Eli Breuer

Transplacental effects of bisphosphonates on fetal skeletal ossification and mineralization in rats

Bisphosphonates are clinically used mainly to reduce bone resorption. We studied the transplacental effects of two bisphosphonates on the fetal skeleton in rats. Pregnant rats were treated during days 11-20 of pregnancy with daily subcutaneous injections of 0.1 mg/kg of alendronate or a newly synthesized bisphosphonate, VS-b6. This period of pregnancy was chosen because the active development of bones from mesenchyme through cartilaginous models occurs during that time. Histological examination of midlongitudinal sections of the 21-day-old fetuses showed an increase in the amount of diaphyseal bone trabeculae with slight shortening of the diaphysis in the experimental fetuses, in comparison to controls. Computerized histomorphometric studies similarly showed an increase in the amount of diaphyseal bone trabeculae with a concomitant decrease in bone marrow volume, but no change in cartilage volume. In addition, chemical analysis of the fetal bones showed an increase in calcium content in the treated fetuses. 14C-alendronate was shown to pass through the rat placenta and accumulate in the fetuses, most probably in their bones. This is presumed because bisphosphonates are known to accumulate in bone, being stored there for long periods of time. It is important, in light of our results, to give careful consideration to the treatment of women with bisphosphonates at childbearing age, whenever this is needed.

Recent non-hydroxamate matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endo-peptidases that have the capacity to degrade all elements of the extracellular matrix (ECM) and are required for homeostatic maintenance of the ECM. Interest in MMPs arose from the accumulating evidence implying that over-activity of MMPs plays a role in mediating or accompanying a diverse array of pathologies. Because of this, there has been an ongoing, and alas unsuccessful, effort for nearly two decades to develop clinically applicable MMP inhibitors (MMPIs) as drugs. The largest family of the inhibitor candidates that failed in clinical trials is that of hydroxamic acids. This review i) attempts to rationalise the failure of hydroxamates as MMPIs, ii) critically reviews publications and patents of the last few years, which report new non-hydroxamate based MMPIs, and iii) summarises factors that are considered important for success in developing clinically useful MMPIs.

Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors.

Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium‐related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2‐(3′‐dimethylaminopyrazinio)ethylidene‐1,1‐bisphosphonic acid betaine (VS‐6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal‐cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix‐maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal‐cell proliferation, and on cell‐mediated mineralization. These BPs differentially interact with cell‐associated phosphohydrolysis, particularly at a concentration of 10−2 of ALP Km, in which alendronate inhibits whereas VS‐6 did not inhibit phosphatase activity. VS‐6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186–194, 1998.

Bisacylphosphonates inhibit hydroxyapatite formation and dissolution in vitro and dystrophic calcification in vivo

Some geminal bisphosphonates are used clinically for a number of important bone/calcium related diseases; however, side effects and lack of selectivity impede their wide use. This work reports the synthesis and evaluation of bisacylphosphonates (e.g., adipoyl- and suberoylbisphosphonate). These compounds were found to inhibit significantly hydroxyapatite formation and dissolution in vitro and the calcification of bioprosthetic tissue implanted subdermally in rats. These are the first instances of nongeminal bisphosphonates [P–(C)n– P, n ≥ 2] that have been reported to be active in calcium-related disorders. The reported bisacylphosphonates possess apparent lower toxicity, and their calcium complexes/salts have improved solubility properties. Therefore, they are of potential importance for clinical applications.

Anticalcification and antiresorption effects of bisacylphosphonates

Some geminal bisphosphonates are used clinically in a number of important bone and calcium-related diseases. This work reports the anticalcification and antiresorption effects of a series of bisacylphosphonates, nongeminal compounds with varying chain lengths having oxo groups in alpha positions relative to the phosphonic functions. We compared the activity of the novel compounds to clinically used geminal bisphosphonates, and to a bisphosphonate devoid of the oxo groups. The interaction of the compounds with calcium was studied by various in vitro and in vivo models. We found that keto groups in alpha positions to the phosphonic functions render activity. The bisacylphosphonates with a shorter chain [(CH2)n, = 4, 6] were found significantly to inhibit hydroxyapatite formation and dissolution in vitro, the calcification of bioprosthetic tissue implanted subdermally in rats, and bone resorption in the intact young animal model. The various in vitro results were found to be in good correlation with the in vivo results. Structure-activity relationship studies indicate that both bisacylphosphonates and geminal bisphosphonates are active only when at least three ionizable groups are present in the molecule. The role of the keto groups is related to their contribution to chelating calcium and/or to their electron-withdrawing influence on acidity.

Carbamoylphosphonate-based matrix metalloproteinase inhibitor metal complexes: solution studies and stability constants. Towards a zinc-selective binding group

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-(N,N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of 1 and 2. For comparison with carbamoylphosphonates 1 and 2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of 1 and 2 with those from 3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both 1 and 2 are Ca(II)8 the dimethylamino group of compound 2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid 2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.

Efficient and selective dealkylation of phosphonate dilsopropyl esters using Me3SiBr

Abstract Diisopropyl phosphonates, having a variety of functional groups in the molecule, can be dealkylated with complete chemoselectivity by Me 3 SiBr in dioxane at 60 °C

The preparation of some cyclic phosphonates and their use in olefin synthesis

Abstract The preparation and the use in olefin synthesis of two 5-membered cyclic phosphonates, 2-carbethoxy-methyl 4,5-dimethyle-2-oxo-1,3,2-dioxaphospholane (4b) and 2-cyanomethyl-4,5-dimethyl-2-oxo-1,3,2-diox-aphospholane (4c) and the 6-membered phosphonates, 2-carbalkoxymethyl-5,5-dimethyl-2-oxo-1,3,2-diox-aphosphorinanes (5a and 5b) and 2-cyanomethyl-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinane (5c) are described. Reactions of 4b with aromatic and aliphatic aldehydes lead to preferential formation of cis-olefins. Reactions of the other cyclic phosphonates with aldehydes lead to various mixtures of cis and trans olefins.

Carbamoylphosphonate matrix metalloproteinase inhibitors 6: cis-2-aminocyclohexylcarbamoylphosphonic acid, a novel orally active antimetastatic matrix metalloproteinase-2 selective inhibitor--synthesis and pharmacodynamic and pharmacokinetic analysis.

cis-2-Aminocyclohexylcarbamoylphosphonic acid ( cis-ACCP) was evaluated in vitro and in two in vivo cancer metastasis models. It reduced metastasis formation in mice by approximately 90% when administered by a repetitive once daily dosing regimen of 50 mg/kg via oral or intraperitoneal routes and was nontoxic up to 500 mg/kg, following intraperitoneal administration daily for two weeks. Pharmacokinetic investigation of cis-ACCP in rats revealed distribution restricted into the extracellular fluid, which is the site of action for the antimetastatic activity and rapid elimination ( t 1/2 approximately 19 min) from blood. Sustained and prolonged absorption ( t 1/2 approximately 126 min) occurred via paracellular mechanism along the small and large intestine with overall bioavailability of 0.3%. The in vivo concentrations of cis-ACCP in the blood in rats was above the minimal concentration for antimetastatic/MMP-inhibitory activity, thus explaining the prolonged action following once daily administration. Finally, 84% of the intravenously administered cis-ACCP to rats was excreted intact in the urine.

Solid‐state NMR of bisphosphonates adsorbed on hydroxyapatite

Solid‐state 31P and 13C magic angle spinning (MAS) NMR spectra were used to characterize pure bisphosphonates and also bisphosphonates adsorbed on hydroxyapatite. Four geminal bisphosphonates, including the clinically used compounds ethane‐1‐hydroxy‐1,1‐diphosphonic acid, 3‐amino‐1‐hydroxypropane‐1,1‐diphosphonic acid and 4‐amino‐1‐hydroxybutane‐1,1‐diphosphonic acid, five α,ω‐bisphosphonates and phosphonoacetic acid were investigated. NMR spectra of pure and adsorbed bisphosphonates differ in the observed linewidths and in the isotropic chemical shifts. The broad lines reflect mainly the poor crystallinity of the adsorbed compounds. Shifts of δiso in both directions do not reveal a correlation with the molecular structures. The molar ratio of phosphonates adsorbed on hydroxyapatite determined by 31P spectra without cross‐polarization (CP) is approximately two times larger for geminal bisphosphonates than for α,ω‐bisphosphonates and phosphonoacetic acid. 13C CP/MAS spectra of pure and adsorbed bisphosphonates recorded in two cases for identification of adsorbed compounds give additional information about the state of adsorbed compounds. Disodium α,ω‐dihydroxypolymethylene‐α,ω‐bisphosphonates in the solid state show characteristic 13C chemical shifts which are indicative of either odd or even numbers of CH2 groups. Copyright