Purpose: Demonstrating efficacy of potential disease-modifying OA drugs (DMOAD) requires long treatment times and a large number of subjects, resulting in expensive clinical trials.An important risk of such studies is the absence of beneficial effect of the drug.Accurate prediction of the doses that would result in cartilage protection and joint preservation would help minimize this risk.Although dose may be extrapolated from pre-clinical studies, the lack of approved DMOADs obscures the clinical predictive capability of the OA animal models.Here we describe a translational pharmacology strategy using TIINE (a type II collagen neoepitope assay) to evaluate MMP-13 inhibitors for the treatment of OA Methods: The MMP-13 inhibitors belong to the class of compounds that bind the S 1 pocket of the enzyme providing good potency (Ki 2-6 nM) and selectivity (>4000 fold over 15 other MMPs-, ADAMTS-4 and -5, and TACE).TIINE was measured using a sandwich immunoassay and an LC-MS/MS assay specific for the 45-mer peptide.Rat medial meniscal tear (MMT) and dog partial medial meniscectomy (pMx) were performed at Bolder BioPath as previously described.All studies were approved by the Institutional Animal Care and Use Committees.Results: Studies in vitro showed that TIINE was generated by addition of active MMP-13 to de-vitalized cartilage in a time-and concentrationdependent manner, which was inhibited by MMP-13 inhibitors.Because TIINE was also generated by addition of other MMPs, this biomarker is not specific for MMP-13 activity.In vivo, TIINE was upregulated about two fold in the urine from OA patients in contrast to age-matched controls.To understand the role of MMP-13 in the generation of such OA-associated TIINE elevation, TIINE modulation was evaluated in animal models of OA, and in response to MMP-13 selective inhibitors.In the rat MMT, TIINE was elevated in the synovial fluid (SF) from the operated knee, but not the contralateral knee, and it was inhibited in a time-and dose-dependent manner following oral administration of an MMP-13 inhibitor to a maximum of ~50%.Similar TIINE inhibition in the SF and urine was observed at the end of a 4-wk study (BID, prophylactic), and maximal inhibition correlated with cartilage protection as evaluated by histology.All animals and human subjects evaluated to date excrete some amount of TIINE in their urine (which is age-dependent).Single dose oral administration of MMP-13 inhibitor to skeletally-mature beagle dogs (naive) inhibited baseline urinary TIINE in a time-and dose-dependent manner up to ~50%.In the 4-wk dog pMx model, MMP-13 inhibitors (BID, prophylactic) protected cartilage degeneration at exposures that inhibited TIINE ~45% at 2, 3 and 4 wks.These results suggest that doses that result in ~45% inhibition of TIINE in early clinical trials may protect cartilage degradation and joint structure in Phase 2-3 studies.In previous clinical studies with broader spectrum MMP inhibitors, urinary TIINE was found to be inhibited in both human healthy volunteers and OA patients.Conclusions: The proposed translational pharmacology strategy includes: (1) determine drug plasma and/or SF concentration required for maximum TIINE inhibition in urine of naive and/or OA animals, (2) establish correlation between TIINE inhibition and cartilage protection in animal models of OA, (3) evaluate TIINE inhibition in healthy volunteers and/or OA patients in early clinical trials, and (4) in POC studies, use the doses that resulted in sufficient inhibition of TIINE from the FIH studies and that was associated with cartilage protection in pre-clinical studies.

We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels (neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate, and lung cancer biopsy tissue. COX-1 is broadly distributed in normal, as well as in neoplastic, tissues. The contribution of COX-2 to human tumor growth was indicated by the ability of celecoxib, an agent that inhibits the COX-2 enzyme, to suppress growth of lung and colon tumors implanted into recipient mice. Mechanistically, celecoxib demonstrated a potent antiangiogenic activity. In a rat model of angiogenesis, we observe that corneal blood vessel formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These and other data indicate that COX-2 and COX-2-derived prostaglandins may play a major role in development of cancer through numerous biochemical mechanisms, including stimulation of tumor cell growth and neovascularization. The ability of celecoxib to block angiogenesis and suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer.

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.

Osteoclastic bone degradation requires intimacy between the matrix and the resorptive cell. While the precise role the integrin alpha(v)beta3 plays in the process is not yet understood, occupancy of the heterodimer by soluble ligand or by blocking antibody effectively inhibits bone resorption in vitro and in vivo, suggesting that alpha(v)beta3 blockade may prevent postmenopausal osteoporosis. Thus, we identified a synthetic chemical peptide mimetic, beta-[2-[[5-[(aminoiminomethyl)amino]-1-oxopentyl]amino]-1-+ ++oxoethyl]amino-3-pyridinepropanoic acid, bistrifluoroacetate (SC56631) based upon the alpha(v)beta3 ligand, Arg-Gly-Asp (RGD), which recognizes the isolated integrin, and its relative, alpha(v)beta5, as effectively as does the natural peptide. The mimetic dampens osteoclastic bone resorption in vitro and in vivo. Most importantly, intravenous administration of the mimetic prevents the 55% loss of trabecular bone sustained by rats within 6 wk of oophorectomy. Histological examination of bones taken from SC56631-treated, oophorectomized animals also demonstrates the compound's bone sparing properties and its capacity to decrease osteoclast number. Thus, an RGD mimetic prevents the rapid bone loss that accompanies estrogen withdrawal.

The cell surface integrin, alphaVbeta3, is important for the attachment of osteoclasts to bone matrix and the subsequent resorption of bone. The present study was designed to determine the effects of F11, a monoclonal antibody to the rat beta3 subunit, on calcium mobilization in a rat model of bone resorption. Male Sprague Dawley rats became hypocalcemic within 18 h after thyroparathyroidectomy. Synthetic PTH-related protein (PTHrP(1-34)) administered to control rats caused serum calcium to return to normal. Anti-beta3 treatment of rats after thyroparathyroidectomy inhibited the calcemic response to PTHrP by 65%. Circulating F11 was biologically active as demonstrated by osteoclast retraction and by the inhibition of adenosine diphosphate-induced platelet aggregation via inhibition of the platelet integrin alphaIIbbeta3 in ex vivo assays. F11 antibody was localized by immunohistological staining to osteoclasts in long bones, suggesting that the mechanism of action of the antibody was via a direct effect upon osteoclasts. Echistatin and calcitonin also inhibited calcemic responses to PTHrP in this in vivo model, whereas an isotype-matched, control antibody was ineffective. These studies provide the first direct evidence in vivo that osteoclast-mediated bone resorption is regulated via beta3 integrin.

Nitric oxide (NO) produced by the vascular endothelium is an endogenous contributor to the regulation of vascular relaxation and the maintenance of blood pressure. The effective half-life of NO and the relaxation of aortic rings by NO is enhanced by a reduction in the concentration of superoxide radicals with superoxide dismutase (SOD). In the current study, SC52608, a newly synthesized SOD mimic with a manganese core, was tested for its ability to potentiate the activity of NO both in vitro and in vivo. SC52608 relaxation of rat aortic segments was endothelium dependent as well as concentration dependent. The maximum relaxation following KCl contraction was 44% with 300 microM SC52608. Cyclic GMP concentrations in the segments were increased 1.6- and 3.2-fold with 5 and 300 microM SC52608, respectively. N-monomethyl-I-arginine pretreatment of aortic rings abolished the relaxation and cyclic GMP accumulation mediated by SC52608. In a smooth muscle cell reporter system of nitric oxide synthase activity, SC52608 potentiated the increase in cyclic GMP elicited by NO in a concentration-dependent manner with a maximum increase of 5.2-fold at 100 microM. Injection of SC52608 into conscious, restrained rats resulted in a dose-dependent decrease of blood pressure. Therefore, the data suggest that SC52608 potentiates the actions of nitric oxide on vascular tone, cyclic GMP, and blood pressure by enhancing the half-life of NO through a mechanism that mimics the action of SOD.

Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10-12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl-L-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl-L-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl-L-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase.

1 Endotoxin E. Coli lipopolysaccharide (LPS)‐treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo‐oxygenase (COX‐2) and nitric oxide synthase (iNOS) expression accounted for the LPS‐induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg−1) increased the plasma levels of nitrite/nitrate from 14 ± 1 to 84 ± 7 μm within 3 h and this rise was inhibited to 35 ± 1 μm by dexamethasone. Levels of 6‐keto PGF1α in the plasma were below the detection limit of the assay (< 0.2 ng ml−1). However, 3 h after the injection of LPS these levels rose to 2.6 ± 0.2 ng ml−1 and to 0.7 ± 0.01 ng ml−1 after LPS in rats that received dexamethasone.2 The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS‐treated animals. For instance, the LPS‐induced increased in plasma nitrite/nitrate and 6‐keto PGF1α at 3 h was decreased to 18 ± 2 μm and 0.5 ± 0.02 ng ml−1, 23 ± 1 μm and 0.7 ± 0.01 ng ml−1, 29 ± 2 μm and 1 ± 0.01 ng ml−1 in rats treated with LPS in the presence of the NOS inhibitors NG‐ monomethyl‐l‐arginine, NG‐nitro arginine methyl ester and aminoguanidine, respectively.3 The intravenous infusion of the NO donors sodium nitroprusside (SNP) or glyceryl trinitrate (GTN) increased prostaglandin production in normal animals (for instance urinary PGE2 excretion was increased from 96 ± 10 to 576 ± 12 pg min−1 and 400 ± 24 pg min−1 in the presence of GTN or SNP respectively).4 Proteinuria was measured in order to evaluate the roles of NO and PG in renal damage associated with the in vivo injection of LPS. Interestingly, dexamethasone and the NOS inhibitors attenuated proteinuria in the LPS‐treated rats. The COX inhibitors had no effect. It therefore appears that NO and not PG contributes to the LPS‐induced renal damage; these findings support the potential use of NOS inhibitors in the treatment of renal inflammation.5 This study demonstrates the regulatory contribution of NO on the in vivo production of prostanoids and suggests that in inflammatory diseases that are driven by both NO and the prostaglandins, NOS inhibitors may act to reduce inflammation by the dual inhibition of cytotoxic NO and pro‐inflammatory PG.

The half-life of nitric oxide (NO) and the relaxation of aortic rings are enhanced by superoxide dismutase. Manganese and manganese-containing preparations have been reported to mimic superoxide dismutase activity. In the current study, manganese was tested for its ability to potentiate the activity of NO both in vitro and in vivo. Manganese relaxation of aortic segments was endothelium dependent as well as concentration dependent. Cyclic GMP concentrations in the segments were increased 2- and 4-fold with 5 and 300 μM manganese, respectively. N-Monomethyl-L-arginine pretreatment of aortic rings abolished the relaxation and cyclic GMP accumulation mediated by manganese. Infusion of manganese into conscious, restrained rats resulted in a decrease of blood pressure which was abolished by N-nitro-L-arginine pretreatment. Therefore, manganese may prolong the half-life of NO by a mechanism that mimics the action of superoxide dismutase resulting in potentiation of NO actions in vascular tissue.

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.

1 The present studies compared the renal and hypotensive response to (a) exogenous atrial natriuretic peptide (ANP) (99-126), (b) an endopeptidase-24.11 inhibitor (candoxatrilat) and (c) an antagonist of ANP clearance receptors (SC 46542) in conscious rats.2 Infusion of low-dose-ANP (100 ng kg-1 min-1) produced a gradual increase in urinary sodium and guanosine 3':5'-cyclic monophosphate (cyclic GMP) excretion without significant change in glomerular filtration rate (GFR) or fractional lithium clearance (FeLi). There was a significant fall in blood pressure.3 Infusion of high-dose ANP (300 ng kg-1 min-1) produced a brisk, 3 fold increase in urinary sodium and cyclic GMP excretion along with a rise in GFR, but had no significant effect on FeLi compared to the control group. The renal response was accompanied by a pronounced fall in blood pressure.4 Candoxatrilat or SC 46542, alone, had no significant effect on sodium excretion compared to control animals. Both compounds enhanced the natriuretic and cyclic GMP responses to a low-dose ANP infusion (100 ng kg-1 min-1) to levels similar to, or greater than, those observed with the high-dose ANP (300 ng kg-1 min-1). However, unlike high-dose ANP, these renal effects were not accompanied by a significant change in GFR and neither compound potentiated the hypotensive effect of the low-dose ANP infusion. Only candoxatrilat when given with ANP produced a marked rise in FeLi.5 Similarly, combined administration of candoxatrilat and SC 46542 (without exogenous ANP) induced an increase in sodium and cyclic GMP excretion comparable to high-dose ANP but did so without a significant increase in GFR and with a significantly smaller fall in blood pressure. Interestingly, there was no increase in FeLi with the combination of the two compounds, suggesting that the major contribution to sodium excretion came from SC 46542.6 Both candoxatrilat and SC 46542 increased sodium and cyclic GMP excretion in the rat A-V fistula model of heart failure, a model hyporesponsive to infusions of ANP, without significant change in blood pressure.7 These data show that candoxatrilat and SC 46542 do not simply reproduce the effects of an ANP infusion but preferentially enhance the natriuretic response to ANP. Inhibition of E-24.11 may potentiate a tubule action of ANP while the renal mechanism of action of the C-ANP receptor ligand needs further study. Both manipulations are of potential value in the management of heart failure.

1. The observation that the area postrema expresses a high level of nerve-growth-factor-receptor immuno-reactivity prompted an investigation of the effects of nerve growth factor on autonomic function in the rat.2. Bolus injection of pharmacological doses of the peptide via the femoral vein led to reproducible, dose-dependent falls in blood pressure.3. Administration via the vertebral artery, a more direct route to the brainstem, did not appear to lower the threshold dose required to induce hypotension. Furthermore, pretreatment with hexamethonium did not inhibit the hypotensive response to nerve growth factor.4. Administration of a second dose of nerve growth factor after recovery from the first injection produced little or no fall in blood pressure. Similarly, pretreatment with the mast cell degranulating agent, compound 48/80, rendered the animal refractory to nerve growth factor.5. The fall in blood pressure induced by nerve growth factor was markedly attenuated by pretreatment with chlorpheniramine.6. It is concluded that the fall in blood pressure induced by intravenous administration of pharmacological doses of nerve growth factor is mediated by vasoactive substances, particularly histamine, released from mast cells.