Effects of odanacatib on bone-turnover markers in osteoporotic postmenopausal women: a post hoc analysis of the LOFT study.

This post hoc analysis and modeling study examined the mechanism of action of odanacatib using a statistical model to explain sCTx response in ODN-treated patients as a function of other bone-turnover biomarkers that, with other observed biomarker changes, showed that odanacatib persistently inhibited osteoclastic bone removal activity without preventing osteoclastogenesis. INTRODUCTION: Odanacatib (ODN) is an oral selective cathepsin K (CatK) inhibitor, previously in development for osteoporosis treatment. A post hoc analysis examined ODN's mechanism of action on bone-turnover biomarkers. METHODS: A subset of patients who completed 60 months' treatment in the Long-Term Odanacatib Fracture Trial (LOFT; NCT00529373) (N = 112 [57 ODN, 55 placebo]) were evaluated. Serum (s) and urine (u) samples were assayed at baseline and months 6-60 for 10 known bone-remodeling biomarkers: sCTx, uαα- and ußßCTx/Cr, uNTx/Cr, sNTx, uDPD/Cr, sICTP, sTRAP5b, sPINP, and sBSAP. Because the CrossLaps® CTx assay identifies the CTx peptide as well as larger molecular weight CTx-containing peptides, including ICTP, a best-fit model was developed to explain the transient sCTx reduction in ODN-treated patients. RESULTS: ODN persistently reduced the bone-resorption markers sNTx, uNTx/Cr, uαα- and ußßCTx/Cr, and uDPD/Cr, and gradually increased the target-engagement marker sICTP and osteoclast number (sTRAP5b), versus placebo from baseline to month 60. sCTx was transiently reduced with ODN within 12 months, returning to baseline by month 48. Modeling suggested that sCTx changes in the ODN group were primarily due to increased accumulation of larger CTx species, including sICTP. The bone-formation markers sPINP and sBSAP showed partial reductions, versus placebo, in the first 6 months but approached baseline by months 48-60. CONCLUSION: Observed changes in bone-turnover biomarkers support the persistent efficacy of ODN in direct inhibition of osteoclastic bone-resorption activity, without inhibition of osteoclastogenesis. Long-term evaluation also underscores the unique mechanism of ODN on osteoclastic collagen processing and subsequently osteoblastic bone formation. TRIAL REGISTRATION: NCT00529373.

Evaluation of Therapeutics for Advanced-Stage Heart Failure and Other Severely-Debilitating or Life-Threatening Diseases.

Severely-debilitating or life-threatening (SDLT) diseases include conditions in which life expectancy is short or quality of life is greatly diminished despite available therapies. As such, the medical context for SDLT diseases is comparable to advanced cancer and the benefit vs. risk assessment and development of SDLT disease therapeutics should be similar to that of advanced cancer therapeutics. A streamlined development approach would allow patients with SDLT conditions earlier access to therapeutics and increase the speed of progression through development. In addition, this will likely increase the SDLT disease therapeutic pipeline, directly benefiting patients and reducing the economic and societal burden of SDLT conditions. Using advanced-stage heart failure (HF) as an example that illustrates the concepts applicable to other SDLT indications, this article proposes a streamlined development paradigm for SDLT disease therapeutics and recommends development of aligned global regulatory guidance.

Validation of a microdose probe drug cocktail for clinical drug interaction assessments for drug transporters and CYP3A.

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.

Effect of the cathepsin K inhibitor odanacatib on bone resorption biomarkers in healthy postmenopausal women: two double-blind, randomized, placebo-controlled phase I studies.

Inhibition of cathepsin K (CatK) is a potential new treatment for osteoporosis. In two double-blind, randomized, placebo-controlled phase I studies, postmenopausal female subjects received odanacatib (ODN), an orally active, potent, and selective CatK inhibitor, once weekly for 3 weeks or once daily for 21 days. Bone turnover biomarkers, safety monitoring, and plasma ODN concentrations were assessed. These studies showed ODN to be well tolerated. Pharmacokinetic (PK) analysis revealed a long half-life (t(1/2); 66-93 h) consistent with once-weekly dosing. Pronounced reductions in C-terminal telopeptide of type I collagen (approximately 62%) and N-terminal telopeptide of type I collagen normalized to creatinine (NTx/Cr) (approximately 62%) at trough (C(168 h)) were seen following weekly administration. Robust reductions in CTx (up to 81%) and NTx/Cr (up to 81%) were seen following daily administration. ODN exhibits robust and sustained suppression of bone resorption biomarkers (CTx and NTx/Cr) at weekly doses > or = 25 mg and daily doses > or = 2.5 mg.

Effect of different durations of ketoconazole dosing on the single-dose pharmacokinetics of midazolam: shortening the paradigm.

Given the prominent role of cytochrome P450 3A (CYP3A) in the metabolism of drugs, it is critical to determine whether new chemical entities will be affected by the inhibition of this enzyme system and result in clinically relevant drug interactions. Ketoconazole interaction studies are frequently performed to determine a given compound's sensitivity to CYP3A metabolism. The present study evaluated whether probing a sensitive substrate (midazolam) with a potent inhibitor (ketoconazole) at earlier time points (days 1 or 2) might be used to reliably gauge the magnitude of a meaningful interaction. The geometric mean ratios (ketoconazole+midazolamday 5/ketoconazole+midazolamday 1 and ketoconazole+midazolamday 5/ketoconazole+midazolamday 2) for midazolam AUC0-infinity were 1.36 and 1.06 with corresponding 90% confidence intervals of (1.17, 1.57) and (0.83, 1.23), respectively. These findings suggest that short-term drug-drug interaction studies can predict the magnitude of change in AUC as reliably as studies using longer duration treatments.

Cathepsin K inhibitors: a novel target for osteoporosis therapy.

Osteoporosis is characterized by low bone mass with skeletal fragility and an increased risk of fracture. This bone loss is brought about by an imbalance between bone resorption and formation. Cathepsin K is the most abundant cysteine protease expressed in the osteoclast and is believed to be instrumental in bone matrix degradation necessary for bone resorption. Cathepsin K inhibitors represent a novel target for developing agents to treat osteoporosis and other disorders characterized by increased bone resorption.

Effect of L-000845704, an alphaVbeta3 integrin antagonist, on markers of bone turnover and bone mineral density in postmenopausal osteoporotic women.

The alphaVbeta3 integrin (vitronectin receptor) plays a pivotal role in bone resorption. We hypothesized that L-000845704, an alphaVbeta3 integrin antagonist, would potently inhibit bone resorption, thereby increasing bone mass as assessed by bone mineral density (BMD) in women with postmenopausal osteoporosis. In a multicenter, randomized, double-blind, placebo-controlled, 12-month study, 227 women (average 63 yr) with low lumbar spine or femoral neck BMD were randomly assigned to receive 100 or 400 mg L-000845704 once daily (qd), 200 mg L-000845704 twice daily (bid), or placebo. L-000845704 increased lumbar spine BMD (2.1, 3.1, and 3.5% for the 100-mg-qd, 400-mg-qd, and 200-mg-bid treatment groups, respectively, vs. -0.1% for placebo; P < 0.01 all treatments vs. placebo). Only 200 mg L-000845704 bid significantly increased BMD at the hip (1.7 vs. 0.3% for placebo; P < 0.03) and femoral neck (2.4 vs. 0.7% for placebo; P < 0.05). No L-000845704 group increased total body BMD. All doses of L-000845704 resulted in a similar approximately 42% decrease from baseline of N-telopeptide cross-links (P < 0.001 vs. placebo). L-000845704 was generally well tolerated; adverse events resulting in discontinuation from the study were relatively infrequent. In conclusion, the antiresorptive effect of the alphaVbeta3 integrin antagonist L-000845704 translated into significant increases in lumbar spine BMD. Furthermore, 200 mg L-000845704 bid provided efficacy at the hip sites. These data suggest that the alphaVbeta3 integrin antagonist L-000845704 could be developed as an effective therapeutic agent for osteoporosis.

Bone loss in men with prostate cancer treated with gonadotropin-releasing hormone agonists.

Prostate cancer is the most common visceral malignancy in men. As the tumor is testosterone dependent, a frequent treatment modality involves therapy with GnRH agonists (GnRH-a) resulting in hypogonadism. Because testosterone is essential for the maintenance of bone mass in men, we postulated that GnRH-a therapy would negatively impact skeletal integrity. We compared bone mineral density (BMD), biochemical markers of bone turnover, and body composition in 60 men with prostate cancer (19 men receiving GnRH-a therapy and 41 eugonadal men) and BMD in 197 community-living healthy controls of similar age. BMD was assessed by dual energy x-ray absorptiometry and ultrasound. Biochemical markers of bone turnover, included markers of bone resorption (urinary N-telopeptide) and bone formation markers (bone-specific alkaline phosphatase and osteocalcin). Body composition (total body fat and lean body mass) was assessed by dual energy x-ray absorptiometry. Significantly lower BMD was found at the lateral spine (0.69 +/- 0.17 vs. 0.83 +/- 0.20 g/cm(2); P < 0.01), total hip (0.94 +/- 0.14 vs. 1.05 +/- 0.16 g/cm(2); P < 0.05), and forearm (0.67 +/- 0.11 vs. 0.78 +/- 0.07 g/cm(2); P < 0.01) in men receiving GnRH-a compared with the eugonadal men with prostate cancer. Significant differences were also seen at the total body, finger, and calcaneus (all P < 0.01). BMD values in eugonadal men with prostate cancer and healthy controls were similar. Markers of bone resorption (urinary N-telopeptide) and bone formation (bone-specific alkaline phosphatase) were elevated in men receiving GnRH-a therapy compared with those in eugonadal men with prostate cancer. Men receiving GnRH-a also had a higher percent total body fat (29 +/- 5% vs. 25 +/- 5%; P < 0.01) and lower percent lean body weight (71 +/- 5% vs. 75 +/- 5%; P < 0.01) compared with eugonadal men with prostate cancer. In conclusion, men with prostate cancer receiving androgen deprivation therapy have a significant decrease in bone mass and increase in bone turnover, thus placing them at increased risk of fracture.

Classification of osteoporosis and osteopenia in men is dependent on site-specific analysis.

To examine the diagnosis of osteoporosis and osteopenia in men based on bone density measurements at single or multiple sites using central and peripheral measurements, we studied 206 ambulatory, community-dwelling men over age 50. Bone mineral density of the hip, PA spine, forearm, and finger were assessed by dual-energy X-ray absorptiometry. The diagnosis of osteoporosis based on a single measurement ranged from 1% using the trochanter to 39% using Ward's triangle. Twenty-one percent of men had osteoporosis if the diagnosis was based on at least one osteoporotic value at three central sites (PA spine, total hip, femoral neck). Among these men using T-scores provided by the manufacturers, 51% of osteoporotic patients would be misclassified as normal using the accuDEXA((R)) (finger), and 37% of osteoporotic men would be misclassified as normal using the PA spine. We conclude depending on the number and selection of sites there is considerable variability in the classification and misclassification of osteoporosis and osteopenia in men.

Covalent immobilization of recombinant human alphavbeta3 integrin on a solid support with retention of functionality.

Alphavbeta3 is the major receptor mediating the attachment of osteoclasts to bone surface and plays a critical role in bone resorption and remodeling. Interfering with alphavbeta3 binding inhibits osteoclast-mediated bone resorption, and thus demonstrates the potential utility of alphavbeta3 antagonists for therapy of osteoporosis. This report describes the generation of an alphavbeta3 affinity column which was created to enable screening of collections of large numbers of ligands, e.g., combinatorial libraries (previously prepared by us), to sort and identify ligands with the highest affinity for alphavbeta3. We demonstrate that covalent immobilization of the heterodimeric alphavbeta3 receptor can be achieved with retention of characteristic ligand binding properties. Human alphavbeta3 was isolated from human embryonic kidney cells (HEK 293) that stably express high levels of the recombinant receptor and then affinity purified to homogeneity. Purified alphavbeta3 receptor was linked covalently to activated CH-Sepharose 4B beads. Quantification of immobilized functional receptor was determined by Scatchard analysis. The immobilized functional receptor maintains binding properties similar to the membrane-embedded and soluble receptor. The immobilized receptor also can be used to select the highest affinity ligand from among a mixture of peptides which differ in their binding affinity, structure, and hydrophobicity, both when the peptides are loaded in equimolar concentrations in a mixture and when the concentration of the highest affinity ligand is reduced 10-fold. Liquid chromatography-mass spectrometry was utilized to confirm selective ligand binding and to demonstrate that preferential binding was not due to nonspecific hydrophobic interactions with immobilized alphavbeta3 receptor or the affinity column. This approach may be of general use for affinity-based screening of ligands for other integrins and should enable practical screening of combinatorial libraries containing large numbers of potential ligands for the human alphavbeta3 integrin receptor, including linear peptides, cyclic peptides, and peptidomimetics.

Enrichment of rab11, a small GTP-binding protein, in gastric parietal cells.

Parietal cell secretion of acid requires the coordinated fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing tubulovesicles with a secretory canalicular target membrane. We have previously reported the presence of rab2 on parietal cell tubulovesicles (L. H. Tang, S. A. Stoch, I. M. Modlin, and J. R. Goldenring. Biochem. J. 285: 715-719, 1992). Since 60% of the small GTP-binding protein sequences obtained from parietal cells were > 95% homologous with human rab11 (J. R. Goldenring, K. R. Shen, H. D. Vaughan, and I.M. Modlin. J. Biol. Chem. 268: 18419-18422, 1993), we sought to study rab11 in gastric parietal cells. A complete rab11 sequence was obtained, and the deduced amino acid sequence of rabbit rab11 was identical to that for human. Rab11 mRNA was present throughout the gastrointestinal mucosa. mRNA for both rab11 and rab2 were enriched in isolated parietal cells compared with chief cells. A polyclonal antiserum against rab11 labeled a single 25-kDa band in isolated parietal cells. Immunostaining of rat fundic tissue demonstrated prominent staining of parietal cells. Rab11 staining cosegregated with alpha-H(+)-K(+)-ATPase staining in enriched preparations of rabbit parietal cell tubulovesicles. These results suggest that rab11 is enriched in parietal cells and is associated with intracellular tubulovesicles.

Transforming growth factor-alpha (TGF alpha) inhibition of parietal cell secretion: structural requirements for activity.

Parietal cells of the gastric fundus produce transforming growth factor-alpha (TGF alpha) which functions as a potent inhibitor of acid secretion. We have previously demonstrated that TGF alpha can inhibit aminopyrine uptake in isolated rabbit parietal cells. In this study, we have evaluated the components of TGF alpha structure which determine its ability to inhibit parietal cell function. Both human and rat TGF alpha inhibited histamine stimulation by increasing the EC50 for agonist stimulation. Three fragments containing the third loop domain of TGF alpha (rat TGF alpha 34-43, human TGF alpha 34-43 and human TGF alpha 34-50) all inhibited histamine stimulation with IC50 values 20, 33 and 4-fold higher, respectively, than that of the native molecule. Rat TGF alpha inhibited carbachol stimulation throughout an agonist dose response. Human TGF alpha was only effective in inhibiting carbachol if incubations were performed in the presence of air rather than 100% O2. In air incubation, all three of the TGF alpha fragments inhibited carbachol stimulation but, in contrast to the effects on histamine, the peptides all were virtually equipotent with the native molecule. The human sequence fragments, like the native human TGF alpha, elicited no inhibition when incubations were performed in the presence of 100% O2. The results suggest that there are pharmacological differences in the response of isolated parietal cells to TGF alpha-mediated inhibition of histamine and carbachol. In addition, in contrast with previous investigations on the mitogenic action of TGF alpha, third loop fragments of TGF alpha retain the capacity to inhibit aminopyrine accumulation.

Identification of rab2 as a tubulovesicle-membrane-associated protein in rabbit gastric parietal cells.

Rab proteins, which are ras-like low-molecular-mass GTP-binding proteins, are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. Previously, we reported a 23 kDa tubulovesicle-associated GTP-binding protein in rabbit gastric parietal cells [Basson, Goldenring, Tang, Lewis, Padfield, Jamieson & Modlin (1991) Biochem. J. 279, 43-48]. The major component of the 23 kDa protein is now identified as rab2. Rab2 was co-localized in tubulovesicle membranes from parietal cells. Consistent with GTP-binding activity (as documented before), upon maximal stimulation of parietal cells, rab2 immunoreactivity was redistributed from a 50,000 g to a 4000 g subcellular membrane fraction. The tubulovesicle-associated rab2 behaved as an integral membrane protein, since both 0.5 M-NaCl and 0.1 M-carbonate extraction failed to remove the protein from the tubulovesicle membrane. Utilizing a PCR the rab2 cDNA sequence from rabbit parietal cells was obtained, and it showed only one amino acid difference compared with the human sequence. The results of the present study provide strong evidence that parietal cells possess a rab2 protein which is tightly associated with tubulovesicle membranes.