Preclinical development of AMG 139, a human antibody specifically targeting IL-23.

BACKGROUND AND PURPOSE: AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. EXPERIMENTAL APPROACH: The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. KEY RESULTS: AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4-13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg(-1) and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg(-1) s.c. or i.v.). CONCLUSIONS AND IMPLICATIONS: The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

Size-topology relations in packings of grains, emulsions, foams, and biological cells.

Particulate packings in 3D are used to study the effects of compression and polydispersity on the geometry of the tiling in these systems. We find that the dependence of the neighbor number on cell size is quasilinear in the monodisperse case and becomes nonlinear above a threshold polydispersity, independent of the method of creation of the tiling. These size-topology relations can be described by a simple analytical theory, which quantifies the effects of positional disorder in the monodisperse case and those of size disorder in the polydisperse case and is applicable in two and three dimensions. The theory thus gives a unifying framework for a wide range of amorphous systems, ranging from biological tissues, foams, and bidisperse disks to compressed emulsions and granular matter.

Pharmacokinetics of spinosad and milbemycin oxime administered in combination and separately per os to dogs.

Pharmacokinetic (PK) studies were conducted to determine the potential PK interactions when spinosad and milbemycin oxime (MBO) are administered simultaneously. Investigations used commercial MBO tablets (C-MBO; Interceptor(®) Flavor Tabs, active ingredient MBO, Novartis Animal Health, Greensboro, NC, USA), novel-source (Elanco) MBO (E-MBO) in a gelatin capsule, spinosad API (Active Pharmaceutical Ingredient using registered manufacturing process) in a gelatin capsule, spinosad tablets (Comfortis(®) chewable beef flavored tablets, active ingredient spinosad, Elanco Animal Health, Greenfield, IN, USA), and the recently registered spinosad + E-MBO combination tablets (Trifexis™ chewable beef flavored tablets, active ingredients E-MBO and spinosad, Elanco Animal Health, Greenfield, IN, USA). Regardless of the source of MBO, in the presence of spinosad, greater systemic exposure of MBO was obtained as compared to MBO administered alone. Target animal safety studies conducted with dose multiples of spinosad and MBO indicate the increased exposure of MBO does not have implications on adverse clinical reactions. Further research is required to determine whether the higher levels of MBO have any implications for improved effectiveness as compared to C-MBO. Effectiveness studies conducted with 0.5 mg/kg of E-MBO in combination tablets demonstrated noninterference against C-MBO with both products achieving >99% effectiveness against the dose-limiting nematode, Ancylostoma caninum. No statistical differences were detected in the PK of MBO when comparing animals receiving E-MBO (without spinosad) and C-MBO. Also, the PK of spinosad was unaltered when co-administered with MBO.

Safety evaluation and treatment affect of LY2190416, a CB-1 antagonist/inverse agonist in growing beagle dogs.

The objective of this study was to assess the safe use of LY2190416, a cannabinoid receptor 1 receptor antagonist/inverse agonist, for obesity management in dogs. Twenty-four clinically normal young beagle dogs were administered LY2190416 at doses of 3, 9, or 18 mg/kg or placebo, orally, once daily for 13 weeks. Food consumption and body weight were determined, and dogs were evaluated for changes in hematology, clinical chemistry, urinalysis, and serum cortisol. LY2190416 had no significant effect on hematology, clinical chemistry, urinalysis, and serum cortisol. All dogs consumed 100% of their entire daily allowance throughout the study. All dogs gained weight during the study, but treated dogs gained less than control dogs by the end of the study. During the first month, dogs exhibited a dose-dependent decrease in rate of weight gain (19.7 g/day for control dogs vs. 10.6 g/day for the 18 mg/kg dose group). LY2190416 was found to be safe at doses up to 18 mg/kg administered daily for 3 months. Results suggest that LY2190416 decreases rate of weight gain without affecting appetite or causing significant adverse health effects in normal growing dogs. Possible mechanisms for a proposed metabolic effect are discussed.

Nonlinear mixed effects pharmacokinetic/pharmacodynamic analysis of the anticonvulsant ameltolide (LY201116) in a canine seizure model.

The anticonvulsant ameltolide (LY201116) is a novel potential therapy for the treatment of canine epilepsy. Eight dogs were administered five different oral doses of ameltolide and clinical scoring of the maximal electroshock (MES) induced seizures at 3 and 24 h postdosing were determined in two separate crossover design studies. Plasma ameltolide concentrations were determined at the time of seizures in all dogs and complete plasma concentration-time profiles were also determined in a separate study. A nonlinear mixed effects PK/PD model was fit to the resulting data. A one compartment open model with first order absorption was determined to best fit the ameltolide pharmacokinetics. An effect compartment with a cumulative logistic regression equation was used to establish the PK/PD relationship. The mean bioavailability normalized volume of distribution and the elimination half-life were estimated at 1.20 L/kg and 5.46 h, respectively. The fitted model estimated that from 2 to 15 h following a single 3 mg/kg oral ameltolide dose the mean probability of obtaining a 1 unit reduction in the seizure clinical score severity was greater than 0.80. The utilized PK/PD analysis combined with the canine MES model allowed for the rapid and efficient determination of the plasma ameltolide concentration-anticonvulsant relationship preclinically in dogs.

Development and validation of the maximal electro-shock seizure model in dogs.

The development and validation of the maximal electro-shock (MES) model using phenobarbital (Pb) as the positive control is described. This approach builds on previous work in rodent model systems, and has been adapted to dogs as a tool for pharmaceutical dose selection. Dogs, like rodents, exhibit generalized convulsions which manifest as progressive clinical signs in a dose (electrical current) dependent fashion. At the limit (300 mA, 200 msec) animals underwent clonic-tonic convulsions consistent with complete generalized (Grand Mal) seizures with a grade 3 clinical score (CS) and a menace response time of 98.5 +/- 24.4 sec (n = 8). Pretreatment of animals with Pb at 3, 10, and 30 mg/kg, in a 4-by-4 complete block crossover design (Latin-Square), resulted in a dose-dependant reduction in CS and menace response time. Estimates of plasma Pb concentration taken prior to MES induction showed a similar dose-dependent reduction in CS and menace response time with concentration. Using a cumulative logistic regression model, a predicted 50% probability of a CS = 1 was approximately 11.4 mg/kg. In addition, plasma Pb concentrations predicted a 50% probability of a CS = 1 occurs at plasma Pb concentration of approximately 16.0 mug/mL. Combined these data suggest that MES is a useful model for evaluating generalized convulsions in canines and may provide a tool for dose selection of novel pharmaceutical compounds.

Identification of potential CSF biomarkers in ALS.

BACKGROUND: The clinical diagnosis of ALS is based entirely on clinical features. Identification of biomarkers for ALS would be important for diagnosis and might also provide clues to pathogenesis. OBJECTIVE: To determine if there is a specific protein profile in the CSF that distinguishes patients with ALS from those with purely motor peripheral neuropathy (PN) and healthy control subjects. METHODS: CSF obtained from patients with ALS, disease controls (patients with other neurologic disorders), and normal controls were analyzed using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry proteomics technique. Biomarker sensitivity and specificity was calculated with receiver operating characteristic curve methodology. ALS biomarkers were purified and sequence identified by mass spectrometry-directed peptide sequencing. RESULTS: In initial proteomic discovery studies, three protein species (4.8-, 6.7-, and 13.4-kDa) that were significantly lower in concentration in the CSF from patients with ALS (n = 36) than in normal controls (n = 21) were identified. A combination of three protein species (the "three-protein" model) correctly identified patients with ALS with 95% accuracy, 91% sensitivity, and 97% specificity from the controls. Independent validation studies using separate cohorts of ALS (n = 13), healthy control (n = 25), and PN (n = 7) subjects confirmed the ability of the three CSF protein species to separate patients with ALS from other diseases. Protein sequence analysis identified the 13.4-kDa protein species as cystatin C and the 4.8-kDa protein species as a peptic fragment of the neurosecretory protein VGF. CONCLUSION: Additional application of a "three-protein" biomarker model to current diagnostic criteria may provide an objective biomarker pattern to help identify patients with ALS.

Tolerance of high-dose (3,000 mg/day) coenzyme Q10 in ALS.

An open-label dose-escalation trial was performed to assess the safety and tolerability of high doses of coenzyme Q10 (CoQ10) in ALS. CoQ10, a cofactor in mitochondrial electron transfer, may improve the mitochondrial dysfunction in ALS. In this study, CoQ10 was safe and well tolerated in 31 subjects treated with doses as high as 3,000 mg/day for 8 months.

Projectile-shape dependence of impact craters in loose granular media.

We report on the penetration of cylindrical projectiles dropped from rest into a dry, noncohesive granular medium. The cylinder length, diameter, density, and tip shape are all explicitly varied. For deep penetrations, as compared to the cylinder diameter, the data collapse onto a single scaling law that varies as the 1/3 power of the total drop distance, the 1/2 power of cylinder length, and the 1/6 power of cylinder diameter. For shallow penetrations, the projectile shape plays a crucial role with sharper objects penetrating deeper.

Identification of a superimmunoglobulin gene family member overexpressed in benign prostatic hyperplasia .

BACKGROUND: Benign prostate hyperplasia (BPH), a nonmalignant disease with an increasing rate of occurrence associated with advancing age, requires auxiliary markers to help identify its presence and distinguish its progression from prostate cancer. METHODS: Hybridoma technology was used to generate an antibody against a BPH antigen, which was subsequently characterized by Western blot analysis, sequence homology, and RT-PCR. RESULTS: A BPH-associated protein, designated P25/26, was identified that showed a strong sequence similarity with superimmunoglobulin family members, overexpressed in BPH, with lower expression observed in both normal and prostate cancer tissues. CONCLUSIONS: Further studies appear warranted to assess the role that this and other superimmunoglobulin family members may have in the pathogenesis of BPH, and to determine if these glycoproteins have any clinical utility in the differential diagnosis or therapeutic monitoring of BPH.

Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy.

OBJECTIVES: To determine the expression of prostate-specific membrane antigen (PSMA) before and after androgen-deprivation therapy and to compare PSMA expression with prostate-specific antigen (PSA) expression. METHODS: We studied specimens from 20 patients with prostate cancer undergoing medical or surgical castration or combination androgen-deprivation therapy in whom matched pretreatment and post-treatment tissue specimens were available and 16 patients in whom only a post-treatment specimen was available. The expression of PSMA and PSA in the tissue specimens was determined by immunoperoxidase staining. The extent of staining was calculated by multiplying the percent of antigen-positive tumor cells by the staining intensity to arrive at a stain index for each biomarker. An in vitro study assessed the concentration of PSMA and PSA in extracts of LNCaP cells cultured in the presence or absence of androgen as determined by immunoassays and Western blot analysis. RESULTS: PSMA reactivity was found to be increased in 55% (11 of 20) of post-treatment primary tissues and 100% (4 of 4) of post-treatment metastatic specimens. In contrast, PSA expression was found to be decreased in 70% (14 of 20) of post-treatment primary and 100% (4 of 4) of post-treatment metastatic specimens. Neither type of androgen-deprivation treatment nor tissue sensitivity to androgen deprivation appeared to influence degree of biomarker expression. PSMA was found to be downregulated and PSA upregulated when LNCaP cells were cultured in the presence of testosterone or dihydrotestosterone. CONCLUSIONS: The enhanced expression of PSMA in tissues and LNCaP cells after androgen deprivation suggests that PSMA is upregulated in the majority of prostate carcinomas after androgen treatment. The high expression in metastatic tissues strongly suggests that PSMA may be a clinically useful target for antibody-and genetic-directed therapy of prostate cancer that recurs after androgen deprivation. The mechanism whereby androgens suppress the expression of PSMA, and the association of PSMA with the development of hormone-independent prostate cancers, will require further study.

Lack of magainin-like activity in human cervical tissue.

PROBLEM: The cervix plays an integral role in innate immunity of the human reproductive tract. Magainins are antimicrobial and spermicidal peptides recently described in human submandibular glands. We investigated the human cervix for magainin-like peptides. METHOD: Histologic sections of frozen and paraffin embedded cervical tissue from nine subjects were separately incubated with two rabbit, polyclonal, anti-magainin antibodies (CB-2 and CB-7) to investigate for magainin-like activity in the human cervix. RESULTS: There was no specific staining of cervical columnar cells within the endocervical canal or in the endocervical crypts. Magainin-like immunoreactivity in the human submandibular gland confirmed previous observations. CONCLUSION: Antigen related to magainin-like peptides were not discovered in the human cervix.

Two-site monoclonal antibody-based immunoradiometric assay for measuring prostate secretory protein in serum.

We developed a double-determinant immunoradiometric assay for measuring serum prostate secretory protein (PSP), using monoclonal antibodies (MAb) against two different epitopes: MAb PSP-19 was the capture antibody and MAb PSP-6 was the tracer antibody. Assay sensitivity was 0.1 microgram/L. Analytical recovery of PSP was 93.5-104.6%, whereas the intra- and interassay mean CVs were 4.2% and 6.9%, respectively. In 92 normal men, ages greater than 50 years, the mean PSP concentration was 5.7 micrograms/L, with 10 (10.9%) men having concentrations greater than 10 micrograms/L. In contrast, 20 of 49 (40.8%) patients with benign prostate hyperplasia (BPH; mean PSP concentration 9.4 micrograms/L) and 46 of 100 (46%) patients with prostate cancer (mean PSP concentration 22.2 micrograms/L) had PSP concentrations greater than 10 micrograms/L. Mean serum PSP concentrations of the BPH (P less than 0.05) and prostate cancer (P less than 0.01) groups were significantly different from those of age-matched normal men. In a small group of patients, serial PSP concentrations correlated with the clinical course during therapy. Thus, PSP may be a useful marker for evaluating patients with prostate cancer.

Effects of conjugated estrogen on the calcitriol response to parathyroid hormone in postmenopausal women.

We evaluated the effects of estrogen on the calcitriol response to the active peptide, human PTH(1-34), in postmenopausal women. Fifteen women were studied before and again after at least 1 month of treatment with conjugated equine estrogens, 1.25 mg/day. Six women received two series of four graded peptide infusions, each with the sequence 200, 400, 800, and 1600 USP U hPTH(1-34). Nine women received only one dose of peptide, either 200 or 800 U, before and while taking estrogen. Baseline values and the incremental and percent changes in circulating calcitriol 24 h after the 20-min infusions were evaluated. Estrogen treatment resulted in significant reductions in blood levels of calcium (2.26 +/- 0.03 mmol/L vs. 2.16 +/- 0.02, P less than 0.05) and phosphorus (1.23 +/- .05 mmol/L vs. 1.14 +/- 0.03, P less than 0.005), a rise in serum calcitriol concentrations (42.2 +/- 3.9 pg/mL vs. 28.6 +/- 3.1, P less than 0.005), and no change in circulating PTH. The rise in calcitriol after 200 U hPTH(1-34) was significantly greater on estrogen (17.6 +/- 2.0 pg/mL vs. 9.5 +/- 1.8, P less than 0.01), but estrogen did not alter incremental responses to larger doses. When results were normalized for differences in baseline values, the estrogen-related change in response to 200 U was no longer significant. hPTH(1-34) acutely increased urinary clearance of cAMP and phosphorus, but estrogen did not affect this response. We conclude that exogenous estrogen does not increase renal sensitivity to PTH in postmenopausal women.

Effects of voluntary exercise on bone mineral content in rats.

We used a voluntary running model to explore the relationship between average daily running distance and bone mineral status of rats. A total of 60 male Sprague-Dawley rats were randomly assigned at 6 weeks of age to a sedentary control group (n = 22) or to a group with unlimited access to a running wheel (n = 38). The running distance of exercising rats was monitored daily, and steady-state running levels ranged from 3.2 to 18.1 km/day. At the end of the experimental period, femora and tibiae were dissected and bone mineral content (BMC, g/cm) and bone mineral density (BMD, g/cm2) were measured by single-photon absorptiometry. Cross-sectional morphometry was examined by taking a transverse section of the femoral middiaphysis. Hindlimb percentage fat was significantly higher in controls than in runners (20.0 +/- 1.2 versus 11.1 +/- 0.6, p less than 0.001), and soleus mass was greater in runners than in controls (371 +/- 8.1 versus 320 +/- 0.8 mg, p less than 0.001). Femoral and tibial lengths, weights, and volumes were significantly higher in runners than in controls (p less than 0.005). BMC and BMD were higher in runners than in controls at all sites apart from the distal femur. Cross-sectional areas at the femoral midshaft were greater in running rats than in sedentary controls (6.26 +/- 0.1 versus 5.45 +/- 0.3 mm2, p less than 0.02), as was the polar moment of inertia (15.6 +/- 0.6 versus 12.7 +/- 0.2 mm4, p less than 0.05). No positive correlation was found between distance run and BMC, BMD, cross-sectional area, or polar moment of inertia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of estrogen on daylong circulating calcium, phosphorus, 1,25-dihydroxyvitamin D, and parathyroid hormone in postmenopausal women.

We studied the effects of estrogen on daylong circulating levels of calcium, inorganic phosphorus, parathyroid hormone (PTH), and 1,25-(OH)2D3 (calcitriol) in a group of 10 postmenopausal women (68.5 +/- 1.4 years, mean +/- SEM). The study was conducted under strict dietary control, with mean calcium and phosphorus intakes of 845 and 970 mg. After treatment with conjugated equine estrogens, 1.25 mg/day, for 1 month, significant decreases in fasting (0800 h) serum levels were observed for calcium (9.09 +/- 0.08 versus 9.46 +/- 0.10 mg/dl, p less than 0.01) and phosphorus (3.38 +/- 0.10 versus 3.73 +/- 0.08 mg/dl, p less than 0.01). On the 0800 h fasting specimen, midmolecule PTH concentrations were higher (44.0 +/- 7.9 versus 34 +/- 8.2 pg/ml, p less than 0.05), but intact PTH was unchanged (28.6 +/- 2.7 versus 29.1 +/- 1.7 pg/ml) and a rise in circulating calcitriol (39.8 +/- 4.3 versus 31.6 +/- 2.1 pg/ml) was marginally significant (p = 0.07). When data represented multiple samples averaged over 7 and 15 h, significant estrogen-related reductions in serum calcium and phosphorus concentrations were observed. In addition, estrogen was associated with a significant rise in the daylong (15 h) level of calcitriol (39.4 +/- 4 versus 30.5 +/- 2.4 pg/ml, p less than 0.01). Daylong mid- and intact PTH concentrations were unchanged on estrogen compared to baseline values. No significant correlations were observed between changes in fasting calcitriol level and changes in fasting concentrations of calcium, phosphorus, or PTH. Further, the rise in daylong calcitriol concentration did not correlate significantly with changes in fasting or integrated values of calcium or PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation and characterization of monoclonal antibodies to prostate secretory protein.

Monoclonal antibodies (MAbs) were produced against a highly purified preparation of prostate secretory protein (PSP) isolated from normal seminal plasma. Fifteen antibodies were selected for further evaluation based on their strong reactivity and specificity for PSP. All the MAbs had a specificity for prostate epithelial cells and none reacted to any of a variety of normal tissues as determined by immunoperoxidase staining. Six of the MAbs were selected for further immunohistochemical evaluation based on their ability to recognize different antigenic determinants. Using competitive binding immunoassays, a variety of overlapping specificities were observed with at least 2 distinct epitopes identified. Although some staining variability was noted, the 6 antibodies, in general, gave the same pattern of tissue reactivity. Both the normal prostate and the benign prostate hyperplastic ductal epithelial cells stained intensely, with 78 to 100% and 50-100% of the cells staining, respectively. The number and often the staining intensity of the tumor cells decreased as the tumor became more undifferentiated. Approximately 40 to 100% and 15 to 70% of the tumor cells stained in the moderately-differentiated and well-differentiated carcinoma tissues, respectively, whereas either no staining was observed or less than 20% of the tumor cells stained in the poorly-differentiated and undifferentiated tumors. Most of the metastatic prostate tumors showed either no staining or scattered staining in a few cells (i.e., less than 20%).

Dunning rat prostate tumors and cultured cell lines fail to express human prostate carcinoma-associated antigens.

The objective of this study was to determine if human prostate carcinoma-associated tumor markers were expressed by Dunning rat prostate carcinomas. Frozen and formalin-fixed paraffin-embedded tissues from 12 different sublines of Dunning tumors were evaluated for marker expression by immunoperoxidase staining by using a panel of 9 monoclonal antibodies, including antibodies against human PAP and PSA. None of the Dunning tumors were found to express any of the human prostate tumor markers. Both fixed and live immunofluorescent assays were performed on 5 cultured Dunning tumor cell lines, evaluated either as single cells or as monolayers. As with the Dunning tumor tissues, none of the cell cultures expressed any of the 9 human prostate tumor markers. The lack of antigen expression by the Dunning tumor tissues and cell lines suggests that these human prostate tumor markers are quite species specific. These results limit the use of the Dunning prostate tumors as models to explore the preclinical application of these human prostate carcinoma-associated monoclonal antibodies and their target antigens.

Relationship of body composition, muscle strength, and aerobic capacity to bone mineral density in older men and women.

We evaluated the relationship of body composition, maximal aerobic capacity (VO2max), and muscle strength to bone mineral density in 91 healthy men and women, age 61-84 years. Lean body mass was estimated from two independent measures of fat mass, bioelectrical impedance and skinfold thickness. VO2max was determined by treadmill ergometry with direct measurement of oxygen consumption. Grip and back strength were measured by isometric dynamometry. Mineral density of lumbar spine and midradius were measured by dual- and single-photon absorptiometry. Men had significantly greater lean mass, muscle strength, aerobic capacity, and bone density than women. In women, grip strength correlated with forearm and spine density (r = 0.37, r = 0.28, p less than 0.05). In men, grip strength correlated with forearm density (r = 0.47, p less than 0.05), and back strength was significantly correlated with both spine (r = 0.46, p less than 0.01) and forearm density (r = 0.46, p less than 0.01). In women, neither forearm nor spine density correlated significantly with aerobic capacity. In men, midradius density did not correlate significantly with oxygen consumption, but the simple correlation between spine density and VO2max was significant (r = 0.41, p less than 0.05). Back strength and VO2max were significantly related in men (r = 0.47, p less than 0.01). By stepwise multiple regression, back strength emerged as the most robust predictor of spine mineral, accounting for 19% of the variation in bone density. Addition of VO2max to the regression did not add significant predictive value. However, when VO2max was expressed per kilogram lean body mass, both back strength and VO2max contributed significantly to the prediction of spine density in men, and the coefficient of determination R2 increased to 0.30. We conclude that body mass and grip strength, but not aerobic capacity, significantly predict bone density in elderly women. In elderly men, back strength is a more robust predictor of axial bone density than traditional expressions of aerobic capacity, but VO2max per kilogram lean mass and back strength both make significant contributions to the prediction of spine mineral density. The applicability of these results to younger men and women is uncertain.