IgG2b. Two proteins with average m.w. of 70,000 and 60,000 and a pI range of 3.8 to 4.7 were isolated with immobilized IgG2a. The 60-kd protein appeared to be derived from the 70-kd protein. The IgG1/IgG2b Fc-binding proteins were resistant to trypsin, whereas the IgG2a Fc-binding proteins were sensitive to trypsin. In IgG-binding studies with the isolated proteins, the IgG2a Fc-binding proteins continued to exhibit restricted binding specificity for the IgG2a subclass; however, the IgG1/IgG2b Fc-binding proteins showed a broader specificity and would rebind to IgG1, IgG2a, and IgG2b. The IgG-binding properties and trypsin sensitivity of these Fc-binding proteins were similar to the properties of the IgG binding sites on the intact cells. These data indicate that the IgG subclass-specific binding sites on macrophage-like cell lines are due to the presence of distinct protein molecules on the cell surface.

Structural evidence for distinct IgG subclass-specific Fc receptors on mouse peritoneal macrophages.

Membrane proteins which selectively bind to the Fc portion of IgG were identified in the Nonidet P-40 extracts of radiolabeled thioglycollate- elicited mouse peritoneal macrophages. Affinity columns of various IgG preparations coupled to Sepharose 4B were used to absorb the Fc-binding proteins. Analysis of the acetic acid or sodium dodecyl sulfate (SDS) eluates from aggregated human IgG or antigen-complexed rabbit IgG columns revealed two Fc(gamma)/-specific proteins with apparent 67,000 and 52,000 mol wt. These proteins were not detected in acid or SDS eluates from F(ab')(2) columns or in eluates from IgG column, over which were passed lysates of Fc receptor-negative cells. With the use of affinity columns that contained aggregated mouse myeloma proteins of different IgG subclasses, we found that the 67,000-dahon protein selectively binds to IgG2a, whereas the 52,000-dalton protein binds to IgG1 and IgG2b. Neither protein was found in SDS eluates from IgG3 columns. Trypsin treatment of the macrophages before detergent lysis removed the 67,000-dalton protein, although it leaves intact the 52,000-dalton protein. These results provide structural confirmation for the existence of separate Fc receptors on mouse macrophages and indicate that the two Fc-binding proteins identified in this study represent all or part of the trypsin- sensitive Fc receptor which binds IgG2a and the trypsin-resistant Fc receptor which binds IgG2b and IgG1.

Isolation and partial characterization of the major outer membrane protein of Chromatium vinosum.

The 42,000 major outer membrane protein of Chromatium vinosum was purified by a combination on ion-exchange chromatography, gel filtration, and isoelectric focusing. Upon isoelectric focusing, the final material produced four major hands. Three of the four bands were isolated and analyzed for similarity or differences. Protease peptide maps and cyanogen bromide maps of the three isoelectric species were identical. When the isolated isoelectric species were refocused, each produced multiple isoelectric species, suggesting that the procedure used was generating the multiple charged species. Protease treatment of the isolated outer membrane produced a 31,000 fragment from the 42,000 protein. This fragment was isolated by preparative sodium sulfate-polyacrylamide gel electrophoresis. Although the amino acid compositions of the 42,000 protein and its 31,000 trypsin fragment were different, their polarity index was the same (45%). The amino-terminal sequences of the 42,000 protein and 31,000 trypsin fragment were identical, and it concluded that the amino-terminal was buried in the membrane.