Toxicity and exposure of an adenovirus containing human interferon alpha-2b following intracystic administration in cynomolgus monkeys.

The safety and toxicokinetics of SCH 721015, an adenovirus encoding the human interferon alpha-2b gene, and Syn3 (SCH 209702), a novel excipient, were assessed in cynomolgus monkeys administered intravesical doses of 2.5 × 10E11 or 1.25 × 10E13 particles SCH 721015 in 25 mg Syn3 or 25 mg Syn3 alone on study days 1 and 91. There was no systemic toxicity. Monkeys dosed with SCH 721015 in Syn3 were positive for SCH 721015-specific DNA in the urine for 2 to 3 days following each dose and had interferon alpha-2b protein in the urine for 1-3 days after a single dose and in fewer animals after a second dose. Intracystic administration was associated with inflammation and focal/multifocal ulceration in the urinary bladder and irritation in the ureters and urethra at necropsy. The physical trauma from catheterization and filling/emptying of the bladder was likely a contributing factor and Syn3 exacerbated the trauma. There was nearly complete resolution of these findings 2 months after the last dose. The trauma to the bladder likely contributed to low, transient systemic exposure to Syn3, SCH 721015 and human interferon protein. The results of this study support the clinical investigation of SCH 721015 in Syn3.

Diethyldithiocarbamate prolongs survival of mice in a lipopolysaccharide-induced endotoxic shock model: evidence for multiple mechanisms.

It is now known that overproduction of nitric oxide (NO) by nitric oxide synthase (NOS) is an important contributing factor for the development of cardiovascular collapse and subsequent death in endotoxic shock. Diethyldithiocarbamate (DETC) is a molecular scavenger of NO and can inhibit overexpression of a number of cytokines during shock through inactivation of transcription factors such as nuclear factor (NF)-kappaB. Thus, DETC may be a useful adjunct in the therapy of endotoxic shock. In our study, we examined the effect of DETC on survival time in a murine model of severe endotoxic shock. Our results indicated that selected in vivo dosage regimens of DETC (intraperitoneal: at -2, -1, 3, 6, and 10 h or at -2, -1, 3, 6, 9, 12, 15, and 18 h relative to lipopolysaccharide administration, 180 mg/kg, at t = 0) in endotoxic mice were effective in increasing survival time when compared with untreated animals and DETC pretreatment was more effective than methylprednisolone (p<.05). DETC was shown to exert multiple beneficial mechanisms, including 1) a decrease in circulating NO, as determined by plasma nitrite/nitrate levels, 2) a reduction in plasma tumor necrosis factor-alpha after lipopolysaccharide induction, and 3) decreased expressions of metalloproteinases such as gelatinase A and B which may be responsible for cellular release of cytokines. These results indicate that DETC and its analogs may be useful in the treatment of endotoxic shock.

Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.

Gelatinase B, a matrix metalloproteinase (MMP) of high specific activity, is highly expressed and activated by mouse blastocysts in culture, and inhibition of this enzyme activity inhibits lysis of extracellular matrix (Behrendtsen, O., Alexander, C. M. and Werb, Z. (1992) Development 114, 447-456). Because gelatinase B expression is linked to invasive potential, we studied the expression of gelatinase B mRNA and protein in vivo, in implanting trophoblast giant cells, and found that it was expressed and activated during colonization of the maternal decidua. mRNAs for several other MMPs (stromelysin-1, stromelysin-3 and gelatinase A) and MMP inhibitors (TIMP-1 and TIMP-2) were expressed in the undifferentiated stroma toward the outside of the decidua, and TIMP-3 mRNA was expressed in primary and some mature decidual cells during their differentiation. Both mRNA and TIMP-3 protein were present at high concentrations transiently, and declined from 6.5 days post coitum onward, as the cells underwent apoptosis during the main period of gelatinase B expression and ectoplacental growth and expansion. To assess the function of MMPs during implantation and decidual development, we either injected a peptide hydroxamate MMP inhibitor into normal mice or studied transgenic mice overexpressing TIMP-1. In both cases, decidual length and overall size were reduced, and the embryo was displaced mesometrially. Embryo orientation was less strictly regulated in inhibitor-treated deciduae than in control deciduae. Morphogenesis and development of oil-induced deciduomas were also slowed in the presence of the inhibitor. We conclude that administration of MMP inhibitors retards decidual remodeling and growth, and we suggest that the MMPs expressed in precursor stromal cells promote their differentiation and expansion.

Identification and characterization of human tissue inhibitor of metalloproteinase-3 and detection of three additional metalloproteinase inhibitor activities in extracellular matrix.

We have identified and characterized a novel human tissue inhibitor of metalloproteinase (TIMP). It is found exclusively in the extracellular matrix of a large number of cultured human cells, including: primary embryonal kidney (293), neuroblastoma (SK-N-SH), normal whole embryo (FHs 173We), cervical carcinoma (HeLa S3), colon adenocarcinoma (Caco-2), ileocecal adenocarcinoma (HCT-8), fibrosarcomas (SW 684 and Hs 913T) and normal gingival fibroblasts (GF11 and 1292). It was not detected in the conditioned media from any of these cell lines. Its apparent molecular mass of 24-25 kDa, as determined by its migration on protease-substrate gels, is intermediate between TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa). Like the latter two proteins, human TIMP-3 contains intrachain disulfide bonds and displays altered electrophoretic mobility in the presence of beta-mercaptoethanol. The N-terminal, amino acid sequence of the protein is identical to that of chicken TIMP-3 (ChIMP-3), and its amino acid composition is similar. The protein is not N-glycosylated, as determined by treatment with N-glycosidase-F. Finally, it is recognized by antisera raised against pure ChIMP-3 but not by anti-human TIMP-1 or anti-human TIMP-2 antibodies. Based on these properties, we propose that this protein is TIMP-3 and is the human counterpart of ChIMP-3 (Pavloff et al., J. Biol. Chem. 267: 17321-17326, 1992). Two additional inhibitors detected in the matrix of human cell lines, designated inhibitor of metalloproteinase (IMP)-a and IMP-b, migrate with apparent masses of 29 kDa and 30 kDa. Both are N-glycosylated. A fourth inhibitor activity, which is smaller in mass than TIMP-3 and is also pecifically located in the matrix, is detectable in some cell lines.

A new inhibitor of metalloproteinases from chicken: ChIMP-3. A third member of the TIMP family.

We report cDNA cloning and primary structure of a new metalloproteinase inhibitor (ChIMP-3) produced by chicken embryo fibroblasts. ChIMP-3, formerly called the 21-kDa protein, is one of five ChIMPs (Chicken Inhibitor of MetalloProteinases). In this paper, we report that of the three most abundant ChIMPs, ChIMP-3 and ChIMP-a are extracellular matrix components, whereas ChIMP-2 is found in the media conditioned by the cells. Treatment of ChIMP-3 and ChIMP-a with N-glycosidase-F indicates that ChIMP-a is N-glycosylated whereas ChIMP-3 is not. The deduced amino acid sequence of ChIMP-3 predicts a protein whose properties are consistent with experimental measurements. Analysis of sequence alignments with the two previously described members of the TIMP (tissue inhibitor of metalloproteinases) family, TIMP-1 and TIMP-2, from various species indicates that ChIMP-3 is a related but distinct protein. This conclusion is supported by lack of significant binding with anti-TIMP-1 and anti-TIMP-2 antibodies. Based on these data, its unusual solubility properties, and its exclusive location in the matrix, we propose that ChIMP-3 is a new member of this family of metalloproteinase inhibitors, a TIMP-3.