Immunogenicity assay development and validation for biological therapy as exemplified by ustekinumab.

Introduction of biotherapeutics has been a major milestone in the treatment of different chronic diseases. Nevertheless, the immune system can recognize the administered biological as non-self and respond with generation of anti-drug antibodies (ADA), including neutralizing ADA (nADA). Immunogenic responses may result in altered drug dynamics and kinetics leading to changes in safety and efficacy. However, there are several challenges with standard techniques for immunogenicity testing. Ustekinumab (UST), used in different inflammatory diseases, is a therapeutic antibody directed against the shared p40 subunit of interleukin (IL)-12 and IL-23, interfering in the pathogenically crucial T helper type 1 (Th1)/Th17 pathway. We established and validated different approaches for detection and quantitation of UST, UST-specific ADA and nADA. Addressing the obstacle of complex formation of UST with nADA, we developed an acidification assay to approach the total amount of nADA. Validated methods were based on surface plasmon resonance spectroscopy (SPR), enzyme-linked immunosorbent assay (ELISA) and a cell-based approach to characterize neutralizing capacity of nADA. Parameters assessed were determination and quantitation limits, linearity, range, precision, accuracy and selectivity. Quantitation of ADA and UST was feasible at lower concentrations using ELISA, whereas SPR showed a wider linear range for determination of ADA and UST. Accuracy, precision and linearity for quantitation were comparable using ELISA, SPR and the cell-based approach. All validated parameters fulfill the requirements of regulatory agencies. A combination of the testing approaches could address the increasing demand of precision medicine as it can be suitable for capturing the whole spectrum of immunogenicity and is transferable to other biologicals.

A machine-learned analysis of human gene polymorphisms modulating persisting pain points to major roles of neuroimmune processes.

BACKGROUND: Human genetic research has implicated functional variants of more than one hundred genes in the modulation of persisting pain. Artificial intelligence and machine-learning techniques may combine this knowledge with results of genetic research gathered in any context, which permits the identification of the key biological processes involved in chronic sensitization to pain. METHODS: Based on published evidence, a set of 110 genes carrying variants reported to be associated with modulation of the clinical phenotype of persisting pain in eight different clinical settings was submitted to unsupervised machine-learning aimed at functional clustering. Subsequently, a mathematically supported subset of genes, comprising those most consistently involved in persisting pain, was analysed by means of computational functional genomics in the Gene Ontology knowledgebase. RESULTS: Clustering of genes with evidence for a modulation of persisting pain elucidated a functionally heterogeneous set. The situation cleared when the focus was narrowed to a genetic modulation consistently observed throughout several clinical settings. On this basis, two groups of biological processes, the immune system and nitric oxide signalling, emerged as major players in sensitization to persisting pain, which is biologically highly plausible and in agreement with other lines of pain research. CONCLUSIONS: The present computational functional genomics-based approach provided a computational systems-biology perspective on chronic sensitization to pain. Human genetic control of persisting pain points to the immune system as a source of potential future targets for drugs directed against persisting pain. Contemporary machine-learned methods provide innovative approaches to knowledge discovery from previous evidence. SIGNIFICANCE: We show that knowledge discovery in genetic databases and contemporary machine-learned techniques can identify relevant biological processes involved in Persitent pain.

Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis.

Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the 'symptom-free' intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach. Graphical summary of lysophosphatidic signaling in multiple sclerosis.

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20-30 mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley's sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

Comparison of the effects of inhibitors of cytochrome P-450-mediated reactions on human platelet aggregation and arachidonic acid metabolism.

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactions, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1-14C]thromboxane B2 from [1-14C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.

Clinical pharmacokinetics of molsidomine.

Molsidomine is a prodrug for the formation of nitric oxide (NO). Its pharmacokinetics are characterised by rapid absorption and hydrolysis, taking a short time to achieve maximal systemic concentrations of both the parent compound and its active metabolite, SIN-1. The time to peak plasma drug concentration (tmax) is 1 to 2 hours. The bioavailability of the parent compound after oral administration in tablet form is 44 to 59%, but further metabolism to release NO and form polar metabolites is rapid; the half-life (t-1/2) of SIN-1 is 1 to 2 hours. Urinary excretion accounts for more than 90% of the part of the administered dose of molsidomine which is not excreted unchanged. Protein binding of the parent compound is very low (3 to 11%) and its volume of distribution (Vd) corresponds to the range of bodyweight. Single-dose studies (1, 2 and 4 mg) have revealed linear pharmacokinetics, and multiple dose studies in healthy individuals (2 mg 3 times daily for 7 days) and coronary artery disease (CAD) patients (4 mg 4 times daily for 4 weeks) do not show any accumulation of the drug. A study in young and elderly individuals indicated that the first-pass effect is decreased and t-1/2 prolonged with age, resulting in an increased area under the concentration-time curve (AUC) of molsidomine and SIN-1. In patients with liver disease and congestive heart failure similar changes were observed, but much less so in patients with CAD. Clearance was also impaired in patients with liver disease, but the pharmacokinetics of molsidomine were not markedly altered by impaired renal function. In general, due to a large therapeutic dose range, dosage adjustments are not required on the basis of clinical experience. In certain patients a lower starting dose may be recommended, such as in those with impaired liver or kidney function, in congestive heart failure or in the presence of concomitant treatment with other vasoactive compounds. A linear dose-effect relationship is observed with counterclockwise hysteresis, i.e. a greater effect associated with the decrease of plasma concentrations than during their increase, which may be at least partly due to the metabolic delay in the formation of NO from SIN-1. Accordingly, the duration of action of molsidomine is longer than would be expected on the basis of the elimination half-life. The pharmacokinetics of molsidomine support the recommended dosages for use in angina pectoris.

Time-dependent stimulatory and inhibitory effects of prostaglandin E1 on exudative and tissue components of granulomatous inflammation in rats.

1 The effects of prostaglandin (PGE(1)), following local administration during different phases of developing sponge-induced granulomata, were studied in normal and essential fatty acid deficient (EFAD) rats.2 In normal rats, a single dose of 1 mug PGE(1) on implantation (day 1) increased exudate production without altering total leucocyte counts after 6 h and stimulated granulomatous tissue formation after 8 days.3 Repeated daily administration of the same dose of PGE(1) on days 1 to 3 had no effect, while administration on days 4 to 7 (i.e. when tissue growth is already in progress) inhibited granuloma formation.4 In EFAD rats, which are known to produce only very small amounts of endogenous prostaglandins, acute (6 h) exudate formation was unaffected by 0.05 mug PGE(1). However, early stimulatory and later inhibitory effects of 0.05 mug PGE(1) per day were obtained on the granulomatous tissue, similar to those obtained with the 20 fold higher dose in normal rats.5 The early stimulatory action of PGE(1) on granulomatous tissue formation was enhanced, in normal rats, by concomitant administration of 10 mug theophylline. This latter compound did not influence the later inhibitory effect of PGE(1).6 These results indicate that PGE(1) exerts either pro- or anti-inflammatory actions on the proliferative (tissue) component of the inflammatory process, depending on the time of administration. While the stimulatory effect following early administration may have been secondary to an initial cyclic adenosine 3',5'-monophosphate-mediated, vascular response, such a mechanism is unlikely to have been responsible for the later anti-inflammatory action of PGE(1).7 The implications of these results are discussed in relation to the postulated negative-feedback role of endogenous PGE in chronic inflammation.

Kadsurenone distinguishes between different platelet activating factor receptor subtypes on macrophages and polymorphonuclear leucocytes.

The effects of the antagonist kadsurenone on the platelet activating factor (Paf)-induced chemiluminescence of guinea-pig peritoneal macrophages and on pig peripheral blood leucocyte aggregation were compared. Linearity and slopes of unity of the Schild plots confirmed the competitive nature of the antagonism by kadsurenone. pA2 values indicated a 91 fold lower affinity of kadsurenone for leucocyte Paf receptors than for those in macrophages. It is concluded that these two types of Paf receptors are not identical and are provisionally designated Paf1 and Paf2 receptors, respectively.

The influence of metyrapone on the synthesis and release of prostaglandins from the pregnant rat uterus in vitro.

1 Metyrapone (150 mg/kg, s.c. or i.p.) an inhibitor of corticosteroid biosynthesis, significantly reduced the release of prostaglandins of the F-type from isolated preparations of pregnant rat uteri in vitro, on day 22 - the expected day of delivery. 2 Metyrapone and indomethacin administered in vitro both inhibited the conversion of 14C-arachidonic acid to prostaglandin E2 by homogenates of day 22 pregnant rat uteri. Metyrapone was approximately 150 times less potent than indomethacin. Although indomethacin also inhibited prostaglandin F2alpha production, metyrapone stimulated synthesis of this prostaglandin. The differential inhibition of prostaglandin synthesis by metyrapone may reflect sensitivity of the inhibitor to changes in experimental conditions. 3 Inhibition of prostaglandin synthesis may explain the effects of metyrapone on parturition in the rat.

Antagonism of platelet activating factor-induced chemiluminescence in guinea-pig peritoneal macrophages in differing states of activation.

1. The effects of the platelet activating factor (Paf) antagonists alprazolam, BN 52021, kadsurenone, L 652,731 and SRI 63119 have been studied on Paf-induced chemiluminescence (CL) of guinea-pig, C. parvum-activated peritoneal macrophages in vitro. 2. All antagonists produced a shift to the right in the dose-response curve to Paf (0.001-10 mumol l-1). Schild plots for BN 52021, L 652,731, kadsurenone and SRI 63119 were linear, but only for BN 52021 and kadsurenone did the mean slope not differ significantly from unity. Mean pA2 values for BN 52021 and kadsurenone were 6.60 +/- 0.05 and 6.41 +/- 0.14 (mean + s.e.mean) respectively. Calculation of IC50 values for all antagonists (at 0.1 mumol l-1 Paf) gave an order of potency: L 652731 greater than kadsurenone greater than or equal to BN 52021 greater than alprazolam greater than SRI 63119. 3. When individual pA2 values for BN 52021 and kadsurenone were plotted against the maximal CL response to Paf of cell suspensions in the absence of antagonist (reflecting the degree of activation of the macrophages by the C. parvum), it was found that the affinity of both antagonists for macrophage Paf receptors remained relatively constant irrespective of the activation state of the cells. 4. We conclude that activation of guinea-pig peritoneal macrophages does not account for the increased affinity for macrophage Paf receptors previously observed for kadsurenone. Kadsurenone and BN 52021 presumably bind to a site on Paf receptors which is not affected by the activation process, while alprazolam and SRI 63119 are non-specific antagonists. The reason for the difference between the competitive nature of kadsurenone and its structural analogue L 652,731 is unclear.

Evidence for a possible foetal control of prostaglandin release from the pregnant rat uterus in vitro.

The release of prostaglandin-like material and these spontaneous contractions of individual horns from the pregnant rat uterus in vitro have been studied on day 22 of pregnancy - the expected day of delivery. Removal of foetuses (retaining placentae in utero) from one or both uterine horns on day 16 or 17 significantly reduced prostaglandin F release and spontaneous activity. Rats which had been made unilaterally pregnant after ligation of one uterine horn, exhibited a decrease in prostaglandin F output from both horns. Uterine activity and prostaglandin release were increased in quiescent uteri by the addition of arachidonic acid (5 mug/ml) or phospholipase A (160 mu./ml); the effects were abolished by indomethacin (20 mug/ml). However, the stimulation of uterine activity by PGF2alpha (30-60 ng/ml) was not affected by indomethacin. It is concluded that the release of prostaglandins from the pregnant rat uterus in vitro at term is related to the presence of viable foetuses.

Antirheumatic agents and leukocyte recruitment. New light on the mechanism of action of oxaceprol.

Most anti-inflammatory agents used in the treatment of joint diseases exert inhibitory effects on leukocyte infiltration. Methotrexate, a disease-modifying drug, and corticosteroids also inhibit leukocyte accumulation during inflammation. However, the mechanisms of action of these different compounds on leukocytes vary and in the case of non-steroidal anti-inflammatory drugs (NSAIDs) the mechanism(s) may be indirect. No current drug for inflammatory or degenerative joint disease has been proposed to act specifically by an inhibitory action on neutrophilic leukocytes. Oxaceprol is an amino acid derivative that has been used for several years for the treatment of osteoarthritis and rheumatoid arthritis, ameliorating pain and stiffness and showing good gastrointestinal safety, particularly in comparison with NSAIDs. Recent experimental studies have shown that oxaceprol does not inhibit the synthesis of prostaglandins in vitro, but markedly inhibits neutrophil infiltration into the joints of rats with adjuvant arthritis. These results support earlier screening data showing inhibition by oxaceprol of leukocyte infiltration into sites of acute inflammation. In studies on surgical ischemia reperfusion in hamsters in vivo, oxaceprol was an effective inhibitor of leukocyte adhesion and extravasation. It is proposed that oxaceprol represents a therapeutic agent for degenerative and inflammatory joint diseases, which acts predominantly by inhibiting leukocyte adhesion and migration.

A novel biologically active seleno-organic compound--III. Effects of PZ 51 (Ebselen) on glutathione peroxidase and secretory activities of mouse macrophages.

PZ 51 (2-phenyl-1,2-benzisoselenazol-3(2H)-on), a selenium-containing compound with glutathione peroxidase (GSH-Px)-like activity, was administered to selenium-deficient mice for 5 days. A significant increase in peritoneal macrophage GSH-Px activity after treatment was only observed when basal GSH-Px activity was almost zero (i.e. in 19 weeks selenium-deficient animals), possibly due to binding of PZ 51 to the macrophages. This indicates that PZ 51 releases very little, if any, free selenium for incorporation into endogenous GSH-Px. The compound in vitro exerted a concentration-dependent inhibition of the generation of chemiluminescence by resident mouse peritoneal macrophages and a partial inhibition of the production of prostaglandin E2 by resident peritoneal macrophages. beta-Glucuronidase production by C. parvum-activated peritoneal macrophages was unaffected by PZ 51. These in vitro data can be explained on the basis of a selective peroxide scavenging and/or GSH-Px-like activity of PZ 51, offering a novel approach to anti-inflammatory therapy.

Inflammation: from molecular biology to clinic.

The 3rd World Congress on Inflammation reflected an emphasis on cytokines and cell signaling, but also provided an overview of some of the current hot topics in inflammation research. The chemokines are an exponential growth area for researchers in several fields, and chemokine antagonists are under study for potential use in inflammatory disease. The use of soluble receptors, antibodies and gene deletion has already validated a number of new approaches, notably those targeted at TNF-alpha, IL-8 and, as revealed at this meeting, IL-6, NFkappaB and AP-1. The pharmacological regulation of apoptosis for disease therapy is also becoming a possibility. It is probable that within the next 12 months a number of small-molecule synthetic compounds will be reported as likely candidates for future antiinflammatory agents. The marketing of specific COX-2 inhibitors is likely to provide a significant step forward in therapeutic tolerability. Gene therapy for inflammation still lies several years in the future, but initial clinical studies are under way.

The effect of metyrapone on uterine prostaglandin output and smooth muscle activity.

Metyrapone, at low doses (0.5-1.0 mM), stimulated the output of both PGE and PGF from the isolated uterus of the pregnant rat determined following extraction of bath fluid, chromatographic separation and bioassay of the prostaglandin. At higher doses (2-4 mM), metyrapone inhibited PGF output, but had no effect on PGE output. Uterine activity was rapidly inhibited by metyrapone in a dose-related manner. This inhibition was not related to PG output as, at 1 mM metyrapone, activity was inhibited and PG output stimulated. Both metyrapone and papaverine produced dose-dependent inhibition of the activity of the isolated rabbit ileum, papaverine being 10 times more potent than metyrapone. Propranolol antagonised the response of the ileum to isoprenaline, but had no effect on the response to metyrapone. These observations confirm earlier data, suggsting that metyrapone exerts a differential effect on uterine PGE and PGF production and indicate that metyrapone has a direct inhibitory effect on smooth muscle activity.

Anti-inflammatory effects of macrolide antibiotics.

Macrolides are widely used as antibacterial drugs. Clinical and experimental data, however, indicate that they also modulate inflammatory responses, both contributing to the treatment of infective diseases and opening new opportunities for the therapy of other inflammatory conditions. Considerable evidence, mainly from in vitro studies, suggests that leukocytes and neutrophils in particular, are important targets for modulatory effects of macrolides on host defense responses. This underlies the use of the 14-membered macrolide erythromycin for the therapy of diffuse panbronchiolitis. A variety of other inflammatory mediators and processes are also modulated by macrolides, suggesting that the therapeutic indications for these drugs may be extended significantly in future.

Improvement of the gastric tolerance of non-steroidal anti-inflammatory drugs by polyene phosphatidylcholine (Phospholipon 100).

The effect of co-administration with polyene phosphatidylcholine (Phospholipon 100) on the oral gastrotoxicity of various non-steroidal anti-inflammatory drugs (NSAIDs) was studied in the rat. The highly unsaturated phospholipid reduced gastric mucosal lesions measured 3.5 h after oral administration of aspirin, indomethacin, phenylbutazone, diclofenac, piroxicam and sudoxicam to rats which had received a 3 day bread diet followed by 24 h fasting. The extent of reduction of gastrotoxicity varied amongst the individual NSAIDs. Phospholipon 100 also reduced gastric lesions induced by 3 day oral piroxicam and diclofenac administration. A trend towards reduction of oral diclofenac gastrotoxicity was observed following intravenous Phospholipon 100 administration. Phospholipon 100 H (100% saturated phosphatidylcholine) was less effective than Phospholipon 100 in improving acute gastric tolerance to oral phenylbutazone, diclofenac and piroxicam. Administration of the NSAID-Phospholipon 100 combination produced little change in the anti-inflammatory activities of diclofenac on carrageenan paw oedema and diclofenac and piroxicam on adjuvant arthritis in the rat. Combination with Phospholipon 100 offers a novel means for reducing the gastric side-effects of NSAID therapy.

A multi-centre, double-blind comparative study of the efficacy and safety of aceclofenac and diclofenac in the treatment of rheumatoid arthritis.

A long term multi-centre, double-blind, parallel group study was undertaken to investigate the efficacy and safety of aceclofenac (170 patients, 100 mg b.i.d. and placebo once daily) in comparison to diclofenac (173 patients, 50 mg t.i.d.) given for 6 months to patients of both sexes with active rheumatoid arthritis. Efficacy was evaluated in 131 aceclofenac and 130 diclofenac patients at 15 days, 1, 2, 4 and 6 months. Although both treatment groups showed significant improvement in all evaluations of pain and inflammation (assessed by a Visual Analogue Scale and the Ritchie Index) and a progressive reduction in morning stiffness, there were no significant differences between the groups. There was, however, a trend towards greater improvement in hand grip strength with aceclofenac (22% improvement) than diclofenac (17% improvement). Adverse events in both groups were minor, predominantly gastro-intestinal, and fewer patients tended to experience gastro-intestinal events on aceclofenac (13%) than on diclofenac (17%). The overall assessment of tolerance, however, did not differ significantly between groups. In summary, this study supports a therapeutic role for aceclofenac in the treatment of rheumatoid arthritis, and suggests it is an effective and safe NSAID for the treatment of this disease.

Toxicity screening of liposomes.

Phospholipids are the major components of most liposomes. Extensive testing of these naturally occurring compounds has revealed them to be remarkably safe for pharmaceutical use. Addition of other constituents to liposomes in order to alter stability or kinetics can result in an increase in toxic potential, particularly on parenteral administration of liposomes. This paper describes some simple in vitro cellular tests for direct toxicity of liposomes, particularly following intravenous (i.v.) or topical administration, including tests for haemolysis, thrombosis and cytotoxicity. In addition, an in vivo test for the effects on phagocytosis and for pyrogenicity are described, together with a brief outline of the requirements for the further toxicity testing of liposomal drugs at a later stage of development.

Redistribution of cardiac output during localized granulomatous inflammation and the effect of methysergide in anesthetized rats.

The distribution of cardiac output (CO) to different organs and tissues was determined by the radioactive microsphere technique in anesthetized rats 3, 5, 7, and 10 days after subcutaneous implantation of carrageenan-impregnated sponges to induce granulomatous inflammation. Distribution of CO to organs which usually respond to stress (heart, spleen, adrenals) was increased during the acute-subacute inflammatory response, at the expense of the distribution to the head, brain, abdominal muscle, and hind legs. Later decreases were seen in distribution of CO to the kidneys, tail, forelegs, and urogenitals. Vascular-related skin temperature (measured by thermography) and distribution of CO to the back skin generally showed a decrease, particularly to the skin covering the granuloma. This latter decrease was reversed by systemic treatment with methysergide (40 mg/kg) which also increased distribution of CO to the head and decreased that to the liver. Most of the effects observed can be attributed to the extra burden on the circulation presented by the highly vascularized granuloma tissue and/or to the release of 5-hydroxytryptamine, but may differ from changes in conscious rats.