Discovery of a potent, metabolically stabilized resorcylic lactone as an anti-inflammatory lead.

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.

Characterization of 5'-flanking region of human aggrecanase-1 (ADAMTS4) gene.

Aggrecanase-1, also known as ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), cleaves at the Glu373-Ala374 site of aggrecan, thereby indicating aggrecan degradation. It is thought that ADAMTS4 plays a pivotal role in inflammatory joint diseases and cartilage degradation. To elucidate the mechanisms of regulation of ADAMTS4 gene expression, we cloned the 5'-flanking region of the human ADAMTS4 gene and characterized its promoter activity by means of reporter assay using porcine chondrocytes and NIH3T3 cells. Reporter gene analysis using deletion variants suggested that the region between -383 and +10 relative to the tentative transcription start site is necessary for full promoter activity; this region contains one Sp1 and three AP2 sites. In addition, the segment between -726 and -384 appears to contain silencer element(s). A complete deletion mutant of the nuclear factor I (NFI) binding site at -441 to -429 resulted in recovery of the promoter activity in chondrocytes, but not in NIH3T3 cells. Thus, the NFI site is involved in negative regulation of the human ADAMTS4 promoter activity in chondrocytes.

Evaluation of intestinal pressure-controlled colon delivery capsule containing caffeine as a model drug in human volunteers.

The delivery ability of a pressure-controlled colon delivery capsule (PCDC) containing caffeine as a test drug was evaluated after oral administration to healthy male human volunteers. The driving force causing PCDC disintegration in the intestinal tract is the physiological luminal pressure which results from peristalsis. Three kinds of PCDCs having different thickness of a water-insoluble polymer membrane was prepared by coating the inner surface of the gelatin capsules with ethylcellulose (EC). The mean thickness were 40 +/- 1 (SE) for type 1, 44 +/- 1 for type 2 and 50 +/- 1 micron for type 3 PCDC, respectively. Caffeine was dissolved with a suppository base (PEGs 400 and 1000) and the capsules were filled. Doses were 15, 45 or 75 mg. After blank saliva samples were obtained, test preparations were orally administered to the volunteers and saliva samples were collected for 1 min intervals hourly from 1 to 10 h in the fasted state study, and from 1 to 20 h and at 25 h in the fed state study. Caffeine concentrations in the saliva samples were analyzed by HPLC. The maximum salivary caffeine excretion rate increased as the oral caffeine dose increased. The maximum salivary caffeine excretion rate increased predominantly compared to the pre-dose level in 75 mg dose study. Therefore, all following studies were performed with this dose. The first appearance time of caffeine into the saliva, TI, was used as a parameter to estimate the disintegration time of test preparations in the gastrointestinal tract. The mean TI of types 1, 2, and 3 PCDCs were 3.0 +/- 0.4, 4.0 +/- 0.4 and 4.5 +/- 0.3 h, respectively. After oral administration of 75 mg caffeine in pain gelatin capsule as a reference preparation, caffeine appeared in the saliva within 0.5 h. The mean hardness of the PCDCs were 1.05 +/- 0.10 (type 1), 1.55 +/- 0.06 (type 2) and 2.08 +/- 0.15 newton (type 3), respectively. There were good correlations between three parameters: EC coating membrane thickness, hardness and TI (determination coefficient r2 = 0.935 between TI and thickness, r2 = 0.998 between thickness and hardness, r2 = 0.958 between hardness and TI). The effect of food intake on the delivery ability was examined with type 3 PCDCs. Food intake prolonged the mean TI, from 4.5 +/- 0.3 to 7.8 +/- 1.3 h. This increase is thought to be ascribed to prolonged gastric emptying time. Comparison with reported colon arrival times indicates that the type 3 PCDC functions in colon delivery of caffeine and is thought to be applicable to other drugs.

K1115 A, a new anthraquinone derivative that inhibits the binding of activator protein-1 (AP-1) to its recognition sites. I. Biological activities.

K1115 A, a new anthraquinone derivative, was isolated from the culture broth of Streptomyces griseorubiginosus (Mer-K1115). K1115A inhibited the direct binding of activator protein-1 (AP-1) to AP-1 oligonucleotide (IC50 = 100 microM), and the production of collagenase in IL-1 alpha-stimulated rat synovial cells (IC50 = 60 microM). In vivo, the application of K1115 A decreased phorbol myristate acetate (PMA)-induced mouse ornithine decarboxylase (ODC) activity. These results indicated that K1115 A is able to attenuate the inflammatory response mediated by AP-1.

Cloning of rabbit TR4 and its bone cell-specific activity to suppress estrogen receptor-mediated transactivation.

To clone a new nuclear receptor, we screened a rabbit heart complementary DNA (cDNA) library with degenerate oligonucleotide probes corresponding to the DNA-binding domain of nuclear receptors, which is highly conserved among receptors. One of the cDNA clones, clone 23, encodes a novel protein of 596 amino acids, and predicted molecular mass is 66 kDa. Homology search analysis identified this protein as rabbit TR4 (TR4-0). We also cloned the cDNA encoding a rabbit TR4 isoform (TR4-1), which lacks the putative C-terminal ligand-binding domain (350 amino acids) caused by a 23-bp exon deletion, which probably occurred during messenger RNA (mRNA) splicing. Northern blot analysis showed that TR4s are expressed with two kinds of mRNAs (9.0 kb and 2.8 kb), both of which are relatively abundant in brain, testis, and bone. RT-PCR analysis, using pairs of primers specific for each TR4, showed that both types of receptor express in various tissues. Furthermore, both are present in primary osteoblasts and bone marrow cells, though the mRNA levels of TR4-0 were much higher than those of TR4-1. A functional study, using a transient transfection assay, showed that both receptors suppressed retinoid X receptor (RXR)-retinoid acid receptor, RXR-TR, and RXR-VDR-mediated transactivation significantly in COS-1 and osteosarcoma cells (UMR-106, ROS17/2.8) and that TR4-0 was much more effective than TR4-1. Unexpectedly, we found that the TR4s effectively suppressed estrogen receptor-mediated transactivation in bone cells, but neither in kidney (COS-1) nor breast cancer cells (MCF-7, one of the major target cells of the estrogen action). Thus, the present study shows a novel property of the TR4 orphan receptor, acting as a bone cell-specific repressor in the estrogen receptor-mediated signaling pathway.