Biomedical subjects

D C Blakey

Publications and source records attributed to D C Blakey.

31 records · Page 2Linked to original sources

PubMed1987

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.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

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.

Acetylglucosaminidase↗

PubMed1987

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)

Abrin↗

PubMed1986

Factors responsible for the formation of different N-alkylated porphyrins in rat liver microsomal systems exposed to norethindrone. The role of 3 alpha-hydroxysteroid dehydrogenase.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

Destruction of cytochrome P-450 and formation of green pigments by contraceptive steroids in rat hepatocyte suspensions.

The contraceptive steroid norethindrone caused a rapid time and dose-dependent loss of cytochrome P-450 from rat hepatocytes in suspension cultures. Up to 30% of this cytochrome was lost in the first 5 min of incubation; longer incubations resulted in little further loss even though not all the steroid was metabolised and the cells remained viable. Such cultures were used to investigate the formation of N-alkylated porphyrins (green pigments) which could be extracted from cell incubation mixtures following exposure to norethindrone and separation by HPLC or TLC. The number of N-alkylated porphyrins formed was dependent both on the time of incubation and the concentration of steroid. After 1 min, 1 major green pigment (GP1) was resolved using either high (0.3 mM) or low (0.03 mM) norethindrone concentrations. With longer incubation times (60 min), at high steroid concentrations, only one additional polar adduct (GP2) was formed. At lower steroid levels, 3 more polar components (GP2, 3 and 4) were seen. As judged by HPLC or TLC, GP1 corresponds to the pigment formed in microsomal preparations incubated with norethindrone in vitro, while GP2, 3 and 4 correspond to the pigments extracted from the livers of rats administered this steroid in vivo. Pretreatment of rats with either phenobarbitone or 3-methylcholanthrene induced cytochrome P-450s. Relative to controls, phenobarbitone pretreatment also resulted in a greater accumulation of green pigments in hepatocytes incubated with norethindrone, the more polar forms of green pigments (GP3 and 4), showing a disproportionate increase in concentration. The mixed function oxidase inhibitor SKF 525-A or high concentrations of steroid not containing an ethynyl function, e.g. norethandrolone, when added to cell cultures containing norethindrone, preferentially inhibited the formation of GP3 and 4. When purified green pigments were added to cell incubation mixtures in the absence of norethindrone, preferentially inhibited the formation of GP3 and 4. When purified green pigments were added to cell incubation mixtures in the absence of norethindrone, no interconversion of one form to another could be demonstrated. The results suggest that the more polar norethindrone-protoporphyrin IX adducts (GP2, 3 and 4) arise as a result of metabolic modification of norethindrone rather than the protoporphyrin IX moiety, either prior to or after activation of the ethynyl function. The formation of several green pigment components in hepatocyte suspensions was not unique to norethindrone but occurred with a number of other 17-ethynyl-substituted steroids.

Animals↗

PubMed1986

Purification of ricin A1, A2, and B chains and characterization of their toxicity.

This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains.

Ammonium Chloride↗

PubMed1986

Immunotoxins.

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Bone Marrow Transplantation↗

PubMed1986

Effect of chemical deglycosylation on the in vivo fate of ricin A-chain.

Chemical deglycosylation of ricin A-chain virtually eliminated its entrapment by the liver and delayed its clearance from the bloodstream of mice. Liver entrapment of native ricin A-chain occurred to approximately equal extents in the parenchymal and non-parenchymal cell fractions of the liver. The chemical deglycosylation procedure reduced uptake of the A-chain by both cell fractions.

Animals↗

PubMed1985

Unscheduled DNA synthesis caused by norethindrone and related contraceptive steroids in short-term male rat hepatocyte cultures.

The genotoxic potential of oral contraceptive steroids such as norethindrone was investigated in short-term rat hepatocyte cultures by measurement of unscheduled DNA synthesis. Norethindrone caused a small dose-dependent increase in unscheduled DNA synthesis in male rat hepatocytes as judged by the incorporation of [methyl-3H]thymidine into DNA. This was assessed either by liquid scintillation counting following isolation of DNA or by autoradiography. No increase in unscheduled DNA synthesis could be detected in female rat hepatocytes treated with norethindrone. Pre-treatment of male rats with phenobarbitone prior to hepatocyte preparation decreased the norethindrone mediated unscheduled DNA synthesis relative to control hepatocyte cultures while 3-methylcholanthrene pre-treatment had little effect. Unscheduled DNA synthesis in norethindrone treated control male rat hepatocytes was reduced by the mixed function oxidase inhibitors SKF 525A or metyrapone. In 24- or 52-hour-old hepatocyte cultures in which the cytochrome P-450 content was lower than in freshly prepared cells, or in a hepatocyte-derived cell line lacking cytochrome P-450, unscheduled DNA synthesis due to norethindrone was either decreased or abolished. Structure activity studies showed that only steroids containing a 17 alpha-ethynyl substituent caused an increase in unscheduled DNA synthesis.

Animals↗

PubMedDate not supplied

Antitumor effects of an antibody-carboxypeptidase G2 conjugate in combination with a benzoic acid mustard prodrug.

The F(ab')2 fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme prodrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl)amino]-benzoyl-L-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.

Animals↗

PubMedDate not supplied

Further evidence for different isoenzymic forms of a cell surface protease, guanidinobenzoatase, associated with tumours.

Normal colonic epithelial cells possess a cell surface protease referred to as guanidinobenzoatase (GB) and a corresponding cytoplasmic protein inhibitor of GB. Colonic carcinoma cells possess two isoenzymic forms of GB, the normal and the carcinoma specific form, each of which is recognised by the corresponding inhibitors present in the cytoplasm of colonic carcinoma cells. An affinity-purified inhibitor preparation obtained from the cytoplasm of cultured colonic carcinoma cells inhibited these two colonic carcinoma isoenzymic forms of GB but not the GB associated with other forms of tumour. The data suggest that each cell type possessing isoenzymic forms of cell surface GB also possesses the corresponding cell-specific inhibitors of GB.

Carboxylic Ester Hydrolases↗

PubMedDate not supplied

The interactions of protein inhibitors with tumour proteases studied in solution and immobilised on cell surfaces in frozen sections.

The cell surface protease guanidinobenzoatase (GB) has been purified from human colonic and lung carcinoma tissue by an affinity step involving the binding of the enzyme either onto fibrin fibrils or onto agmatine-sepharose. The inhibitor protein (I) was extracted from the cytoplasm of tumour cells and isolated by an affinity step involving the binding of I to GB on the surface of cultured carcinoma cells. The interaction of GB and I in solution was followed by kinetic studies employing the release of the fluorescent 4-methylumbelliferone (MU) from the synthetic substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUGB). The interaction of soluble I with membrane bound GB was followed by using the yellow fluorescent probe 9-aminoacridine (9AA) which binds to active GB but not to GB-I. The results of these studies demonstrated the presence of isoenzymic froms of GB which were recognized specifically by their appropriate isoinhibitor, isolated from the appropriate cell type. This high degree of selectivity suggests a cell specific regulatory role for the inhibitors and the possibility that they might be used for the delivery of cytotoxic molecules to the surface of specific types of tumour cells.

Carboxylic Ester Hydrolases↗