Conjugation of Methotrexate-Amino Derivatives to Macromolecules through Carboxylate Moieties Is Superior Over Conventional Linkage to Amino Residues: Chemical, Cell-Free and In Vitro Characterizations

Abstract

In this study, we examined the possibility of introducing methotrexate (MTX) to the carboxylate rather than to the ε-amino side chains of proteins. We found that MTX-amino compounds covalently linked to the carboxylate moieties of macromolecules, undergo unusual peptide-bond cleavage, with the release of the MTX amino derivatives from the conjugates. This event takes place at an accelerated rate under acidic conditions, and at a slower rate at physiological pH values. The glutamate portion of MTX is responsible for this behavior, with little or no contribution of the p-aminobenzoate-pteridine ring that is linked to the α-amino side chain of the glutamate. Carboxylate-linked Fmoc-Glu-γ-CONH-(CH2)6-NH2 undergoes hydrolysis in a nearly indistinguishable fashion. A free α carboxylate moiety is essential for this effect. Carboxylate linked Fmoc-glutamic-amide-γ-CONH-(CH2)6-NH2 undergoes no hydrolysis under acidic conditions. Based on these findings, we engineered a cysteine specific MTX containing reagent. Its linkage to bovine serum albumin (BSA) yielded a conjugate with profound antiproliferative efficacy in a MTX-sensitive glioma cell line. In conclusion, carboxylate linked MTX-amino derivatives in particular, and carboxylate linked R-α-GLU-γ amino compounds in general are equipped with'built-in chemical machinery' that releases them under mild acidic conditions.

Fig 1. Decrease in the absorbance of PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX with time during dialysis against 1mM HCl.

PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX and PEG₄₀-CONH-(CH₂)₆-NHCO-MTX (0.2 μmole/ml of each) were dialyzed against one liter of 1mM HCl (pH 3.0). At the indicated time points, 30 μl aliquots were withdrawn from the dialysis tube, diluted 20 folds and their absorbance at 305 nm was monitored. Data are presented as mean±STDEV (n = 3).

Fig 2. MTX and Fmoc-L-glutamic acid structures.

Illustrations of MTX (A) and Fmoc-L-glutamic acid (B).

Fig 3. Dose response inhibition of dihydrofolate-reductase by MTX-hexamethylenamine, released from PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX at acidic conditions.

The indicated concentrations of MTX and its derivatives were analyzed for their potencies to inhibit the reduction of dihydrofolate to tetrahydrofolate by DHFR. In this process NADPH is oxidized to NADP, and the extent of this oxidation is monitored by the decrease in absorbance at 340 nm (experimental section).

Fig 4. Rate of hydrolysis of MTX-CONH-(CH₂)₆-NH₂ from PEG₄₀-CONH-(CH₂)₅-CONH-(CH₂)₆-NHCO-MTX at acidic and neutral pH values.

Rate of hydrolysis was determined by the decrease in the absorbance of the PEG₄₀-MTX conjugate at 305nm as a function of time during dialysis against 1mM HCl (pH 3.0) or 50 mM Hepes buffer (pH 7.4). The experimental protocol described in the experimental part was applied. Data are presented as mean±STDEV (n = 4).

Fig 5. Engineering a cysteine-specific MTX-containing reagent- Rates of hydrolysis following its conjugation to PEG₂₀-SH or to HSA at acidic pH.

PEG₂₀-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX and HSA-S-MAL-(CH₂)₂-CONH(CH₂)₆-NHCO-MTX were dialyzed against 1L of 1mM HCl and the decrease in the absorbance at 305nm in the dialysis tube was recorded applying the protocol as described in the legend for Fig 1. Data are presented as mean±STDEV (n = 4).

Fig 6. Rate of hydrolysis of F-moc-L-glutamic-hexamethylenamine linked to PEG₃₀-----COOH at acidic and neutral pH values.

The rates of hydrolysis were determined by the decrease in absorbance of the conjugates at 280nm as a function of time during dialysis against 1mM HCL (pH 3.0, Fig 5A and 5C) or PBS (pH 7.4, Fig 6B). Data are presented as mean±STDEV (n = 4).

Fig 7. Enhanced antiproliferative efficacy by hydrolysable BSA-MTX conjugate is CNS-1 glioma cell line.

Dose dependent toxicity experiments were conducted on CNS-1 cells as described in the Methods section. MTX (A), BSA---NH-CO-MTX (B) and BSA-S-MAL-(CH₂)₂-CONH-(CH₂)₆- NHCOMTX (C) were added to the cell culture for 72 hrs before MTT toxicity assay was applied to determine their antiproliferative efficacies. Data are presented as mean±SEM of 3–4 experiments in quadruplicates.

Fig 8. Schematic presentation of the proposed mechanisms involved in the chemical hydrolysis of methotrexate-amino derivatives covalently linked to carboxylate moieties of macromolecules (A) at acidic (B) and neutral (C) pH values.