Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.
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PMID:Chemotherapeutic potential of methotrexate peptides. 307 29

Methotrexate-alpha-phenylalanine (MTX-Phe), a second-generation prodrug in the MTX alpha-peptide series designed for activation to MTX by carboxypeptidase-mAb conjugates, was synthesized by reaction of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with L-glutamyl-alpha-L-phenylalanine. Production of MTX from MTX-Phe, catalyzed by bovine pancreas carboxypeptidase A (CPA), was 250-fold faster than the corresponding reaction involving methotrexate-alpha-alanine, previously the best MTX peptide substrate for the enzyme. The amount of CPA required to make MTX-Phe equitoxic with MTX, when tested against UCLA-P3 human lung adenocarcinoma cells in vitro, was more than 10-fold lower than that required to achieve the same result with MTX-alpha-alanine. When the lung tumor cells were treated with CPA conjugated to KS1/4 (a mAb targeted to these cells) and excess conjugate removed by extensive washing, the ID50 for MTX-Phe improved from 2.2 x 10(-6) M (no enzyme present) to 6.3 x 10(-8) M; the latter value was comparable to that of the parent drug MTX (4.5 x 10(-8) M). [3H]MTX-Phe was synthesized and used to investigate the mechanism by which the prodrug exerts its cytotoxic effect in the presence and absence of CPA. The present results demonstrate that, for use in conjunction with CPA-mAb conjugates, the alpha-phenylalanine derivative is the optimal prodrug form of MTX (and probably other antifols that contain the glutamate moiety).
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PMID:Methotrexate-alpha-phenylalanine: optimization of methotrexate prodrug for activation by carboxypeptidase A-monoclonal antibody conjugate. 783 11

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.
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PMID:Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro. 856 31

Selective delivery of lethal concentrations of drugs to tumors, allowing the latter to be eradicated without damage to other tissues, continues to be a major goal in cancer chemotherapy. Prodrugs (i.e. drugs that have been derivatized to prevent uptake into cells or interaction with targets), activated by enzyme-monoclonal antibody conjugates positioned at tumor sites, offer promise for achieving this objective. Methotrexate alpha-peptides (derivatives in which an amino acid is linked to the alpha-carboxyl group of the glutamate moiety) are ideal prodrugs, since they are not transported into cells and can be converted to the parent drug by carboxypeptidases. The L,L-diastereomer of MTX-alpha-Phe, synthesized in good yield by treatment of the p-nitrophenyl ester of 4-amino-4-deoxy-10-methylpteroic acid with Glu-alpha-Phe, was hydrolyzed readily by carboxypeptidase A (CP-A). Conjugate was prepared by derivatizing the enzyme and monoclonal antibody KS1/4 with linkers containing maleimide and sulfhydryl groups, respectively; interaction of these groups to form a stable thioether bond joined the proteins. When administered in vitro to UCLA-P3 human lung adenocarcinoma cells (ca. 5 x 10(4) antibody binding sites/cell) that had been pre-treated with the conjugate (whose antibody KS1/4 is targeted to these cells), and excess conjugate removed by extensive washing, MTX-Phe (ID50 = 6.3 x 10(-8) M) approached the toxicity of MTX (ID50 = 4.5 x 10(-8) M). In the absence of conjugate, MTX-Phe was much less toxic (ID50 = 2.2 x 10(-6) M).
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PMID:Development of methotrexate alpha-peptides as prodrugs for activation by enzyme-monoclonal antibody conjugates. 938 87

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.
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PMID:Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843. 989 62

In antibody-directed enzyme-prodrug therapy (ADEPT), antibody-enzyme conjugates specifically activate non-toxic prodrugs in tumour tissue. The A33 cognate antigen is a promising target for immunotherapy of gastrointestinal cancers. We have explored A33-based ADEPT with carboxypeptidase A (CPA) and the prodrug, methotrexate-phenylalanine (MTX-Phe). In A33-positive SW1222 cells, the toxicity of MTX-Phe was about 3 logarithms lower compared to MTX. Preincubation with a huA33 antibody-CPA conjugate (huA33-CPA), but not with an isotypic control conjugate, rendered MTX-Phe equally toxic to MTX. No toxicity was observed in mice receiving MTX-Phe in 8-fold the LD50 of MTX. Nude mice bearing A33-positive SW1222 colon carcinoma xenografts were injected intravenously (IV) with 125I-labeled huA33-CPA. The conjugate localised to the tumour with a maximum from 6-24 h. Pre-treating these mice with excess A33 substantially reduced subsequent conjugate uptake, demonstrating immunologic specificity of tumour-uptake. Total tumour uptake and ratios of tumour over blood or normal tissues, however, were lower than with unconjugated A33. This may explain in part why no significant tumour responses were observed in xenografted mice. In summary, our results demonstrate in principle the feasibility of A33-based enzyme targeting, but they call for small recombinant antibody-enzyme constructs to facilitate tumour penetration and clearance from the bloodstream.
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PMID:Specific tumour localisation of a huA33 antibody--carboxypeptidase A conjugate and activation of methotrexate-phenylalanine. 1506 53