Identification of novel vascular targets in lung cancer.

BACKGROUND: Lung cancer remains the leading cause of cancer-related death, largely owing to the lack of effective treatments. A tumour vascular targeting strategy presents an attractive alternative; however, the molecular signature of the vasculature in lung cancer is poorly explored. This work aimed to identify novel tumour vascular targets in lung cancer. METHODS: Enzymatic digestion of fresh tissue followed by endothelial capture with Ulex lectin-coated magnetic beads was used to isolate the endothelium from fresh tumour specimens of lung cancer patients. Endothelial isolates from the healthy and tumour lung tissue were subjected to whole human genome expression profiling using microarray technology. RESULTS: Bioinformatics analysis identified tumour endothelial expression of angiogenic factors, matrix metalloproteases and cell-surface transmembrane proteins. Predicted novel tumour vascular targets were verified by RNA-seq, quantitative real-time PCR analysis and immunohistochemistry. Further detailed expression profiling of STEAP1 on 82 lung cancer patients confirmed STEAP1 as a novel target in the tumour vasculature. Functional analysis of STEAP1 using siRNA silencing implicates a role in endothelial cell migration and tube formation. CONCLUSIONS: The identification of cell-surface tumour endothelial markers in lung is of interest in therapeutic antibody and vaccine development.

Liposomal encapsulation enhances the antitumour efficacy of the vascular disrupting agent ZD6126 in murine B16.F10 melanoma.

Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.

Regulatory aspects of genotoxicity testing: from hazard identification to risk assessment.

This symposium focused on the use of tests for chromosomal damage, and other genotoxicity measures, for detection of potentially harmful chemicals. The speakers discussed the information that has been gained over the last three decades about the use of "short-term tests" for genotoxicity in cultured cells and in animals (mainly rodents), and the ongoing debates about the rational use of data from such experimental systems in trying to extrapolate to an understanding of potential human risk. The overall theme was that the field of regulatory toxicology currently is over-reliant on qualitative outcomes of in vitro hazard-screening tests, generally conducted at the maximum achievable exposures, and needs a more realistic approach that incorporates in vivo exposure levels and dose-response information.

Intensity-modulated Radiotherapy for Anal Cancer: Dose-Volume Relationship of Acute Gastrointestinal Toxicity and Disease Outcomes.

AIMS: Intensity-modulated radiotherapy (IMRT) is increasingly used in the treatment delivery of chemoradiotherapy in anal cancer with the ability to reduce toxicity. We report on 4 year outcomes since the introduction of IMRT and identify the most predictive bowel organ at risk that correlates with acute diarrhoea. MATERIALS AND METHODS: Fifty-eight patients receiving definitive chemoradiotherapy for squamous or basaloid cell anal carcinoma (T1-4NanyM0) were reviewed. Fifty-four per cent of patients had stage III disease and most (79%) were treated with a dose of 54 Gy in 30 fractions. Patient acute gastrointestinal toxicity was recorded using Common Terminology Criteria of Adverse Events (CTCAE) diarrhoea grading. Four different methods of bowel were re-contoured for each patient and correlated with acute diarrhoea. Locoregional control and overall survival were analysed. RESULTS: CTCAE grade 3 or more diarrhoea occurred in 11/58 patients (19%). Seven patients did not complete treatment; 10 patients (17%) required a treatment break of 3 or more days. 'Bowel cavity' was the best predictor of acute grade 3 toxicity using volume (P = 0.002) or volume to bowel cavity in 5 Gy bins (V5-V50Gy); P < 0.05. Bowel cavity V30Gy ≤ 300 cm3 predicts a 6% grade 3 diarrhoea risk versus > 300 cm3 predicts a 42% risk. Four year progression-free survival was 84% (95% confidence interval 73-92%) and overall survival was 88% (95% confidence interval 75-95%). CONCLUSION: Chemoradiation using IMRT provides excellent local control and acceptable acute gastrointestinal toxicity. Bowel cavity is the most sensitive predictor for grade 3 versus grade 0-2 diarrhoea, with any volume receiving 5-50 Gy discriminatory.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards.

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test.

A survey conducted as part of an International Workshop on Genotoxicity Testing (IWGT) has identified a number of compounds that appear to be more readily detected in vivo than in vitro. The reasons for this property varies from compound to compound and includes metabolic differences; the influence of gut flora; higher exposures in vivo compared to in vitro; effects on pharmacology, in particular folate depletion or receptor kinase inhibition. It is possible that at least some of these compounds are detectable in vitro if a specific in vitro test is chosen as part of the test battery, but the 'correct' choice of test may not always be obvious when testing a compound of unknown genotoxicity. It is noted that many of the compounds identified in this study interfere with cell cycle kinetics and this can result in either aneugenicity or chromosome breakage. A decision tree is outlined as a guide for the evaluation of compounds that appear to be genotoxic agents in vivo but not in vitro. The regulatory implications of these findings are discussed.

Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different cross-linking agents: antitumor effects, in vivo fate, and tumor cell mutants.

The A-chain of the plant toxin abrin was covalently linked to monoclonal anti-Thy 1.1 antibody (OX7) with the use of either N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane hydrochloride (2IT). The SPDP reagent generates a linkage containing a disulfide bond and an amide bond, whereas the 2IT reagent generates a linkage containing a disulfide bond and an amidinium bond. The two immunotoxins were powerfully and specifically toxic to Thy 1.1-expressing murine AKR-A lymphoma cells in vitro. Both reduced the rate of protein synthesis of the cells by 50% at a concentration of 10(-11) M. However, clonogenic assays revealed that about 1% of the AKR-A cells survived treatment with high concentrations of OX7-SPDP-abrin A, whereas only about 0.1% survived treatment with similar concentrations of OX7-2IT-abrin A. Several clones of the surviving cells were isolated. Of 11 clones of cells that had survived exposure to OX7-SPDP-abrin A, 10 were resistant to further treatment with OX7-SPDP-abrin A but had normal sensitivity to OX7-2IT-abrin A. These clones expressed moderate to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. In contrast, all 10 clones of cells that had survived exposure to OX7-2IT-abrin A were substantially or entirely resistant to both immunotoxins. They expressed low to high levels of the Thy 1.1 antigen and were fully sensitive to abrin. The 2IT-linked immunotoxin was much more effective than the SPDP-linked immunotoxin at protecting nu/nu mice against the growth of AKR-A lymphoma cells in the peritoneal site. A single iv injection of 0.3 nmol OX7-2IT-abrin A eradicated at least 99.99% of the tumor cells, as judged from the extension in the median survival time of the animals, whereas OX7-SPDP-abrin A eradicated only about 99% of the cells. The tumors that developed in the animals that received OX7-2IT-abrin A were Thy 1.1-negative, whereas those in the recipients of OX7-SPDP-abrin A generally expressed normal levels of the Thy 1.1 antigen. The difference in antitumor activity of the immunotoxins was not due to differences in their in vivo fate, inasmuch as they were cleared from the bloodstream at an identical rate and broke down at the same rate to release free antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of induction of nuclear anomalies by gamma-radiation in the colonic epithelium of the mouse.

The induction of karyorrhexis and "nuclear anomalies" in colonic crypt cells has been correlated positively with the induction of colonic tumors by chemical treatment. These nuclear anomalies occur in the proliferative region of the crypt and exhibit a variety of morphological characteristics. Some nuclear anomalies resemble the micronuclei that arise from chromosomal fragments after mitosis. Here, we report that the nuclear anomalies observed within the first few hr of insult with gamma-radiation are independent of mitosis for their expression, as evidenced by failure of colchicine to inhibit their induction, and do not arise from chromosomal material lost during mitosis.

Effect of chemical deglycosylation of ricin A chain on the in vivo fate and cytotoxic activity of an immunotoxin composed of ricin A chain and anti-Thy 1.1 antibody.

The carbohydrate present on ricin A chain causes ricin A chain immunotoxins to be cleared rapidly in animals by the reticuloendothelial system. In an effort to overcome this problem we destroyed the carbohydrate on ricin A chain by treating it with a mixture of sodium metaperiodate and sodium cyanoborohydride and then linked the "deglycosylated" A chain to monoclonal anti-Thy 1.1 antibody. The deglycosylation procedure did not affect the ability of the A chain component of the immunotoxin to inhibit protein synthesis in a cell-free system or the capacity of the immunotoxin to inhibit protein synthesis in Thy-1.1 positive lymphoma cells in vitro. Immunotoxins prepared with deglycosylated A chain were cleared from the bloodstream of mice more slowly than native ricin A chain immunotoxins. The difference in the blood clearance rates of the two immunotoxins could be accounted for by a decreased entrapment of the deglycosylated ricin A chain immunotoxin by the liver. Both immunotoxins broke down in vivo with the appearance of free antibody in the bloodstream. The site of cleavage of the immunotoxin was possibly the liver because immunotoxins taken up by it rapidly became unreactive with antiricin but retained reactivity with anti-mouse immunoglobulin G suggesting that dissociation of the A chain from the antibody had occurred. The immunotoxins taken up by the liver were metabolized further and the acid insoluble radioactive metabolites gradually accumulated in the stomach, thyroid, and salivary gland. The deglycosylated ricin A chain immunotoxin should be a more effective antitumor agent in vivo because it is cleared from the blood more slowly and so has greater opportunity to localize within the tumor target.

Suppression of tumorigenicity in human teratocarcinoma cell line PA-1 by introduction of chromosome 4.

Teratocarcinomas are tumors that develop spontaneously in the gonads and usually contain a rapidly dividing, undifferentiated stem cell population. Immature ovarian teratocarcinomas are highly malignant with only 30-60% of patients surviving for 2 years after diagnosis. We have used microcell fusion to introduce individually tagged normal human chromosomes into the PA-1 human teratocarcinoma cell line. Introduction of human chromosome 4 caused a cell morphology in culture and suppressed PA-1 tumorigenicity in nude mice, whereas addition of portions of either chromosome 7 or 12 had no effect on the cell phenotype. The PA-1 cell line regained its tumorigenicity when the tagged chromosome 4 was lost under negative selection. We conclude that there is a putative tumor suppressor gene on human chromosome 4 whose expression interferes with the tumorigenicity of PA-1 cells.

Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages.

A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.

Comparison of the pharmacokinetics and hepatotoxic effects of saporin and ricin A-chain immunotoxins on murine liver parenchymal cells.

Immunotoxins containing the ribosome-inactivating protein, saporin, are very effective antitumor agents but are highly toxic to mice. They induce severe necrotic lesions in the liver parenchyma of the recipients. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity of the saporin immunotoxins was found in the present study to arise from a combination of two effects. First, saporin and saporin immunotoxins were 30- and 6-fold more toxic to primary cultures of mouse liver parenchymal cells than were ricin A-chain and ricin A-chain immunotoxins, respectively. This was despite the fact that the cells bound 4- to 5-fold less saporin or saporin immunotoxins than ricin A-chain or ricin A-chain immunotoxins. The binding of ricin A-chain and its immunotoxin to the cells was mediated through the carbohydrate residues present on the A-chain whereas saporin is not glycosylated and thus must bind to other sites on the cell surface which result in transport of saporin relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half-life (t 1/2 alpha = 1.1 h; t 1/2 beta = 17.1 h) than the ricin A-chain immunotoxin (t 1/2 = 0.52 h; t 1/2 beta = 9.7 h). Analyses using a two-compartment pharmacokinetic model showed that the two immunotoxins broke down in vivo to give free antibody at a similar rate (t 1/2 = 10-12 h) but that the ricin A-chain immunotoxin was eliminated 11 times more rapidly than the saporin immunotoxin by routes other than breakdown. It was calculated that, in mice given a median lethal dose of saporin immunotoxin, the blood levels of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain our previous finding that it had antitumor activity superior to that of a ricin A-chain immunotoxin in mice.

Detection of nuclear anomalies in the colonic epithelium of the mouse.

Colon carcinogens produce a variety of nuclear anomalies including pyknotic, karyorrhectic, and micronucleated cells in the colonic epithelium within a few hr. Two model carcinogens, 1,2-dimethylhydrazine and gamma-rays, have been used to determine appropriate techniques, conditions, and scoring criteria for detecting such nuclear anomalies most efficiently. The results show that a rapid and sensitive assay for nuclear anomalies can be conducted with a variety of preparation techniques. We anticipate that the assay will be useful as a screen for potential colon carcinogens in the diet or elsewhere. The assay as recommended by us requires at least five animals per group and takes about 1 hr to analyze. A single sample at 6 or 24 hr after treatment should detect most carcinogens.

New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts.

ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.

Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo.

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution.

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.

Radiotherapy treatment for isolated loco-regional recurrence of rectosigmoid cancer following definitive surgery: Peter MacCallum Cancer Institute experience, 1981-1990.

PURPOSE: To assess the success of external beam radiation treatment in the management of loco-regional recurrence of rectosigmoid cancer. METHODS AND MATERIALS: A retrospective analysis of 135 patients with locally recurrent rectosigmoid cancer presenting to Peter MacCallum Cancer Institute between January 1981 and December 1990 was undertaken. Patients were treated with three different dose ranges of radiotherapy: 50-60 Gy ("Radical" group), 45 Gy ("High-dose palliative" group), and <45 Gy ("Low-dose palliative" group). Symptomatic response rates and overall survival for each group were determined. RESULTS: Symptomatic response rates of 85, 81, and 56% were achieved in the radical, high-dose palliative, and low-dose palliative groups, respectively. Estimated median survival times were 17.9, 14.8, and 9.1 months for the radical, high-dose palliative, and low-dose palliative groups, respectively.

Optimization of alkylating agent prodrugs derived from phenol and aniline mustards: a new clinical candidate prodrug (ZD2767) for antibody-directed enzyme prodrug therapy (ADEPT).

Sixteen novel potential prodrugs derived from phenol or aniline mustards and their 16 corresponding drugs with ring substitution and/or different alkylating functionalities were designed. The [[[4-]bis(2-bromoethyl)-(1a), [[[4-[bis(2-iodoethyl)-(1b), and [[[4-[(2-chloroethyl)-[2-(mesyloxy)ethyl]amino]phenyl]oxy] carbonyl]-L-glutamic acids (1c), their [[[2- and 3-substituted-4-[bis(2-chloroethyl)amino]phenyl]oxy]carbonyl]-L- glutamic acids (1e-1), and the [[3-substituted-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl]-L- glutamic acids (1o-r) were synthesized. They are bifunctional alkylating agents in which the activating effect of the phenolic hydroxyl or amino function is masked through an oxycarbonyl or a carbamoyl bond to a glutamic acid. These prodrugs were designed to be activated to their corresponding phenol and aniline nitrogen mustard drugs at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in antibody-directed enzyme prodrug therapy (ADEPT). The synthesis of the analogous novel parent drugs (2a-r) is also described. The viability of a colorectal cell line (LoVo) was monitored with the potential prodrugs and the parent drugs. The differential in the cytotoxicity between the potential prodrugs and their corresponding active drugs ranged between 12 and > 195 fold. Compounds 1b-d,f,o exhibited substantial prodrug activity, since a cytotoxicity differential of > 100 was achieved compared to 2b-d,f,o respectively. The ability of the potential prodrugs to act as substrates for CPG2 was determined (kinetic parameters KM and kcat), and the chemical stability was measured for all the compounds. The unsubstituted phenols with different alkylating functionalities (1a-c) proved to have the highest ratio of the substrates kcat:KM. From these studies [[[4-[bis(2-iodoethyl)amino]phenyl]oxy]carbonyl]-L-glutamic acid (1b) emerges as a new ADEPT clinical trial candidate due to its physicochemical and biological characteristics.

New mustard prodrugs for antibody-directed enzyme prodrug therapy: alternatives to the amide link.

Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step approach for the treatment of cancer which seeks to generate a potent cytotoxic agent selectively at a tumor site. In this work described the cytotoxic agent is generated by the action of an enzyme CPG2 on a relatively nontoxic prodrug. The prodrug 1 currently on clinical trial is a benzamide and is cleaved by CPG2 to a benzoic acid mustard drug 1a. We have synthesized a series of new prodrugs 3-8 where the benzamide link has been replaced by, for example, carbamate or ureido. Some of these alternative links have been shown to be good substrates for CPG2 and therefore new candidates for ADEPT. The active drugs 3a and 4a derived from the best of these prodrugs are potent cytotoxic agents (1-2 microM) some 100 times more than 1a. The prodrugs 3 and 4 are some 100-200-fold less cytotoxic, in a proliferating cell assay, than their corresponding active drugs 3a and 4a.