Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants.

OBJECTIVE: MMP-13 is highly upregulated in arthritis and therefore strongly implicated in the pathogenesis of osteoarthritis (OA). Selective inhibition of MMP-13 may provide the desired cartilage degradation protection, while overcoming the musculoskeletal toxicity seen with nonselective inhibition of MMPs. METHODS: Activity and selectivity of novel MMP-13 inhibitors were determined in enzymatic and collagenase assays. Inhibition kinetics and competitive binding experiments were performed. The inhibition of collagen degradation was studied in cartilage explants from OA patients and in bovine and human articular cartilage systems. RESULTS: We have identified a new class of very potent and highly selective non-zinc-binding MMP-13 inhibitors. Selective MMP-13 inhibitors completely blocked type II collagen degradation in bovine explants and showed up to 80% inhibition in human OA cartilage. CONCLUSIONS: These results indicate MMP-13 as the primary collagenase in the human OA cartilage and in the IL-1/OSM-induced cartilage degradation process and suggest that selective MMP-13 inhibitors may be a potential treatment of OA.

A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models.

OBJECTIVE: Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity. METHODS: Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors. RESULTS: A number of non-hydroxamic acid-containing compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompetitive inhibition, and Dixon plot analysis from competition studies with a zinc chelator (acetoxyhydroxamic acid) and ALS 1-0635 demonstrated nonexclusive binding. ALS 1-0635 inhibited bovine articular cartilage degradation in a dose-dependent manner (48.7% and 87.1% at 500 nM and 5,000 nM, respectively) and was effective in inhibiting interleukin-1alpha- and oncostatin M-induced C1,C2 release in human OA cartilage cultures. ALS 1-0635 modulated cartilage damage in the rat MIA model (mean +/- SEM damage score 1.3 +/- 0.3, versus 2.2 +/- 0.4 in vehicle-treated animals). Most significantly, when treated twice daily with oral ALS 1-0635, rats with surgically induced medial meniscus tear exhibited histologic evidence of chondroprotection and reduced cartilage degeneration, without observable musculoskeletal toxicity. CONCLUSION: The compounds investigated in this study represent a novel class of MMP-13 inhibitors. They are mechanistically distinct from previously reported broad-spectrum MMP inhibitors and do not exhibit the problems previously associated with these inhibitors, including selectivity, poor pharmacokinetics, and MSS liability. MMP-13 inhibitors exert chondroprotective effects and can potentially modulate joint pain, and are, therefore, uniquely suited as potential disease-modifying osteoarthritis drugs.

Functional characterization and comparison of the outer blood-retina barrier and the blood-brain barrier.

PURPOSE: To determine efflux systems of the outer blood-retina barrier (oBRB) and compare the oBRB with the blood-brain barrier (BBB). METHODS: Porcine oBRB structure and transport characteristics of freshly dissected intact tissue sheets were investigated with scanning electron microscopy, immunocytochemistry, vital dye labeling, and pharmacological agents, using HPLC/mass spectrometry. To compare drug permeation across the oBRB and the BBB, three different systems were used: (1) oBRB tissue sheets in a two-chamber device in vitro; (2) an in vitro BBB model composed of purified astrocytes and brain capillary endothelial cells on transfilter membranes; and (3) an in vivo model based on the brain-plasma ratio of drugs in mice. RESULTS: Efflux pumps (multidrug resistance protein [P-gp] and multidrug resistance-associated protein [MRP]) were demonstrated by antibody staining. Side-specific application of three P-gp and MRP substrates and selective transport inhibition suggested that both membrane proteins were preferentially located on the choroidal side of the oBRB. Therefore, the efflux was directed toward the blood, as in the BBB. To relate the transport characteristics of the oBRB to the BBB, up to nine different test compounds were used. The ranking of the permeability coefficients (P(e)) and the brain-plasma ratios of test compounds indicated that the oBRB has barrier and carrier features similar to those of the BBB in vitro and in vivo. CONCLUSIONS: Despite the fact that epithelial oBRB and endothelial BBB have developed as separate entities with many site-specific functions, their transport and permeation characteristics display surprising similarities, that include the polarized expression of the two major efflux pumps P-gp and MRP.

Mass spectrometric analysis of surfactant metabolism in human volunteers using deuteriated choline.

Surfactant reduces surface tension at pulmonary air-liquid interfaces. Although its major component is dipalmitoyl-phosphatidylcholine (PC16:0/16:0), other PC species, principally palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC, and palmitoyloleoyl-PC, are integral components of surfactant. The composition and metabolism of PC species depend on pulmonary development, respiratory rate, and pathologic alterations, which have largely been investigated in animals using radiolabeled precursors. Recent advances in mass spectrometry and availability of precursors carrying stable isotopes make metabolic experiments in human subjects ethically feasible. We introduce a technique to quantify surfactant PC synthesis in vivo using deuteriated choline coupled with electrospray ionization tandem mass spectrometry. Endogenous PC from induced sputa of healthy volunteers comprised 54.0 +/- 1.5% PC16:0/16:0, 9.7 +/- 0.7% palmitoylmyristoyl-PC, 10.0 +/- 1.0% palmitoylpalmitoleoyl-PC, and 13.1 +/- 0.3% palmitoyloleoyl-PC. Infusion of deuteriated choline chloride (3.6 mg/kg body weight) over 3 hours resulted in linear incorporation into PC over 30 hours. After a plateau of 0.61 +/- 0.04% labeled PC between 30 and 48 hours, incorporation decreased to 0.30 +/- 0.02% within 7 days. Compared with native PC, fractional label was initially lower for PC16:0/16:0 (31.9 +/- 8.3%) but was higher for palmitoyloleoyl-PC (21.0 +/- 1.2%), and equilibrium was achieved after only 48 hours. We conclude that infusion of deuteriated choline and electrospray ionization tandem mass spectrometry is useful to investigate surfactant metabolism in humans in vivo.