Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

FrzB-2: a human secreted frizzled-related protein with a potential role in chondrocyte apoptosis.

OBJECTIVE: To characterize a novel secreted frizzled-related protein (SFRP) and determine its tissue distribution at the mRNA and protein level. METHODS: The FrzB-2 gene was identified by expressed sequence tag (EST) analysis of human tissue-derived libraries. Tissue distribution of FrzB-2 mRNA was determined by Northern blot analysis and in situ hybridization. FrzB-2 protein reactivity was localized in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody against a peptide sequence unique to FrzB-2. Apoptosis was detected in articular cartilage sections using Tunel staining. RESULTS: ESTs corresponding to FrzB-2 were found in osteoblast, chondrosarcoma, osteosarcoma, osteoclastoma and synovial fibroblast libraries. FrzB-2 mRNA is expressed in a number of tissues and cell types including bone-related cells and tissues such as primary human osteoblasts and osteoclastoma. In situ hybridization studies showed strong FrzB-2 mRNA expression in human chondrocytes in human osteoarthritic (OA) cartilage but negligible levels in normal cartilage chondrocytes. The FrzB-2 cDNA encodes a secreted 40 kDa protein consisting of 346 amino acids. FrzB-2 is 92. 5% identical to the rat orthologue, DDC-4, which has been shown to be associated with physiological apoptosis. FrzB-2 protein was selectively detected in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody. Consistent with its potential role in apoptosis, positive FrzB-2 staining and Tunel positive nuclei staining were detected in chondrocyte clones in sections of human OA cartilage. CONCLUSION: These data suggest that FrzB-2 may play a role in apoptosis and that the expression of this protein may be important in the pathogenesis of human OA.

Microsphere distribution in normal and tumor-bearing (DMBA-induced carcinogenesis) hamster cheek pouch.

This study evaluates, for the first time by direct visualization, the microvascular distribution of microspheres in normal hamster cheek pouch and in hamster cheek pouch bearing tumor induced by 7, 12 Dimethylbenz (A) Anthracene solution (DMBA). In contrast to the results of the previously used open-chest technique, this carotid injection technique does not lead to irregular distribution of 15-mu carbonized microspheres, chain, or impaction phenomena. It is concluded that methodology differences may account for different results.

Contrast media tonicity. Part II. Isotonic ioxaglate vs. standard renografin for IV-DSA evaluation of the carotid bifurcation, a double-blind prospective clinical trial.

Laboratory research has suggested that isotonic contrast media may be optimal for intravenous digital subtraction angiography (IV-DSA) by generating taller, narrower time-concentration curves. Clinical investigation of low osmolality contrast media has suggested that less patient discomfort is encountered with low-osmolality contrast media than with standard, high-osmolality agents. In order to directly compare isotonic contrast media with a standard hypertonic contrast media, isotonic ioxaglate (Hexabrix-20) was compared with Renografin-76 in a double-blind prospective clinical trial for IV-DSA examination of the carotid artery bifurcation. Isotonic ioxaglate produced superior contrast medium time-opacification curves and produced superior images across four scales of image quality: anatomic "openness" of the carotid bifurcation, contrast level within the carotid vessels, bone misregistration artifact over the bifurcation, and air (soft tissue) misregistration. The bilateral overall score for isotonic ioxaglate was 1.68 vs. 1.37 for Renografin-76, a 23% superiority. The bulk of the superiority occurred in the contralateral carotid artery. Over the four scales, isotonic ioxaglate was 37% better in image quality of the contralateral carotid artery bifurcation. Since the contralateral carotid artery is very often difficult to visualize during IV-DSA, isotonic ioxaglate represents a significant improvement for this imaging modality.

Variability of myocardial CT measurements in vivo.

Variability of myocardial CT measurements, as indicated by standard deviations of mean CT numbers from four myocardial regions, was compared in 12-second scans, 3-second scans, and gated end-diastolic and end-systolic images, all from the same 12 seconds of scan data, both without and with radiographic contrast enhancement in experimental animals. There were statistically significant differences (P less than 0.05) in standard deviations of myocardial CT measurements when comparing 3-second and 12-second scans without contrast (10.4 vs. 7.7 CT#s), and 12-second scans without and with contrast (7.7 vs. 11.2 CT#s). Standard deviations of mean myocardial CT measurements were significantly greater (P less than 0.01) in gated images (end-diastolic) when compared with 12-second scans, both without contrast (22.2 vs. 7.7 CT#s) and with contrast (20.2 vs. 11.2 CT#s). In this study variability of myocardial CT measurements increased as scan time decreased, with radiographic contrast enhancement and with gating cardiac images.

Effect of volume and rate of contrast medium injection on intravenous digital subtraction angiographic contrast medium curves.

The image quality of temporal (mask mode) intravenous digital subtraction angiography is directly dependent on the shape of arterial time-concentration curves produced by the intravenous injection of contrast medium. Curves that are narrow and tall minimize motion artifact (misregistration) and maximize contrast enhancement (pre- and postcontrast differences). To determine the effects of rate and volume of injection of contrast medium on intravenous digital subtraction angiographic curves, ioxaglate (Hexabrix), a monoacidic ionic dimer, was injected into large mongrel dogs. Quantitative measurements of opacification were made over time in the femoral arteries using a modified General Electric CT/T scanner. Peak opacification was directly proportional to the volume of contrast medium injected. Curve width was not affected by increasing volume of injection. At rates below a critical point, slower injection rates produced progressively shorter and wider arterial time-concentration curves. Above that critical point, increasing the rate of injection did not affect either curve width or curve peak.

An experimental evaluation of central vs. peripheral injection for intravenous digital subtraction angiography (IV-DSA).

At a given radiation dosage and field of view, five variables are under meaningful control for intravenous digital subtraction angiography (IV-DSA): concentration and quantity of contrast media injected, volume of injectate, rate of injection, and site of injection. Some controversy exists regarding the selection of a central vs. a peripheral injection site for IV-DSA. This study determined the influence of the site of injection on the peak and width of the arterial time-concentration curve produced by contrast media. Using a noninvasive, in vivo, quantitative x-ray measurement method, 36 separate injections (10 ml of ioxaglate at 8 ml/sec) were administered into the cephalic vein, subclavian vein, and main pulmonary artery in dogs. Injection sites were varied using a Latin-square experimental design. Cardiac output, central blood volume and the peak and width of the contrast media time-concentration curves were measured. The average peak enhancement was greatest for the pulmonary artery injection site. Normalizing peak and width values to make the pulmonary artery values 100%, the average peak values for injections into the subclavian vein and cephalic vein were 93% and 56%, and the average widths were 141% and 163%, respectively. These data support the use of a more central injection site for optimizing IV-DSA examinations.

Diameter of arterial microvessels trapping 8--10 micron, 15 micron and 25 micron microspheres as determined by vital microscopy of the hamster cheek pouch.

An assumption inherent in the use of microsphere methodology for measuring regional blood flow is that microspheres are removed from the circulation by impacting in arterial vessels of approximately their own diameter. We investigated the in vivo relationship between the diameter of varying size microspheres and the calibre of trapping microvessels within the hamster cheek pouch. Intracardiac injection of 8-10, 15 +/- 5, 25 +/- 5 micron carbonized microspheres with diversion of the cardiac output into the bracheocephalic trunk provided direct, in vivo visualization of the impaction sites of these various size microspheres within the cheek pouch microvasculature. Fifteen micron microspheres usually impacted at the orifice or neck of small arterioles and protruded into the lumen of the parent vessel. Eight to ten micron microspheres lodged in vessels with a mean diameter of 11.5 +/- SD 3.4 micron indicating that they usually impacted primarily in terminal arterioles.