Jiajiu Shaw

Prospective of colon cancer treatments and scope for combinatorial approach to enhanced cancer cell apoptosis

Colorectal cancer is the leading cause of cancer-related mortality in the western world. It is also the third most common cancer diagnosed in both men and women in the United States with a recent estimate for new cases of colorectal cancer in the year 2012 being around 103,170. Various risk factors for colorectal cancer include life-style, diet, age, personal and family history, and racial and ethnic background. While a few cancers are certainly preventable but this does not hold true for colon cancer as it is often detected in its advanced stage and generally not diagnosed until symptoms become apparent. Despite the fact that several options are available for treating this cancer through surgery, chemotherapy, radiation therapy, immunotherapy, and nutritional-supplement therapy, but the success rates are not very encouraging when used alone where secondary complications appear in almost all these therapies. To maximize the therapeutic-effects in patients, combinatorial approaches are essential. In this review we have discussed the therapies previously and currently available to patients diagnosed with colorectal-cancer, focus on some recent developments in basic research that has shaded lights on new therapeutic-concepts utilizing macrophages/dendritic cells, natural killer cells, gene delivery, siRNA-, and microRNA-technology, and specific-targeting of tyrosine kinases that are either mutated or over-expressed in the cancerous cell to treat these cancer. Potential strategies are discussed where these concepts could be applied to the existing therapies under a comprehensive approach to enhance the therapeutic effects.

The entirely carbohydrate immunogen Tn-PS A1 induces a cancer cell selective immune response and cytokine IL-17

The tumor-associated carbohydrate antigen/hapten Thomsen-nouveau (Tn; α-D-GalpNAc-ONH2) was conjugated to a zwitterionic capsular polysaccharide, PS A1, from commensal anaerobe Bacteroides fragilis ATCC 25285/NCTC 9343 for the development of an entirely carbohydrate cancer vaccine construct and probed for immunogenicity. This communication discloses that murine anti-Tn IgG3 antibodies both bind to and recognize human tumor cells that display the Tn hapten. Furthermore, the sera from immunization of mice with Tn-PS A1 contain cytokine interleukin 17 (IL-17A), which is known to possess anti-tumor function and represents a striking difference to an IL-2, and IL-6 profile obtained with anti-PS A1 sera.

Comparison of two molecular scaffolds, 5-methylisoxazole-3-carboxamide and 5-methylisoxazole-4-carboxamide.

Leflunomide is a disease-modifying antirheumatic drug (DMARD) for the treatment of rheumatoid arthritis (RA). Structurally, it is a derivative of 5-methylisoxazole-4-carboxamide. Upon metabolism, the N-O bond in the isoxazole ring is cleaved to form the active metabolite, teriflunomide, which was recently approved by the FDA for the treatment of multiple sclerosis. Both leflunomide and teriflunomide inhibit dihydroorotate dehydrogenase (DHODH) thereby inhibiingt the synthesis of pyrimidine. For both drugs, the two major concerns are potential liver toxicity and teratogenicity. It was suspected that these undesirable effects might be related to the cleavage of the N-O bond. We herein summarize the metabolites-toxicity issues related to leflunomide/teriflunomide and discuss two related molecular platforms, UTL-4 and UTL-5. UTL-4 compounds are based on the same scaffold of leflunomide; their toxicological and pharmacological effects are not significantly different from those of leflunomide/teriflunomide. In UTL-5 series, the leflunomide scaffold is changed into 5-methylisoxazole-3-carboxamide. Unlike leflunomide, the N-O bond of a UTL-5 compound, UTL-5b, is not cleaved upon metabolism; instead, the peptide bond is cleaved to form its major metabolites. UTL-5b and its metabolites do not inhibit DHODH in vitro. In addition, UTL-5b and all other UTL-5 compounds have lower acute toxicity than leflunomide/teriflunomide. Furthermore, from leflunomide to UTL-5b/UTL-5g, the potential liver toxicity becomes liver protective effect. With the reduced toxicity, UTL-5 compounds still maintain significant pharmacological effects including anti-inflammatory and antiarthritic effects. In summary, our observations provide a valuable direction in drug optimization based on the modification of the leflunomide scaffold.

Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase

UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA.

A liquid chromatography with tandem mass spectrometry method for simultaneous determination of UTL-5g and its metabolites in human plasma.

UTL-5g is a novel small-molecule TNF-α inhibitor under investigation as both a chemoprotective and radioprotective agent. Animal studies showed that pretreatment of UTL-5g protected kidney, liver, and platelets from cisplatin-induced toxicity. In addition, UTL-5g reduced liver and lung injuries induced by radiation in vivo. Although a number of preclinical studies have been conducted, a validated bioanalytical method for UTL-5g in human plasma has not been published. In this work, a sensitive and reproducible reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the determination of UTL-5g and its metabolites, 5-methylisoxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA), in human plasma. The method involves a simple methanol precipitation step followed by injection of the supernatant onto a Waters 2695 HPLC system coupled with a Waters Quattro Micro™ triple quadrupole mass spectrometer. Chromatographic separation was accomplished using a Waters Nova-Pak C18 column maintained at 30°C, running at gradient mode with mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.2mL/min. The analytes were monitored under positive electrospray ionization (ESI). Quantitation of these compounds in plasma was linear from 0.05 to 10μM. The lower limit of quantitation (LLOQ) was 0.05, 0.1, and 0.2μM for UTL-5g, ISOX and DCA, respectively. The accuracy and intra-and inter-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method provides a practical tool to measure and characterize the plasma concentration-time profiles for UTL-5g and its metabolites, ISOX and DCA.

In vitro metabolism and drug-drug interaction potential of UTL-5g, a novel chemo- and radioprotective agent.

N-(2,4-dichlorophenyl)-5-methyl-1,2-oxazole-3-carboxamide (UTL-5g), a potential chemo- and radioprotective agent, acts as a prodrug requiring bioactivation to the active metabolite 5-methylisoxazole-3-carboxylic acid (ISOX). UTL-5g hydrolysis to ISOX and 2,4-dichloroaniline (DCA) has been identified in porcine and rabbit liver esterases. The purpose of this study was to provide insights on the metabolism and drug interaction potential of UTL-5g in humans. The kinetics of UTL-5g hydrolysis was determined in human liver microsomes (HLM) and recombinant human carboxylesterases (hCE1b and hCE2). The potential of UTL-5g and its metabolites for competitive inhibition and time-dependent inhibition of microsomal cytochrome P450 (P450) was examined in HLM. UTL-5g hydrolysis to ISOX and DCA in HLM were NADPH-independent, with a maximum rate of reaction (Vmax) of 11.1 nmol/min per mg and substrate affinity (Km) of 41.6 µM. Both hCE1b and hCE2 effectively catalyzed UTL-5g hydrolysis, but hCE2 exhibited ∼30-fold higher catalytic efficiency (Vmax/Km) than hCE1b. UTL-5g and DCA competitively inhibited microsomal CYP1A2, CYP2B6, and CYP2C19 (IC50 values <50 µM), and exhibited time-dependent inhibition of microsomal CYP1A2 with the inactivation efficiency (kinact/KI) of 0.68 and 0.51 minute−1·mM−1, respectively. ISOX did not inhibit or inactivate any tested microsomal P450. In conclusion, hCE1b and hCE2 play a key role in the bioactivation of UTL-5g. Factors influencing carboxylesterase activities may have a significant impact on the pharmacological and therapeutic effects of UTL-5g. UTL-5g has the potential to inhibit P450-mediated metabolism through competitive inhibition or time-dependent inhibition. Caution is particularly needed for potential drug interactions involving competitive inhibition or time-dependent inhibition of CYP1A2 in the future clinical development of UTL-5g.

Development and Validation of a Simple Method for the Detection of Fascaplysin in Plasma

Fascaplysin is a cytotoxic natural product isolated from a variety of Indo-Pacific marine organisms, primarily Fascaplysinopsis sponges and Didemnum tunicates. Positive xenograft studies involving this alkaloid structural class have indicated that fascaplysin may serve as an important lead compound for preclinical development. This study was undertaken as a prelude to a full pharmacokinetics and therapeutic assessment of fascaplysin. We describe here a simple plasma preparation and a rapid HPLC method for the detection of fascaplysin in mice. The method was validated by parameters including good linear correlation, a limit of quantification of 107.1 μg/mL, and a good precision with a coefficient of variation of 0.92% for 10 μg/mL. This method provides excellent sensitivity and visualization of fascaplysin as a single peak allowing for rapid analysis of plasma samples involving absorption, distribution, and metabolism studies. A preliminary pharmacokinetics study in C57Bl/6 mice using 20.6 mg/kg fascaplysin yielded a biphasic curve with T½α=16.7 min, T½β=11.7 h, and C0 of 17.1 μg/mL.

Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation

UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca2+ and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR < 1%) as was phosphorylation of Stat3, consistent with the IL-6 results in the transcription factor assay. Neither analysis indicated that LPS-induced activation of the NF-kB, cAMP/PKA and JNK signaling pathways were affected by UTL-5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects.