A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophagederived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin– Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2–activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

Background and purpose: Hydrogen sulphide (H2S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H2S in blood and tissues. Experimental approach: Concentrations of reactive sulphide in saline and freshly drawn whole blood were quantified by reaction with the thio-specific derivatization agent monobromobimane, followed by reversed-phase fluorescence HPLC and/or mass spectrometry. In pharmacokinetic studies, male rats were exposed either to intravenous infusions of sodium sulphide or to H2S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H2S/HS -were concomitantly measured using an amperometric sensor. Key results: Monobromobimane was found to rapidly and quantitatively derivatize sulphide in saline or whole blood to yield the stable small molecule sulphide dibimane. Extraction and quantification of this bis-bimane derivative were validated via reversed-phase HPLC separation coupled to fluorescence detection, and also by mass spectrometry. Baseline levels of sulphide in blood were in the range of 0.4-0.9 mM. Intravenous administration of sodium sulphide solution (2-20 mg·kg) or inhalation of H2S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg·kg -1 ·h -1 of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H2S gas inhalation. Conclusions and implications:The monobromobimane derivatization method is a sensitive and reliable means to measure reactive sulphide species in whole blood. Using this method, we have established a bioequivalence between infused sodium sulphide and inhaled H2S gas.

With the discovery of hydrogen sulfide as a signaling molecule and a potential therapeutic, measurement of free sulfide in blood – as hydrogen sulfide or hydrosulfide anion – has taken on importance. Here, we demonstrate and validate a method of free sulfide measurement whereby the free sulfide in whole blood is derivatized with excess monobromobimane. The resulting sulfide‐dibimane is subsequently extracted into ethyl acetate, followed by quantitation of sulfide‐dibimane via reverse‐phase HPLC with fluorescence detection. Reaction conditions are validated through 1) characterization of rate of conversion from sulfide to sulfide‐dibimane, 2) analysis of reaction in the presence of potential interferants, and 3) recovery of standard samples from a whole‐blood matrix. We found that reaction conditions of a mixture of acetonitrile and HEPES buffer (50 mM pH 8) gave rapid, clean conversion of sulfide to sulfide‐dibimane in the presence of excess monobromobimane. For whole blood, a 1:1:1 reaction mixture of 200 μl each acetonitrile:HEPES:blood proved optimal. Using this protocol, standard samples were consistently recovered in approximately 76% yield over the range of the assay. Baseline levels of free sulfide in rat blood were found to be about 0.3 – 0.5 μM. Subsequent work has proved the method effective in generating whole‐blood sulfide PK data in multiple species.

Hydrogen sulfide is best known as an environmental pollutant and human health hazard. Recently, sulfide has gained recognition as an endogenous biological mediator and is being examined for use as a therapeutic agent. As sulfide may be administered by both inhaled and parenteral routes, the question arises of how exposure to sulfide compares between the two modes of administration. Sprague Dawley rats were exposed for up to two hours of hydrogen sulfide gas up to 400 ppm or received up to 20 mg/kg sodium sulfide by continuous intravenous infusion. Sulfide concentrations in venous blood were quantified as sulfide dibimane derivative. Both modes of administration lead to a dose‐dependent elevation in blood sulfide concentrations over baseline concentrations and reached a new steady‐state concentration within two hours. Steady state blood sulfide concentrations show a simple linear relationship to hydrogen sulfide gas concentration or sodium sulfide dose. Identical blood sulfide concentrations can be achieved by either route of administration suggesting bioequivalency of hydrogen sulfide inhalation and sodium sulfide intravenous infusion in modulating blood sulfide concentrations. This suggests that both modes of administration may exert similar therapeutic effects.

Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division in response to genotoxic stress by arresting cell cycle progression in the S & G2 phases. Pharmacological inhibition of Chk1 is proposed to target tumor cells with increased DNA replication stress, resulting in the uncoupling of DNA replication checkpoint function and the induction of DNA damage and cell death. These properties make Chk1 inhibition a novel therapeutic approach as a single agent in cancers with high replication stress that is driven by oncogenic signaling and loss of parallel DNA damage response pathway function. Methods and Results: This report highlights the activity of the orally bioavailable, selective small molecule Chk1 inhibitor, CASC-578, in solid tumor derived cell lines. CASC-578 is a sub-nanomolar enzymatic inhibitor of Chk1 with limited off-target activity against a panel of protein kinases. When evaluated in a large cell line panel in vitro, CASC-578 demonstrated a broad potency range as a single agent in solid tumor derived cells lines, with IC50s ranging from 30 nM to greater than 50 μM. Several solid tumor types demonstrated enriched sensitivity to CASC-578 in vitro, including gastric, non-small cell lung and ovarian cancers. Treatment of sensitive cell lines with CASC-578 resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX, and the induction of cell death. CASC-578 was active as a single agent in SK-MES-1 and NCI-H727 NSCLC tumor xenograft models in vivo with minimal effects on body weight in treated mice. In addition to the potent single agent activity of CASC-578, combination with the Wee1 inhibitor AZD-1775 was highly synergistic in vitro in multiple solid tumor cell lines and the combination was more efficacious than either agent alone in NSCLC tumor xenograft models. These data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of solid tumor diseases. Experiments are ongoing to identify biomarkers associated with sensitivity to CASC-578 as a single agent in solid tumor cell lines to prospectively identify tumor genotypes that are more responsive to the drug. Citation Format: Alex Vo, Janelle Taylor, Robert Rosler, Julia Piasecki, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Bob Boyle, Rich Boyce, Scott Peterson. CASC-578, a novel Chk1 inhibitor, is active as a single agent in solid tumors and displays synergistic anti-tumor activity in combination with Wee1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2017-295