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 sulphide (H(2)S) 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 H(2)S in blood and tissues.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 H(2)S gas inhalation, and levels of available blood sulphide were measured. Levels of dissolved H(2)S/HS(-) were concomitantly measured using an amperometric sensor.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 microM. Intravenous administration of sodium sulphide solution (2-20 mg x kg(-1) x h(-1)) or inhalation of H(2)S gas (50-400 ppm) elevated reactive sulphide in blood in a dose-dependent manner. Each 1 mg x kg(-1) x h(-1) of sodium sulphide infusion into rats was found to be pharmacokinetically equivalent to approximately 30 ppm of H(2)S gas inhalation.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 H(2)S gas.
Abstract Background: Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division by arresting progression in the S & G2 phases of the cell cycle in response to genotoxic stress. Pharmacological inhibition of Chk1 uncouples the completion of DNA replication from G2/M phase transition in tumor cells that have impaired DNA damage response networks, resulting in DNA damage induction, mitotic catastrophe and cell death. These properties make Chk1 inhibition an attractive therapeutic approach as a single agent, or to enhance the efficacy of other cancer drugs that target DNA damage response pathways. Methods and Results: This report highlights the activity of the orally bioavailable, selective, small molecule Chk1 inhibitor, CASC-578, in multiple mantle cell lymphoma (MCL) models in vitro and in tumor mouse xenograft studies. MCL have the common genetic biomarker t(11;14)(q13;q32), a chromosomal translocation leading to the constitutive expression of the oncogene CCND1 which encodes cyclin D1, a cell cycle-regulating protein. This genotypic characteristic has been observed in the clear majority of patients with MCL in the clinic and may provide a rationale for sensitivity to Chk1 inhibition. Consistent with this idea, CASC-578 demonstrated remarkable potency as a single agent in blocking the proliferation of MCL cell lines in vitro, with an average IC50 of 61 nM. Treatment with CASC-575 as a single agent resulted in the induction of DNA damage, as measured by phosphorylated histone H2AX and activation of apoptosis, as measured by cleaved caspase. In a JEKO-1 xenograft tumor study, CASC-578 as a single agent completely regressed subcutaneously implanted tumors in the majority of mice after oral drug administration using 20 mg/kg daily or when dosed on an intermittent schedule at 25 mg/kg, with minimal effects on body weight. In addition to the observed single agent activity of CASC-578, the combination of CASC-578, and the Wee1 inhibitor AZD-1775, was highly synergistic in MCL cell lines including JEKO-1, REC-1, Z-138 and MAVER-1 in vitro. CASC-578 was also highly potent as a single agent, and in combination with AZD-1775, in blocking the proliferation of a variety of leukemia derived cell lines in vitro. Taken together, these data support the advancement of CASC-578 into clinical development as a potential therapeutic agent for the treatment of MCL, as well as other hematological malignancies. Citation Format: Robert Rosler, Janelle Taylor, Dina Leviten, Teresa Sierra, Ashley Dozier, Kevin Klucher, Rich Boyce, Bob Boyle, Alex Vo, Scott Peterson. The novel orally available sub-nanomolar potent and selective checkpoint kinase 1 inhibitor CASC-578 is highly active in mantle cell lymphoma as a single agent and 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 297. doi:10.1158/1538-7445.AM2017-297
Abstract Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division by arresting progression in the S & G2 phases of the cell cycle in response to genotoxic stress. Pharmacological inhibition of Chk1 selectively uncouples the completion of DNA replication from G2/M phase transition in tumor cells lacking parallel cell cycle checkpoint controls, resulting in mitotic catastrophe and cell death. These properties make Chk1 inhibition a unique therapeutic approach as a single agent or as a means to enhance the efficacy of DNA-targeted chemotherapeutic drugs. In this report, we describe our progress in the development of orally bioavailable, potent and selective Chk1 inhibitors derived from an aminopyrazole chemical scaffold. Starting with the lead compound, ONT-2409 (IC50 of <0.1 nM against the Chk1 enzyme), a series of Chk1 inhibitors have been developed. Lead candidates have been generated with potent biochemical IC50s against Chk1 (IC50s = 0.1-0.3 nM). In a panel of 125 protein kinases the top candidates were shown to be highly selective for Chk1, with little appreciable activity against Chk2 or other kinases involved in cell cycle control/DNA damage response. The lead candidates have potent single agent activity against a panel of diverse cancer cell lines (EC50s = 0.03-0.4 μM) and show synergistic enhancement in the activity of clinically relevant DNA targeted chemotherapeutics. Mechanistically, the lead candidates abrogate gemcitabine-induced cell cycle arrest and induce apoptotic cell death. Biomarker studies show the lead candidates inhibit gemcitabine-induced auto-phosphorylation of Chk1 at S296 and reduce phosphorylation of CDK1 at Y15. Compared with ONT-2409, the optimized candidates display improved cell permeability/efflux ratios, superior intrinsic microsomal and hepatic half-lives, and have excellent oral bioavailability in mice and rats. Lead candidate Chk1 inhibitors have also demonstrated potent potentiation of DNA targeted chemotherapies and single agent activity in xenograft tumor models Citation Format: Alex C. Vo, Janelle Taylor, Robert Rosler, Dina Leviten, Teresa Sierra, Richard Boyce, Robert Boyle, Scott Peterson, Kevin Klucher. Discovery and development of orally available subnanomolar potent checkpoint kinase 1 inhibitors as potential anticancer therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2721.
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.
Abstract Introduction: 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 selectively uncouple the completion of DNA replication from G2/M phase transition in tumor cells that have impaired DNA damage response networks, resulting in mitotic catastrophe and cell death. CASC-578 is a novel small molecule inhibitor of Chk1 that is selective, highly potent and orally bioavailable in multiple preclinical species. Methods: The in vitro ADME properties of CASC-578 were evaluated, including in vitro intrinsic microsomal clearance, Caco2 permeability, plasma protein binding and stability, blood to plasma partitioning, cytochrome P450 inhibition and induction, and transporter inhibition. Pharmacokinetic studies of CASC-578 were conducted in mice, rats, and cynomolgus monkeys as either single dose (IV and/or PO) or repeat dose (PO only). Multiple oral dose pharmacokinetic studies were conducted in mice, rats and cynomolgus monkeys for 5-7 days. Results: CASC-578 is highly bound in plasma protein across all species. The apparent permeability is high in Caco2 bi-directional transport study and correlated well with a rapid absorption profile observed in vivo. CASC-578 is not a substrate of P-glycoporotein (efflux =1). There was no direct nor time dependent inhibition on human CYP450 enzymes, and only a slight induction of CYP3A4 was seen at 10 uM drug concentration in a transporter cell-based induction assay. In all animal species, CASC-578 exhibited species-dependent systemic clearance resulting from both phase I and phase II metabolism, and a moderate to high volume of distribution. The elimination kinetics appeared to be monophasic. Oral bioavailability was high in all species studied (>60% F). There was no significant difference in the pharmacokinetics of the drug between genders. Maximal plasma concentration and total drug exposure (AUC) appeared to be proportional from repeat dose studies. Overall, CASC-578 has very desirable drug-like properties and ideal pharmacokinetics for an oral once daily drug, and represents a suitable candidate for clinical development as a novel potential therapeutic approach for the treatment of solid and hematological malignancies Citation Format: Dina Leviten, Teresa Sierra, Ashley Dozier, Richard Boyce, Bob Boyle, Scott Peterson, Alex C. Vo. Preclinical pharmacokinetics of CASC-578, a novel, selective, potent, and orally bioavailable small molecule checkpoint kinase 1 inhibitor [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 4090. doi:10.1158/1538-7445.AM2017-4090
Hydrogen sulfide exhibits many characteristics of an endogenous biological mediator and is present at low baseline levels in tissues and blood. A large body of evidence suggests that exogenously applied sulfide may also be suitable as a therapeutic agent. Therefore, a method using the rare 34S sulfur isotope was developed and validated to distinguish exogenously administered sulfide from endogenous sulfide which is predominantly 32S‐sulfide. Sodium 34S‐sulfide was prepared and administered intravenously to Sprague Dawley rats. Concentrations of 32S and 34S‐sulfide were measured in blood and tissues after derivatization with monobromobimane as monobromobimane derivative, separation by reverse phase HPLC and quantification by mass spectroscopy. On intravenous infusion of sodium 34S‐sulfide into rats, blood sulfide concentrations of 34S‐sulfide, but not 32S‐sulfide increased in a dose‐dependent manner indicating that exogenously administered sulfide can be traced and distinguished from endogenous sulfide. The method will be useful in monitoring of the distribution of therapeutic sulfide in tissues and organs.
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.
