Population pharmacokinetics of ustekinumab in patients with active psoriatic arthritis.

PURPOSE: To characterize the population pharmacokinetics of subcutaneous ustekinumab, a human IgG1Kappa; monoclonal antibody against interleukin-12/23p40, using data from a randomized, double-blind, placebo-controlled Phase II study in patients with active psoriatic arthritis (PsA). METHODS: A total of 786 quantifiable serum ustekinumab concentrations from 130 patients were analyzed using a nonlinear mixed-effects modeling approach. A 1-compartment model with first-order absorption and elimination was selected as the structural model. RESULTS: The population typical mean (percent relative standard error (%RSE)) values for apparent clearance (CL/F), apparent volume of distribution (V/F), and absorption rate constant (ka) obtained from the final covariate model were 0.465 l × day-1 (5.1%), 14.3 l (4.4%), and 0.427 day-1 (3.9%), respectively. The between-subject variability in CL/F, V/F, and ka were 53.9%, 42.3%, and 82.4%, respectively. Patient body weight and antibody-to-ustekinumab status were significant covariates affecting the CL/F and/or V/F of ustekinumab. None of the other factors evaluated, such as age, sex, race, baseline disease characteristics, concomitant methotrexate or nonsteroidal anti-inflammatory drugs, and past use of immunosuppressives, biologics, systemic corticosteroids, or disease-modifying antirheumatic drugs, were found to have significant effects on the pharmacokinetics of ustekinumab. CONCLUSION: The pharmacokinetics of ustekinumab in patients with PsA are comparable to those in patients with moderate-to-severe plaque psoriasis which was previously investigated.

Population pharmacokinetics of golimumab in patients with ankylosing spondylitis: impact of body weight and immunogenicity.

AIMS: To develop a population pharmacokinetic (PK) model of subcutaneously administered golimumab, a human anti-tumor necrosis factor monoclonal antibody, in patients with ankylosing spondylitis (AS), estimate typical fixed and random population PK parameters, and identify significant covariates on golimumab pharmacokinetics. METHODS: Serum concentration data through Week 24 of a randomized, double-blind, placebo-controlled Phase III trial of golimumab (50 or 100 mg every 4 weeks) were analyzed using a nonlinear mixed-effects modeling approach. The effects of potential covariates on golimumab were evaluated. RESULTS: A one-compartment PK model with first-order absorption and elimination was chosen to describe the observed golimumab concentration-time data in patients with AS. Population estimates obtained from the final model for a typical 70-kg patient were: apparent systemic clearance (CL/F), 1.41 l/day (95% confidence interval (CI): 1.31 - 1.51) and apparent volume of distribution (V/F), 22.6 L (95% CI: 20.7 - 24.4). The first-order absorption rate constant (Ka) was estimated to be 1.01 day-1 (95% CI: 0.760 - 1.46). The between-subject variabilities for CL/F, V/F, and Ka were 35.2%, 38.6%, and 78.6%, respectively. Body weight was the most significant covariate, affecting both CL/F and V/F. Antibody-to-golimumab status, baseline C-reactive protein level, and sex were also identified as significant covariates on CL/F. CONCLUSIONS: A one-compartment model with first-order absorption and elimination adequately described the PK of golimumab following subcutaneous administrations in patients with AS. Body weight and anti-golimumab antibody status were found to significantly influence golimumab clearance. When a patient does not respond to the prescribed golimumab therapy, the possibility of the development of antibodies to golimumab has to be considered.

A one-step, competitive electrochemiluminescence-based immunoassay method for the quantification of a fully human anti-TNFalpha antibody in human serum.

A quantitative, one-step, competitive electrochemiluminescence (ECL)-based immunoassay for the determination of a fully human, anti-TNFalpha monoclonal antibody in human serum has been developed. A biotinylated, mouse anti-variable region-specific antibody and a ruthenium-labeled anti-TNFalpha antibody were the only specific reagents needed to develop the assay. A single incubation step of 2 h followed by ECL detection was used. The assay was capable of measuring the analyte in neat serum over approximately a 1600-fold range with higher concentrations measured following a single dilution. Assay accuracy, precision, and reproducibility were suitable to support pharmacokinetic studies of the analyte. This competitive assay format offers an alternative approach to the development of immunoassays for the measurement of macromolecules in complex matrices to support preclinical and clinical studies.

Development of high-affinity 5-HT3 receptor antagonists. 1. Initial structure-activity relationship of novel benzamides.

This report describes the development of novel benzamides which are orally active, highly potent, specific antagonists of 5-HT3 receptors. Described in this first report are the structure-activity relationships that led to novel structures with improved potency and selectivity. From this series of compounds, (S)-28 was identified and selected for further evaluation as a 5-HT3 receptor antagonist. Compared with 5-HT3 antagonists such as GR 38032F, BRL 43694, and metoclopramide, (S)-28 was most active in (a) inhibiting binding to 5-HT3 receptor binding sites in rat entorhinal cortex with an Ki value of 0.19 nM and (b) blocking cisplatin-induced emesis in the ferret with an ED50 value determined to be 9 micrograms/kg po.

Pharmacological properties of ureido-acetamides, new potent and selective non-peptide CCKB/gastrin receptor antagonists.

We present here the pharmacological properties of 3 ureido-acetamide members of a novel family of non-peptide cholecystokinin-B (CCKB) receptor antagonists. RP 69758 (3-(3-[N-(N-methyl N-phenyl-carbamoylmethyl) N-phenyl-carbamoylmethyl] ureido)phenylacetic acid), RP 71483 ((E)-2-[3-(3-hydroxyiminomethyl phenyl) ureido] N-(8-quinolyl) N-[(1,2,3,4-tetrahydro 1-quinolyl)carbonylmethyl]acetamide) and RP 72540 ((RS)-2-[3-(3-[N-(3-methoxy phenyl) N-(N-methyl N-phenyl-carbamoylmethyl) carbamoylmethyl] ureido) phenyl] propionic acid) displayed nanomolar affinity for guinea-pig, rat and mouse CCKB receptors labelled with [3H]pCCK-8 or with the selective CCKB receptor ligand [3H]pBC264. RP 69758 and RP 72540 showed selectivity factors in express of 200 for CCKB versus CCKA receptors. All three compounds had also high affinity for gastrin binding sites in the stomach. The ureido-acetamides behaved as potent antagonists of CCK-8-induced neuronal firing in rat hippocampal slices in vitro, a functional model of brain CCKB receptor mediated responses. RP 69758 is also a potent gastrin receptor antagonist in vivo that dose dependently inhibits gastric acid secretion induced by i.v. injection of pentagastrin in the rat. None of the three ureido-acetamides, at concentrations up to 1 microM, significantly blocked CCK-8-evoked contractions of the guinea-pig ileum in vitro, a CCKA receptor bioassay. In ex vivo binding studies, i.p. administration of RP 69758 and RP 72540 resulted in a dose-dependent inhibition of [3H]pCCK-8 binding in mouse brain homogenate. However, the relative penetration of these ureido-acetamides into the forebrain after peripheral administration was below 0.01%. RP 71483 did not appear to cross the blood-brain barrier in quantities sufficient to prevent [3H]pCCK-8 binding at low doses, a property that makes it suitable for the exploration of the peripheral versus central origin of the behavioural effects observed following systemic administration of CCK. RP 69758, RP 71483 and RP 72540 are highly potent and selective non-peptide CCKB receptor antagonists which are useful tools to explore the physiological functions of CCKB receptors.

An LTC4 binding site in gastric mucosa is shared with glutathione.

Recent results from our laboratory and others have suggested a possible physiological functional role(s) for leukotrienes in gastric mucosa. In the present study 3H-LTC4 binds to washed rabbit gastric mucosal membranes at 4 degrees C with a Kd of 5 nM and a Bmax of 31.3 pmol/mg protein. Leukotrienes D4, E4, B4, oxidized glutathione (GSSG), cysteine, and mercaptoethanol were unable to displace 3H-LTC4 at 1 microM concentrations, while GSH inhibited binding with a Ki of 47 nM. Differential centrifugation of the membrane preparation to remove mitochondria resulted in Ki values for LTC4 and GSH of 14 and 23 nM, respectively. The similar binding affinities and competitive receptor binding kinetics for GSH and LTC4, the low affinity for other leukotrienes, and a Ki of 7 microM for hematin, a substrate for glutathione S-transferase, suggest that 3H-LTC4 binds to a GSH site which does not discriminate between LTC4 and GSH. Membranes fractionated to remove mitochondria were assayed for glutathione peroxidase, gamma-glutamyltranspeptidase, and glutathione S-transferase as possible binding sites for LTC4. We were unable to detect enzyme activity for any of the three enzymes. The binding of LTC4 in gastric mucosa differs from other tissues with respect to the high affinity for GSH, and thus becomes an appropriate tissue in which to investigate the relationships between LTC4 and GSH.

Effect of dichloromethylene diphosphonate on morphology, enzyme activity, and ash content of bones of thyroparathyroidectomized rats.

Dichloromethylene diphosphonate (Cl2MDP) was given at doses of 4 mg/kg and 10 mg/kg daily for 7 days to adult thyroparathyroidectomized rats fed a low calcium diet. Primary metaphyseal trabeculae in Cl2MDP-treated rats were more numerous and longer than in controls. The light and electron microscopic appearance of osteoblasts, osteocytes and osteoclasts were unaltered by Cl2MDP. Bone alkaline phosphatase was significantly elevated in rats given Cl2MDP but adenosine triphosphatase activity was unchanged. Bone fat-free weight, fat-free minus ash weight, and bone calcium and phosphorus concentration were reduced significantly in rats given 10 mg/kg Cl2MDP compared to controls. Bone magnesium concentration was significantly elevated in rats given 10 mg/kg Cl2MDP. Serum calcium and phosphorus concentration were lower in Cl2MDP-treated rats. These results suggest that Cl2MDP is capable of altering bone remodeling, enzyme activity and mineral content, without significantly altering bone cell morphology, independent of the effects of parathyroid hormone, calcitonin, and dietary calcium.

Interaction of dichloromethylene diphosphonate and vitamin D on bone of thyroparathyroidectomized rats.

Dichloromethylene diphosphonate (Cl2MDP) antagonized the action of vitamin D on bone in thyroparathyroidectomized rats by reducing the metabolic activity of osteoblasts and osteocytes and decreasing the number of osteoclasts. Ultrastructurally, osteoblasts in Cl2MDP-treated rats were interpreted to be less active in bone matrix synthesis. Osteocytes in Cl2MDP-treated rats were interpreted ultrastructurally to be inactive; there was no evidence of bone resorption when compared to osteocytes in rats given vitamin D alone. Abnormal osmiophilic densities in the pericellular bone matrix of rats given vitamin D alone were not present in rats given vitamin D and Cl2MDP. The ultrastructure of osteoclasts was unaltered by Cl2MDT. These cellular changes were associated with a decrease in serum calcium and increase in bone ash and magnesium concentration in rats given high levels (10 mg/kg) of Cl2MDP. Bone adenosine triphosphatase and alkaline phosphatase activities were not affected by Cl2MDP. These results suggest that Cl2MDP may limit the hypercalcemia of hypervitaminosis D by directly inhibiting bone cells in addition to its physicochemical action.

2,3-Diphosphoglycerate phosphatase/synthase: a potential target for elevating the diphosphoglycerate level in human red blood cells.

To exploit the well documented effect of 2,3-diphosphoglyceric acid (2,3-DPG) in enhancing oxygen delivery by human erythrocytes, we have investigated whether the DPG synthase/phosphatase enzyme system can be targeted to increase DPG levels in the cell. The hydrolytic activity (phosphatase) of the DPG metabolizing enzyme complex exhibits a marked dependence on a physiological effector, 2-phosphoglycolate. Little phosphatase activity is detected in the absence of this activator irrespective of the concentrations of the substrate. The phosphoglycolate-dependent phosphatase activity is competitively inhibited by a glycolytic intermediate, 3-phosphoglyceric acid (3-PGA). The 3-PGA inhibition persists when the 2,3-DPG concentration is raised to saturation level. In contrast, 3-PGA enhances the DPG synthase activity in a dose-dependent manner. In intact red cells, one-half of the cellular DPG content is depleted after 6 hr at 37 degrees C in glucose-free medium. The rate of 2,3-DPG degradation is accelerated when the cellular level of phosphoglycolate is increased by incubation with exogenous glycolate. Together, these results indicate that 2,3-DPG content in erythrocytes can be directly regulated through modulation of phosphatase/synthase activities. In support of this notion, a pyruvate kinase inhibitor, L-alanine, increases by 2-fold the cellular 3-PGA level. This is accompanied by a significant increase (30%) in 2,3-DPG content in human red blood cells. It is postulated that the DPG-promoting action of 3-PGA is mediated through simultaneous phosphatase inhibition and synthase activation. Furthermore, as a result of increased DPG accumulation, the oxygen-hemoglobin dissociation curve in L-alanine-treated cells is rightward shifted by 2.5 torr.

Separation of phospholipids by high-performance liquid chromatography: all major classes, including ethanolamine and choline plasmalogens, and most minor classes, including lysophosphatidylethanolamine.

High-performance liquid chromatographic methods for the separation and quantitation of phospholipids were developed and shown to give sensitive, reliable measurements of tissue phospholipids, including difficult-to-resolve pairs such as choline plasmalogen (plasmenylcholine) and phosphatidylcholine, choline glycerophospholipids and sphingomyelin, phosphatidylinositol and phosphatidylserine, and phosphatidylserine and lysophosphatidylethanolamine. Separations of most phospholipids including those mentioned above are more complete than in existing procedures, and require only 40 min per injection. Utilization of the hexane-2-propanol-water system has an advantage over separation techniques that employ acidic solvents in that the plasmalogens are not hydrolyzed and a less degradative environment for labile lipids is provided. Further, a rapid high-performance liquid chromatographic procedure for the separation of intact ethanolamine plasmalogen (plasmenylethanolamine) from phosphatidylethanolamine was developed. Previous procedures have required derivatized samples or acid hydrolysis of the plasmalogen vinyl ether linkage. A slight modification of the primary method (method I) increases the resolution of lysophosphatidylethanolamine from other classes (method II). A third modification (method III) can replace the standard silicic acid column separation of lipids into neutral, glycolipid, and phospholipid fractions.

The gastrin/cholecystokinin-B receptor antagonist L-365,260 reduces basal acid secretion and prevents gastrointestinal damage induced by aspirin, ethanol and cysteamine in the rat.

L-365,260, a nonpeptide antagonist of gastrin/CCK-B receptors, was evaluated in receptor binding, antisecretory and gastrointestinal damage assays. L-365,260 binds potently and stereo-selectively to gastrin and CCK-B sites in guinea pig tissue. In contrast, L-365,260 binds to the isolated canine parietal cell gastrin receptor weakly, and without stereoselectivity. In the pylorus-ligated rat, low doses of L-365,260, given i.v., attenuated pentagastrin-stimulated acid secretion, whereas higher doses were required to inhibit both histamine-stimulated and basal acid secretion. In an aspirin-induced gastric damage model, L-365,260 was 2.4-fold less potent than the standard histamine H2 antagonist cimetidine in preventing gastric damage when given i.v., and was 8.3-fold less potent than cimetidine when given p.o. Moreover, the ED50 value for L-365,260, given i.v., in prevention of aspirin-induced gastric damage (11.5 mg/kg) agreed well with its ED50 value for inhibition of basal acid secretion (12.6 mg/kg). At doses as great as 100 mg/kg p.o., neither L-365,260 nor cimetidine had an effect on ethanol-induced gastric damage. L-365,260, although p.o. less bioavailable relative to cimetidine in the aspirin gastric damage model, was as potent as cimetidine in the prevention of cysteamine-induced duodenal ulcers in the rat. We conclude that the gastrin/CCK-B receptor antagonist L-365,260, at doses supramaximal for the inhibition of pentagastrin-stimulated secretory responses in vivo, inhibits gastrointestinal damage in models of peptic ulcer disease by an antisecretory mechanism of action.

Spectrophotometric determination of lipases, lysophospholipases, and phospholipases.

Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester substrate analogs as well as omega-nitrophenyl derivatives of the corresponding lipids. The most promising results are obtained with the thioester substrate analogs. Mono- and diacylglycerol lipases are assayed by using rac-1-S-decanoyl-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-1-mercapto-2,3-propanediol, respectively. Phospholipases A1 and A2 are determined by using rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and 2-hexadecanoylthio-1-ethyl-phosphocholine, respectively. Lysophospholipases are measured by using 2-hexadecanoylthio-1-ethyl-phosphocholine. Phospholipase C is assayed with rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol. Thioester substrate analog assay procedures are more rapid, sensitive, convenient, continuous, and less expensive than the classical radiochemical techniques.

RG 12915: a potent 5-hydroxytryptamine-3 antagonist that is an orally effective inhibitor of cytotoxic drug-induced emesis in the ferret and dog.

RG 12915 [4-[N-(1-azabicyclo[2.2.2.]octan-3-(S)-yl)]2-chloro-cis 5a-(S)-9a-(S)-5a,6,7,8,9,9a-hexahydrobenzofurancarboxamide hydrochloride] is a potent and effective agent against cisplatin-induced emesis in the ferret after i.v. or p.o. administration. This agent (p.o.) is also highly protective against cisplatin-induced emesis in the dog, as well as cyclophosphamide/doxorubicin-induced emesis in the ferret. When administered either p.o. or i.v., RG 12915 has a lower ED50 value (0.004 mg/kg) than GR 38032F, BRL 43694 and metoclopramide for attenuating cisplatin-induced emetic episodes in the ferret. It also has a long duration of action against cisplatin-induced emesis in the ferret. In contrast to metoclopramide, RG 12915 lacks significant antidopaminergic activity both in vitro [( 3H]spiroperidol displacement), as well as in vivo (apomorphine-induced emesis). In radioligand binding assays, RG 12915 is a potent and selective displacer of binding of 5-hydroxytryptamine (5-HT)3 binding sites (IC50 value = 0.16 nM), whereas failing to displace binding of ligands for the alpha-1, alpha-2 and beta adrenergic, 5-HT1 or 5-HT2 or cholinergic-muscarinic sites with IC50 values less than 1 microM. At a p.o. dose (1 mg/kg) in which RG 12915 is highly protective against cisplatin-induced emesis in the dog, RG 12915 has no significant gastroprokinetic activity in the same species. In summary, RG 12915 is a potent and p.o. effective agent against cytotoxic drug-induced emesis in animal models. The antiemetic potency of RG 12915 against cisplatin is unrelated to antidopaminergic or gastroprokinetic activity, but may be related to its affinity for 5-HT3 binding sites.

RP 73870, a gastrin/cholecystokinin-B receptor antagonist with potent anti-ulcer activity in the rat.

RP 73870, the racemic potassium salt of (([N-(methoxy-3-phenyl)-N-(N-methyl-N-phenyl-carbamoylmethyl)- carbamoylmethyl]-3-ureido)-3-phenyl)-2-ethylsulfonate-(RS) is a potent, reversible antagonist of both gastrin and cholecystokinin-B receptors in guinea pig and rat tissues. This compound is a potent inhibitor of pentagastrin-stimulated gastric acid secretion in the perfused rat stomach. RP 73870 also inhibits basal gastric acid secretion in the rat, although at doses higher than that required for inhibition of pentagastrin-stimulated gastric acid secretion. RP 73870 is a potent inhibitor of aspirin-induced gastric damage in the rat. In the prevention of aspirin-induced gastric damage, RP 73870, given p.o., was 10-fold less potent than when given i.v. RP 73870 was as potent as a H2 receptor antagonist or proton pump inhibitor in the prevention of cysteamine-induced duodenal ulcers in the rat. Relative to other gastrin/cholecystokinin-B antagonists, RP 73870 demonstrates greater affinity to gastrin binding sites, and possesses a unique spectrum of in vivo biological activities appropriate for an anti-ulcer indication.