Alison M. Badger

p38 Mitogen-Activated Protein Kinase Inhibitors— Mechanisms and Therapeutic Potentials

The pyridinylimidazole compounds, exemplified by SB 203580, originally were prepared as inflammatory cytokine synthesis inhibitors. Subsequently, the compounds were found to be selective inhibitors for p38 mitogen-activated protein kinase (MAPK), a member of the MAPK family. SB 203580 inhibits the catalytic activity of p38 MAPK by competitive binding in the ATP pocket. Four homologues of p38 MAPK have been identified to date, and interestingly, their biochemical properties and their respective sensitivities to the inhibitors are distinct. X-ray crystallographic analysis of p38-inhibitor complexes reinforces the observations made from site-directed mutagenesis studies, thereby providing a molecular basis for understanding the kinase selectivity of these inhibitors. The p38 MAPK inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury.

Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis

OBJECTIVE To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

p38 MAP kinase: molecular target for the inhibition of pro-inflammatory cytokines.

Publisher Summary This chapter discusses that the network of immune and inflammatory responses is comprised of a variety of cell types. Coordination of this network occurs through both direct cell–cell contact and by way of intercellular signalling molecules. These signalling molecules regulate the growth, differentiation and function of a variety of target cells. Understanding the structure and function of these molecules has provided new and important insights into the fundamental biology of immunity and inflammation, and has led to the identification of new strategies for the development of more effective medicines for the treatment of a variety of autoimmune and inflammatory diseases. The chapter reviews that the pluripotent pro-inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor alpha (TNFα) appear to play particularly important roles in disease. Although, these proteins and their cellular receptors are structurally unrelated, they elicit a similar profile of pro-inflammatory responses. Because IL-1 and TNF are produced early in response to pro-inflammatory signals, and because of their central role in mediating this response, they have often been termed the master cytokines. While the success of these therapies has validated the importance of IL-1 and TNF in promoting disease, they also highlight the need for improved therapies that do not suffer from the disadvantages of proteinaceous macromolecules, which must be administered parenterally and are inherently more expensive to produce than small molecule drugs. To date, no orally active low molecular weight cytokine receptor antagonist has emerged from clinical trials. However, in the last decade several new strategies to interrupt the synthesis and signalling of these cytokines have emerged. One of the first of these targets to be elucidated is the stress-activated protein kinase, p38.

Disease‐modifying activity of SB 273005, an orally active, nonpeptide αvβ3 (vitronectin receptor) antagonist, in rat adjuvant‐induced arthritis

Objective To evaluate the effects of SB 273005, a potent, orally active nonpeptide antagonist of the integrin αvβ3 vitronectin receptor, on joint integrity in rats with adjuvant-induced arthritis (AIA). Methods Male Lewis rats with AIA were orally dosed either prophylactically (days 0–20) or therapeutically (days 10–20) with SB 273005. Efficacy was determined by measurement of paw inflammation, assessment of bone mineral density using dual-energy x-ray absorptiometry (DEXA), magnetic resonance imaging (MRI), and histologic evaluation. Results SB 273005 is a potent antagonist of the closely related integrins, αvβ3 (Ki = 1.2 nM) and αvβ5 (Ki = 0.3 nM). When SB 273005 was administered prophylactically to AIA rats twice per day, it inhibited paw edema at doses of 10, 30, and 60 mg/kg, by 40%, 50%, and 52%, respectively. Therapeutic administration twice daily was also effective, and a reduction in paw edema was observed at 30 mg/kg and 60 mg/kg of the antagonist (by 36% and 48%, respectively). SB 273005 was also effective when administered once per day, both prophylactically and therapeutically. Significant improvement in joint integrity in treated rats was shown using DEXA and MRI analyses. These findings were confirmed histologically, and significant protection of bone, cartilage, and soft tissue was observed within the joint. Conclusion Symptoms of AIA in rats were significantly reduced by either prophylactic or therapeutic treatment with the αvβ3 antagonist, SB 273005. Measurements of paw inflammation and of bone, cartilage, and soft tissue structure indicated that this compound exerts a protective effect on joint integrity and thus appears to have disease-modifying properties.

Culture conditions affecting induction and release of lymphocyte produced proliferation inhibitory factor (PIF)

Abstract Studies on the factors affecting the production of a proliferation inhibitory factor (PIF) by human lymphocytes are presented. Maximal PIF production occurred with mitogen stimulation of blood lymphocytes cultured at 1 × 106/ml. Optimal cultures contained 10% fetal calf serum, but PIF could be produced in the absence of serum, and after only a 6-hr pulse exposure to PHA. PIF production was found to correlate with lymphocyte activation in response to the mitogen PHA but was not related to lymphocyte proliferation (DNA synthesis). Inhibitory activity could be detected as early as 3 hr after mitogen addition, long before DNA synthesis occurs. The mitogens Con A and PWM initiated different intensities of DNA synthesis in these cultures, but similar quantities of PIF. Antigenic stimulation of sensitive human peripheral lymphocyte populations resulted in the release of PIF. Cells from donors that gave a strong positive skin test to tuberculin (PPD) responded in tissue culture to PPD by producing PIF, while the cells from skin test negative donors did not. A small quantity of PIF was also evident in the supernatants from cultures with no known stimulus (“unstimulated”), this was found to result from activation of the lymphocytes by nonlymphoid elements and by fetal calf serum. An investigation of the PIF-producing capabilities of other lymphoid tissues showed that lymph node cells produced this humoral factor, whereas thymus cells did not. Thymus cell supernatants, in fact, were found to contain an extremely labile cytotoxin which degraded rapidly upon storage.

Studies on the mechanism of action of proliferation inhibitory factor (PIF)

Abstract Preliminary studies on the mechanism of action of the proliferation inhibitory factor (PIF) are described. The proliferation inhibitory factor does not appear to alter the culture medium or to interfere with the growth-enhancing effect of serum. Inhibition of cellular proliferation is not absolute and can readily be reversed if the PIF is removed. There is a rapid attainment of the normal doubling time with a lag period of about 24 hr or less. A synchronized population of cells was utilized to show that all treated cells go through one cell division before the inhibitory effect is seen. Experiments designed to examine the effect of PIF at different times in the cell cycle demonstrated that the inhibitor was most effective when present during mitosis, and that treating cells during G1 had negligible effect.

Immunostimulatory and immunosuppressive factors in human cancer ascites fluids. Effect on the primary plaque-forming response in vitro.

Abstract Immunosuppressive and immunostimulatory activity of human cancer ascitic fluids has been examined using the in vitro primary plaque-forming cell response (PFR) to sheep erythrocytes (Mishell-Dutton assay). We have prepared three fractions of ascitic fluid by precipitation with ammonium sulfate. Suppressive activity occurred in the fraction which was insoluble at 50% of saturation but not those fractions which precipitated at 30 or 80% of saturation. The fractions which were precipitated at 30 and 80% were stimulatory in the assay system. Normal human serum also had suppressive activity in the fraction precipitated at 50% of saturation but not as much as was found in ascitic fluid. Serum did not yield any fractions with stimulatory activity.

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB2) receptor. The results were consistent with changes in cAMP content of hCB2-transfected human embryonic kidney (HEK) cells challenged with the same CB2-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors.

An immunostimulatory substance in the ascites fluid of patients with cancer metastatic to the peritoneum.

Abstract The ascitic fluids from patients with cancer metastatic to the peritoneum contain a factor(s) which stimulates the primary antibody response to sheep red blood cells (SRBC) in vitro . This enhancement is manifested by an increase in the number of plaque-forming cells per culture and a slight increase in plaque size. This factor has a molecular weight in the range 30,000–100,000 as determined by Sephadex gel filtration. The factor, which we have called “stimulatory factor” (SF), will completely replace the requirement for fetal calf serum in the Mishell-Dutton type of assay. Enhancement of the antibody response is most apparent at suboptimal culture conditions. SF does not increase the number of plaque-forming cells to the T-independent antigen Escherichia coli but there is a marked increase in the size of the plaques produced to the lipopolysaccharide using coated SRBC as targets. The stimulation induced by this factor is not due to endotoxin contamination since endotoxin is heat stable and the SF is heat inactivated at 80 °C for 10 min. In addition endotoxin does not act in a manner similar to SF. Thus, the SF appears to influence both T and B cells. With thymus-dependent antigen the factor results in increased numbers of antibody cells being generated; with thymus-independent antigen the factor results in increased quantity of antibody being produced.

A method for the selection of a synchronized population of cloning HeLa cells.

Summary A method is described in which individual clones of cells in synchrony can be selected out of a nonsynchronized population of cells. These cells have progressed no more than 3 hr into the G1 phase of the cell cycle. The clonal growth of these colonies can be observed microscopically for up to 72 hr before they begin to lose synchronous cell division.