Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

A phase I biological and pharmacologic study of the heparanase inhibitor PI-88 in patients with advanced solid tumors

Purpose: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study. Experimental Design: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2). Results: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C-max correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure, No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >= 6 months. Conclusion: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

Attenuation of cardiovascular remodeling in DOCA-salt rats by the vasopeptidase inhibitor, omapatrilat

Omapatrilat, a vasopeptidase inhibitor, inhibits both neutral endopeptidase and angiotensin-converting enzyme with similar potency. The aim of this study was to investigate whether omapatrilat prevents or reverses cardiovascular remodeling and hypertension in deoxycorticosterone acetate (DOCA)-salt rats. Male Wistar rats (313 2 g, n=114) were uninephrectomized (UNX) with or without further treatment with DOCA and 1% NaCl in the drinking water. Compared with UNX control rats, DOCA-salt rats developed hypertension, cardiovascular hypertrophy, perivascular and interstitial cardiac fibrosis and inflammation, endothelial dysfunction, and the prolongation of ventricular action potential duration within four weeks. The administration of omapatrilat (40 mg/kg/day po) for two weeks commencing two weeks after surgery attenuated the development of cardiovascular hypertrophy, inflammation, fibrosis, and ventricular action potential prolongation. In contrast, omapatrilat treatment did not lower systolic blood pressure nor improve endothelial dysfunction. This study concludes that the renin-angiotensin-aldosterone, natriuretic peptide, and bradykinin systems are directly involved in the pathogenesis of cardiovascular remodeling in the DOCA-salt model of hypertension in rats, which may be independent of their effects on blood pressure.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Perindopril, an angiotensin converting enzyme inhibitor, in pulmonary hypertensive rats: comparative effects on pulmonary vascular structure and function

1 Hypoxic pulmonary hypertension in rats (10% O-2, 4 weeks) is characterized by changes in pulmonary vascular structure and function. The effects of the angiotensin converting enzyme inhibitor perindopril (oral gavage, once daily for the 4 weeks of hypoxia) on these changes were examined. 2 Perindopril (30 mg kg(-1) d(-1)) caused an 18% reduction in pulmonary artery pressure in hypoxic rats. 3 Structural changes (remodelling) in hypoxic rats included increases in (i) critical closing pressure in isolated perfused lungs (remodelling of arteries (50 mu m 0.d.) and (ii) medial wall thickness of intralobar pulmonary arteries, assessed histologically (vessels 30-100 and 101-500 mu m o.d.). Perindopril 10 and 30 mg kg(-1) d(-1) attenuated remodelling in vessels less than or equal to 100 mu m (lungs and histology), 30 mg kg(-1) d(-1) was effective in vessels 101-500 mu m but neither dose prevented hypertrophy of main pulmonary artery. 3 mg kg(-1) d(-1) was without effect. 4 Perindopril (30 mg kg(-1) d(-1)) prevented the exaggerated hypoxic pulmonary vasoconstrictor response seen in perfused lungs from hypoxic rats but did not prevent any of the functional changes (i.e. the increased contractions to 5-HT, U46619 (thromboxane-mimetic) and K+ and diminished contractions to angiotensins I and II) seen in isolated intralobar or main pulmonary arteries. Acetylcholine responses were unaltered in hypoxic rats. 5 We conclude that, in hypoxic rats, altered pulmonary vascular function is largely independent of remodelling. Hence any drug that affects only remodelling is unlikely to restore pulmonary vascular function to normal and, like perindopril, may have only a modest effect on pulmonary artery pressure.

Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappa B

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκBα levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκBα, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκBα from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK.

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2 = 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10-5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the 'chymotrypsin-like' enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign.

Non-aqueous silicone elastomer gels as a vaginal microbicide delivery system for the HIV-1 entry inhibitor maraviroc

Aqueous semi-solid polymeric gels, such as those based on hydroxyethylcellulose (HEC) and polyacrylic acid (e.g. Carbopol®), have a long history of use in vaginal drug delivery. However, despite their ubiquity, they often provide sub-optimal clinical performance, due to poor mucosal retention and limited solubility for poorly water-soluble actives. These issues are particularly pertinent for vaginal HIV microbicides, since many lead candidates are poorly water-soluble and where a major goal is the development of a coitally independent, once daily gel product. In this study, we report the use of a non-aqueous silicone elastomer gel for vaginal delivery of the HIV-1 entry inhibitor maraviroc. In vitro rheological, syringeability and retention studies demonstrated enhanced performance for silicone gels compared with a conventional aqueous HEC gel, while testing of the gels in the slug model confirmed a lack of mucosal irritancy. Pharmacokinetic studies following single dose vaginal administration of a maraviroc silicone gel in rhesus macaques showed higher and sustained MVC levels in vaginal fluid, vaginal tissue and plasma compared with a HEC gel containing the same maraviroc loading. The results demonstrate that non-aqueous silicone gels have potential as a formulation platform for coitally independent vaginal HIV microbicides.

Fixed-dose combination therapy for type 2 diabetes:sitagliptin plus pioglitazone

Type 2 diabetes is typically associated with insulin resistance and dysfunction of insulin-secreting pancreatic beta-cells. Addressing these defects often requires therapy with a combination of differently acting antidiabetic agents. A potential novel combination in development brings together the dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin with the thiazolidinedione pioglitazone into a fixed-dose single-tablet combination. The former component acts mainly to increase prandial insulin secretion; the latter improves insulin sensitivity.

Effect of dapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control with metformin:a randomised, double-blind, placebo-controlled trial

Correction of hyperglycaemia and prevention of glucotoxicity are important objectives in the management of type 2 diabetes. Dapagliflozin, a selective sodium-glucose cotransporter-2 inhibitor, reduces renal glucose reabsorption in an insulin-independent manner. We assessed the efficacy and safety of dapagliflozin in patients who have inadequate glycaemic control with metformin.

Interaction of peptides, peptidomimetics and other drug candidates with the DI/tripeptide transport system in intestinal epithelial cells, using the in vitro caco-2 cell culture system

An uptake system was developed using Caco-2 cell monolayers and the dipeptide, glycyl-[3H]L-proline, as a probe compound. Glycyl-[3H]L-proline uptake was via the di-/tripeptide transport system (DTS) and, exhibited concentration-, pH- and temperature-dependency. Dipeptides inhibited uptake of the probe, and the design of the system allowed competitors to be ranked against one another with respect to affinity for the transporter. The structural features required to ensure or increase interaction with the DTS were defined by studying the effect of a series of glycyl-L-proline and angiotensin-converting enzyme (ACE)-inhibitor (SQ-29852) analogues on the uptake of the probe. The SQ-29852 structure was divided into six domains (A-F) and competitors were grouped into series depending on structural variations within specific regions. Domain A was found to prefer a hydrophobic function, such as a phenyl group, and was intolerant to positive charges and H+ -acceptors and donors. SQ-29852 analogues were more tolerant of substitutions in the C domain, compared to glycyl-L-proline analogues, suggesting that interactions along the length of the SQ-29852 molecule may override the effects of substitutions in the C domain. SQ-29852 analogues showed a preference for a positive function, such as an amine group in this region, but dipeptide structures favoured an uncharged substitution. Lipophilic substituents in domain D increased affinity of SQ-29852 analogues with the DTS. A similar effect was observed for ACE-NEP inhibitor analogues. Domain E, corresponding to the carboxyl group was found to be tolerant of esterification for SQ-29852 analogues but not for dipeptides. Structural features which may increase interaction for one series of compounds, may not have the same effect for another series, indicating that the presence of multiple recognition sites on a molecule may override the deleterious effect of anyone change. Modifying current, poorly absorbed peptidomimetic structures to fit the proposed hypothetical model may improve oral bioavailability by increasing affinity for the DTS. The stereochemical preference of the transporter was explored using four series of compounds (SQ-29852, lysylproline, alanylproline and alanylalanine enantiomers). The L, L stereochemistry was the preferred conformation for all four series, agreeing with previous studies. However, D, D enantiomers were shown in some cases to be substrates for the DTS, although exhibiting a lower affinity than their L, L counterparts. All the ACE-inhibitors and β-lactam antibiotics investigated, produced a degree of inhibition of the probe, and thus show some affinity for the DTS. This contrasts with previous reports that found several ACE inhibitors to be absorbed via a passive process, thus suggesting that compounds are capable of binding to the transporter site and inhibiting the probe without being translocated into the cell. This was also shown to be the case for oligodeoxynucleotide conjugated to a lipophilic group (vitamin E), and highlights the possibility that other orally administered drug candidates may exert non-specific effects on the DTS and possibly have a nutritional impact. Molecular modelling of selected ACE-NEP inhibitors revealed that the three carbonyl functions can be oriented in a similar direction, and this conformation was found to exist in a local energy-minimised state, indicating that the carbonyls may possibly be involved in hydrogen-bond formation with the binding site of the DTS.