Application of Gene-Directed Enzyme Prodrug Therapy in Cancer Treatment

Gene-directed enzyme prodrug therapy (GDEPT) is an advanced cancer therapy that has potential use against localized and metastasized cancer. This strategy aims to improve the limitations of chemotherapy and existing cancer treatments by specific gene delivery, which allows the conversion of systemically administered nontoxic prodrugs to active chemotherapeutic drugs inside the target tumor cells, thereby resulting in a significant therapeutic index by introducing high concentrations of cytotoxic compounds to the tumor cells while limiting the systemic toxicity. The main attraction of GDEPT is by expanding the toxicity to adjacent non-expressing target cancer cells through local and distal bystander effects, leading to tumor regression. This review focused on the application of the six main GDEPT systems for treating cancer, including herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV), cytosine deaminase (CD) from bacteria or yeast with 5-fluorocytosine (5-FC), E. coli nitroreductase (NfsB) with 5-(aziridin-1-yl)-2,4- initrobenzamide (CB1954), hepatic cytochrome P4l50 (CYP450) with cyclophosphamide (CPA), purine nucleoside phosphorylase (PNP) from E. coli with 6-methylpurine deoxyriboside (MEP), and bacterial carboxypeptidase G2 (CPG2) with 4-[(2-chloroethyl)(2-mesloxyethyl)amino] benzoyl-L-glutamic acid (CMDA). In each system, the mechanism of action, clinical trials for the past decades, limitations, and areas that need improvement are discussed.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

Methotrexate overdose in clinical practice

Background: A folic-acid antagonist, methotrexate, is one of the most commonly prescribed drugs with its expanding use in clinical practice. The drug requires regular monitoring given its wide range of adverse effects including bone marrow suppression, hepatic or renal dysfunction, gastrointestinal distress, mucocutaneous damage, and neurotoxicity. The toxicity usually occurs rapidly and leads to severe neutropenia, sepsis, and advanced renal failure that are difficult to manage. Methods: This review is an update for the clinicians to understand the pharmacology, clinical features, laboratory evaluation, and treatment of patients with methotrexate overdose. High-quality literature of the past six decades was collected and reviewed in this article. Several landmark articles were reviewed using PubMed, EMBASE Ovid, and the Cochrane Library, that have important implications in current clinical practice. Results: Methotrexate overdose has complex toxicokinetic and produces myriad clinical features mimicking conditions of lesser severity. Organ dysfunction related to bone marrow, kidney or central nervous system is lifethreatening. The management should focus on high-quality supportive care, antidotal therapy (folinic acid and carboxypeptidase- G2) and plasma alkalization. Conclusion: In accordance with the dictum “prevention is better than cure”, the author emphasizes on the role of patient education, regular clinical observation, and laboratory monitoring for prompt recognition and diagnosis of methotrexate overdosing at the earliest stage.