Mighty morphin' materials management.
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Cost Control↗
Biomedical subjects
J Kralovec
Publications and source records attributed to J Kralovec.
A simple procedure for sulfation and 35S radiolabelling of paralytic shellfish poisoning (PSP) gonyautoxins.
A method is described to sulfate PSP toxins at various positions in the molecule and to prepare 35S labelled compounds using H2(35)SO4 in the presence of dicyclohexylcarbodiimide (DCC). The 11-sulfates of saxitoxin and neosaxitoxin, known as gonyautoxins, are often the most abundant of the PSP toxins in algae and contaminated shellfish. Receptor site binding and antibody assays based on these analogues should, therefore, better reflect toxicity than those in which saxitoxin is used. Although the specific activity of 35S-gonyautoxins is lower than that of commercially available 3H-saxitoxin, the label is strongly bound and is not lost through proton exchange with water as occurs with tritiated saxitoxin. The labelling procedure is rapid, inexpensive and can be done on a small scale. Sulfate can be removed from the 11-position of GTX's in methanolic-HCl and from the 21-position by mild acid hydrolysis and H2(35)SO4 added in 5-10-fold excess. Addition or exchange occurs rapidly on mixing DCC in dimethylformamide with dry toxin and sulfate. Reaction conditions were optimized and reaction products identified by capillary electrophoresis, autoradiography and ionspray mass spectrometry. Together with methods for selective removal of sulfate, the sulfation reaction provides an additional way to prepare some of the naturally occurring derivatives of saxitoxin, many of which are sulfates.
Animals↗
Inhibition of Epstein-Barr-virus-transformed human chronic lymphocytic leukaemic B cells with monoclonal-antibody-adriamycin (doxorubicin) conjugates.
The anthracyclin antineoplastic agent doxorubicin (Adriamycin) was linked by four different methods of linkage to DalB02, an IgG1 kappa murine monoclonal antibody (mAb) against surface-associated antigens on human chronic lymphocytic leukaemia (CLL) B cells. All the four conjugates fully retained the immunoreactivity of the parent DalB02. When the inhibitory effect of these conjugates was evaluated in vitro against the target D10-1 cells (a clone derived from an Epstein-Barr-virus-transformed human CLL B cell line that binds DalB02) it was observed that one conjugate was more potent than the free drug but the others were not. When 131I-labelled unmodified DalB02 and the 131I-labelled DalB02-containing conjugate that was found to be potent were injected i.v. into nude mice bearing a subcutaneous D10-1 xenograft, the percentages of the injected dose (%ID) of both 131I-DalB02 and the 131I-DalB02-containing conjugate that localized in the tumour were much higher than the %ID of the respective preparations that localized in normal tissues of D10-1-xenografted mice. The systemic toxicity of the conjugate was less than that of the free drug. At an equitoxic dose level, this conjugate was a more effective inhibitor of established D10-1 xenografts than the free drug.
Animals↗
Inhibition of human renal cancer by monoclonal-anti-body-linked methotrexate in an ascites tumor model.
The monoclonal antibody DAL K29 against a cell-surface antigen associated with a human renal cell carcinoma was covalently linked to the antifolate methotrexate with full retention of antibody activity and partial retention of drug activity. Using an ascites tumor model, developed after intraperitoneal (i.p.) inoculation of 5 x 10(6) cells of the human kidney cancer line Caki-1 per pristane-primed nude mouse, we showed that the methotrexate-Dal-K29 conjugate was a more potent tumor inhibitor (P less than 0.0005) of human renal cell carcinoma (which is resistant to currently available modalities including chemotherapy) than the drug or mAb alone, the drug linked to an isotype-matched nontumor-specific IgG or a mixture of the drug and the mAb. Only the conjugate could produce tumor-free survival in a proportion of the mice during the period of observation (i.e. 150 days after tumor inoculation).
Animals↗
Targeting of methotrexate-containing liposomes with a monoclonal antibody against human renal cancer.
The potential of monoclonal antibody-linked small unilamellar vesicles containing methotrexate [(MTX)SUVs] in cancer chemotherapy was investigated. (MTX)SUVs prepared by probe sonication [50 +/- 20 (SD) nm in diameter] were linked covalently to Dal K29 (an IgG1 monoclonal antibody against human renal cancer), normal mouse IgG, or a nonspecific mouse myeloma IgG1. After incubation with a human kidney cancer cell line, CaKi-1, for 2 h, Dal K29-linked (MTX)SUVs showed 6 and 8 times more binding to CaKi-1 cells than nonspecific mouse myeloma IgG1-linked (MTX)SUV or (MTX)SUVs unlinked. After incubation with Dal K29-linked ([3H]MTX)SUVs, a higher amount of radioactivity was associated with CaKi-1 cells at 37 degrees C than at 4 degrees C. Membrane immunofluorescence revealed aggregation and capping of Dal K29-linked SUVs around CaKi-1 cells after incubation at 37 degrees C for 2 h and endocytosis at 4 and 6 h. Electron microscopic examination confirmed the aggregation of Dal K29-linked SUVs on the surface of CaKi-1 cells and their presence in endocytic vesicles at 4 and 6 h. After incubation with Dal K29-linked gold containing SUVs at 37 degrees C, gold-containing SUVs were seen on the surface as well as inside endocytic vesicle of CaKi-1 cells at 2 and 4 h. A colony inhibition assay showed that Dal K29-linked (MTX)SUVs were 5 and 40 times more potent than Dal K29-MTX and equimolar amounts of free untrapped MTX in inhibiting the growth of the target CaKi-1 cells but were not toxic to a human melanoma line (that did not react with Dal K29).
Antibodies, Monoclonal↗
Synthesis of site-specific methotrexate-IgG conjugates. Comparison of stability and antitumor activity with active-ester-based conjugates.
With a view to increasing drug incorporation without loss of antibody activity, tritium-labeled methotrexate (MTX) was covalently linked to a polyclonal rabbit IgG antibody against bovine serum albumin and a monoclonal mouse IgG antibody against human renal cancer (Dal K20) by a site-specific method based on hydrazone bond formation between MTX hydrazide and the aldehyde groups generated by periodate oxidation of carbohydrate moieties in IgG (which are uncommon in the antigen-binding region). These conjugates were compared with the corresponding non-site-specific MTX-IgG conjugates produced by the N-hydroxysuccinimide active-ester method with regard to synthesis, stability, retention of antibody activity, inhibition of the target enzyme dihydrofolate reductase and antitumor effect. Incorporation levels achieved with the hydrazide method were no greater than with the active-ester method, typically 6-7 mol MTX/mol IgG. Approximately the same dihydrofolate-reductase-inhibitory capacity was observed for MTX bound by either method. Hydrazide conjugates lost bound drug more rapidly than active-ester conjugates on freezing and thawing, on incubation at 37 degrees C and 51 degrees C, and in the presence of serum or rat liver homogenates. Exposure to rat liver homogenates at 37 degrees C, pH 4.6, for 24 h led to the loss of 50%-60% of the bound drug from hydrazide conjugates compared to 20%-30% from the active ester conjugates. Bio-Gel P-2 chromatography of low-molecular-mass fractions, obtained after exposure of each of the conjugates to liver homogenates, revealed the presence of a compound that had the same elution volume and RF on thin-layer chromatography as free MTX. Enzyme-linked immunosorbent assay showed loss of antibody activity of both types of conjugates at 51 degrees C and on freezing and thawing. In a clonogenic assay, the active-ester conjugate of Dal K20 appeared to be equally effective or slightly better as a tumor inhibitor than the corresponding hydrazide conjugate. The hydrazide method may be useful in linking MTX to those monoclonal antibodies that tend to denature when subjected to the active-ester method of linkage.
Antibodies, Monoclonal↗
Inhibition of human renal cancer by methotrexate linked to a monoclonal antibody.
The monoclonal antibody DAL K29 against a human renal cell carcinoma associated cell surface antigen was covalently linked to the antifolate methotrexate with full retention of antibody reactivity and partial retention of drug activity. In a colony inhibition assay, antibody-conjugated methotrexate was 400% more potent in inhibiting the growth of the human kidney cancer line Caki-1 than equimolar amounts of the free drug. Comparable amounts of the antifolate linked to normal mouse IgG did not inhibit the growth of Caki-1 cells. Furthermore, the methotrexate-DAL K29 conjugate had no effect on the two nontarget human cell lines tested, melanoma M21 and B cell leukemia D10-1 cells, even when the conjugate contained amounts of methotrexate equivalent to the 50% inhibitory concentration of the free drug for the nontarget cell lines or an amount equivalent to the 50% inhibitory concentration of the conjugated drug for Caki-1 cells.
Antibodies, Monoclonal↗
Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities.
In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.
Antibodies, Monoclonal↗
Synthesis and testing of antibody-antifolate conjugates for drug targeting.
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Animals↗