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Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms, We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimer’s disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors.

The roots of Salvia miltiorrhiza ("Danshen") are used in traditional Chinese medicine for the treatment of numerous ailments including cardiovascular disease, hypertension, and ischemic stroke. Extracts of S. miltiorrhiza roots in the formulation "Compound Danshen Dripping Pill" are undergoing clinical trials in the United States. To date, the active components of this material have not been conclusively identified. We have determined that S. miltiorrhiza roots contain potent human carboxylesterase (CE) inhibitors, due to the presence of tanshinones. K(i) values in the nM range were determined for inhibition of both the liver and intestinal CEs. As CEs hydrolyze clinically used drugs, the ability of tanshinones and S. miltiorrhiza root extracts to modulate the metabolism of the anticancer prodrug irinotecan (CPT-11) was assessed. Our results indicate that marked inhibition of human CEs occurs following incubation with both pure compounds and crude material and that drug hydrolysis is significantly reduced. Consequently, a reduction in the cytotoxicity of irinotecan is observed following dosing with either purified tanshinones or S. miltiorrhiza root extracts. It is concluded that remedies containing tanshinones should be avoided when individuals are taking esterified agents and that patients should be warned of the potential drug-drug interaction that may occur with this material.

Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.

Background and purpose: Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11. Experimental approach: The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined. Key results: In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results. Conclusions and implications: These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.

Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P R enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P S isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P S isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.

Platelet producing capacity was examined at intervals after acute platelet depletion by reinduction of acute thrombocytopenia and assessment of the subsequent platelet recovery rates of 35S-sulfate incorporation into platelets. Platelet producing capacity was increased on days 1 and 2, somewhat decreased on days 3 and 4 and had returned to baseline by day 5. These studies suggest that there is an initial increase followed by a decrease on days 3 and 4 in cells which can respond to thrombopoietic stimulating factor after induction of acute thrombocytopenia.

Doxazolidine (Doxaz), a formaldehyde-doxorubicin (Dox) conjugate, exhibits markedly increased tumor toxicity with respect to Dox without a concurrent increase in toxicity to cardiomyocytes. Pentyl PABC-Doxaz (PPD) is a Doxaz carbamate prodrug that is hydrolyzed by carboxylesterases. Here, we identify human intestinal carboxylesterase (hiCE) as the agent of activation for PPD. Upon prodrug treatment, cells that express higher levels of hiCE responded with lower IC50 values for growth inhibition. Exposing MCF-7 human breast cancer cells, which respond poorly and express little hiCE, to PPD together with hiCE resulted in a dramatic decrease in the IC50, a decrease that was absent when human carboxylesterase 1 was added to prodrug treatment. Finally, U373MG glioblastoma cells overexpressing hiCE displayed approximately 100-fold reduction in the IC50 for PPD compared to cells lacking the carboxylesterase. Overall, our studies indicate that PPD is selectively hydrolyzed to the active metabolite by hiCE.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived K(i) values with the fluorobenzoins.

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents).

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.

Carboxylesterases are ubiquitous proteins responsible for the detoxification of xenobiotics. However, these enzymes also activate prodrugs, such as the anticancer agents capecitabine and CPT-11. As a consequence, overexpression of carboxylesterases within tumor cells sensitizes these cells to CPT-11. We have recently identified two classes of carboxylesterase inhibitors based on either a benzil (diphenylethane-1,2-dione) or a benzene sulfonamide scaffold and showed that these compounds inhibit carboxylesterases with Kis in the low nanomolar range. Because both classes of inhibitors show reversible enzyme inhibition, conventional in vitro biochemical assays would not accurately reflect the in situ levels of carboxylesterase activity or inhibition. Therefore, we have developed a novel assay for the determination of intracellular carboxylesterase activity using 4-methylumbelliferone as a substrate. These studies show that benzil and a dimethylbenzil analogue efficiently enter cells and inhibit human intestinal carboxylesterase and rabbit liver carboxylesterase intracellularly. This inhibition results in reduced cytotoxicity to CPT-11 due to the lack of carboxylesterase-mediated conversion of the prodrug to SN-38. These results suggest that intracellular modulation of carboxylesterase activity with benzil or its analogues may be applied to minimize the toxicity of normal cells to CPT-11. [Mol Cancer Ther 2006;5(9):2281–8]

Pyrethroid chemicals are attractive alternatives to the organophosphates (OPs) because of their selective toxicity against pests rather than mammals. The carboxylesterases (CEs) are hepatic enzymes that metabolize ester-containing xenobiotics such as pyrethroids. The primary aim of this study was to gain insight into the catalytic properties of the CE enzymes in humans that metabolize pyrethroids, while a secondary aim was to investigate pyrethroid metabolism using CEs from other mammalian species. Pure human CEs (hCE-1 and hCE-2), a rabbit CE (rCE), and two rat CEs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids: 1Rtrans-resmethrin (bioresmethrin), 1RStrans-permethrin, and 1RScis-permethrin. hCE-1 and hCE-2 hydrolyzed trans-permethrin 8- and 28-fold more efficiently than cis-permethrin (when k(cat)/K(m) values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCE-1, but not by hCE-2. The kinetic parameters for the pure rat and rabbit CEs were qualitatively similar to the human CEs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species, which further supports the use of rodent models for pyrethroid metabolism studies. An eight-fold range in hydrolytic rates for 11 individual human liver samples toward trans-permethrin was also found, although this variability was not related to the levels of hCE-1 protein in each sample. We also determined that the CE inhibitor 2-chloro-3,4-dimethoxybenzil blocked hCE-2-catalyzed trans-permethrin hydrolysis 36 times more potently than hCE-1. Thus, this inhibitor will be useful in future studies that examine CE-mediated metabolism of pyrethroids. While there are likely other esterases in human liver that hydrolyze pyrethroids, the results of this study clearly demonstrate that hCE-1 and hCE-2 are human pyrethroid-hydrolyzing CEs.

Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2)0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

There was an error in the sixth author's name. The correct name is Steven M. Lewis. The corrected author contributions are: "Conceived and designed the experiments: ACH RAC MRR. Performed the experiments: ACH TCO RAC MW JSE SML CCE LT CLC SAK. Analyzed the data: ACH RAC JRC SLM PMP DMC MRR. Contributed reagents/materials/analysis tools: ACH TCO RAC MW JSE SML CCE LT CLC SAK. Wrote the paper: ACH TCO RAC MW JSE SML CCE LT CLC SAK JRC SLM PMP DMC MRR."

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: One containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X = CH2, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X =N), or conversion of the compounds into substrates for the enzymes (when X = S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH3 to C8H17] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH3 to C6H13], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.

Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 μM; and kcat/Km, 1.2 and 0.93 μM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.

The activation of the anticancer prodrug CPT-11, to its active metabolite SN-38, is primarily mediated by carboxylesterases (CE). In humans, three CEs have been identified, of which human liver CE (hCE1; CES1) and human intestinal CE (hiCE; CES2) demonstrate significant ability to hydrolyze the drug. However, while the kinetic parameters of CPT-11 hydrolysis have been measured, the actual contribution of each enzyme to activate the drug in biological samples has not been addressed. Hence, we have used a combination of specific CE inhibition and conventional chromatographic techniques to determine the amounts, and hydrolytic activity, of CEs present within human liver, kidney, intestinal and lung specimens. These studies confirm that hiCE demonstrates the most efficient kinetic parameters for CPT-11 activation, however, due to the high levels of hCE1 that are expressed in liver, the latter enzyme can contribute up to 50% of the total of drug hydrolysis in this tissue. Conversely, in human duodenum, jejunum, ileum and kidney, where hCE1 expression is very low, greater than 99% of the conversion of CPT-11 to SN-38 was mediated by hiCE. Furthermore, analysis of lung microsomal extracts indicated that CPT-11 activation was more proficient in samples obtained from smokers. Overall, our studies demonstrate that hCE1 plays a significant role in CPT-11 hydrolysis even though it is up to 100-fold less efficient at drug activation than hiCE, and that drug activation in the intestine and kidney are likely major contributors to SN-38 production in vivo.

CPT-11 is an antitumor prodrug that is hydrolyzed by carboxylesterases (CE) to yield SN-38, a potent topoisomerase I poison. However, the dose limiting toxicity is delayed diarrhea that is thought to arise, in part, from activation of the prodrug by a human intestinal CE (hiCE). Therefore, we have sought to identify selective inhibitors of hiCE that may have utility in modulating drug toxicity. We have evaluated one such class of molecules (benzene sulfonamides), and developed QSAR models for inhibition of this protein. Using these predictive models, we have synthesized a panel of fluorene analogues that are selective for hiCE, demonstrating no cross reactivity to the human liver CE, hCE1, or towards human cholinesterases, and have Ki values as low as 14nM. These compounds prevented hiCE-mediated hydrolysis of the drug and the potency of enzyme inhibition correlated with the clogP of the molecules. These studies will allow the development and application of hiCE-specific inhibitors designed to selectively modulate drug hydrolysis in vivo.

CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As activation of CPT-11 (irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC(50) value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.

Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.

Appropriate low dosages of vincristine stimulate megakaryocytopoiesis and produce thrombocytosis. In this study of the thrombocytotic action of vincristine, administration of a single dose of 0.1 mg/kg to rats produced an increase in megakaryocyte concentration, diameter and 24 h [3H]thymidine labelling index. Transfusion of one body equivalent of platelets from normal donors prevented stimulation of megakaryocytopoiesis by vincristine whereas platelets from vincristine-treated donors did not. These results suggest that vincristine stimulates megakaryocytopoiesis by altering the functional role of circulating platelets in the regulation of thrombopoiesis.

Hypertransfusion can enhance recovery from neutropenia in certain clinical and experimental situations. We have studied the pattern of myeloid recovery in mice hypertransfused after receiving 350 rads whole body irradiation. Both hypertransfused and control groups showed the degenerative phase, abortive rise, and regenerative phase that has been described following sublethal irradiation. The blood granulocyte counts in the hypertransfused group returned to normal more rapidly and were maintained at a significantly higher level during the regenerative phase. This difference is not the result of a shift in granulocytes from the marrow granulocyte reserve or marginal granulocyte pool to the circulating pool, but is associated with significantly enhanced bone marrow granulopoiesis. While the total bone marrow cellularity of the hypertransfused mice is less than that of the control mice, the hypertransfused group contains more CFU-GM and myeloid cells during the regenerative phase. The enhanced granulopoiesis is not due to increased colony-stimulating activity (CSA) levels in the hypertransfused mice, as the CSA levels were significantly lower in this group compared to the controls prior to and during the initial phase of granulopoietic recovery. This study suggests that hypertransfusion increases the rate of recovery of myelopoiesis by increasing the number of precursors available for myeloid differentiation from an earlier stem cell compartment.

Initial progress has been made toward the development of an experimental vaccine or immunostimulant for Pneumocystis carinii. Antigen derived from cell culture propagated P. carinii proved to be a potent immunogen in the rabbit and antibody thus produced demonstrated identity with intact murine and human lung-derived P. carinii organisms. Reactivity of the antibody with P. carinii soluble antigen in the blood of rats and human subjects with P. carinii pneumonitis (PCP) was demonstrated by the Ouchterlony technique, by counterimmunoelectrophoresis (CIE) and by latex particle agglutination (LPA). Murine-derived P. carinii antigen was utilized in an enzyme-linked immunosorbent assay (ELISA) for anti-P. carinii IgG and IgM produced in immunized rabbits and for human IgG antibody against P. carinii. Preliminary biochemical analysis of whole and solubilized cysts has been carried out, as well as slab gel electrophoresis and immunoblot profiling of solubilized organisms and naturally-occurring P. carinii antigen(s) in the blood of humans and rats with PCP. These studies represent an initial step toward the development of a vaccine or immunostimulant against PCP.

Pneumocystiscarinii infections have been detected both serologically and histologically in untreated, germ-free and conventional rats killed immediately upon arrival from two commercial sources. Pneumocystis antigenemia was detected by counterimmunoelectrophoresis (CIE) and antibody was titered by indirect immunofluorescence. The data suggest that 1) P.carinii is enzootic in certain rat colonies; 2) in utero transmission is a distinct possibility; and 3) paucity of cysts does not rule out P.carinii, as trophozoites predominate in early or subclinical infections. Histologic data support the validity of CIE for noninvasive detection of P.carinii antigen. Infection with this agent may be missed by basing diagnosis upon the presence of cyst forms alone, and it may be more common than previously supposed. These data have significant implications for the natural history, diagnosis, and epidemiology of P.carinii with regard to the human host.

Hypertransfusion can enhance myeloid recovery after bone marrow depletion, but its influence on thrombopoietic recovery has not been previously defined. We have studied the pattern of platelet and megakaryocyte recovery in mice hypertransfused after receiving 350 rad whole body irradiation. The platelet counts of the hypertransfused group showing an initial fall due to hemodilution in the expanded blood volume and then fell to a lower nadir than that of the control mice. The rate of platelet recovery was more rapid in the hypertransfused mice. Bone marrow megakaryocyte concentrations in both groups showed a degenerative phase, abortive rise, and regenerative phase. The decrease in megakaryocytes was the same in both groups. The hypertransfused mice showed a greater abortive rise in megakaryocyte concentration preceded by the appearance of a greater number of large megakaryocytes in the bone marrow. However, the most striking effect of hypertransfusion was on megakaryocyte recovery. Although the time of onset of recovery was not different, the rate of recovery was approximately twice as rapid in the hypertransfused group. Administration of daily erythropoietin to hypertransfused mice abolished this more rapid recovery. Thus, the presence of a simultaneous demand for erythroid precursors does affect the rate of megakaryocyte regeneration. Just as the more rapid recovery of granulopoiesis following hypertransfusion may be clinically beneficial, the more rapid reconstitution of thrombopoiesis may also offer clinical advantage in some circcumstances.

Heterosexual controls were found to have significantly higher titers of immunoglobulin G antibody to Pneumocystis carinii than did patients with the acquired immunodeficiency syndrome (AIDS) and P. carinii pneumonitis, human immunodeficiency virus (HIV) antibody-positive or -negative homosexual male "gay bar" patrons, and HIV antibody-positive or -negative commercial plasma donors. The T-helper/T-suppressor lymphocyte ratios of HIV antibody-negative homosexual male gay bar patrons were slightly depressed (mean = 1.31 +/- 0.54) compared with those of heterosexual controls (mean = 1.79 +/- 0.32). In addition to other recognized factors, preexisting humoral as well as cell-mediated immune deficits before infection with HIV may help to explain the prevalence of and morbidity and mortality associated with P. carinii pneumonitis in AIDS patients.

Pentamidine isethionate is a trypanocidal drug used for the treatment of Pneumocystis carinii pneumonitis. Hematological complications have occasionally been reported and include anemia, leukopenia, and thrombocytopenia. We report here several qualitative abnormalities of in vitro platelet function and coagulation that have not been described previously. Platelets were exposed in vitro to concentrations of pentamidine isethionate ranging from 0.5 to 100 μg/ml of platelet-rich plasma. Clot retraction, platelet adhesiveness to glass beads, and platelet aggregation (adenosine 5′-diphosphate [ADP], thrombin, epinephrine, collagen, and ristocetin) were inhibited in a dose-dependent fashion. The addition of pentamidine isethionate after aggregation had been initiated with ADP reversed both primary and, to a lesser degree, secondary aggregation. Platelet factor 3 availability and serotonin uptake and release (using collagen as the releasing agent) were not inhibited. Serotonin release with 10 −4 M ADP was slightly inhibited. Pentamidine isethionate prolonged the thrombin time of plasma at concentrations of 5 μg/ml and greater. The prothrombin time was prolonged at concentrations greater than 10 μg/ml of plasma. The inhibition of aggregation was reversed by washing and resuspension in plasma or by the addition of calcium or magnesium ions.

Thrombopoiesis has been studied during and after an 11 d exposure to discontinuous hypobaric hypoxia. Exposure of rats to 0.4 atmospheres for 16--17 h daily initially caused an increase in platelet count which reached a peak of 1.5 times baseline on days 4 and 5. This thrombocytosis was followed by a decrease in platelets to a nadir of 50--60% of baseline on days 12 and 13. That thrombocytosis results from increased platelet production is supported by increased [35S]sulphate incorporation into platelets and increased megakaryocyte size and turnover. The thrombocytopenia with continued hypoxia seems to result from decreased platelet production since 51Cr-platelet survival was normal while megakaryocyte concentration was decreased to one-half that of untreated controls. These observations suggest that differentiation of precursors into megakaryocytes was decreased during the thrombocytopenic period, although the fewer remaining megakaryocytes appeared stimulated because of their larger size and increased [3H]thymidine labelling. Thus, hypobaric hypoxia had a biphasic effect on thrombopoiesis with increased platelet production in the first few days of exposure followed by subnormal production.

Decreased platelet survival and function have been observed in patients with chronic hypoxemia. To develop a model for investigating these problems, we have studied the mouse exposed to severe hypobaric hypoxia. Mice were exposed to 0.3 or 0.4 atmospheres (ATM) of hypobaric hypoxia 16-19 hours daily for 18 days. Platelet counts first increased with a peak of 1.5 times baseline on day 3 followed by a decline below baseline during the second week of exposure. The greatest effect occurred at 0.3 ATM with a decline of platelets to 15% of baseline as compared to a nadir of 50-80% of baseline at 0.4 ATM. Survival of 51Cr- labeled platelets from untreated donors infused on day 13 of hypoxic exposure was decreased to 1/2 of control in the 0.3 ATM group but normal in the 0.4 ATM group. Megakaryocyte concentration showed the same trend with a decrease to 1/2 of control at 0.3 ATM with only a slight decrease at 0.4 ATM. Platelet aggregation with ADP was markedly reduced after two weeks exposure to 0.3 ATM. Aggregation of control platelets suspended in plasma from the 0.3 ATM group was depressed. Aggregation of control platelets resuspended in equal volumes of hypoxic and control plasmas was also decreased. Resuspension of control platelets in control plasma after a 1 hour incubation with hypoxic plasma partially restored the platelet aggregating activity. These studies indicate that exposure of mice to 0.3 ATM of hypobaric hypoxia for two weeks produces thrombocytopenia and decreased platelet aggregation. The decrease in platelet count is associated with decreased platelet survival and megakaryocytes. Decreased platelet survival is associated with an extrinsic rather than an intrinsic platelet defect since normal platelets have a shortened survival in hypoxic mice at 0.3 ATM. The decrease in platelet aggregation is likewise due to a plasma inhibitor since plasma from hypoxic mice inhibits aggregation of normal platelets. The changes in platelet kinetics and aggregation of the mouse exposed to 0.3 ATM for two weeks mimic in many ways those seen in patients with chronic hypoxemia and provide a laboratory model to study these alterations.

• Pneumocystis carinii antigen and IgG antibody profiles were prepared on 17 pediatric patients with acquired immunodeficiency syndrome (AIDS) with pneumonia who were examined by a variety of invasive methods for P carinii organisms. Overall, the accuracy of the antigen assay in invasively examined pediatric patients with AIDS with pneumonia was 94% (sensitivity, 100%; specificity, 90%), as antigen and invasive test results agreed in 16 of 17 patients. No statistically significant differences in IgG titer were observed between controls and patients invasively examined for P carinii , whether the organism was observed in the specimen or not. Since 38% of all serum samples referred were derived from "blood-borne" cases of AIDS, including patients who contracted AIDS as a result of both transfusion and hemophilia A, this suggests that P carinii pneumonia or P carinii pneumonia—like pneumonias may be more common in these individuals. ( AJDC 1988;142:36-39)

Serologic profiles of Pneumocystis carinii (PC) based upon incidence of antigenemia & IgG antibody (AB) titers in pediatric patients in Papua, New Guinea (NG) & Memphis, TN support the ubiquity of PC. PC IgG AB was titered by an enzyme-linked immunosorbent assay & antigenemia was detected by a counterimmunoelectrophoresis test, both developed in our laboratory. Antigen (AG) used in the ELISA test was derived from cell culture-grown PC. NG children with pneumonia (N=188) had higher anti-PC titers than U.S. controls (N=50). Those with PC AG (+) pneumonia had highest titers (geometric mean titer = 277), suggesting a specific immune response to PC. Those with PC AG (-) pneumonia (GMT=139) had higher titers than matched U.S. controls (GMT=60), perhaps reflecting more environmental exposure or differences due to socio-economic status. The extent to which PC is responsible for morbidity in multiple-agent pneumonias remains to be elucidated. Since PC is treatable, this represents a high priority goal, as the decision to treat might influence survival in critical cases. Mean AB titers were all significantly different (p=