Abstract
The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan; CPT-11) is delayed diarrhea. This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine. After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity. Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent. In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE). Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase. Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental Ki values (r2 = 0.944) for hiCE. Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses. These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications.
7-Ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin) (irinotecan, CPT-11) is a camptothecin-derived prodrug that is activated by esterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor (Tsuruo et al., 1988; Satoh et al., 1994; Tanizawa et al., 1994; Sawada et al., 1996). This drug has demonstrated remarkable antitumor activity in human tumor xenograft models and consequently has been developed for clinical studies in a variety of tumor histotypes (Houghton et al., 1995, 1996; Thompson et al., 1997a,b; Furman et al., 1999). Currently, CPT-11 is approved for use in colorectal cancer, but is in phase I, II, and III trials for treatment of many solid tumors in both adults and children (Furman et al., 1999).
In humans, CPT-11 activation is primarily mediated by carboxylesterases (CE) present in the liver and intestine. Drug hydrolysis is required for antitumor efficacy, and because the parent molecule and metabolites are cleared from the circulation via the bile, very high concentrations of CPT-11 and SN-38 can be detected in this fluid (Morton et al., 2002). Because the bile duct opens into the duodenum, the region of the gut with the highest level of CE activity (Morton et al., 2002), direct activation of CPT-11 by CEs would result in increased levels of SN-38 and hence enhanced toxicity to the small intestine. This would account for the delayed diarrhea caused by mucosal damage that is observed in patients undergoing therapy with this drug.
To alleviate this dose-limiting and potentially life threatening toxicity, we hypothesized that the development of specific human intestinal esterase inhibitors that would prevent the conversion of CPT-11 to SN-38 in the gut might ameliorate these side effects. Recently, we identified the enzyme responsible for CPT-11 activation in the small intestine, human intestinal CE (hiCE), and demonstrated that expression of this protein in mammalian cells sensitized them to the drug (Khanna et al., 2000). This enzyme is highly homologous to the human liver CE 2, and both proteins are probably the products of the same gene (Potter and Danks, 2000). Therefore, in the current study, we have attempted to identify selective inhibitors of hiCE that would not inhibit other human CEs, acetylcholinesterases (AcChE), or butyrylcholinesterase (BuChE). Using a directed screening procedure based upon Telik's Target-Related Affinity Profiling (TRAP) technology (Kauvar et al., 1995; Dixon and Villar, 1998; Beroza et al., 2002) with purified CEs, we have identified a novel class of enzyme inhibitors that demonstrates selectivity and specificity for hiCE.
Materials and Methods
Drugs and Inhibitors. CPT-11 was kindly provided by Dr. J.P. McGovren (Pharmacia Upjohn, Piscataway, NJ). Bis(4-nitrophenyl) phosphate (BNPP) was obtained from Sigma Biochemicals (St. Louis, MO).
Purified Enzymes. Pure rabbit liver CE (rCE) and hCE1 were prepared from baculovirus-infected cell culture media as described previously (Morton and Potter, 2000). hiCE was prepared in a similar fashion, although the samples were only ∼60% pure. Because serum-free media harvested from insect cells is essentially devoid of CE activity (Morton and Potter, 2000), the only enzyme present in the preparation was recombinant hiCE. The cDNA sequences encoding the above proteins have the following GenBank accession numbers; rCE, AF036930 (Potter et al., 1998); hCE1, M73499 (Munger et al., 1991); and hiCE, Y09616 (Schwer et al., 1997). All enzymes were characterized using o-nitrophenyl acetate (o-NPA; Sigma Chemicals, St. Louis, MO) as a substrate (Beaufay et al., 1974; Potter et al., 1998), and preparations were stored at 4°C in 50 mM HEPES, pH 7.4. Human AcChE (hAcChE) and BuChE (hBuChE) were purchased from Sigma and stored at 4°C.
Library of Inhibitors. The compounds for screening were selected from Telik's library of small molecules using the TRAP methodology (Dixon and Villar, 1998). TRAP characterizes a molecule by its binding affinities to a panel of proteins (i.e., its “affinity fingerprint”). The screening process is iterative, with ∼70 compounds being tested at each iteration. The first set of compounds is chosen because their fingerprints represent the diversity of fingerprints in the entire compound collection. Subsequent iterations are based on computational analysis of the fingerprints of the compounds that were screened in previous rounds of analysis. This process allows identification of active compounds of multiple chemotypes by assaying as few as 200 compounds (Dixon and Villar, 1998; Beroza et al., 2002).
Carboxylesterase Assays. CE activity was determined as described previously (Beaufay et al., 1974; Potter et al., 1998). One unit is the amount of enzyme that converts 1 μmol of o-NPA to o-nitrophenol per minute.
Carboxylesterase Enzyme Inhibition Assays. All inhibitors were dissolved in DMSO, typically at 10 mM, before analysis. For screening enzyme inhibitors, 100 μl of HEPES, pH 7.4, containing 6 mM o-NPA and 200 μM test compound were added to each well of a 96-well plate. The reaction was initiated by adding 100 μl of the appropriate CE containing 20 to 30 units of enzyme, and the absorbance was monitored at 15-s intervals for 5 min. All compounds were assayed in duplicate, and all plates included positive (50 μM BNPP) and negative (no enzyme, DMSO alone) controls. DMSO concentrations never exceeded 2% and routinely were 1% or less. Compounds that demonstrated greater than 50% inhibition of CE activity were further characterized by determining their Ki values with the appropriate enzyme.
Determination of Ki Values.Ki values for the inhibitors were determined with either 3 mM o-NPA or 100 μM CPT-11 as substrates. At least eight inhibitor concentrations were used for the analyses, and the data were fitted to one of six equations describing competitive, partially competitive, noncompetitive, partially non-competitive, mixed, or uncompetitive inhibition (Webb, 1963). These equations are described below:
Assume α = ∞
Assume 1 < α < ∞; β = 1
Assume α = 1; β = 0
Assume α = 1; 0 < β < 1
Assume 1 < α < ∞; β = 0
Assume α < 1; β < 1.
In each case, i is fractional inhibition, [s] is substrate concentration, [I] is inhibitor concentration, α is change in affinity of substrate for enzyme, β is change in the rate of enzyme substrate complex decomposition, and Ks is the dissociation constant for the enzyme substrate complex.
After fitting the data to these equations using Prism (GraphPad Software, San Diego, CA), r2 values were determined for each set of results. Using both the r2 value and the statistical analysis of these curve fits, assignment of the mode of enzyme inhibition was performed.
Acetylcholinesterase Enzyme Assays. AcChE assays were performed using a spectrophotometric multiwell plate assay (Doctor et al., 1987) using acetylthiocholine as a substrate (Ellman et al., 1961). Compounds were assayed at a final concentration of 100 μM with the DMSO concentration never exceeding 2%.
Butyrylcholinesterase Enzyme Assays. BuChE assays were performed as described above for AcChE, except that butyrylthiocholine was used as a substrate.
Determination of Irreversible Inhibition. To assess whether the inhibitors produced irreversible inhibition of hiCE, compounds were added to enzyme and incubated for 1 h on ice. Samples were then diluted in HEPES, pH 7.4, and CE activity was determined using a spectrophotometric assay with o-NPA as a substrate (Beaufay et al., 1974; Potter et al., 1998). Final concentrations of inhibitor in the assay reactions did not exceed 40 nM. CE activity was calculated as determined above, and results were expressed relative to control enzyme samples that were incubated with DMSO alone. The irreversible inhibitor BNPP was used as a positive control for these reactions.
Quantitation of CPT-11 and SN-38 by HPLC. CPT-11 and SN-38 concentrations were determined using an HPLC-based assay with a fluorescent detector as described previously(Guichard et al., 1998; Danks et al., 1999).
QSAR Analysis. 3D-QSAR analyses were performed using Quasar 4.0 (Vedani and Dobler, 2002a,b) running on a Macintosh G4. Structures for each analog were initially constructed with Chem3D for Macintosh. Partial atomic charges from the bond charge correction method (Jakalian et al., 2002) and AMBER atom types were assigned using the antechamber module of AMBER7 (University of California, San Francisco, CA). To select optimal conformers for 3D-QSAR, a short molecular dynamics run (250 ps) was performed at 500 K using the sander module of AMBER7. Snapshots were captured at 25-ps intervals, and the sulfonamide structures minimized with respect to energy with the AMBER95 force field.
Synthesis of 4-Chloro-N-(4-{[(4-chlorophenyl)sulfonyl] amino}-2,3,5,6-tetrafluorophenyl) benzenesulfonamide (10). To a stirred solution of 2,3,5,6-tetrafluorobenzene-1,4-diamine (0.2 g, 1.1 mmol) in anhydrous dichloromethane, 4-chlorobenzyl sulfonylchloride (1.4 g, 6.6 mmol), 4-(dimethylamino)-pyridine (0.13 g, 1.1 mmol), and pyridine (1.1g, 13.9 mmol) were added under an atmosphere of argon. The reaction mixture was stirred for 12 h at room temperature, and thin-layer chromatography was performed to confirm that the reaction had gone to completion. After extraction with dichloromethane (3 × 20 ml), the organic layer was washed with 0.5 N HCl, followed by saturated sodium bicarbonate. After drying over anhydrous sodium sulfate, the crude product was purified by column chromatography using petroleum ether/ethyl acetate (4:1; v:v) to give an off-white solid (0.1 g). Melting point, 231 to 233°C; anal., (C18H10Cl2F4N2O4S2); calc., (%) C 40.84, H 1.90, N 5.29; found, (%) C 41.03, H 1.92, N 5.23; 1H NMR (300 MHz, CDCl3) δ 4.4 (s, 2H, NH x 2), 7.56 (d, 4H, J = 13.8 MHz, Ar-H), 7.95 (d, 4H, J = 13.8 MHz, Ar-H); 13C NMR (75.5 MHz, CDCl3) δ 147.2, 139.8, 140.8 126.8, 133.9, 129.6, 128.9; mass spectroscopy (electrospray ionization) m/z 527 (M-1).
Results
Identification of Selective hiCE Inhibitors. Selective hiCE inhibitors were identified from four iterations of screening, a total of 235 compounds. Each compound was assayed against hiCE, hCE1, and rCE. After each round of screening, compounds were selected based upon their ability to selectively inhibit hiCE compared with hCE1. After four iterations of screening, nine novel sulfonamide analogs were identified that demonstrated hiCE-specific enzyme inhibition; 6 of these demonstrated at least 250-fold selectivity compared with hCE1 by examination of the Ki values. The ID, chemical name, and structures of these compounds are shown in Table 1.
The Ki values were determined for several different enzymes, including two human CEs, hiCE and hCE1, rCE, hAcChE, and hBuChE. Because accurate calculation of the Ki value is dependent upon the mode of inhibition, we fitted the assay results to six different equations describing the various mechanisms of enzyme inhibition. Analysis of the curve fits for these data and subsequent statistical calculations demonstrated that partially competitive inhibition (eq. 2) was the most appropriate model describing the interaction of the sulfonamides with hiCE (Table 2). As can be seen, r2 values of 0.94 or above were generated for all compounds using this equation, and the calculated Ki values ranged from 53.3 to 1310 nM.
As indicated in Table 3, a 2-log range for the Ki values for hiCE was obtained. The majority of the compounds were bis-benzene sulfonamides, with the most potent compound (1: 4-chloro-N-(4-{[(4-chlorophenyl)sulfonyl]amino}phenyl) benzenesulfonamide) containing p-chloro-substituted benzene rings at either end of the molecule. This same compound demonstrated inhibition of hCE1 catalysis at concentrations below 100 μM; however, the Ki for 1 with hCE1 was at least 250-fold greater than that for hiCE (13,700 nM). More importantly, none of the compounds demonstrated any inhibition of hAcChE, or hBuChE, at concentrations up to 100 μM.
As indicated in Table 3, there was some overlap in the inhibition pattern for rCE. Because both hiCE and rCE can efficiently metabolize CPT-11, we expected some of the sulfonamide derivatives to inhibit both enzymes. However, even within this small series of compounds, apparent selectivity can be observed. For example, analogs 2, 3, and 4 were selective for hiCE, demonstrating greater than 220-fold selectivity compared with all other enzymes tested. In contrast, compounds 5 and 7 were effective inhibitors of both hiCE and rCE.
Determination of Reversibility of Enzyme Inhibition. To determine whether any of the inhibitors resulted in irreversible inhibition of hiCE, we preincubated enzyme preparations with the inhibitors at a concentration of 10 μM for 1 h, before CE activity assays. Because the enzyme and inhibitor were diluted at least 250-fold in the CE assay, loss of activity can only occur from direct inactivation of the protein by the sulfonamide analog during the preincubation period. None of the inhibitors resulted in permanent inhibition of the enzyme (Fig. 1), whereas 10 μM BNPP resulted in greater than 90% inhibition of hiCE. These results suggest that the sulfonamide analogs inhibited in a reversible fashion.
Inhibition of CPT-11 Metabolism of the Sulfonamide Analogs. Because the sulfonamide analogs are partially competitive inhibitors of hiCE, their Ki values will be dependent upon the substrates used in the kinetics analyses (see eq. 2). Therefore, to assess the ability of these compounds to inhibit hiCE-mediated activation of CPT-11, we determined the Ki values using the drug as a substrate. CPT-11 concentration was fixed at 100 μM, and inhibitor concentrations varied from 1 nM to 100 μM. After incubation for 10 min at 37°C, the levels of SN-38 in the reactions were determined by HPLC. Table 4 indicates that the Ki values for the inhibition of metabolism of CPT-11 are similar to those for the inhibition of o-NPA. However, several analogs did not inhibit drug metabolism. Because the sulfonamides are partially competitive inhibitors and the Ki is dependent upon the ratio of substrate concentration and Km ([s]/Km), it is likely that these discrepancies are a result of the differences in the Km values for the substrates. The Km values for hiCE with CPT-11 and o-NPA are significantly different at 3.2 and 355 μM, respectively. Overall, these results suggest that although the sulfonamide analogs are potent CE inhibitors, it is apparent that their potency is substrate-dependent.
3D-QSAR Analysis of the Sulfonamide Analogs. Because we expected that the sulfonamides must fit into the active site gorge of the carboxylesterase for inhibition, and given the steric constraints on the diameter of this gorge, for 3D-QSAR we selected the most extended sulfonamide conformer from the pool of 10 conformers generated from the molecular dynamics simulation. These were aligned around the sulfonamido group of each inhibitor and model that predicted the activity of the inhibitors was then constructed. The pseudo-receptor model that best reproduced the sulfonamide Ki values is shown in Fig. 2. The properties of the pseudo-receptor are shown as color-coded spheres surrounding the sulfonamide compound 2. The residues in the active site gorge are taken from our homology model of hiCE (Wadkins et al., 2001) and are matched in color-coding to the pseudo-receptor model. Note that the pseudo-receptor model reproduces the distribution of residue types very well, including the charge asymmetry within the gorge. The cationic His, Lys, and Arg residues (red) are located at the opening of the gorge and are clustered on one side of the opening. The anionic Asp and Glu residues (blue) are at the bottom of the gorge, and are clustered on the side of the gorge opposite that where the cationic residues are located. The gorge is lined with hydrophobic residues, which coincide with the predicted hydrophobic surface (gray lines) in the pseudoreceptor model. In summary, the 3D-QSAR captures the essential features of the hiCE active site gorge and can be used to predict activities of novel sulfonamides as hiCE inhibitors. Based on this information, we predicted that halogen substitution of the benzene rings would increase the potency of the inhibitors; hence, we synthesized a fluoro analog to test the validity of the QSAR approach.
Synthesis of Compound 10. Validation of QSAR Analysis. QSAR analysis indicated that compound 10 would be a very good inhibitor of hiCE, with a predicted Ki of ∼390 nM when using o-NPA as a substrate. To assess the validity of this model, we synthesized compound 10 (Table 1) by condensation of 2,3,5,6-tetrafluorobenzene-1,4-diamine with p-chlorobenzenesulfonyl chloride. The ability of 10 to inhibit hiCE, hCE1, rCE, hAcChE, and hBuChE was then assessed using o-NPA for the CEs, acetylthiocholine for hAcChE, or butyrylthiocholine for hBuChE as substrates.
As indicated in Table 5, sulfonamide 10 was a very good inhibitor of hiCE, with a Ki value of 41.5 nM. This compound did not inhibit hCE1, hAcChE, or hBuChE, consistent with results seen with the other sulfonamide analogs. In addition, compound 10 acted in a partially competitive fashion, similar to that observed with sulfonamides 1 to 9. Inhibition was observed when using rCE as an enzyme; as with the other sulfonamide-analogs, however, the Ki value (522 nM) was greater than that observed with hiCE. The Ki value predicted from the QSAR analyses for compound 10 with hiCE (393 nM) was near the observed value (41.5 nM), demonstrating both the validity of the technique and the potential use of the data to design inhibitors with greater specificity. It is noteworthy that sulfonamide 10 had the lowest Ki for the inhibition of CPT-11 metabolism by hiCE (110 nM; Table 5), for all of the compounds analyzed.
Discussion
We have identified a novel class of CE inhibitors based upon the sulfonamide chemotype. These compounds are selective for hiCE, demonstrating no inhibition of a human liver CE, hCE1, hAcChE, or hBuChE. In addition, these sulfonamides are very potent inhibitors, with Ki values in the low nanomolar range. The analogs were identified through application of Telik's TRAP technology after screening of a diverse chemical library. One class of molecules identified in the TRAP screen typically contained two sulfonamide moieties, usually present as a symmetrical molecule. In addition, the majority of these analogs contained phenyl rings both in the central and terminal domains of the structures.
Halogen substitution of benzene rings tended to increase the potency of the inhibitors (e.g., 1 > 2 > 8), suggesting that withdrawal of the π electrons from the rings may influence binding of the sulfonamide within the CE active site. We therefore hypothesized that sulfonamide 10 might be a better inhibitor of hiCE. Subsequent studies confirmed this prediction; compound 10 had a Ki for hiCE with o-NPA of 41.5 nM. These results suggest that chemical substitutions that alter the localization of electrons within the sulfonamide moiety significantly affect the level of enzyme inhibition. Furthermore, we propose that the overall molecular structure of the sulfonamides contributes to their activity. As shown in Fig. 2C, the compounds adopt a conformation that is complementary to the computer-predicted model of the active site gorge of hiCE (shown as a surface representation). Hence, the combination of electronic and structural elements for this class of compounds allows for localization within, and interaction with, the residues that form the boundaries of the active site.
The Ki values for the inhibition of CE-mediated metabolism of CPT-11 by the sulfonamides were significantly greater compared with those derived from using o-NPA as a substrate. This is partly because the Km for hiCE for CPT-11 is approximately 100-fold lower than that for o-NPA (3.2 μversus 355 μM). Because these inhibitors are competitive, the efficacy of inhibition is dependent on the affinity of both the sulfonamide and the substrate for the enzyme. However, as indicated in Tables 4 and 5, several of the analogs were effective inhibitors of CPT-11 metabolism; the greatest inhibition was observed from compound 10. Because this was proposed as an effective agent from the analysis the SAR studies, these results support the development of novel sulfonamide-based CE inhibitors using computational methods.
Esterase-mediated catalysis occurs in a four-stage reaction initiated by the nucleophilic attack of the carbonyl carbon by the deprotonated OH group in the serine amino acid at the active site (Fig. 3). This is followed by elimination of the alcohol and water, after attack by an HO- moiety. Potentially, the sulfur atom present in the inhibitors could act as a site of initial attack by the O- nucleophile, resulting in a square pyramidal intermediate. However, nucleophilic attack of the sulfur atom in sulfonamides requires very harsh chemical conditions; therefore, the formation of the covalently bound transient intermediate is not favored. Although this mechanism has been proposed for sulfonamide-based inhibitors of matrix metalloproteinases and thrombin, crystallographic and computer homology studies with these compounds in complex with the proteins indicate that this does not occur (Brandstetter et al., 1992; MacPherson et al., 1997; Kiyama et al., 1999; Whittaker et al., 1999). Rather, a network of hydrogen bonding between the sulfonyl oxygen atoms and protons exposed within the active site was observed. This stabilizes the sulfonamide-protein complex and presumably prevents access of the substrate to the catalytic amino acids. Thrombin has a catalytic triad of amino acids (Ser, His, and Asp rather than Glu) and a mechanism of substrate cleavage similar to observed in CEs. Hence, it is likely that the mechanism of inhibition of thrombin by sulfonamides would be comparable with inhibition of CEs by this class of compounds.
On the other hand, the sulfonamides may act as inhibitors by mimicking the CE transition state. The first stage in CE-mediated catalysis is the formation of a negatively charged tetrahedral intermediate involving nucleophilic attack by Ser toward the carbonyl carbon of the ester (see Fig. 3). As the sulfonamide chemotype in the compounds described in Table 1, would also adopt a tetrahedral and partially negatively charged conformation at physiological pH, this might act to inhibit catalysis by binding to the active site and blocking enzyme function. Inhibition by transition state analogs is a well validated drug design strategy that has led to the clinical development of protease and glycosidase inhibitors.
Because the sulfonamide-derived hiCE inhibitors contain multiple phenyl rings, they would prefer to localize within regions of the protein that are hydrophobic. Because the active sites of CEs are lined with aromatic amino acids, the gorge leading to the catalytic triad is very hydrophobic, thereby providing a suitable binding pocket for the sulfonamide analogs. Because compounds containing structurally similar functional groups (e.g., benzyl-substituted sulfoxides and sulfones) demonstrated no inhibition of CEs (data not shown), it is likely that resonance of the protons between the sulfur and nitrogen atoms is important for biological activity. Studies to determine the mechanism of inhibition by the sulfonamides using NMR and X-ray crystallography are currently underway.
Previously identified esterase inhibitors have included carbamates (physostigmine, pirimicarb), organophosphates (sarin, VX gas), acridines (e.g., tacrine, 9-amino-6-chloro-2-methoxyacridine), and piperidines (e.g., donepezil, CPT-11). None of these classes of compounds contains the sulfonamide function. Proteases, which demonstrate active site geometry and catalytic amino acid residues very similar to those of esterases, can be inhibited by sulfonyl chlorides and fluorides (e.g., phenylmethylsulfonyl fluoride), and these compounds have been used as protease inhibitors for many years. These agents are known to react directly with the serine at the active site, with the loss of the halogen, to yield a sulfonylated adduct and an inactive protein. Although, in theory, the sulfonylated enzyme could be reactivated, this requires harsh chemical conditions (pH 2.0, 40°C, 3 h) followed by neutralization and extensive dialysis. We propose that the sulfonamide-CE interaction occurs via a different mechanism, because our results indicate that the compounds identified in this report are reversible esterase inhibitors and do not form stable long-lived intermediates. More likely, they yield transient complexes stabilized by hydrogen bonding between the sulfonyl oxygen atoms and protons within the active site gorge (Kiyama et al., 1999). This mechanism would allow for the rapid reversal of the complexes to yield the free enzyme and inhibitor and would constitute a novel mechanism of CE inhibition by the sulfonamide analogs.
For clinical application, we predict that a poorly bioavailable compound would prove the most efficacious for reducing hiCE-mediated CPT-11 activation. The sulfonamide would be administered orally with the intention of maintaining high concentrations of inhibitor within the lumen of the gut. Systemic absorption of the compound would be low, so no change in the antitumor efficacy of CPT-11 would be encountered. However, inhibition of hiCE present within the gut epithelia would occur, eliminating in situ activation of drug and hence the delayed diarrhea associated with CPT-11 administration. We are currently assessing the efficacy of enzyme inhibition by the sulfonamides in animal models. In addition, we are further analyzing the mechanism of enzyme inhibition by conducting structural studies with selected sulfonamides in complex with hiCE.
Acknowledgments
We thank Dr. J. P. McGovren for the gift of CPT-11.
Footnotes
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This work was supported in part by National Institutes of Health Cancer Center Core grant P30-CA21765 and by the American Lebanese Syrian Associated Charities.
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ABBREVIATIONS: CPT-11, irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; SN-38, 7-ethyl-10-hydroxycamptothecin; CE, carboxylesterase; hiCE, human intestinal carboxylesterase; AcChE, acetylcholinesterase; BuChE, butyrylcholinesterase; TRAP, Target Related Affinity Profiling; BNPP, bis(4-nitrophenyl) phosphate; rCE, rabbit liver carboxylesterase; hCE1, human carboxylesterase 1; o-NP, o-nitrophenol; o-NPA, o-nitrophenyl acetate; hAcChE, human acetylcholinesterase; hBuChE, human butyrylcholinesterase; DMSO, dimethyl sulfoxide; 3D, three dimensional; QSAR, quantitative structure-activity relationship; HPLC, high performance liquid chromatography.
- Received October 13, 2003.
- Accepted February 19, 2004.
- The American Society for Pharmacology and Experimental Therapeutics