John B. Cheng

Validation of a Standardized Method for Enumerating Circulating Endothelial Cells and Progenitors: Flow Cytometry and Molecular and Ultrastructural Analyses

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16+CD45−CD31+CD146+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P < 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

Regulation of tumour necrosis factor production by adrenal hormones in vivo: insights into the antiinflammatory activity of rolipram.

1 The role of adrenal hormones in the regulation of the systemic and local production of tumour necrosis factor (TNFα) was examined in male Balb/c mice. 2 Intraperitoneal injection of 0.3 mg E. coli lipopolysaccharide (LPS, 0111:B4) led to high levels of circulating TNFa without stimulating TNFa production in the peritoneal cavity. Systemic production of TNFα in response to LPS was increased in adrenalectomized animals and in normal animals treated with the β‐adrenoceptor antagonist, propranolol. The glucocortoid antagonist, RU 486, did not modify systemic TNFα production. These results indicate that systemic TNFα production is regulated by adrenaline but not by corticosterone. 3 When mice were primed with thioglycollate, TNFα was produced in the peritoneal cavity in response to low dose LPS (1 μg). The levels of TNFα in the peritoneal cavity were not enhanced by adrenalectomy or by treatment with either propranolol or RU 486, indicating local production of TNFα in the peritoneal cavity is not regulated by adrenaline or corticosterone. 4 The phosphodiesterase type IV (PDE‐IV) inhibitor, rolipram, inhibited both the systemic production of TNFa in response to high dose endotoxin (ED50 = 1.3 mg kg−1) and the local production of TNFα in the peritoneal cavity in response to low dose endotoxin (ED50 = 9.1 mg kg−1). In adrenalectomized mice there was a slight reduction in the ability of rolipram to inhibit the systemic production of TNFα (ED50 = 3.3 mg kg−1) while the ability of rolipram to inhibit the local production of TNFα in the peritoneal cavity was virtually abolished (24% inhibition at 30 mg kg−1). The glucocorticoid antagonist, RU 486, also reduced the ability of rolipram to inhibit local TNFα production while propranolol was without effect. 5 Systemic treatment with rolipram increased the plasma concentrations of corticosterone in normal mice but not in adrenalectomized mice indicating that rolipram can cause adrenal stimulation in vivo. 6 In summary, these data indicate that systemic production of TNFα in response to high dose endotoxin is controlled differently from the local production of TNFα in response to low dose endotoxin. The systemic production of TNFα is regulated by catecholamines, but not by corticosterone, while the local production of TNFα in the peritoneal cavity is not regulated by basal levels of either catecholamines or corticosterone. 7 These data also show that the ability of rolipram to inhibit the local production of TNFα is dependent on the release of corticosterone from the adrenal glands.

Synthesis and in vitro profile of a novel series of catechol benzimidazoles. The discovery of potent, selective phosphodiesterase type IV inhibitors with greatly attenuated affinity for the [3H]rolipram binding site

Abstract The synthesis and biological properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors are described. These catechol benzimidazoles were designed from rolipram and initial compounds reflected a similarly high affinity for the [ 3 H]rolipram b binding site (500 to 1000X greater affinity for the [ 3 H]rolipram binding site over the PDE IV inhibitory site). However, SAR studies on the 3-alkoxy position revealed that this [ 3 H]rolipram binding site affinity could be attenuated, while potentiating the PDE IV inhibitory activity. This resulted in the 2-indanyl analog 13 which is a potent, selective PDE IV inhibitor with a 15X differential in favor of PDE IV binding.

Anti-IL21 receptor monoclonal antibody (ATR-107): Safety, pharmacokinetics, and pharmacodynamic evaluation in healthy volunteers: a phase I, first-in-human study.

Safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ATR‐107, a fully human monoclonal anti‐IL‐21 receptor (IL‐21R) antibody, administered as ascending single doses, subcutaneously or intravenously, was evaluated in a placebo‐controlled, double‐blind trial in healthy subjects. The dose levels were 3–300 mg by SC and 30–120 mg by IV. The most important adverse events were hypersensitivity reactions occurring in three out of six subjects in 300 mg SC cohort and considered as dose limiting toxicity. More than 75% of the subjects who received ATR‐107 developed anti‐drug antibodies (ADAs), which had no discernible impact on PK or safety. The PK of ATR‐107 appeared to be dose ‐proportional. T1/2 was shorter than typical therapeutic antibodies. Bioavailability of ATR‐107 was about 30%. IL‐21R occupancy was measured in circulating B cells in the 60 and 120 mg IV cohort. The data indicated that single dose of ATR‐107 was able to maximally occupy IL‐21Rs through at least Day 42. Further escalation in the FIH study was halted partially due to the high rates of ADA formation. In conclusion, ATR‐107 had a prolonged PD effect measured by IL‐21R occupancy; was highly immunogenic after single dose administration and had PK properties with rapid clearance and low bioavailability.

Discovery of micromolar PDE IV inhibitors that exhibit much reduced affinity for the [3H]rolipram binding site: 3-norbornyloxy-4-methoxyphenylmethylene oxindoles

Abstract The synthesis and the in vitro properties of a novel series of potent and selective phosphodiesterase type IV (PDE IV) inhibitors is described. Despite bearing structural similarity to rolipram, several of these compounds have much reduced affinity for the [3H]rolipram binding site.

Biarylcarboxamide inhibitors of phosphodiesterase IV and tumor necrosis factor-α

Abstract Tumor necrosis factor-α (TNF-α) has been implicated as a key mediator in the progression of rheumatoid arthritis. Inhibitors of phosphodiesterase IV (PDE IV) have been shown to inhibit the production of TNF-α by elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Our efforts in a series of biarylcarboxamides have led to the identification of 8j (CP-353,164) as a potent inhibitor of PDE IV and TNF-α production.

pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood

BackgroundIL-21 has been shown to play an important role in autoimmune diseases. ATR-107 is an antibody which directly targets the IL-21 receptor (IL-21R). To aid the clinical development of ATR-107, there is a need for understanding the mechanism of action (MOA) of this antibody when assessing target engagement in human subjects.MethodsTo determine ATR-107 biological activity and potency in human blood, its inhibitory function against IL-21 induced STAT3 phosphorylation in human peripheral T and B cells was measured.ResultsThe data show that IL-21 induces STAT3 phosphorylation in a concentration-dependent manner, consistent with its migration to the nuclear. Using a flow cytometry based functional whole blood assay, ATR-107 is demonstrated to be a potent IL-21 pathway inhibitor. It competes with IL-21 for receptor binding in a competitive manner, but once it binds to the receptor it behaves like a non-competitive inhibitor, most probably due to the long observed koff. The concentration-dependent inhibition observed with ATR-107 correlates inversely with the levels of receptor occupancy, both in ex vivo whole blood assays and directly in human blood when ATR-107 was given to healthy volunteers.ConclusionsIL-21 induced phosphorylation of STAT3 in T and B cells can be used as a biomarker to evaluate the target engagement of ATR-107 in human whole blood. The antibody behaves like a potent non-competitive inhibitor blocking IL-21 induced STAT3 phosphorylation for a long period of time. These results may help with the translation of preclinical information and dose selection towards ATR-107 clinical efficacy.

Development of new chromanol antagonists of leukotriene D4

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

Discovery of CP-199,330 and CP-199,331: Two potent and orally efficacious cysteinyl LT1 receptor antagonists devoid of liver toxicity

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

Chapter 8. Emerging Opportunities in the Treatment of Asthma and Allergy

Publisher Summary This chapter discusses the two molecular targets, such as modulators of intracellular cyclic adenosine monophosphate (cAMP) and inhibitors of lymphocyte-derived cytokines. Current asthma therapies, such as theophylline and steroids, are used to stimulate the cAMP pathways. Cyclic nucleotide PDEs are viewed as attractive molecular targets for the treatment of asthma and inflammation. PDE isozymes are classified into five families based on their specificity, for either cAMP or cGMP, inhibitor sensitivity and calciumcalmodulin dependence. PDE IV and PDE III/lV inhibitors possess both bronchodilatory and anti-inflammatory activity in animal models. A major obstacle in the development of PDE IV inhibitors is the elimination of emetic side effects. PDE IV inhibitor 1 reduces the abnormal levels of IL-4 production, for controlling the defective immune function in atopic dermatitis. Eosinophil counts in conjunctival epithelium in LT-challenged guinea pigs that receive are reduced compared to the LT-challenged controls. The pulmonary anti-allergic activity of theophylline results in part from the inhibition of PDE IV and the bronchodilator activity of theophylline results in part from the inhibition of PDE 111. Several isoforms of the PDE IV isozyme have been cloned, from rat and from man, and access to these isoforms lead to more selective PDE inhibitors. Elevated cAMP binds the two regulatory subunits of the protein kinase A (PKA) holoenzyme, promoting dissociation of two monomeric catalytic subunits.