ABSTRACT(Ϫ)-Deprenyl and structurally related propargylamines increase neuronal survival independently of monoamine oxidase B (MAO-B) inhibition, in part by decreasing apoptosis. We found that deprenyl and two other propargylamines, one of which does not inhibit monoamine oxidase B, increased survival in trophically withdrawn 6-day nerve growth factor (NGF)-and 9-day NGF-differentiated PC-12 cells but not in NGF naive or 3-day NGF-differentiated PC-12 cells. Four days of prior NGF exposure were required for the propargylamine-mediated antiapoptosis. Studies using actinomycin D, cycloheximide, and camptothecin revealed that the maintenance of both transcription and translation, particularly between 2 and 6 h after trophic withdrawal, was required for propargylamine-mediated antiapoptosis. Metabolic labeling of newly synthesized proteins for two-dimensional protein gel autoradiography and scintillation counting showed that the propargylamines either increased or reduced the levels of new synthesis or induced de novo synthesis of a number of different proteins, most notably proteins in the mitochondrial and nuclear subfractions. Western blotting for whole cell or subcellular fraction lysates showed that the timing of new protein synthesis changes or subcellular redistribution of apoptosis-related proteins induced by the propargylamines were appropriate to antiapoptosis. The apoptosisrelated proteins included superoxide dismutases (SOD1 and SOD2), glutathione peroxidase, c-JUN, and glyceraldehyde-3-phosphate dehydrogenase. Most notable were the prevention of apoptotic decreases in BCL-2 levels and increases in mitochondrial BAX levels. In general, (Ϫ)-deprenyl-related propargylamines appear to reduce apoptosis by altering the levels or subcellular localization of proteins that affect mitochondrial membrane permeability, scavenge oxidative radicals, or participate in specific apoptosis signaling pathways. The propargylamine (Ϫ)-deprenyl (DEP) inhibits monoamine oxidase B (MAO-B). DEP was first shown to reduce the death of primate nigrostriatal dopaminergic neurons exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Cohen et al., 1984) and to slow the clinical progress of human Parkinson's disease (Parkinson, 1993). Both actions appeared to depend on MAO-B inhibition. Subsequently, DEP and deprenylrelated propargylamines (DRPs) were demonstrated to reduce neuronal loss independently of MAO-B inhibition in a variety of experimental models including cortical catecholaminergic neurons exposed to N-(-2-chloroethyl)-Nethyl-2-bromobenzylamine, murine or primate substantia nigra dopaminergic neurons exposed to MPTP, rat facial motoneurons after axotomy, dopaminergic cells treated with the 1-methyl-4-phenylpyridinium ion (MPP ϩ ) or nitric oxide, and hippocampal neurons exposed to kainate (see for details and references). The MAO-B-independent increases in neuronal survival by DRPs were shown to involve decreased apoptosis in a number of the above models (e.g., kainate-exposed hippocampal neurons, nitric oxide, o...

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.

Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)2 fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)2 fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)2 compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)2-antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)2antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)2AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)2 fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)2AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)2AELG (0.005 less than P less than 0.01).

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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.