TRV734, an oral G-protein biased ligand at the μ-opioid receptor has demonstrated differentiated pharmacology in preclinical studies compared to unbiased ligands. First-time-in-human data suggested that TRV734 was safe and well tolerated and caused effective pain relief after single doses of 150 to 250 mg. In this study, safety and tolerability of multiple ascending doses of TRV734, and single doses of TRV734 125 mg following various administration paradigms, in healthy subjects were evaluated. In both parts of the study, TRV734 was generally well tolerated with no serious adverse events. Pharmacokinetics of TRV734 were similar when TRV734 125 mg was administered following a high-fat or standard meal. Compared to either of the fed conditions, maximum concentration and area under the plasma concentration-time curve did not change, and time to maximum concentration was 1.5 hours later when TRV734 125 mg was administered as 3 split portions over 120 minutes under fasted conditions. Split doses of TRV734 delayed time to peak decrease in pupil diameter. Following multiple-dose administration of TRV734 60 to 175 mg every 6 hours, there was a trend of slightly less-than-dose proportional increase of maximum concentration, and area under the plasma concentration-time curve and accumulation was modest. Time to maximum concentration was ≈1 to 2 hours and elimination half-life ≈1.9 to 2.5 hours. The analgesic effect of TRV734 on the cold pain test was generally dose proportional and similar to that of oxycodone 10 mg immediate release, after both the first and last doses. There was a dose-related decrease in pupil diameter following administration of TRV734 up to TRV734 125 mg every 6 hours. A favorable trend in bowel function index for TRV734 warrants continued study.

TRV734 is an orally bioavailable G‐protein–biased ligand at the μ‐opioid receptor. In nonclinical studies it was potently analgesic while causing less gastrointestinal dysfunction than morphine, suggesting unique benefits in acute pain management. A 2‐part, first‐in‐human study was conducted with ascending doses of TRV734 to explore its tolerability, pharmacokinetics, and pharmacodynamics in healthy volunteers. TRV734 was well tolerated over the dose range 2 to 250 mg when administered orally. Plasma TRV734 maximum concentration and area under the plasma concentration–time curve generally increased with dose, while time to maximum concentration was similar across doses (0.5–1.3 h). The half‐life increased with dose from 10 mg through 150 mg (0.75–2.28 h) but was similar from 150 mg through 250 mg. Pupil constriction, confirming central nervous system μ‐opioid receptor engagement, correlated with higher plasma TRV734 concentrations; the greatest reductions in pupil diameter occurring between 0 and 4 hours after dosing (–2.9 mm/h, with reduction peaking at 1 hour, and returning to baseline by 8 hours). Following administration of TRV734 125 mg under fasted or fed conditions, there was no significant difference in bioavailability when given as a solution or drug in capsule to fasted subjects. When drug in capsule was given to subjects following a high‐fat meal, absorption was slowed, resulting in decreased peak concentrations, but area under the plasma concentration–time curve was not affected.

The delta opioid receptor (DOR) has been identified as a therapeutic target for migraine, with DOR agonists exhibiting low abuse potential compared with conventional µ-opioid agonists. TRV250 is a novel small molecule agonist of the DOR that is preferentially selective for G-protein signaling, with relatively little activation of the β-arrestin2 post-receptor signaling pathway. This selectivity provides reduced susceptibility to proconvulsant activity seen with non-selective DOR agonists. TRV250 significantly reduced nitroglycerin-evoked hyperalgesia in rodents, indicating a potential utility in acute migraine without the risk of seizure activity or abuse potential. This trial evaluated the safety, tolerability, and pharmacokinetics of ascending dose levels of TRV250 administered subcutaneously (SC) and the relative bioavailability of TRV250 administered orally compared with SC administration. This was a two-part, single ascending dose study. Part A included four cohorts of healthy adults (N = 38). Each cohort was dosed on three occasions (placebo and two different dose levels of TRV250, allocated in randomized order and administered by SC route). In Part B, a single cohort of nine subjects received an oral dose of either TRV250 (n = 7) or placebo (n = 2) in a fed or fasted state. Serial blood samples were obtained for pharmacokinetic determination across a 24-h post-dose period. Safety assessments included clinical laboratory measures, vital signs, 12-lead electrocardiogram (ECG), and electroencephalogram (EEG) pre- and post-dosing. TRV250 was well tolerated. There were no serious adverse events (SAEs), and all AEs were mild in severity. Injection-site reactions and headache were the most common AEs. One subject was withdrawn from the study due to a TRV250-related AE of postural orthostatic tachycardia. There were no clinically relevant changes in physical examination, hematology, clinical chemistry, urinalysis, suicidal ideation, or vital signs, with the exception of orthostatic changes in some subjects. No subject experienced abnormalities in EEGs or experienced a change from baseline in heart-rate-corrected QT interval (QTcF) > 60 ms, or an absolute QTcF interval > 480 ms at any post-dosing observation. Peak and total plasma exposure to TRV250 increased in a dose-proportional manner following 0.1–30 mg SC doses, with the mean half-life ranging from 2.39 to 3.76 h. Oral bioavailability of TRV250 ranged from 14% (fasting) to 19% (fed) relative to SC dosing, while administration with food increased the AUC but decreased the rate of absorption as reflected by a modest delay in median time to maximum concentration and a slight reduction in maximum concentration. The findings from the first-in-human study support further evaluation of TRV250, a G-protein selective DOR agonist, in the treatment of acute migraine.

Opioids provide powerful analgesia but also efficacy-limiting adverse effects, including severe nausea, vomiting, and respiratory depression, by activating μ-opioid receptors. Preclinical models suggest that differential activation of signaling pathways downstream of these receptors dissociates analgesia from adverse effects; however, this has not yet translated to a treatment with an improved therapeutic index. Thirty healthy men received single intravenous injections of the biased ligand TRV130 (1.5, 3, or 4.5mg), placebo, or morphine (10mg) in a randomized, double-blind, crossover study. Primary objectives were to measure safety and tolerability (adverse events, vital signs, electrocardiography, clinical laboratory values), and analgesia (cold pain test) versus placebo. Other measures included respiratory drive (minute volume after induced hypercapnia), subjective drug effects, and pharmacokinetics. Compared to morphine, TRV130 (3, 4.5mg) elicited higher peak analgesia (105, 116 seconds latency vs 75 seconds for morphine, P<.02), with faster onset and similar duration of action. More subjects doubled latency or achieved maximum latency (180 seconds) with TRV130 (3, 4.5mg). Respiratory drive reduction was greater after morphine than any TRV130 dose (-15.9 for morphine versus -7.3, -7.6, and -9.4 h*L/min, P<.05). More subjects experienced severe nausea after morphine (n=7) than TRV130 1.5 or 3mg (n=0, 1), but not 4.5mg (n=9). TRV130 was generally well tolerated, and exposure was dose proportional. Thus, in this study, TRV130 produced greater analgesia than morphine at doses with less reduction in respiratory drive and less severe nausea. This demonstrates early clinical translation of ligand bias as an important new concept in receptor-targeted pharmacotherapy.

OBJECTIVE:Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha.METHODS:Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6.RESULTS:In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation.CONCLUSION:Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Patients treated with recombinant human Epo demonstrate an improvement in insulin sensitivity. We aimed to investigate whether CNTO 530, a novel Epo receptor agonist, could affect glucose tolerance and insulin sensitivity. A single administration of CNTO 530 significantly and dose-dependently reduced the area under the curve in a glucose tolerance test in diet-induced obese and diabetic mice after 14, 21, and 28 days. HOMA analysis suggested an improvement in insulin sensitivity, and this effect was confirmed by a hyperinsulinemic-euglycemic clamp. Uptake of (14)C-2-deoxy-D-glucose indicated that animals dosed with CNTO 530 transported more glucose into skeletal muscle and heart relative to control animals. In conclusion, CNTO530 has a profound effect on glucose tolerance in insulin-resistant rodents likely because of improving peripheral insulin sensitivity. This effect was observed with epoetin-α and darbepoetin-α, suggesting this is a class effect, but the effect with these compounds relative to CNTO530 was decreased in duration and magnitude.

Cathepsin K is an osteoclast-derived cysteine protease that has been implicated as playing a major role in bone resorption. A substantial body of evidence indicates that cathepsin K is critical in osteoclast-mediated bone resorption and suggests that its pharmacological inhibition should result in inhibition of bone resorption in vivo. Here we report the pharmacological characterization of SB-462795 (relacatib) as a potent and orally bioavailable small molecule inhibitor of cathepsin K that inhibits bone resorption both in vitro in human tissue and in vivo in cynomolgus monkeys. SB-462795 is a potent inhibitor of human cathepsins K, L, and V (K(i, app)=41, 68, and 53 pM, respectively) that exhibits 39-300-fold selectivity over other cathepsins. SB-462795 inhibited endogenous cathepsin K in situ in human osteoclasts and human osteoclast-mediated bone resorption with IC50 values of approximately 45 nM and approximately 70 nM, respectively. The anti-resorptive potential of SB-462795 was evaluated in normal as well as medically ovariectomized (Ovx) female cynomolgus monkeys. Serum levels of the C- and N-terminal telopeptides of Type I collagen (CTx and NTx, respectively) and urinary levels of NTx were monitored as biomarkers of bone resorption. Administration of SB-462795 to medically ovariectomized or normal monkeys resulted in an acute reduction in both serum and urinary markers of bone resorption within 1.5 h after dosing, and this effect lasted up to 48 h depending on the dose administered. Our data indicate that SB-462795 potently inhibits human cathepsin K in osteoclasts, resulting in a rapid inhibition of bone resorption both in vitro and in vivo in the monkey. These studies also demonstrate the therapeutic potential of relacatib in the treatment of postmenopausal osteoporosis and serves to model the planned clinical trials in human subjects.

Although bone is composed primarily of extracellular matrix (ECM), the dynamic role that the ECM plays in regulating bone remodeling secondary to estrogen loss is relatively unexplored. Previous studies have shown that mice deficient in the matricellular protein thrombospondin-2 (TSP2-null) form excess endocortical bone; thus, we postulated that enhanced bone formation in TSP2-null mice could protect against ovariectomy (OVX)-induced bone loss. Wild-type (WT) OVX mice showed a significant loss of both midfemoral endocortical and proximal tibial trabecular bone, but OVX did not significantly alter TSP2-null bone. TSP2-null mice showed an increase in bone formation, as indicated by a 70% increase in serum osteocalcin two weeks post OVX and a two-fold increase in bone formation rate (BFR) five weeks post OVX as measured by dynamic histomorphometry. WT animals showed only a 20% increase in serum osteocalcin at two weeks and no change in BFR at five weeks. This increase in bone formation in TSP2-null OVX mice was accompanied by a three-fold increase in osteoprogenitor number. Although these results provide a partial explanation for the maintenance of bone geometry post-OVX, TSP2-null mice five weeks post-OVX also showed a significantly lower level of bone resorption than OVX WT mice, as determined by serum levels of the amino-terminal telopeptide of type I collagen (NTx). We conclude that the absence of TSP2 protects against OVX-induced bone loss by two complementary processes: increased formation and decreased resorption.

The extension of a previously reported cathepsin K azepanone-based inhibitor template to the design and synthesis of potent and selective inhibitors of the homologous cysteine protease cathepsin L is detailed. Structure-activity studies examining the effect of inhibitor selectivity as a function of the P3 and P2 binding elements of the potent cathepsin K inhibitor 1 revealed that incorporation of either a P3 quinoline-8-carboxamide or a naphthylene-1-carboxamide led to increased selectivity for cathepsin L over cathepsin K. Substitution of the P2 leucine of 1 with either a phenylalanine or a beta-naphthylalanine also resulted in an increased selectivity for cathepsin L over cathepsin K. Molecular modeling studies with the inhibitors docked within the active sites of both cathepsins L and K have rationalized the observed selectivities. Optimization of cathepsin L binding by the combination of the P3 naphthylene-1-carboxamide with the P2 beta-naphthylalanine provided 15, which is a potent, selective, and competitive inhibitor of human cathepsin L with a K(i) = 0.43 nM.

Objective: It has been suggested that chondrocyte death by apoptosis may play a role in the pathogenesis of cartilage destruction in osteoarthritis, but the results of in-vivo and in-vitro investigations have been conflicting. To investigate further the cell death in our in-vitro model for traumatic joint injury, we performed a quantitative analysis by electron microscopy (EM) of cell morphology after injurious compression. For comparison, the TUNEL assay was also performed.Design: Articular cartilage explant disks were harvested from newborn calf femoropatellar groove. The disks were subjected to injurious compression (50% strain at a strain rate of 100%/s), incubated for 3 days, and then fixed for quantitative morphological analysis.Results: By TUNEL, the cell apoptosis rate increased from 7+/-2% in unloaded controls to 33+/-6% after injury (P=0.01; N=8 animals). By EM, the apoptosis rate increased from 5+/-1% in unloaded controls to 62+/-10% in injured cartilage (P=0,02, N=5 animals). Analysis by EM also identified that of the dead cells in injured disks, 97% were apoptotic by morphology.Conclusions: These results confirm a significant increase in cell death after injurious compression and suggest that most cell death observed here was by an apoptotic process. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.

OBJECTIVETraumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation.METHODSArticular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1alpha [IL-1alpha] or tumor necrosis factor alpha [TNFalpha]).RESULTSDuring the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n = 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1alpha or 100 ng/ml TNFalpha caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1alpha and TNFalpha, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1alpha.CONCLUSIONThe cytokines IL-1alpha and TNFalpha can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.

Pharmacokinetic evaluation of the potent azepanone-based cathepsin K inhibitor 3 showed that it has an oral bioavailability of 42% in the rat and 4.8% in the monkey. The less than optimal oral bioavailabilities of 3 in the rat and the monkey precluded this analogue from being subjected to more detailed pharmacokinetic and pharmacodynamic analyses. In vitro and in vivo studies aimed at identifying the mechanisms which may be limiting the bioavailability of 3 in these species served to guide the syntheses of subsequent analogues for further evaluation. These studies have led to the identification of azepanone 6 that possesses improved oral bioavailability in both the rat (66.3%) and the monkey (23.4%).

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (Ki = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-{3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl}-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (Ki = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (Ki = 100 nmol/L), L (0.48 nmol/L), or S (Ki = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC50 ∼ 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC50 ∼ 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (μCT; ∼60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.