Phase II and gene expression analysis trial of neoadjuvant capecitabine plus irinotecan followed by capecitabine-based chemoradiotherapy for locally advanced rectal cancer: Hoosier Oncology Group GI03-53.

PURPOSE: We designed this study in locally advanced rectal cancer to determine the pathological response, toxicity, and disease-free survival (DFS) with induction capecitabine plus irinotecan followed by capecitabine-based chemoradiotherapy (CRT) and analyze the gene expression of enzymes involved in the metabolism of capecitabine and irinotecan for associations with response and toxicity. METHODS: Patients with T3/T4 or node positive rectal cancer were treated with capecitabine 1,000 mg/m(2) twice daily (BID) days 1-14, and irinotecan 200 mg/m(2) on day 1 every 21 days for 2 cycles, followed by capecitabine 825 mg/m(2) BID days 1-5 per week with concurrent radiotherapy 50.4 Gy in 28 fractions. Surgical resection occurred a median of 7.4 weeks after CRT. Gene expression levels or sequencing were used to analyze carboxylesterase-converting enzymes (CES1, CES2), thymidylate synthase (TS), thymidine phosphorylase (TP), dehydropyrimidine dehydrogenase (DPD), topoisomerase I (TOPO I), and uridine-diphosphate (UDP) glucuronosyl transferase 1A1 in pre- and post-treatment tumor and normal tissue samples. RESULTS: Twenty-two patients were enrolled, and 18 completed neoadjuvant therapy and underwent R0 resection. Two patients with UGT1A1 7/7 had grade 3 and 4 neutropenic fever and sepsis. Pathological complete response (pCR) occurred in 6 of 18 patients (33 %) and 10 (56 %) had tumor and/or nodal downstaging. The 3-year DFS was 75.5 % (95 % CI, 39.7-91.8 %). Locoregional control rate was 100 %. We observed higher TP gene expression in pCR patients, but no correlations with toxicity. CONCLUSIONS: This neoadjuvant regimen was safe and demonstrated significant antitumor activity. High TP tumor gene expression was associated with obtaining pCR.

A key role for orexin in panic anxiety.

Panic disorder is a severe anxiety disorder with recurrent, debilitating panic attacks. In individuals with panic disorder there is evidence of decreased central gamma-aminobutyric acid (GABA) activity as well as marked increases in autonomic and respiratory responses after intravenous infusions of hypertonic sodium lactate. In a rat model of panic disorder, chronic inhibition of GABA synthesis in the dorsomedial-perifornical hypothalamus of rats produces anxiety-like states and a similar vulnerability to sodium lactate-induced cardioexcitatory responses. The dorsomedial-perifornical hypothalamus is enriched in neurons containing orexin (ORX, also known as hypocretin), which have a crucial role in arousal, vigilance and central autonomic mobilization, all of which are key components of panic. Here we show that activation of ORX-synthesizing neurons is necessary for developing a panic-prone state in the rat panic model, and either silencing of the hypothalamic gene encoding ORX (Hcrt) with RNAi or systemic ORX-1 receptor antagonists blocks the panic responses. Moreover, we show that human subjects with panic anxiety have elevated levels of ORX in the cerebrospinal fluid compared to subjects without panic anxiety. Taken together, our results suggest that the ORX system may be involved in the pathophysiology of panic anxiety and that ORX antagonists constitute a potential new treatment strategy for panic disorder.

Loss of SIMPL compromises TNF-alpha-dependent survival of hematopoietic progenitors.

OBJECTIVE: Emerging work has revealed an integral role of the tumor necrosis factor-alpha (TNF-alpha) nuclear factor (NF)-kappaB pathway in the regulation of hematopoiesis. TNF-alpha inhibition of hematopoietic stem/progenitor cell growth involves type I TNF-alpha receptor (TNF-RI) and type II TNF-alpha receptor (TNF-RII). However, the role of TNF-RI vs TNF-RII in mediating this response is less clear. Full induction of NF-kappaB-dependent gene expression through TNF-RI requires the transcriptional coactivator SIMPL (substrate that interacts with mouse pelle-like kinase). To address the role of SIMPL in TNF-alpha-dependent signaling in hematopoiesis, endothelial cells and hematopoietic progenitors expressing SIMPL short hairpin RNA were characterized. MATERIAL AND METHODS: In vitro gene expression and progenitor assays employing SIMPL short hairpin RNA were used to examine the requirement for SIMPL in TNF-alpha-dependent effects upon cytokine gene expression and hematopoietic progenitor cell growth. Competitive repopulation studies were used to extend these studies in vivo. RESULTS: SIMPL is required for full TNF-RI-dependent expression of NF-kappaB-controlled cytokines in endothelial cells. Hematopoietic progenitor cell expansion is not affected if progenitors lacked SIMPL or if progenitors are treated with human TNF-alpha, which signals through TNF-RI. In the absence of SIMPL, human TNF-alpha leads to a dramatic decrease in progenitor cell expansion that is not due to apoptosis. Loss of SIMPL does not affect the activity of transforming growth factor-beta1 and interferon-gamma, other known suppressors of hematopoiesis. CONCLUSIONS: Suppression of myeloid progenitor cell expansion requires signaling through TNF-RI and TNF-RII. Signals transduced through the TNF-alpha-TNF-RI-SIMPL pathway support hematopoietic progenitor cell survival, growth and differentiation.

Kinetic and cellular characterization of novel inhibitors of S-nitrosoglutathione reductase.

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of beta-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-kappaB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.

Human carboxylesterases: an update on CES1, CES2 and CES3.

Carboxylesterases belong to Phase I group of drug metabolizing enzymes. They hydrolyze a variety of drug esters, amides, carbamates and similar structures. There are five 'carboxylesterase' genes listed in the Human Genome Organization database. In this review, we will focus on the CES1, CES2 and CES3 genes and their protein products that have been partially characterized. Several variants of these three CESs result from alternate splicing, single nucleotide polymorphisms and multiple copy variants. The three CESs, are largely localized to tissues that are major sites of drug metabolism like the mucosa of the gastrointestinal tract, lungs and liver but, they differ in tissue-specific expression. The amino acid alignment of the three CESs reveals important conserved catalytic and structural residues. There are interesting insertions and deletions that may affect enzymatic function as determined by homology modeling of CES3 using the CES1 three-dimensional structure. A comparison of the substrate specificity of CES1 versus CES2 reveals broad but distinct substrate preferences. There is little information on the substrate specificity of CES3 but it seems to have a lower catalytic efficiency than the other two CESs for selected substrates.

Expression of carboxylesterase and lipase genes in rat liver cell-types.

Approximately 80% of the body vitamin A is stored in liver stellate cells with in the lipid droplets as retinyl esters. In low vitamin A status or after liver injury, stellate cells are depleted of the stored retinyl esters by their hydrolysis to retinol. However, the identity of retinyl ester hydrolase(s) expressed in stellate cells is unknown. The expression of carboxylesterase and lipase genes in purified liver cell-types was investigated by real-time PCR. We found that six carboxylesterase and hepatic lipase genes were expressed in hepatocytes. Adipose triglyceride lipase was expressed in Kupffer cells, stellate cells and endothelial cells. Lipoprotein lipase expression was detected in Kupffer cells and stellate cells. As a function of stellate cell activation, expression of adipose triglyceride lipase decreased by twofold and lipoprotein lipase increased by 32-fold suggesting that it may play a role in retinol ester hydrolysis during stellate cell activation.

Expression and characterization of a human carboxylesterase 2 splice variant.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Delta(458-473)) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Delta(458-473) transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Delta(458-473) protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Delta(458-473) protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Delta(458-473) protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Delta(458-473) protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.

Maternal separation and handling affects cocaine self-administration in both the treated pups as adults and the dams.

Repeated maternal separation of pups from dams is often used as an early life stressor that causes profound neurochemical and behavioral changes in the pups that persist into adulthood. The effects of maternal separation on both the dams and the treated pups as adults on cocaine self-administration were examined using four separation conditions: 15- or 180-min separation (MS15 and MS180), brief handling without separation (MS0), and a nonhandled group (NH). The separations and handling occurred daily on postnatal days 2 to 15. The acquisition of cocaine self-administration (0.0625-1.0 mg/kg/infusion) was evaluated in the treated pups as adults. The MS180 group acquired cocaine self-administration at the lowest dose tested (0.0625 mg/kg/infusion), whereas the MS15s did not respond for cocaine at rates greater than that seen with saline administration. The NH group received the greatest number of infusions and intake at the highest doses. After self-administration, no differences were observed between groups in activity of two liver carboxylesterases involved in the inactivation of cocaine, ES10 and ES4. Maternal separation affected cocaine self-administration in the dams as well. Although there was an overall significant affect of treatment on cocaine self-administration, the length of separation (15 or 180 min) did not affect cocaine self-administration on the dams. The MS0 dams averaged a greater number of infusions per session than NH group during the 1st week of acquisition. These data suggest that in addition to the profound changes that occur in pups as result of maternal separation, the dams are also susceptible to alterations in behaviors.

Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1.

Methylphenidate is an important stimulant prescribed to treat attention-deficit hyperactivity disorder. It has two chiral centers, but most current commercial formulations consist of the racemic mixture of the threo pair of methylphenidate isomers (d-, l-threo-methylphenidate). The d-isomer is the pharmacologically active component. Numerous studies reported that oral administration of the methylphenidate racemate undergoes first-pass, stereoselective clearance in humans with l-methylphenidate being eliminated faster than d-methylphenidate. Accordingly, the kinetics of hydrolysis of individual enantiomers by purified native and recombinant human liver carboxylesterases CES1A1 and CES2 and a colon isoenzyme CES3 were examined with a liquid chromatography/mass spectrometry assay. The expression of CES1A1, CES2, and CES3 in Sf9 cells and the methods for purification of the three isoenzymes are reported. CES1A1 has a high catalytic efficiency for both d- and l-enantiomers of methylphenidate. No catalytic activity was detected with CES2 and CES3 for either enantiomer. The catalytic efficiency of CES1A1 for l-methylphenidate (k(cat)/K(m) = 7.7 mM(-1) min(-1)) is greater than that of d-methylphenidate (k(cat)/K(m) = 1.3-2.1 mM(-1) min(-1)). Hence, the catalytic efficiency of CES1A1 for methylphenidate enantiomers agrees with stereoselective clearance of methylphenidate reported in human subjects. Both enantiomers of methylphenidate can be fit into the three-dimensional model of CES1A1 to form productive complexes in the active site. We conclude that CES1A1 is the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate.

Hydrolysis of irinotecan and its oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases CES1A1, CES2, and a newly expressed carboxylesterase isoenzyme, CES3.

Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min(-1) microM(-1) among the three carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min(-1) microM(-1). APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 x 10(-3) min(-1) microM(-1) for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 >> CES3, for all three substrates.

Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis.

PURPOSE: The purpose is to develop new analytical methods to study the expression profile of CPT-11 carboxylesterases and topoisomerase I in colon tumor samples and understand the impact of their expression on CPT-11 metabolism in chemotherapy. EXPERIMENTAL DESIGN: We investigated 24 colon tumors for expression of carboxylesterases CES1A1, CES2, CES3, hBr-3, and topoisomerase I genes by real-time PCR and correlated the gene expression with activity assays. The relative abundance of the carboxylesterase isoenzymes and topoisomerase I genes was determined by real-time PCR. Activity assays performed on colon tumor extracts included CPT-11 hydrolase, 4-methylumbelliferyl acetate hydrolase, and topoisomerase I activity assays. Additionally, nondenaturing activity gel electrophoresis with activity staining showed the distribution of carboxylesterases. RESULTS: We detect the expression of CES1A1, CES2, and CES3 carboxylesterase genes in human colon tumors. We were unable to detect the hBr-3 (also called hCE-3) in human liver, colon, or brain. We find large interindividual variation, >/=150-fold, for both CES1A1 and CES3 genes, 23-fold for CES2, and 66-fold for topoisomerase I. Only CES2 gene expression correlated with the carboxylesterase activity assays (P < 0.01) with CPT-11 and 4-methylumbelliferyl acetate as substrates. Nondenaturing activity gel electrophoresis showed that CES2 was the most predominant activity. Topoisomerase I gene expression significantly correlated with topoisomerase I activity (P < 0.01) in the colon tumors, but interindividual variation was very high. CONCLUSIONS: We conclude that CES2 is the most abundant carboxylesterase in colon tumors that is responsible for CPT-11 hydrolysis. This pilot study reinforces the hypothesis that there is a large interindividual variation in expression of carboxylesterases that may contribute to variation in therapeutic outcome and/or toxicity of CPT-11 therapy for colon cancer.

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

Current progress on esterases: from molecular structure to function.

This article reports on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 2001 Experimental Biology meeting. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of enzyme activity.