The synthesis and biological evaluation of non-peptidic matrix metalloproteinase inhibitors.

Novel sulfonamide matrix metalloproteinase inhibitors of general formula (9) were synthesised by a route involving a stereoselective conjugate addition reaction. Enzyme selectivity was found to be dependant on the nature of the sulfonamide substituents. Compounds (9f, 9q) are potent selective collagenase inhibitors with good oral bioavailability.

Involvement of matrix metalloproteinases in human immunodeficiency virus type 1-induced replication by clinical Mycobacterium avium isolates.

The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%-95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.

Preclinical and clinical studies of MMP inhibitors in cancer.

The role of matrix metalloproteinases in tumor angiogenesis and growth is now well recognized for models of both human and animal cancer. Clinical studies currently under way with the prototype matrix metalloproteinase inhibitor, marimastat, will establish whether inhibitors of these enzymes are of benefit in the treatment of different types of human cancer. On chronic therapy in humans, marimastat induces a reversible tendinitis that can also be detected in certain animal species. This paper compares the ability of broad-spectrum and various types of selective matrix metalloproteinase inhibitors to induce tendinitis and to exhibit anticancer effects in an animal cancer model. Under conditions in which both systemic exposure and inhibitor potency are controlled, selective inhibitors are less pro-tendinitic, but are weaker anticancer agents than broad-spectrum agents such as marimastat. The clinical relevance of these findings is discussed.

The synthesis of novel matrix metalloproteinase inhibitors employing the Ireland-Claisen rearrangement.

Matrix metalloproteinase inhibitors of general formula (1) were synthesised by a route involving an Ireland-Claisen rearrangement which enables systematic modification of the substituent alpha to the hydroxamic acid. An analogue (12c) possessing an alpha-cyclopentyl group is a potent broad spectrum inhibitor that displays high and sustained blood levels following oral dosing in both the rat and marmoset ex-vivo bioassays. This compound and analogues are also potent inhibitors of TNF alpha release.

Role of metalloproteases in the release of the IL-1 type II decoy receptor.

The IL-1 type II receptor (decoy RII) is a nonsignaling molecule the only established function of which is to capture IL-1 and prevent it from interacting with signaling receptor. The decoy RII is released in a regulated way from the cell surface. Here, we reported that hydroxamic acid inhibitors of matrix metalloproteases inhibit different pathways of decoy RII release, including the following: (a) the slow (18 h) gene expression-dependent release from monocytes and polymorphonuclear cells exposed to dexamethasone; (b) rapid release (minutes) from myelomonocytic cells exposed to tumor necrosis factor, chemoattractants, or phorbol myristate acetate; (c) phorbol myristate acetate-induced release from decoy RII-transfected fibroblasts and B cells. Inhibition of release was associated with increased surface expression of decoy RII. Inhibitors of other protease classes did not substantially affect release. However, serine protease inhibitors increased the molecular mass of the decoy RII released from polymorphonuclear cells from 45 to 60 kDa. Thus, irrespective of the pathway responsible for release and of the cellular context, matrix metalloproteases, rather than differential splicing, play a key role in production of soluble decoy RII.

Fas ligand in human serum.

The Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis in Fas-bearing cells. The membrane-bound human FasL was found to be converted to a soluble form (sFasL) by the action of a matrix metalloproteinase-like enzyme. Two neutralizing monoclonal anti-human FasL antibodies were identified, and an enzyme-linked immunosorbent assay (ELISA) for sFasL in human sera was established. Sera from healthy persons did not contain a detectable level of sFasL, whereas those from patients with large granular lymphocytic (LGL) leukemia and natural killer (NK) cell lymphoma did. These malignant cells constitutively expressed FasL, whereas peripheral NK cells from healthy persons expressed FasL only on activation. These results suggested that the systemic tissue damage seen in most patients with LGL leukemia and NK-type lymphoma is due to sFasL produced by these malignant cells. Neutralizing anti-FasL antibodies or matrix metalloproteinase inhibitors may be of use in modulating such tissue damage.

BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.

Four different ways of philanthropic aid to the blind in medieval eastern Christendom.

The care of the blind, either as medical treatment or as divine therapy, has probably been the most ancient form of help for ill people. However, it was during the Byzantine Empire (325-1453 AD) that the state organized a 'blindness relief' plan as part of a widespread public health system. Our sources for the subject include medical writings, state decrees, Saint's 'vitae' and representations of relevant works of art. Based on the above data we classify the health care for the blind in Byzantium as: (a) support of ophthalmological education as evidenced by an abundance of medical writings on the subject; (b) establishment of charitable institutions exclusively or partially for the blind, where there was not only medical care but also provision for a wide range of social aid - the most advanced being specially trained escorts for each blind person; and (c) support by the state of an extended chain of religious institutions where miraculous help for the blind was promised. We conclude that the public health policy in Byzantium made adequate and very early provision for the blind.

Matrix metalloproteinases and processing of pro-TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is released from a cell membrane-anchored precursor by proteolytic cleavage. We have shown that broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs) prevent the processing of the TNF precursor but do not inhibit the release of other cytokines. Purified MMPs, stromelysin, matrilysin, collagenase, and the gelatinases can all cleave a recombinant pro-TNF substrate to yield mature TNF. MMP inhibitors prevent the rise in blood levels of TNF after endotoxin administration in rats and are effective in animal models of inflammatory disease such as adjuvant arthritis. Drugs that inhibit MMP action and TNF release show great promise for the treatment of autoimmune inflammatory diseases.

Processing of tumour necrosis factor-alpha precursor by metalloproteinases.

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions such as rheumatoid arthritis, Crohn's disease, multiple sclerosis and the cachexia associated with cancer or human immunodeficiency virus infection. TNF-alpha is initially expressed as a 233-amino-acid membrane-anchored precursor which is proteolytically processed to yield the mature, 157-amino-acid cytokine. The processing enzyme(s) which cleave TNF-alpha are unknown. Here we show that the release of mature TNF-alpha from leukocytes cultured in vitro is specifically prevented by synthetic hydroxamic acid-based metalloproteinase inhibitors, which also prevent the release of TNF-alpha into the circulation of endotoxin challenged rats. A recombinant, truncated TNF-alpha precursor is cleaved to biologically active, mature TNF-alpha by several matrix metalloproteinase enzymes. These results indicate that processing of the TNF-alpha precursor is dependent on at least one matrix metalloproteinase-like enzyme, inhibition of which represents a novel therapeutic mechanism for interfering with TNF-alpha production.

Supernumerary eyes and man's search for hyper-vision: a historical review of relevant representational arts.

In this paper we present examples of man's identification of superiority with visual hyper-efficiency. From Babylonian, Egyptian, Minoan and Biblical times, the eye was the symbol of the master or the inspector. Similarly, a being or deity that was endowed with multiple eyes--with or without multiple heads--was considered to be extra powerful. An example is the crest of the British College of Optometrists, which is surmounted by a bird with three heads and hence supernumerary eyes, linking it to the College's motto 'aequis oculis videre' denoting equal vision. We present here photographic and textual data from several historical periods extending from the fourth millennium BC to the sixteenth century AD; and from different religious sources, both Christian and non-Christian, to support this thesis. However, these are only a few examples, selected from a larger on-going study of the subject.

Pathways of dephosphorylation of 1-D-myo-inositol 1,4,5-trisphosphate in GH3 pituitary tumor cells.

Previous work in [3H]inositol-labelled GH3 pituitary tumor cells stimulated with thyrotropin-releasing hormone (TRH) reported the existence of at least ten distinct [3H]inositol-containing substances which were identified as different inositol mono-, bis- and tris-phosphate isomers [1]. Here a complete kinetic study of the dephosphorylation pathways of the second messenger Ins(1,4,5)P3 is reported in GH3 cell homogenates, identifying a new intermediate, Ins(4,5)P2, in the metabolism of the second messenger. in vitro results obtained with exogenous substrates are compared with in vivo results obtained measuring levels of the endogenous [3H]inositol-labelled isomers that participate in the dephosphorylation pathways of Ins(1,4,5)P3 in resting and TRH-stimulated GH3 cells. The effect of Li+ on the activity of the different phosphatases involved in these pathways is studied as well.

Metalloproteinase inhibitors as therapeutics.

Matrix metalloproteinases (MMPs) are a family of enzymes which contain zinc at their active site and can degrade most of the matrix macromolecules found in connective tissues. These MMPs are secreted by connective tissue cells and infiltrating leucocytes in response to inflammatory mediators. There is now widespread recognition that MMPs are the major class of proteinases responsible for the excessive degradation of cartilage that leads to joint dysfunction in rheumatoid arthritis. The properties of the MMPs are reviewed and a therapeutic role for synthetic, zinc-binding pseudopeptide MMP inhibitors in the treatment of arthritis is proposed.

Chronic lithium treatment inhibits basal and agonist-stimulated responses in rat cerebral cortex and GH3 pituitary cells.

Li+ is used clinically in the management of bipolar-disordered (manic-depressive) illness, but the mechanism of its clinical efficacy remains unclear. Li+ inhibits the metabolism of certain inositol phosphates, leading to a decreased cycling of inositol that may be sufficient to reduce phosphoinositide metabolism. We have tested this hypothesis in slices of rat cerebral cortex and in rat pituitary GH3 cells grown in the presence of low extracellular inositol. We show that basal and stimulated mass levels of inositol-1,4,5-trisphosphate were reduced in rat cerebral cortex and in GH3 cells after chronic, but not acute, treatment with a therapeutic concentration of Li+. In GH3 cells chronic treatment with Li+ also decreased basal levels of intracellular Ca2+ and secretion of prolactin, effects that were prevented by the presence of myo-inositol. Agonist-stimulated mobilization of Ca2+ and prolactin release were also reduced in Li(+)-treated cells. These findings show that chronic perturbation of the phosphoinositide pathway by Li+ is sufficient to reduce basal and agonist-stimulated cellular responses, an action that may underlie its effectiveness in the alleviation of affective disorders.

Purification and analysis of proteinase-resistant mutants of recombinant platelet-derived growth factor-BB exhibiting improved biological activity.

Recombinant platelet-derived growth factor (PDGF)-BB was expressed and secreted from yeast in order to study the structure-function relationships of this mitogen. A simple purification scheme has been developed which yields greater than 95% pure PDGF-BB. Analysis of this recombinant PDGF-BB shows partial proteolysis after arginine-32. Substitution of this arginine residue, or arginine-28 [a potential KEX2 (lysine-arginine endopeptidase) cleavage site], prevents or reduces cleavage of PDGF-BB respectively. These mutations result in a 5-fold increase in expression levels of PDGF-BB, and the resulting mutant proteins show higher activity in a number of biological assays than the cleaved wildtype PDGF-BB. These data are in accord with previous work by Giese, LaRochelle, May-Siroff, Robbins & Aaronson [(1990) Mol. Cell Biol. 10, 5496-5501] suggesting that the region isoleucine-25-phenylalanine-37 is involved in PDGF-receptor binding.

Two PDGF-B chain residues, arginine 27 and isoleucine 30, mediate receptor binding and activation.

PDGF may be involved in the pathogenesis of a variety of disorders including atherosclerosis and certain types of cancer. There is currently little understanding of the molecular structure of PDGF and of the critical amino acid residues involved in receptor binding and cell activation. Two such PDGF-B chain residues, arginine 27 and isoleucine 30, have been identified by a site-directed mutagenesis programme. Substitutions in these positions can lead to PDGF mutants defective in both receptor affinity and cell activation as judged by displacement of [125I]PDGF-BB, mitogenic assay and inositol lipid turnover. Circular dichroism and fluorescence spectroscopy show that such mutations do not disrupt the structure of PDGF.

The interaction of benzodiazepines with thyrotropin-releasing hormone receptors on clonal pituitary cells.

1. Seven benzodiazepines were investigated for their ability to interact with receptors for thyrotropin-releasing hormone (TRH) on GH3 and GH4C1 pituitary tumour cells. 2. Midazolam and chlordiazepoxide were the most potent inhibitors of TRH-induced [3H]-inositol phosphate formation with Ki values in the low micromolar range. The antagonism was competitive in nature and was increased in potency at sub-physiological temperatures. 3. None of the agents examined antagonized bombesin-induced [3H]-inositol phosphate formation in GH4C1 cells. 4. While the ability of benzodiazepines to interact with the GABA receptor-chloride channel ionophore is markedly stereospecific, little difference was evident in the ability of (+)- and (-)-4-methylmidazolam (Ro 21-5656 and Ro 21-5657) to compete with TRH at its receptor. 5. Recently it has been suggested that, in contrast to phosphatidylinositol hydrolysis, the TRH-induced breakdown of phosphatidylinositol polyphosphates is transient in clonal pituitary cells. Addition of chlordiazepoxide to TRH-stimulated GH3 cells up to 60 min after initiating the reaction leads, however, to an immediate decline in the cellular content of inositol trisphosphate. This indicates that TRH-induced phosphatidylinositol 4,5-bisphosphate hydrolysis is not transient.

Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions.

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.