Sarcopenia is a recently defined medical condition described as age-associated loss of skeletal muscle mass and function. Recently, a transgenic mouse model was described linking dispersal of the neuromuscular junction caused by elevated agrin degradation to the rapid onset of sarcopenia. These mice show a significant elevation of serum levels of a C-terminal agrin fragment (CAF) compared to wild-type littermates. A series of experiments was designed to ascertain the significance of elevated agrin degradation in the development of human sarcopenia. A quantitative Western blot method was devised to detect CAF in sera of humans. A first trial on consenting blood donors (n=169; age 19-74 years) detected CAF in the limited range of 2.76 ± 0.95 ng/ml. In sarcopenia patients (diagnosed according to clinical and instrumental standards) mean CAF levels were significantly elevated (p=9.8E10-9; n=73; age 65-87 years) compared to aged matched controls. Of all sarcopenia patients, 40% had elevated, non-overlapping CAF levels compared to controls. Evidence is presented for a pathogenic role of the agrin/neurotrypsin system in a substantial subset of sarcopenia patients. These patients are characterized by elevated CAF blood levels compared to aged-matched healthy volunteers suggesting the identification of an agrin-dependent form of sarcopenia. Elevated CAF levels in a large subpopulation of sarcopenic patients suggest the existence of a specific form of sarcopenia for which CAF could become a biomarker and a new target for therapeutic interventions. The feasibility of this approach was demonstrated by the development of a small molecule capable of inhibiting neurotrypsin in vitro and in vivo.

IntroductionInhibitors of bone resorption can preserve bone mass and maintain normal bone architecture, thereby preventing fractures in later life (1).The bone-resorption process requires secretion of protons by the osteoclasts (2). This allows the dissolution of the bone mineral and the maintenance of the acidic environment required by proteolytic enzymes to degrade the bone matrix (3). Protons are extruded by a proton pump belonging to the class of vacuolar-type H + -ATPases (V-H + -ATPases (4) that is present in high concentration on the ruffled border (apical surface) of the resorbing osteoclast.The osteoclast V-H + -ATPase therefore represents a previously unexploited molecular target for the design of novel agents for reducing osteoclast activity. However, since V-H + -ATPase is an ubiquitous component of eukaryotic organisms and is the major electrogenic pump of endomembranes (5), the selective inhibition of the osteoclast V-H + -ATPase must be achieved in order to afford novel drugs that may constitute useful and safe agents for the treatment of osteoporosis and related osteopenic diseases.Bafilomycin A 1 , a potent and specific inhibitor of V-H + -ATPases (6), inhibits bone resorption both in vitro (7) and in vivo (8). However, since bafilomycin is not selective for any particular type of V-H + -ATPases, when administered to animals it inhibits all the essential V-H + -ATPases leading to systemic alteration of cellular physiology and to unacceptable toxicity (9). Recently, a novel class of small molecules endowed with potent osteoclast V-H + -ATPase inhibitory activity has been discovered (10), whose optimization resulted in (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide (SB 242784; Figure 1) (11). The in vitro V-H + -ATPase inhibitory profile of SB 242784 has been reported previously (11). SB 242784 was a low-nanomolar inhibitor (IC 50 = 26.3 nM) of the bafilomycin-sensitive (vacuolar) Mg 2+ -ATPase in membrane preparations of osteoclasts obtained from egg-laying hens. In addition, SB 242784 was a very potent inhibitor of bone resorption (IC 50 = 3.4 nM) in an assay measuring the collagen fragments released from bone slices after a 48-hour incubation with human osteoclasts.In this paper we characterize further the biochemical and pharmacological actions of SB 242784 and demonstrate that it is highly selective for the V-H + -ATPase of human osteoclasts when compared with V-H + -ATPases from a number of other human tissues. Most importantly, we show that oral administration of SB 242784 reduces the retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats and prevents the A potent and selective inhibitor of the osteoclastic V-H + -ATPase, (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl) -2,4-pentadienamide (SB 242784), was evaluated in two animal models of bone resorption. SB 242784 completely prevented retinoid-induced hypercalcemia in thyroparathyroidectomized (TPTX) rats when administered orally ...

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A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

The macrolide antibiotic bafilomycin A1 is a highly potent and selective inhibitor of all the vacuolar ATPases (V-ATPases). With the aim of obtaining novel analogues specific for the osteoclast subclass of vacuolar ATPase, 31 derivatives of bafilomycin A1 were synthesized and tested for their ability to inhibit differentially the V-ATPase-driven proton transport in membrane vesicles derived from chicken osteoclasts (cOc) and bovine chromaffin granules (bCG). Although none of the new analogues were more potent than the parent compound, the obtained data provided a significant amount of information about the structural requirements for the inhibitory activity of bafilomycin A1. The different effects of a few analogues on the two enzymes could also suggest the possibility of a selective modulation of the V-ATPases in different tissues.