Translational development of an ADAMTS-5 antibody for osteoarthritis disease modification.

OBJECTIVE/METHOD: Aggrecanase activity, most notably ADAMTS-5, is implicated in pathogenic cartilage degradation. Selective monoclonal antibodies (mAbs) to both ADAMTS-5 and ADAMTS-4 were generated and in vitro, ex vivo and in vivo systems were utilized to assess target engagement, aggrecanase inhibition and modulation of disease-related endpoints with the intent of selecting a candidate for clinical development in osteoarthritis (OA). RESULTS: Structural mapping predicts the most potent mAbs employ a unique mode of inhibition by cross-linking the catalytic and disintegrin domains. In a surgical mouse model of OA, both ADAMTS-5 and ADAMTS-4-specific mAbs penetrate cartilage following systemic administration, demonstrating access to the anticipated site of action. Structural disease modification and associated alleviation of pain-related behavior were observed with ADAMTS-5 mAb treatment. Treatment of human OA cartilage demonstrated a preferential role for ADAMTS-5 inhibition over ADAMTS-4, as measured by ARGS neoepitope release in explant cultures. ADAMTS-5 mAb activity was most evident in a subset of patient-derived tissues and suppression of ARGS neoepitope release was sustained for weeks after a single treatment in human explants and in cynomolgus monkeys, consistent with high affinity target engagement and slow ADAMTS-5 turnover. CONCLUSION: This data supports a hypothesis set forth from knockout mouse studies that ADAMTS-5 is the major aggrecanase involved in cartilage degradation and provides a link between a biological pathway and pharmacology which translates to human tissues, non-human primate models and points to a target OA patient population. Therefore, a humanized ADAMTS-5-selective monoclonal antibody (GSK2394002) was progressed as a potential OA disease modifying therapeutic.

Quantitation OF ARGS aggrecan fragments in synovial fluid, serum and urine from osteoarthritis patients.

OBJECTIVE: To characterise ARGS neoepitope concentrations in various matrices from patients with knee osteoarthritis (OA) and assess performance of an immunoassay to facilitate clinical development of therapeutics affecting the A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) pathway. DESIGN: Matched sera, urine, and synovial fluid (SF) (surgical subjects only) were collected from healthy subjects, subjects with knee OA (non-surgical OA), and OA subjects undergoing total knee replacement (OA-TKR; n = 20 per group). Diurnal and inter-day variation was evaluated in the non-surgical OA group over 3 separate visits. Serum and urine samples were collected on two visits for the OA-TKR group with SF taken only at the time of surgery. ARGS neoepitope was quantitated using an optimized immunoassay. RESULTS: Serum ARGS neoepitope concentrations were elevated in OA-TKR subjects compared to non-surgical OA subjects (P = 0.005) and healthy subjects (P = 0.0002). Creatinine corrected urinary ARGS neoepitope concentrations were more variable, but were also elevated in the OA-TKR subjects compared to healthy subjects (P = 0.008). No significant diurnal effect or inter-day variance was observed in serum or urine. Serum ARGS neoepitope concentrations correlated with age (P = 0.0252) but not with total number of joints with OA involvement. SF ARGS neoepitope concentrations correlated with Western Ontario and MacMaster OA Index (WOMAC) stiffness score (P = 0.04) whereas a weaker, non-significant trend towards positive correlation with combined WOMAC score and the number of concurrent joints was observed. CONCLUSIONS: This study utilized a sensitive and robust assay to evaluate ARGS neoepitope concentrations in various matrices in OA patients and healthy volunteers. ARGS neoepitope appears promising as a prognostic/stratification marker to facilitate patient selection and as an early pharmacodynamic marker for OA therapeutic trials.

Evidence for reversible sequestration of morphine in rat liver.

The residence of morphine in the systemic circulation is prolonged despite a high systemic clearance, suggestive of significant extravascular sequestration. The present study was conducted to test the hypothesis that morphine binds significantly in tissues, and that the liver plays an important role in morphine binding. [14C]Morphine was administered to male Sprague-Dawley rats 55 min before unlabeled morphine or saline. Blood 14C increased immediately after injection of unlabeled morphine; the area under the blood concentration-time curve (AUC) for 14C increased approximately 2-fold after morphine compared with saline injection. Residual radioactivity in the liver was lower in morphine-treated rats than in controls, suggesting that unlabeled drug displaced [14C]morphine (or a metabolite) from binding sites. To examine this phenomenon more directly, a recirculating isolated perfused liver system was employed. [14C]Morphine was added to the perfusate reservoir 15 min before unlabeled morphine or saline; perfusate and bile samples were collected for 120 min. Upon termination of perfusion, the liver was fractionated to identify the hepatic subcellular fraction(s) in which morphine was sequestered. The perfusate AUC for [14C]morphine was increased approximately 2-fold in response to unlabeled drug, consistent with the in vivo experiment. Morphine was associated preferentially with the cytosolic fraction, and [14C]morphine in all relevant fractions was reduced after administration of unlabeled morphine. In contrast, unlabeled drug had no influence on derived [14C]morphine-3-beta,D-glucuronide. These data are consistent with significant, reversible binding of morphine in hepatic tissue.

Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation.

P-glycoprotein (P-gp) is a cell membrane-associated protein that transports a variety of drug substrates. Although P-gp has been studied extensively as a mediator of multidrug resistance in cancer, only recently has the role of P-gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined. P-glycoprotein is present in organ systems that influence drug absorption (intestine), distribution to site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues. Many marketed drugs inhibit P-gp function, and several compounds are under development as P-gp inhibitors. Similarly, numerous drugs can induce P-gp expression. While P-gp induction does not have a therapeutic role, P-gp inhibition is an attractive therapeutic approach to reverse multidrug resistance. Clinicians should recognize that P-gp induction or inhibition may have a substantial effect on the pharmacokinetics and pharmacodynamics of concomitantly administered drugs that are substrates for this transporter.

A pilot study of estrogen's effects on bronchial myocyte adhesion molecule expression.

We examined the effects of estrogen on tumor necrosis factor alpha (TNF-alpha)-induced expression of intracellular adhesion molecule (ICAM-1) and vascular adhesion molecule (VCAM-1) in cultured human bronchial smooth muscle cells (BSMC). Experiments were performed in triplicate in T-75 tissue culture flasks containing normal human BSMC. Four experiments were carried out: untreated BSMC cells (control); TNF-alpha 1000 U/ml stimulation of BSMC; forskolin 5 microM before TNF-alpha stimulation of BSMC; and estradiol 30 microM before TNF-alpha stimulation of BSMC. Cyclic adenosine monophosphate was measured by a commercially available radioimmunoassay kit. Cell expression of ICAM-1 and VCAM-1 was quantified by flow cytometry Incubation of cells with TNF-alpha 1000 U/ml for 24 hours elicited a 27-fold increase in basal expression of ICAM-1 and a 2-fold increase in VCAM-1 (p>0.05). Incubation of BSMC with forskolin 5 microM, for 1 hour before TNF-alpha, decreased TNF-alpha-induced expression of ICAM-1 by 62% and VCAM-1 slightly by 17%. The BSMC incubated with estradiol 30 microM, 1 hour before TNF-alpha, decreased TNF-alpha-induced expression of ICAM-1 by 21%; VCAM-1 remained unchanged (p>0.05). We found a trend toward inhibition of TNF-alpha-stimulated ICAM-1 expression in cultured BSMC with pretreatment with estradiol. However, due to large variability within the cell culture model, statistical significance was not reached.

Pharmacokinetics of flunisolide administered via metered dose inhaler with and without a spacer device and following oral administration.

BACKGROUND: After inhalation of a glucocorticoid from a meter dose inhaler (MDI), a certain portion of the delivered dose is deposited in the lungs, and the remainder is deposited in the oropharynx. OBJECTIVE: To examine the absolute bioavailability of flunisolide given orally via metered dose inhaler, and metered dose inhaler with a commercially available spacer device as well as to determine the fraction of drug deposited in the lungs following inhalation. METHODS: Twenty-four healthy volunteers were enrolled in the study; twenty-two completed the study. The IRB approved the study protocol, and informed consent was obtained. Volunteers received four treatments: treatment A (MDI), 1.0 mg inhaled flunisolide; treatment B (MDI-S), 1.0 mg inhaled flunisolide with a spacer device; treatment C, 1.0 mg of orally administered flunisolide with 240 mL of water; and treatment D, 1.0 mg intravenous flunisolide by IV push in the antecubital vein over 60 seconds. Plasma and urine flunisolide were quantified by HPLC/mass spectrometry/mass spectrometry. RESULTS: Flunisolide is a corticosteroid with low oral bioavailability (6.7%), which was found to be lower than previously reported. Similar AUCs were observed between the MDI and MDI-S groups, but by using mass balance equations, it appears that more flunisolide was delivered to the lungs in the MDI-S group (410 microg versus 280 microg). Oropharyngeal deposition was an important difference between the two inhaler groups. Approximately an 11-fold reduction in the oropharyngeal deposition of flunisolide through use of the spacer device was observed. CONCLUSIONS: Use of a spacer device improved pulmonary delivery of flunisolide by almost 50% and significantly decreased the oropharyngeal exposure to drug.