Elizabeth A. Gullen

The Four-Herb Chinese Medicine PHY906 Reduces Chemotherapy-Induced Gastrointestinal Toxicity

A traditional Chinese medicine reduces the intestinal toxicity of chemotherapy by inhibiting inflammation and promoting cell proliferation. Flower Power for Intestinal Healing Peonies and a pretty purple flower called skullcap, together with licorice and fruit from a buckthorn tree, might conjure up an enchanting still life, but this combination of plants can heal more than the spirit. These four ingredients form Huang Qin Tang, a centuries-old traditional Chinese medicine used to treat intestinal disorders such as diarrhea, nausea, and vomiting. A Western-style phase 1/2 trial confirmed that this drug reduces the intestinal side effects of chemotherapy in colon or rectal cancer patients. Now Lam et al. use a carefully prepared, consistent formulation of this medicine and show that it exerts its salutary effects by stimulating gut cell division and reducing inflammation. Mice with colon tumors were given CPT-11, a powerful cytotoxic agent used for cancer chemotherapy, which shrank their tumors but also caused massive destruction of the intestinal lining. However, when the colon tumor–carrying mice received a combination of CPT-11 and standardized Huang Qin Tang (called PHY906), they lost less weight than did mice treated with CPT-11. Closer examination of the intestines of the doubly treated mice revealed PHY906-induced restoration of the normal architecture by 4 days after treatment, with a faster disappearance of apoptotic cells and an increase in proliferating progenitor cells, particularly at the bottom of the intestinal crypts, relative to mice that received CPT-11 only. Markers of the Wnt signaling pathway also increased, suggesting that PHY906 functioned by stimulating Wnt signaling, possibly at multiple points. So it was unexpected when the authors found that PHY906 itself did not stimulate Wnt. An explanation emerged when they discovered that, after mixing either PHY906 or the extract from skullcap alone with a bacterial enzyme common in the human gut, Wnt activity became apparent. PHY906 has other critical actions: In addition to stimulating gut cell repopulation, it blocked the migration of inflammatory cells to the gut and inhibited inducible nitric oxide synthase and cyclooxygenase-2, two signs of inflammation, as well as the proinflammatory transcription factor NF-κB. Although all of the mechanistic details have not yet been deciphered, these effects seem to be caused by multiple actions of PHY906. Currently, drug developers are eagerly testing combinations of agents that they hope will heal patients better than any one alone. Traditional Chinese medicine may have been the original proponent of this approach, as these results by Lam et al. reveal. PHY906, a four-herb Chinese medicine formula first described 1800 years ago, decreases gastrointestinal toxicity induced by the chemotherapeutic drug CPT-11 (irinotecan), as shown in a phase I/II clinical study. Similarly, in a murine colon 38 allograft model, PHY906 increased the antitumor activity of CPT-11 while decreasing animal weight loss caused by CPT-11. Here, we have further examined the effect of PHY906 on the intestinal toxicity caused by CPT-11 in mice. PHY906 did not protect against the initial DNA damage and apoptosis triggered by CPT-11 in the intestine, but by 4 days after CPT-11 treatment, PHY906 had restored the intestinal epithelium by promoting the regeneration of intestinal progenitor or stem cells and several Wnt signaling components. PHY906 also potentiated Wnt3a activity in human embryonic kidney–293 cells. Furthermore, PHY906 exhibited anti-inflammatory effects in mice by decreasing the infiltration of neutrophils or macrophages, tumor necrosis factor–α expression in the intestine, and proinflammatory cytokine concentrations in plasma. Chemical constituents of PHY906 potently inhibited nuclear factor κB, cyclooxygenase-2, and inducible nitric oxide synthase. Our results show that the herbal medicine PHY906 can counteract the toxicity of CPT-11 via several mechanisms that act simultaneously.

Novel 4′-Substituted Stavudine Analog with Improved Anti-Human Immunodeficiency Virus Activity and Decreased Cytotoxicity

ABSTRACT The antiviral drug 2′,3′-didehydro-3′-deoxythymidine (D4T; also know as stavudine and Zerit), which is used against human immunodeficiency virus (HIV), causes delayed toxicity (peripheral neuropathy) in long-term use. After examining a series of 2′,3′-didehydro-3′-deoxy-4′-substituted thymidine (4′-substituted D4T) analogs, 4′-ethynyl D4T was found to have a fivefold-better antiviral effect and to cause less cellular and mitochondrial toxicity than D4T. The antiviral activity of this compound can be reversed by dThd but not by dCyd. The compound acted synergistically with β-l-2′,3′-deoxy-3′-thiacytidine (also known as lamivudine) and β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (also known as elvucitabine) and additively with 2′,3′-dideoxyinosine (also known as didanosine and Videx) and 3′-azido-3′-deoxythymidine (also known as Retovir and zidovudine) against HIV. 4′-Ethynyl D4T is phosphorylated by purified human thymidine kinase 1 (TK-1) from CEM cells with a faster relative Vmax and a lower Km value than D4T. The efficiency of TK-1 in the phosphorylation of 4′-ethynyl D4T is fourfold better than that of D4T. While D4T is broken down by the catabolic enzyme thymidine phosphorylase, the level of breakdown of 4′-ethynyl D4T was below detection. Since 4′-ethynyl D4T has increased anti-HIV activity and decreased toxicity and interacts favorably with other currently used anti-HIV drugs, it should be considered for further development as an anti-HIV drug.

Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold.

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.

Butyrate mediates nucleotide‐binding and oligomerisation domain (NOD) 2‐dependent mucosal immune responses against peptidoglycan

The interaction between digestive tract microbiological flora and food has an important influence on human health. Butyrate is produced during the fermentation of dietary fibres by intestinal bacteria and plays an important role in the regulation of mucosal immunity. In this report, we studied the impact of butyrate on the defence mechanism against the bacterial membrane component peptidoglycan (PGN). Butyrate was found to enhance PGN‐mediated IL‐8 and GRO‐α production. The expression of these chemokines required the activation of NF‐κB and was dependent on the concentrations of butyrate and PGN. Butyrate was found to up‐regulate nucleotide‐binding and oligomerisation domain (NOD) 2, but not NOD1 or TLR2. NOD2 up‐regulation was mediated by an increase in histone acetylation in the Nod2 promoter region, leading to enhanced PGN‐induced IL‐8 and GRO‐α secretion. Knockdown of NOD2 and TLR2 by siRNA significantly reduced PGN‐mediated chemokine production, suggesting that both NOD2 and TLR2 are required for maximal response. Our findings provide a better understanding of the mechanism by which butyrate regulates mucosal immunity for normal intestinal function. Based on the results of this study, we infer that dietary fibres can impact inflammatory bowel diseases.

Favorable interaction of β-l(−) nucleoside analogues with clinically approved anti-HIV nucleoside analogues for the treatment of human immunodeficiency virus

Abstract The combination of l (−)-2-′,3′-dideoxy-3′-thiacytidine ( l (−)SddC, 3TC), l (−)-2′,3′-dideoxy-5-fluorocytidine ( l (−)FddC), or l (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine ( l (−)FTC) with 3′-azido-3′-deoxythymidine (AZT) synergistically inhibited replication of human immunodeficiency virus (HIV) in vitro . Similar synergistic activity was also obtained when these compounds were used in combination with 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T). In terms of 2′,3′-dideoxyinosine (ddI) and 2′,3′-dideoxycytidine (ddC), only additive anti-HIV activity was observed. None of the β- l (−) nucleoside analogues had additive toxicity in cell culture, and they could protect against the delayed mitochondrial toxicity associated with AZT, D4T, ddC, and ddI in drug-treated cells. Thus, combinations of β- l (−) nucleoside analogues with any of the approved anti-HIV drugs could have a potentially beneficial outcome.

Inhibition of Hepatitis B Virus Gene Expression and Replication by Helioxanthin and its Derivative

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-α (IFN-α) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 μM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 μM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.

Synthesis and enantioselectivity of the antiviral effects of (R,Z)-,(S,Z)-methylenecyclopropane analogues of purine nucleosides and phosphoralaninate prodrugs: influence of heterocyclic base, type of virus and host cells.

A series of R and S enantiomers of 2-aminopurine methylenecyclopropane analogues of nucleosides was synthesized. Two diastereoisomeric lipophilic phosphate prodrugs derived from R and S enantiomers of 2,6-diaminopurine analogue were also prepared. Enantioselectivity (diastereoselectivity in case of prodrugs) of in vitro antiviral effects was investigated with human and murine cytomegalovirus (HCMV and MCMV, respectively), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), Epstein—Barr virus (EBV) and varicella zoster virus (VZV). Strong differences in enantioselectivity were found between the R and S enantiomers of adenine analogue and enantiomeric 2-aminopurine analogues. Thus, the enantiomers of adenine analogue were equipotent against HCMV but not MCMV, where the S enantiomer is strongly preferred. The same S preference was found throughout the 2-aminopurine series for both HCMV and MCMV. In contrast, R-synadenol in HIV-1 assays was the best agent, whereas the S enantiomers of moderately effective 2-amino-6-cyclo-propylamino and 2-amino-6-methoxypurine analogues were preferred. Little enantiomeric preference was found for R and S enantiomers of synadenol and the corresponding enantiomers of 2,6-diaminopurine analogue against HBV. A mixed pattern of enantioselectivity was observed for EBV depending on the type of host cells and assay. Against VZV, the R and S enantiomers of adenine analogue were equipotent or almost equipotent, but throughout the series of 2-aminopurine analogues a distinct preference for the S enantiomers was found. The stereoselectivity pattern of both diastereoisomeric prodrugs mostly followed enantioselectivity of the parent analogues. The varying enantioselectivities in the series of purine methylenecyclopropane analogues are probably a consequence of differences in the mechanisms of action in different virus/host cell systems.

Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library

Amentoflavone has been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library. In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo. Molecular modeling and kinetic experiments suggested that the analogues may function as Type II inhibitors of JAK2.

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Synthesis and comparative evaluation of two antiviral agents: β-l-Fd4C and β-d-Fd4C

The synthesis of beta-D-Fd4C was achieved in a stereoselective fashion from D-xylose. The antiviral activity and cytotoxicity of beta-D-Fd4C was compared with that of beta-L-Fd4C and 3TC (Lamivudine). Of the three agents compared, beta-L-Fd4C was found to be the most potent antiviral agent.