Loretta A. Bober

Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists.

Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor‐specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor—specific inverse agonist, the triaryl bis‐sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G‐protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis‐sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte‐specific actin bundling protein L‐plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen‐induced mono‐articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice

BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

The Toxicity of SCH 351591, a Novel Phosphodiesterase-4 Inhibitor, in Cynomolgus Monkeys

SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.

The effect of GM-CSF and G-CSF on human neutrophil function

A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil-function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB4. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. albicans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (Fc gamma R1, CR-1 and CR-3), and adhesion signalling (ICAM-1), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lepob/Lepob (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1–3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils

IL‐4 is a T‐helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL‐4 on neutrophilic maturation using the cell line HL‐60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL‐60 cells with recombinant hu IL‐4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL‐60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL‐4 increased surface expression of the neutrophil‐lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL‐4 treated HL‐60 cells demonstrated enhanced Fc‐ and complement‐mediated phagocytic capacity and increased hexose‐monophosphate shunt activity. In addition, IL‐4 was capable of sustaining the neutrophil maturation of HL‐60 cells that had been pre‐treated for 24h with DMSO. To investigate the effect of IL‐4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL‐4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti‐human IL‐4 abolished phagocytic stimulation. Finally, IL‐4 treatment also stimulated resting neutrophils to migrate toward zymosan‐activated serum (ZAS) and human IL‐5. The results demonstrate that IL‐4 is a potent maturation factor for myelocytes to become neutrophils and that IL‐4 can stimulate resting mature neutrophils.

Targeting the CB2 receptor for immune modulation

Early work on the biology of the components of Cannabis sativa showed evidence for a potential influence on immune regulation. With the discovery of a peripheral cannabinoid receptor associated with immune cells, many laboratories have sought to link the immunoregulatory activities of cannabinoid compounds with this receptor, hoping that such compounds would lack the psychoactive effects of marijuana and other nonspecific cannabinoid agonists. In this report, the authors investigate the role of the cannabinoid CB2 receptor in immune regulation, with particular emphasis on compounds shown to regulate immune cell recruitment. The authors conclude by using the immune cell recruitment model to rationalise cannabinoid CB2 receptor-specific effects in modulating immune disease, particularly the increasing evidence for its role in experimental autoimmune encephalomyelitis and in influencing bone density.

The effects of colony stimulating factors on human monocyte cell function.

We used a panel of functional assays to compare directly the pattern and potency of GM-CSF and M-CSF on monocyte activity associated with cell-mediated immune defense. GM-CSF and M-CSF were found to be equivalent both in their capacity to stimulate human monocyte functions in vitro and in their pattern of monocyte activation. The two CSFs were effective in inducing monocyte chemotaxis towards either fMLP or LTB4 at equivalent concentrations across a panel of donors. GM-CSF and M-CSF demonstrated equipotency in the induction of monocyte phagocytosis of heat-killed bakers yeast and in the regulation of the hexose-monophosphate shunt (NBT reduction). Both were also found to be equivalent in preventing steroid (dexamethasone)-induced suppression of monocyte anti-bacterial (Candida albicans) and anti-fungal (Staphylococcus aureus) phagocytic capacities. GM-CSF was somewhat more effective than M-CSF in stimulating monocyte C. albicans killing at a lower E:T ratio.

Regulatory effects of interleukin‐4 and interleukin‐10 on human neutrophil function ex vivo and on neutrophil influx in a rat model of arthritis

OBJECTIVE To assess the capacity of interleukin-4 (IL-4) and IL-10 to block polymorphonuclear neutrophil (PMN) activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis. METHODS The ex vivo phagocytic capacity of cytokine-activated human PMNs was assessed by use of assays for measuring the ingestion of heat-killed yeast and by subsequent hexose-monophosphate shunt activation using nitroblue tetrazolium reduction. The in vivo activity of IL-4 and IL-10 was measured using a rat adjuvant arthritis model in which the mycobacterial antigen concentration was titrated to modify disease intensity. RESULTS IL-4 and IL-10 suppressed the ex vivo activation state of interferon-gamma- and tumor necrosis factor alpha-activated human neutrophils. In the rat adjuvant arthritis model, treatment with systemic murine IL-10 (mIL-10) effectively suppressed all disease parameters in rats that received the lower concentrations of mycobacteria, whereas systemic mIL-4 was effective against even the most severe disease. Both cytokines were effective in lowering the absolute PMN cell number recovered and the PMN activation state in the joint synovia. We also observed lower levels of the messenger RNA transcript for CINC protein (cytokine-induced neutrophil chemoattractant; a rat homolog for human IL-8) in the synovia. CONCLUSION IL-10 is an effective antiarthritic agent and has a major effect on the presence and function of PMNs in the joint synovia when disease intensity is not severe. IL-4 has an inhibitory profile that is similar to that of IL-10, but is effective in modifying even the most severe disease. Both cytokines reduced the phagocytic activation of human PMNs in response to proinflammatory cytokines. These data demonstrate that IL-4 and IL-10 can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis. Treatment with these cytokines alone or in combination may therefore be very useful in the management of patients with rheumatoid arthritis.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [35S]mesyl chloride (synthesized from commercially obtained [35S]methane sulfonic acid) to generate [35S]Sch225336. [35S]Sch225336 has high specific activity (>1400 Ci/mmol) and affinity for hCB2 (65 pm). Using [35S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [35S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [35S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.