*Article* **Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells**

**Itzik Cooper 1,2,3,\* , Michal Schnaider-Beeri 1,2,4, Mati Fridkin <sup>5</sup> and Yoram Shechter <sup>6</sup>**


**Abstract:** A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTXinhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells.

**Keywords:** cancer; albumin; disulfide; glioma; prodrug; conjugate

## **1. Introduction**

Methotrexate (MTX) is an antiproliferative and immunosuppressive agent widely and effectively used against a broad spectrum of diseases, primarily cancerous tumors but also certain autoimmune diseases such as psoriasis and rheumatoid arthritis [1]. It facilitates its antiproliferative effects by inhibiting dihydrofolate-reductase (DHFR), a ubiquitous enzyme that catalyzes the reduction of folate to dihydrofolate (DHF) and then to tetrahydrofolate. The latter is a cofactor for de novo synthesis of thymidylate, purine, and glycine [2].

As with most antimetabolites, MTX is only partially selective for tumor cells and is toxic to all rapidly dividing normal cells, such as those of the intestinal epithelium and bone marrow [3]. Although relatively less toxic compared with other therapeutic agents, it is not devoid of side effects. In addition, a variety of tumor cells develop resistance to MTX upon treatment with agents, affecting several mechanistic pathways, including impaired transport of MTX into cells [4,5]. Solving these deficiencies and increasing MTX selectivity toward inflamed, diseased, or malignant tissues is therefore of primary clinical significance.

One of the approaches undertaken in recent years as an attempt to increase selectivity of chemotherapeutic agents was to link them covalently to macromolecules such as albumin.

**Citation:** Cooper, I.; Schnaider-Beeri, M.; Fridkin, M.; Shechter, Y. Albumin–Methotrexate Prodrug Analogues That Undergo Intracellular Reactivation Following Entrance into Cancerous Glioma Cells. *Pharmaceutics* **2022**, *14*, 71. https://doi.org/10.3390/ pharmaceutics14010071

Academic Editor: Katona Gábor

Received: 30 November 2021 Accepted: 23 December 2021 Published: 28 December 2021

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Extravasation of albumin is upregulated about six-fold in inflamed, diseased, and malignant tissues [6,7]. If the covalently-linked MTX has low antiproliferative activity, this would create a rather profound clinical advantage, provided that the conjugate undergoes efficient intracellular reactivation following entrance into tumorigenic cells. The amide bond of MTX linked to the ε-amino group of lysine is chemically and enzymatically stable [8], and reactivation appears to be dependent on extensive intracellular proteolysis, as only MTX linked to short peptide fragments through its γ-carboxylate moiety regain significant antiproliferative efficacy (this study). Similarly, binding MTX to other macromolecules, such as anti-TNFα antibodies, might be clinically beneficial in autoimmune and inflammatory diseases such as arthritis, for example, where MTX is administrated at low dosages separately from anti-TNFα antibodies. Generating such an antibody–MTX conjugate may reduce side effects and improve efficacy as well as provide better treatment tolerance.

We thus initially searched for a chemical procedure to covalently link MTX to aminocontaining molecules through the γ-carboxylate moiety of MTX. The linkage of such molecules to the α-carboxylate of MTX was documented to yield inactive analogues [9,10]. Secondly, we examined the possibility of introducing MTX to albumin through spacers containing disulfide bonds. Malignant tissues have elevated de novo synthesis of glutathione [11–13], and we wondered whether this would lead to increased selectivity and enhanced reduction-dependent release of active MTX in tumorigenic cells.

In this study, we describe our synthetic procedures for obtaining a variety of BSA–MTX conjugates, their cell-free DHFR-inhibitory potencies in the absence and in the presence of glutathione or cysteine, and their antiproliferative efficacies in several lines of cancerous cells relative to that of methotrexate. Two glioma cell lines are investigated here, for the first time, in the context of albumin conjugates to align with other research in our lab, in which new compounds and methods that enable blood–brain barrier (BBB) opening, and thus, the entrance of large therapeutic molecules such as albumin are being developed [14–18].

#### **2. Experimental Procedures**

#### *2.1. Materials*

Methotrexate, bovine serum albumin, cystamine dihydrochloride, hexamethyl diaminedihydrochloride, dihydrofolate (DHF), *N*,*N*0dicyclohexylcarbodiimide (DCC), β-nicotinamide adenosine dinucleotide 20 phosphate (NADPH), reduced glutathione (GSH) oxidized glutathione (GSSG), and L-cysteine were purchased from Sigma (St. Louis, MO, USA). Methotrexate linked to a peptide (MTX–AYGRKKRRQRRR) was synthesized by the manual conventional solid-phase synthesis. An Fmoc (N-9-flurenylmethoxy carbonyl) strategy was employed through the peptide chain assembly. All other materials used in this study were of analytical grade.

#### *2.2. Synthesis of MTX–Anhydride*

Methotrexate (45.4 mg, 100 µM) dissolved in 0.7 mL dimethylformamide (DMF) and 95 µL from a solution of 1 M DCC in DMF (95 µM) was added. The reaction was carried out for 3 h at 25 ◦C. Dicyclohexylurea was filtered out. The MTX–anhydride formed was kept at 4 ◦C until used.
