Calcification inhibitors

It has been hypothesized that phosphate accelerates this chondro-osteogenic conversion by inducing expression of runt-related transcription factor 2 (RUNX2), osteocalcin and tissue-nonspecific alkaline phosphatase (TNAP) [2]. Elevated TNAP further favors vascular calcification by hydrolyzing the calcification inhibitor inorganic pyrophosphate (PPi) [3-6]. In the Enppl mouse model of GACI, inhibition of TNAP can restore PPj to sufficient levels to maintain normal mineralization [3]. 

Howell, D. S., Pita, J. C., Marquez, J. F., Gatter, R. A. Demonstration of macromolecular inhibitor (s) of calcification and nucleational factor (s) in fluid from calcifying sites in cartilage. J. Clin. Invest. 48, 630 (1969)... 

If corals are kept in darkness and in the presence of carbonic anhydrase inhibitors, calcification will still continue but at a much lower rate. The following conclusions can be drawn from these data ... 

Inhibition of abnormal calcification initials explorations of BPs as inhibitors of calcification showed promise, and early applications of etidronate included use in myositis ossificans, as well as in patients who had undergone total hip replacement surgery, in order to prevent subsequent heterotopic ossification and to improve mobility [10]. 

The enzyme has been found in corals (Goreau, 1959), annelids (Clark, 1975), crustaceans (Costlow, 1959), molluscs (Wilbur, 1972, for references), and echinoderms (Heatfield, 1970). In all cases, sulfanilamide inhibitors of the enzyme added to the medium reduced the rate of calcification at least 50%, indicating that catalysis by carbonic anhydrase is required for the normal rate of mineralization. In theory, the reactions should occur without enzyme catalysis and this appears to be true. Various mechanisms of action have been suggested (e.g., Goreau, 1959 Istin and Girard, 1970b) but the exact role of carbonic anhydrase remains uncertain. 

The fact that plaque is a region in the mouth relatively free from salivary inhibitors is probably one reason why calcification, leading to calculus formation, is associated with plaque. A second reason is the presence of certain bacteria that calcify, e.g. Corynebacterium (formerly Bacterionoma) matruchotii and S. sanguis I/II (more recently S. gordonii). The former bacterium contains cell wall components that can nucleate crystal growth [44, 45, 73], whilst the latter bacterium can metabolise basic amino acids, [74] and thereby, locally raise pH, which favours calcium phosphate precipitation. 

Narisawa S, Harmey D, Yadav MC et al (2007) Novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification. J Bone Miner Res 22 1700-1710... 

Here, we describe methods to evaluate the ability of small molecules inhibitors of TNAP and PHOSPHOl in preventing mineralization of primary cultures of murine vascular smooth muscle cells. The procedures are also applicable to primary cultures of calvarial osteoblasts. These cell-based assays are used to complement kinetic testing during structure-activity relationship studies armed at improving scaffolds in the generation of pharmaceuticals for the treatment for medial vascular calcification. 

VSMC isolated from mice were used for in vitro calcification studies in the presence of PHOSPHOl inhibitors as well as the potent TNAP inhibitor MLS-0038949 [16]. The PHOSPHOl and TNAP inhibitors alone or in combination were able to significant reduce calcification in VSMC culture and alizarin red staining was used for the quantification of mineralization reduction (Fig. 2). 

Other reports (27-29) have focused on the role of citric acid, as a source of carboxylate anions, during precipitation of calcium phosphates from electrolyte solutions. It has been found that citrate anions inhibit the ciystal growth of calcium phosphates and hinder their transformation into hydroxyapatites. This was attributed to the adsorption of citrate anions into the crystals and the displacement of an equivalent amount of phosphate anions. Interestingly, Rhee and Tanaka (30) found that the presence of a collagen membrane in the medium changed the behavior of citrate anions from being an inhibitor to becoming a promoter of calcification, provided that the molar ratios of calcium to citric acid were between 2 and 12. 

The compound SAT (83), inhibitor of calcification in vitro, was required for pharmacokinetic studies in vivo to evaluate its therapeutic potential in prevention and treatment of kidney stones. It has been produced in high purity in a rapid and simple two-step synthesis (equation 34)64,65. 

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