Recent advances in 5-fluorouracil: Co-crystal synthesis, prodrug derivatives and modulation strategies to enhance anti-cancer activity

Aqsa Aslam; Farhat Jubeen; Misbah Sultan; Muhmmad K. Saleemi; Munawar Iqbal; Naveed Ahmad; Wissem Mnif; Kald Mohammed A. Algarni; Farooq Anwar

doi:10.25259/AJC_247_2024

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Review Article

ARTICLE IN PRESS

doi:

10.25259/AJC_247_2024

Recent advances in 5-fluorouracil: Co-crystal synthesis, prodrug derivatives and modulation strategies to enhance anti-cancer activity

Aqsa Aslam^a, Farhat Jubeen^a,*, Misbah Sultan^b, Muhmmad K. Saleemi^c, Munawar Iqbal^d,b,*, Naveed Ahmad^e,*, Wissem Mnif^f, Kald Mohammed A. Algarni^g, Farooq Anwar^h,i

aDepartment of Chemistry, Government College Women University, Arfa Kareem Road Faisalabad, Pakistan

bSchool of Chemistry, University of Punjab, Lahore, Pakistan

cDepartment of Pathology, University of Agriculture Faisalabad, Pakistan

dRenewable Energy and Environmental Technology Center, University of Tabuk, Tabuk, Saudi Arabia

eDepartment of Chemistry, Division of Science and Technology, University of Education, Lahore, Pakistan

fDepartment of Chemistry, College of Science, University of Bisha, Bisha, Saudi Arabia

gCollege of Medicine, University of Bisha, Bisha, Saudi Arabia

hInstitute of Chemistry, University of Sargodha, Sargodha, Pakistan

iFaculty of Health Sciences, Shinawatra University, Bang Toey, Pathum Thani, Thailand

* Corresponding authors: E-mail addresses: mnavedahmad@yahoo.com (N. Ahmad), dr.farhatjubeen@gcwuf.edu.pk (F. Jubeen)

Received: 2024-12-11, Accepted: 2025-11-20, Epub ahead of print: 2026-03-09,

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Abstract

5-Fluorouracil (5-FU), a fluoropyrimidine analog of uracil, is an extensively employed chemotherapy drug to treat many cancers, including solid malignancies, melanoma, colorectal cancer, pancreatic cancer, and gastric cancer. Its mechanism of action involves disrupting mRNA translation and DNA synthesis. However, the effectiveness of 5-FU is limited by several factors, including its rapid breakdown into inactive forms, loss of site selectivity, and fast elimination from the body when used as a standalone therapy. To address these challenges and improve its therapeutic potential, researchers have explored various strategies over time. This review paper presents a comprehensive overview of various strategies aimed at enhancing the therapeutic efficacy of 5-FU. It covers the development of co-crystals, prodrugs, modulation approaches with co-administration, and derivatization techniques. Additionally, the review provides valuable insights into different carrier systems utilized for the targeted delivery of 5-FU, offering potential advancements in cancer treatment. By evaluating the rationale, advantages, and challenges associated with each approach, this paper highlights the ongoing efforts to optimize the use of 5-FU in cancer treatment.

Keywords

5-Fluorouracil

Modulation strategies

Nanoparticles/drug carriers

Pharmaceutical co-crystals

Prodrugs

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1. Introduction

Cancer remains a major, formidable global disease, imposing a substantial burden on both individuals and societies. In the quest for effective treatments, numerous chemotherapeutic drugs have been employed to combat this devastating illness. Among them, 5-Fluorouracil (5-FU) plays a crucial role; it is a uracil analog in which fluorine replaces hydrogen atoms at the C-5 position. First synthesized in 1957, 5-FU stands as one of the earliest synthetic anti-cancer medications [1]. Nucleoside analogs, including 5-FU, are key therapeutic agents in the development of antiviral and anti-cancer treatment, predominantly used in the treatment of various solid malignant tumors, with single-agent response rates ranging from 10–30%. When employed topically, 5-FU has proven effective in the treatment of superficial basal cell carcinomas. Although, 5-FU can dysfunction DNA and RNA synthesis to exhibit its anti-cancer potential, though there are several pivotal challenges in its clinical applications; i) non- target specificity of 5-FU as well as swift DNA repair mechanisms can result in development of resistance against the drug [2] ii) narrow therapeutic index inducing toxicities like myelosuppression, inflammation of mucosa etc. [3], iii) short biological half-life is critical application and iv) challenge of poor physicochemical attributes as well as side-effects like nausea, alopecia, and others [4].

Numerous structural modifications of 5-FU have been made to address these drawbacks and to improve the physicochemical, pharmacological, and pharmacokinetic properties [5]. One prominent strategy that has gained substantial attention is co-crystallization, where 5-FU is combined with other biologically active coformers. This approach aims to enhance drug stability, solubility, and bioavailability, ultimately improving its therapeutic efficacy [6]. A notable example of co-crystallization is Entresto, a heart failure medication developed by Novartis, which combines valsartan and sacubitril in a 1:1 ratio. In 2015, the FDA approved this approach, highlighting the promising potential of co-crystal drugs [7]. Furthermore, extensive research on prodrug derivatives of 5-FU has been utilized to develop numerous approved compounds of medicinal importance in clinical trials. These prodrugs achieved reduced toxicity by evading certain degradation pathways (with the prodrug not acting as a substrate for degradation by enzymes) or by selectively delivering the active agent to the tumor site (prodrugs that release the active component exclusively within tumor cells).

Modulation strategies for 5-FU involve diverse approaches aimed at enhancing the drug’s effectiveness while mitigating associated toxicities. The coordinated administration of 5-FU with specific modulators or combination therapies can enhance its anti-tumor activity, either by targeting multiple signaling pathways or by synergistic effects [8]. Furthermore, advanced drug delivery systems, including microspheres, viruses, liposomes, and nanoparticles, offer a transformative solution. Utilization of these drug carriers allows for controlled release, targeted delivery, and improved intracellular uptake of 5-FU, thereby enhancing its therapeutic index [9]. Within the realm of optimization strategies for 5-FU, notable modifications are often implemented at the N₁ or N₃ position. These alterations commonly involve the integration of pharmacologically active compounds possessing anti-cancer activity, resulting in what is referred to as a “hybrid molecule.” Additionally, functionalization with different chemical groups, termed “conjugates,” is another prevalent approach. A key advantage of incorporating alkylated metabolites is the extension of 5-FU’s plasma half-life. However, it is significant to remember that these metabolites are not directly responsible for the observed increase in cytotoxicity in tumor tissues. Instead, the enhanced cytotoxic effects are attributed to other mechanisms of action, making these modifications a promising avenue for refining the 5-FU therapeutic potential.

This review article provides a comprehensive and insightful analysis of the innovative strategies employed to optimize the 5-FU efficacy for the treatment of cancer. Delving into the rationale, advantages, and recent breakthroughs, we explore four key areas: co-crystallization, prodrug derivatives, co-administration with other active agents, and advanced drug delivery systems. By examining these approaches, the review aims to highlight ongoing efforts to address and overcome the limitations of 5-FU therapy.

2. 5-Fluorouracil

5-FU, initially developed as a systematically designed anti-cancer drug three decades ago, remains extensively employed in the treatment of various prevalent malignancies, including colorectal, breast, and skin cancers. Dushinsky and colleagues made a significant contribution to humanity by successfully synthesizing the antimetabolite anti-cancer 5-fluoro-2, 4-pyrimidinedione for the first time and identified its anti-tumor properties against colorectal cancer treatment in 1957 [1]. The synthesis of 5-FU involves the reaction of uracil with fluorine in the presence of appropriate catalysts, resulting in the substitution of a H-atom with a fluorine at the fifth C–C position, as given in Figure 1 [10]. The same synthetic method was used to produce 5-FU commercially and to use it as an open-chain starting material. 5-FU exerts its anti-cancer effects through different mechanisms. Notably, 5-FU effectiveness extends beyond its direct cytotoxic effects, as it also displays immunomodulatory properties, supporting the capacity of the immune system to identify followed by remove cancer cell lines. Although 5-FU is beneficial, it only generates a minimal response in 15–28% of those who have severe illness, neutropenia, alopecia, and neurotoxic effects. Commonly observed as typical adverse effects of 5-FU therapeutic action are digestive issues, such as gastrointestinal disturbances.

Chemical synthesis of 5-FU from uracil by fluorination.

2.1. 5-FU anti-cancer: Mechanism of action

Although the precise processes by which 5-FU exerts its cytotoxic potential are not yet fully understood, one of its main modes of action is to interfere with the synthesis of mRNA and DNA. 5-FU is undoubtedly the most effective medication approved for cancer treatment, as it shares similarities with uracil, a component of RNA and DNA; it exerts its anti-cancer effects through three distinct mechanisms that interfere with cell proliferation. Firstly, it stops the enzyme thymidylate synthase (TS), thereby halting DNA synthesis and the necessary repairs essential for cell division. Secondly, 5-FU possesses the ability to directly attach to DNA, potentially inducing damage to the nucleotide sequence. Finally, it impacts repair processes and RNA synthesis, disrupting the cellular proteins and ultimately causing cell death.

The anti-cancer mechanism of action of 5-FU is multifaceted and involves its conversion into active metabolites that disrupt crucial processes in cancer cells. Once administered, 5-FU is enzymatically changed into active forms, including 5-fluoro-2’-deoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP), as shown in Figure 2 [2]. FdUMP inhibits TS, an enzyme essential for DNA synthesis, leading to the depletion of thymidine, a vital building block of DNA. Furthermore, FUTP incorporates into RNA instead of Uridine Triphosphate (UTP), impairing RNA function and cellular processes. By inhibiting DNA repair mechanisms and causing DNA damage through incorporation into DNA, 5-FU disrupts cell cycle progression and induces apoptosis, ultimately impeding cancer cell growth and survival. 5-FU induces apoptosis through various mechanisms, including the activation of caspases, which are responsible for executing the cell death program [1]. The prevalence of any of these pathways in human tumors is uncertain and is likely to vary among types of tumors, and with diverse methods and doses of drug administration. Recent research indicates that extended exposure to low doses of 5-FU tends to induce cell death primarily through the TS-directed mechanism, while bolus administration of 5-FU primarily leads to an RNA-mediated process of cell death [11].

Multifaceted anti-cancer mechanism of 5-FU in cancer treatment.

3. Pharmaceutical co-crystallization

Co-crystallization is a versatile technique used in pharmaceutical research to modify and optimize drug properties [12]. It involves the formation of crystalline structures by combining an active pharmaceutical ingredient (API) with coformers [6]. Co-crystals are composed of distinct chemicals in a specific stoichiometric ratio, connected by non-ionic and non-covalent linkages. Unlike other solid-state techniques like salts, polymorphs, solvates, and hydrates, as represented in Figure 3 [12], co-crystallization offers advantages for a wider range of APIs. Pharmaceutical co-crystals have gained attention for their capacity to improve physicochemical properties while maintaining pharmacological activity. These stable co-crystals enhance solubility, dissolution, and compressibility, offering a promising approach for drug formulation.

Leveraging solid-state techniques for enhanced drug development.

3.1. Co-crystal preparation techniques

Several methods of producing co-crystals have been reported so far. The following categories describe the most frequently employed co-crystallization techniques:

3.1.1. Solution-based method

In solution-based approaches, supersaturation of ternary phases (API, coformer, and solvent) is desired, requiring a significant amount of solvent [11]. The choice of solvent influences co-crystallization outcomes and the interaction between components.

a)

Solvent evaporation: Solvent evaporation is a technique for forming co-crystals with desired properties by dissolving the components in a solvent and subsequently removing the solvent. Examples are nebivolol hydrochloride and nicotinamide co-crystals [13].
b)

Anti-solvent method: Anti-solvent crystallization, conducted in continuous processes or semi-batch, is a valuable technique for monitoring co-crystal quality and particle size, as demonstrated in the creation of indomethacin-saccharin co-crystals [1].
c)

Cooling crystallization: Cooling crystallization enables controlled crystal growth and the production of pure, well-defined crystals by gradually cooling a supersaturated solution beyond its solubility limit. It has attained increased attention recently due to its potential for large-scale co-crystal production. Co-crystals of caffeine and p-hydroxybenzoic acid were found through cooling crystallization experiments [14].
d)

Slurry conversion: This method involves the transformation of the solution-mediated phase and gradual dissolution of additional co-crystal components, enabling the formation and growth of co-crystals, e.g., pharmaceutical compound [15].
e)

Freeze drying: Freeze drying, also known as lyophilization, is a technique where a solution is rapidly frozen below the freezing point and then subjected to high vacuum, the frozen solvent is separated through sublimation and leaving behind a low-density amorphous solute/material. Amorphous forms are principally obtained through freeze drying, but crystalline forms can be achieved spontaneously if the glass transition temperature of the amorphous form and the ambient temperature are close to each other or by an additional annealing step. This method has been reported to prepare oxalic acid and theophylline co-crystals [16].
f)

Spray drying: The coformers are dissolved in a solvent and then atomized into fine droplets. In a hot compartment, the solvent is evaporated, and droplets are promptly desiccated, leaving behind solid co-crystals [11]. This method is appropriate for industrial manufacturing, as co-crystals of specific morphologies and particle sizes can be synthesized. Stable inhalable co-crystals are synthesized through this method, like those of itraconazole with suberic acid, which were prepared by rotary evaporation and spray drying.
g)

Ultrasonic method: As the name implies, high-frequency ultrasonic sound waves are used for co-crystallization. The ultrasound waves are applied at specified high temperatures to, coformer solution, which accomplishes nucleation and cavitation for crystal growth. Co-crystals of maleic acid and caffeine, norfloxacin and urea are reported through this method [17,18]. It is environmentally friendly, and co-crystals with potentially improved properties are obtained.
h)

Gas phase diffusion: In the gas or vapor phase diffusion process of co-crystallization, the coformers are in solution and vapor phases, respectively. Once coming in contact, the vapor phase diffuses into the solution phase, leading to supersaturation and subsequent formation of the co-crystals. Organic solvents can serve a catalytic role, and pharmaceutical co-crystals not achievable via the direct solution method can be synthesized through this technique. In drug delivery systems, gas phase diffusion also regulates the release rates of volatile compounds. Co-crystals of diflunisal and pyrimethamine were synthesized through this method to modify the extreme needle shape morphology of the former and to improve the solubility of the latter [19].

3.1.2. Solid-based method

Solid-based methods facilitate the direct interaction between co-crystal components, resulting in the formation of desired solid-state structures without the need for solvents.

a)

Solid-state grinding: Solid-state grinding involves the mechanical grinding of co-crystal components, promoting their intimate contact and enabling the formation of desired solid-state structures, e.g., racemic bis-β-naphthol and benzoquinone co-crystals [20].
b)

Neat grinding: Neat grinding, also called dry grinding, utilizes high vapor pressure and mechanical grinding to create a movable solid surface, enabling the formation of co-crystals through enhanced diffusion coefficients, as demonstrated by aromatic and picric acid hydrocarbon co-crystals [21].
c)

Liquid-assisted grinding: Liquid-assisted grinding technique (also referred to as kneading, solvent-drop, wet cogrinding) improves the yield and crystallinity of co-crystal products by incorporating a small amount of liquid, enhancing molecular diffusion, and acting as a catalyst for co-crystal formation. An example is kaempferol with 5-FU co-crystals [22].
d)

Hot-melt extrusion: Hot-melt extrusion (HME) is evolving as a continuous, single-step, scalable, and industrially viable method for producing co-crystals. HME innovates drug formulation, enabling the creation of co-crystals with enhanced properties and customized drug delivery capabilities, e.g., used for the copolymer of lactide and glycolide [23]. The techniques for the preparation of pharmaceutical co-crystals are depicted in Figure 4 [24].

Techniques for pharmaceutical co-crystal preparation.

4. Co-crystallization of 5-FU

5-FU is a common chemotherapeutic drug with potent anti-cancer properties. However, it faces limitations such as poor solubility, instability, and dose-related toxicities [8]. To address these challenges, researchers have explored the concept of utilizing 5-FU co-crystals. These co-crystals, formed by linking 5-FU with other substances, offer enhanced properties in terms of absorption rate, stability, and solubility. From the perspective of crystal engineering, the 5-FU possesses two H-bonding donors (N–H) and two H-bonding acceptors (C=O) in the structure of its molecule. It serves as a robust H-bonding synthon with adjacent molecules in the assembly of its crystals. Various molecules, including acridine, phenazine, piperazine, urea, thiourea, acetanilide, aspirin, malic acid, benzoic acid, succinic acid, and cinnamic acid have been successfully used to construct co-crystals with 5-FU. Table 1 lists these anti-cancer 5-FU co-crystals. In these co-crystals, 5-FU acts as the API, while the coformer forms a stable crystalline lattice, improving the drug’s performance.

Table 1. Recently synthesized anti-cancer 5-FU co-crystals with bioactive coformers.

S. no	Coformers	Co-crystal preparation method	Cell lines	References
1	Urea, Thiourea, 2,2′-bipyridine, 4,4′bipyridine	Mechanochemical grinding and the normal solution method	Colorectal cancer	[8]
2	Hydroquinone	Liquid-assisted grinding method	Human cervical cancer cell line (HeLa)	[25]
3	Piperazine	Liquid-assisted ball milling and slurry crystallization methods		[36]
4	5-Fluorocytosine	Neat grinding and solvent-drop grinding		[28]
5	Urea, Thiourea, Acetanilide, Aspirin	Solid-state grinding method	HCT 116 colorectal cell lines in vitro	[1]
6	Gentisic acid, 3,4-dihydroxybenzoic acid, 4-aminopyridine	Solvent-assisted grinding	MCF-7, Hela, and Caco-2 cancer cell lines	[30]
7	Succinic acid, cinnamic acid, malic acid, benzoic acid	Solid state grinding slow evaporation solution	HCT-116 Colorectal Cell Lines	[29]
8	Caffeic acid	liquid-assisted grinding combined with a solution vaporization method		[37]
9	Kaempferol	liquid-assisted grinding and slurry conversion crystallization evaporation		[22]
10	Nicotinamide	solvent evaporation technique and liquid phase-assisted grinding	HCT116 cells.	[32]
11	Ferulic acid	Solvent-assisted co-grinding	(MDA-MB-231), osteosarcoma (MG-63), and lung cancer cell lines	[31]
12	Phenylalanine	Water-assisted grinding and slurry method	B16, F10 cells	[33]
13	Glycine, Tryptophane, Leucine, Alanine	Non-grinding solution method	MCF7 breast and SW480 colon cancer cell lines	[11]
14	Cytarabine	solution evaporation and a liquid-assisted grinding method	for promyelocytic leukemia (HL-60), leukemia (K562), and colon cancer cells (HT-29)	[34]
15	Schiff bases (benzylidene-urea, benzylidene-aniline, salicylidene-aniline,salicylidene-phenylhydrazine, para-hydroxy benzylideneaniline)	Grinding method	SW480 colon cancer cells	[6]
	Gallic acid	Solvent-assisted grinding method	4T1- breast cancer cell line of a mouse	[35]

In 2016, Nadzri and his research mates synthesized a series of 5-FU co-crystals containing 2,2′-bipyridine, 4,4′-bipyridine, urea, and thiourea as coformers via the normal solution method and mechanochemical grinding. Results indicated that both methods produced similar products. The development of H-bonds, facilitated by the N-H group of 5-FU, served as the primary synthons in these co-crystals. Hydrogen bonds have been identified as primary interactions in all the structures of its crystals, except in the case of 2,2′-bipyridine, where the position of the N atoms in its molecule prevents the formation of hydrogen bonds. Molecular modeling rationalized co-crystal activities and predicted their binding to colorectal cancer target protein via hydrogen bonds. Calculated energies highlighted the potential anti-cancer effects of co-crystals [8]. Later, another research group presented a comprehensive study to improve the physicochemical character of 5-FU. The researchers employed a liquid-assisted grinding technique to synthesize a novel co-crystal of 5-FU and hydroquinone as the coformer. The crystal packing of the resulting co-crystal, named 5-fluorouracil hydroquinone (5FUHYQ), was elucidated, revealing the presence of intricate N-H⋯O and O-H⋯O classical H-bonds that form a sheet-like molecular arrangement. Anti-cancer efficacy of 5FUHYQ was evaluated against the HeLa cell line, representing human cervical cancer, demonstrating promising improvements in both physicochemical and pharmacokinetic properties compared to the original drug [25].

A novel co-crystal of 5-FU with anthelmintic piperazine was synthesized employing advanced techniques such as slurry crystallization and liquid-aided ball milling. The distinctive interactions of H-bonding between the N-H group of 5-FU and the C=O group of piperazine served as the driving force for co-crystal formation [26]. Building on this, the same group extended their research on these novel solid forms, providing deeper insights through X-ray photoelectron spectroscopy (XPS) investigation. Both 5-FU and piperazine, as pharmaceuticals, hold significant potential across biomedical applications, enhancing anti-cancer effects. In 2014, Da Silva and co-workers initially reported the supramolecular synthesis of a co-crystal involving two APIs: 5-Fluorocytosine and 5-FU using slow evaporation of the solvent (SES). Unfortunately, SES did not yield purity across the entire sample, thereby preventing a comprehensive evaluation of the new co-crystal’s physical properties at that time [27]. In 2019, the same group introduced a solvent-drop grinding (SDG) method for synthesis. Through SDG, a satisfactory yield of the co-crystal was achieved, enabling thermal and spectroscopic evaluations, as well as an assessment of its physical stability. The successful synthesis of this co-crystal provides a pathway for further assessments, such as exploring its potential anti-cancer effects [28].

Jubeen et al. [1] reported the synthesis of 5-FU co-crystals with four safe coformers: acetanilide, aspirin, urea, and thiourea. Two fabrication methods, solution approach and solid-state grinding, were employed, resulting in successful co-crystal formation at room temperature. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) assay results demonstrated the effectiveness of all co-crystals against colorectal cancer cell line HCT 116 [1]. Notably, these co-crystals showcased simplicity, cost-effectiveness, and successful active pharmaceutical ingredient (API) derivatization. In 2020, the same group of researchers further enhanced the anti-cancer effectiveness of 5-FU through co-crystallization using acetone as a solvent. Supramolecular interactions were enhanced with the addition of four coformers (malic acid, succinic acid, benzoic acid, and cinnamic acid). Solid-state grinding was used after a slow evaporation solution approach, yielding colorless pure crystals. The synthesized supramolecular synthons were tested against the hepatocellular carcinoma cell line HCT-116, showing promise for further evaluation in in vivo tests and investigation of their membrane-crossing abilities [29]. In 2022, they reported 5-FU co-crystals with multiple cyclic dimers of the amino acid residues (Leucine, Glycine, Tryptophane, and Alanine) using the co-crystallization technique, with a goal to enhance its anti-cancer efficiency and to eliminate its associated downsides. By using a non-grinding solution approach, co-crystals were made. Using the MTT assay, anti-cancer activity was assessed against the MCF7 and SW480 cancer cell lines. The outcomes indicated that the newly generated co-crystals had higher anti-cancer potential than previously published 5-FU co-crystal and 5-FU alone [11]. A year later, the same group reported anti-cancer 5-FU co-crystals with five different Schiff bases as salicylidene-aniline, benzylidene-phenyl hydrazine, benzylidene-urea, para-hydroxy benzylideneaniline, and benzylidene-aniline, as given in Figure 5 [1, 6, 11, 29], which were synthesized by the condensation method. After the successful synthesis of Schiff bases, co-crystals were prepared with 5-FU by the grinding method. The structures of the synthesized Schiff bases and co-crystals were assessed through suitable analytical techniques, suggesting no side product formation. MTT was used to test the anti-cancer effectiveness of 5-FU derivatives against SW480 colon tumor cells, which demonstrated effective results. To further evaluate the binding mode of TS in many 5-FU complexes, molecular docking was performed [6].

Co-crystallization of 5-FU with (a) thiourea and urea, (b) succinic acid and malic acid, (c) alanine and glycine, and (d) benzylidene-urea and salicylidene-phenylhydrazine.

In 2019, Gautam et al. [30] reported the effects of three crystal forms of 5-FU incorporating 3,4-dihydroxybenzoic acid (5-FUBA), gentisic acid (5-FUGA), and 4-aminopyridine (5-FUPN) using an advanced technique known as mechanochemical solvent-assisted grinding. The biological evaluation of these innovative co-crystals was conducted on various cancer cell types, including MCF-7, HeLa, and Caco-2 cell lines. The results revealed exceptional and potent anti-cancer activity. The discovery of these co-crystals has far-reaching implications for enhancing drug permeability and effectiveness [30].

4.1. Two-component synergy in co-crystals

As two-component synergy can significantly contribute towards improvement of physicochemical properties as well as cytotoxic potential, i.e., drug-drug co-crystals are synthesized to improve cytotoxic potential as well as with pharmacologically active components to improve physicochemical properties. Motivated by earlier literature findings suggesting that the co-crystallization of 5-FU with acids holds greater potential, a novel 5-FU co-crystal with caffeic acid, FL-CF-2H₂O, has been successfully synthesized through a combination of the solution vaporization and liquid-assisted grinding process by a research group in 2020. Rigorous analyses have unequivocally confirmed that the asymmetric part of this newly developed co-crystal contains one molecule of 5-FU, one molecule of CF, and co-solvent water molecules. Promising results emerged from biological evaluation on breast (MDA-MB-231), colon (HCT-116), and lung (A549) cancer cells, prompting further in vivo assessment. This breakthrough not only creates potential for synergistic anti-cancer co-crystals but also introduces a novel crystalline phase for FL, opening doors to exciting future applications in cancer therapeutics. Within the following year, the same group advanced their work by incorporating another acid, ferulic acid, into a 5-FU co-crystal. This enhancement improved binding affinity and anti-tumor activity, marking a significant milestone in narcotic co-crystals and showcasing synergistic anti-tumor effects. To evaluate the efficacy of co-crystal, an in vitro anti-cancer study was conducted on lung (A549), breast (MDA-MB-231), and osteosarcoma (MG-63) cancer cells, yielding highly promising results, supported by encouraging in vivo results [31].

In 2020, a breakthrough was achieved by successfully combining 5-FU and kaempferol to form a co-crystal using liquid-assisted grinding, evaporation crystallization, and slurry conversion, as shown in Figure 6 [22]. This achievement resulted in a co-crystal that not only increases stability but also significantly improves dissolution behavior. The synergistic properties of this co-crystal hold immense potential, paving the way for its use as a highly effective oral formulation. By addressing the limitations of the individual parent drugs, this novel co-crystal represents a major advancement in the field of pharmaceutical research, offering new possibilities for enhanced drug delivery and improved therapeutic outcomes [22].

Proposed H-bonding interactions between 5-FU and kaempferol.

Also, Zang et al. [32] synthesized the co-crystal, namely 5-FU-nicotinamide, featuring a distinctive 2D layer structure formed through hydrogen bonding. This co-crystal was successfully produced at room temperature utilizing a combination of solvent evaporation and liquid process grinding techniques. Extensive evaluations encompassing toxic effects, oil-water partition coefficient, solubility, and in vivo and in vitro anti-tumor impact, and pharmacokinetic characteristics were performed, comparing the co-crystal to the individual 5-FU compound. The results demonstrated that this co-crystal exhibited remarkable anti-cancer activities specifically against the HCT116 cells [32]. Later, Hao reported the 5-FU co-crystal with phenylalanine synthesized by solvent-assisted co-grinding, as shown in Figure 7 [33]. The cytotoxicity assessment against B16 cells was performed using the MTT assay, revealing the superior cytotoxicity of the 5-FU co-crystal compared to individual components or physical mixtures. To comprehensively understand the cytotoxic mechanisms, a non-targeted metabolomics approach was utilized. MS/MS-UHPLC-Q-TOF technology with information dependent acquisition (IDA) data acquisition was employed, enabling detailed analysis of the metabolites [33].

Proposed H-bonding interaction between 5-FU and phenylalanine co-crystal.

To address the challenges of short half-life, poor permeability, and low oral bioavailability associated with the marine anti-cancer drug cytarabine (ARC), and to capitalize on the synergistically enhanced anti-tumor effects, a co-crystal of ARC with 5-FU was synthesized as given in Figure 8 [34]. Through a cyclic hydrogen bonding arrangement and a molar ratio of 1:1, co-crystals of 5-FU and ARC were successfully formed. The synthesis process involved a liquid-assisted grinding method and solution evaporation to create the co-crystals. To evaluate the biological activity, leukemia (K562), promyelocytic leukemia (HL-60), and colon cancer cells (HT-29) were subjected to the assay for sulforhodamine B (SRB). Notably, the co-crystals of 5-FU and ARC lead to reduced IC₅₀ value, indicating enhanced anti-tumor activity against cell proliferation. The experimental investigation, coupled with theoretical co-crystal structure analysis, showcased the enhanced permeability and decreased solubility of the co-crystals [34].

Synthesis of 5-FU and ARC co-crystal with H-bonding interaction.

Another potential contribution towards two-component synergy in co-crystals was reported by Hao and co-workers. Keeping in view the anti-cancer potential of polyphenolic compound, gallic acid, its hydrate co-crystal was synthesized, as 5-FU-GA-H₂O. Co-crystallization was based on van der Waals interaction between the carbonyl of 5-FU and the hydroxyl of gallic acid. A decrease in dissolution rate and solubility was observed with increased membrane permeance and higher cytotoxicity against mouse breast cancer, for the synthesized co-crystal in comparison to 5-FU and a physical mixture [35].

5. 5-FU prodrug derivatives

Prodrugs are chemically modified compounds designed to undergo biotransformation within the body, converting into 5-FU and optimizing drug delivery and efficacy. By strategically modifying the chemical structure of 5-FU, prodrugs aim to improve drug stability, bioavailability, and targeted activation, ultimately enhancing treatment outcomes for cancer patients [36]. Different strategies have been used in the design of 5-FU prodrug derivatives, including the incorporation of specific chemical moieties that are selectively activated by tumor-associated enzymes or physiological conditions unique to cancer cells. These modifications allow for site-specific conversion of the prodrugs into 5-fluorouracil (5-FU) [37]. Various derivatives of 5-FU prodrugs, including TS inhibitors like capecitabine (CAP), ftorafur (FTO; tegafur), furtulon, or doxifluridine, have been formulated to enhance therapeutic efficacy. In different countries, these prodrugs are currently employed in clinical experiments, either as standalone or in combined therapy with other anti-cancer agents (Table 2). Other derivatives include topoisomerase inhibitors like gemcitabine (2,2-difluorodeoxyribofuranosylcytosine) and the ethynyl analog, often known as eniluracil, a powerful inhibitor of dihydropyrimidine dehydrogenase.

Table 2. Clinically approved prodrug derivatives of 5-FU

Sr. no	Drug	Route of Administration	Protein/Enzymes	Cell lines	References
1	Tegafur	Oral administration	cytochrome P-450 enzyme, dihydropyrimidine dehydrogenase	Various types of cancer as solid tumors and hematological malignancies.	[45]
2	Carmofur	Oral administration		gastrointestinal, breast, and lung cancers	[46]
3	Gemcitabine	Intravenousadministration		solid tumors, including pancreatic, lung, breast, and bladder cancers	[47]
4	Capecitabine	Oral administration	Carboxylesterase, cytidine deaminase, Thymidine phosphorylase	colorectal, breast, and gastric cancers	[48]
5	Doxifluridine	Oral administration	thymidine phosphorylase (TP)	metastatic breast and colorectal cancers	[49]
6	Eniluracil	Oral administration	dihydropyrimidine dehydrogenase (DPD)	severe colorectal cancer patients, breast cancer, and pancreatic cancer	[50]

5.1. Tegafur

Ftorafur, also known as 1-(2-tetrahydrofuryl)-5-fluorouracil, Tegafur, or Futraful, represents the initial prodrug of 5-FU formulated in the former Soviet Union in the 1960s. It has been used for over 30 years to treat solid tumors and hematological malignancies. It offers improved drug delivery, tolerability, and convenience for patients, making it widely used in clinical practice. Originally evaluated in the USA for intravenous administration, its efficacy was established through oral administration [11]. Oral administration provides benefits like self-administration, patient compliance, and fewer healthcare facility visits. To maximize Tegafur’s effectiveness, treatment regimens often involve multiple doses. The liver plays a crucial role in metabolizing tegafur through cytochrome P450. Additionally, the liver and upper intestine contain pyrimidine-nucleoside phosphorylase, which converts tegafur into 5-FU [1].

5.2. Carmofur

Carmofur, developed and manufactured by Hoshi in Japan in 1975, belongs to the fluoropyrimidine family and plays a crucial part in inhibiting tumor growth and metastasis. It has been clinically used for various malignancies, including gastrointestinal, breast, and lung cancers. Clinical trials have demonstrated their effectiveness in improving patient outcomes, showcasing benefits in overall survival and response rates [36]. Carmofur is administered orally, allowing convenient at-home use. It is available as capsules or tablets that can be easily swallowed with water. Carmofur undergoes a process where a hexyl group is added to the carbamoyl bond, leading to the production of 5-FU without the need for enzymes that help medicines be metabolized. Upon entering the bloodstream, it gradually disperses 5-FU throughout tissues, lymph, and ascites. Additionally, Carmofur successfully inhibits acid ceramidase, impacting cancer cells’ survival, growth, and death through its effects on ceramide [38].

5.3. Gemcitabine

Gemcitabine, also known as 2’,2’-difluorodeoxycytidine, is an effective treatment for various solid tumors, including pancreatic, lung, breast, and bladder cancers. It acts as a topoisomerase inactivator and a nucleoside analog, exerting its anti-cancer effects through multiple mechanisms of action. Typically administered intravenously, gemcitabine can be used in combination with other chemotherapy agents or targeted therapies, depending on the specific cancer type and treatment goals. It is a prodrug that undergoes intracellular phosphorylation to form its active triphosphate form, gemcitabine triphosphate. The intravenous delivery of gemcitabine varies in dose, ranging from one to 1.2 g m^-2, depending on the tumor being treated. This medication is absorbed quickly through the gastrointestinal tract [1]. Possible side effects of gemcitabine include myelosuppression (reduced bone marrow activity), gastrointestinal disturbances, flu-like symptoms, and skin rashes. Close monitoring and appropriate management of these side effects are necessary during gemcitabine treatment.

5.4. Capecitabine

CAP or Xeloda®, officially known as N⁴-pentyloxycarbonyl-5’-deoxy-5-fluorocytidine, holds significant importance in the treatment of gastrointestinal and breast malignancies [39]. As an orally administered cytotoxic agent, it is designed to be preferentially activated in tumors. Developed in the 1990s by Japanese scientists, fluoropyrimidine carbamate serves as an oral formulation aimed at overcoming the unacceptable toxicity associated with 5’dFUrd (doxifluridine) [40]. The major problem associated with 5’dFUrd stems from its toxicity to the gastrointestinal tract, linked to the release of 5-FU in the small intestine under the influence of Thymidine phosphorylase (TP). CAP was thus formulated as a 5’dFUrd prodrug that remains unmetabolized by TP in the intestine [41]. Results from various trials highlighted improved overall survival, response rates, and patient outcomes. CAP undergoes a three-step activation process to produce 5-FU. Firstly, the liver’s carboxylesterase breaks down capecitabine, with minimal impact on the gut epithelium. Then, the cytidine deaminase enzyme, highly expressed in the tumors and liver, sequentially converts the tissue. Finally, TP produces 5-FU, which shows more activity in tumor tissue. This multi-step metabolism ensures superior tumor selectivity while minimizing gastrointestinal and bone damage [1]. This prodrug of 5-FU has two main advantages: first, it increases the concentration of the active principle at the tumor site; second, it decreases the concentration of the drug in healthy tissues with a consequent reduction in systemic toxicity.

5.5. Doxifluridine

In 1979, Cook and his colleagues formulated 5’-deoxy-5-fluorouridine (5’dFUrd), known as doxifluridine or Furtulon®. This compound is characterized by a molecular structure comprising a molecule of 5-FU bonded to a pseudopentose at position 1. It lacks phosphorylation capability, necessitating metabolic transformation into 5-FU before being incorporated into nucleic acids. The rationale behind its development lies in the enzymatic action of thymidine phosphorylase (TP), a tumor-associated angiogenesis factor, which cleaves it into 5-FU, thereby activating the compound [42]. Subsequently, 5-FU disrupts processes important for the growth and proliferation of cancer cells. Initial clinical trials involving intravenous administration of 5’dFUrd demonstrated promising potential against colorectal cancers, but were accompanied by significant neurological and cardiac toxicity. The development of this compound was halted until it could be made available in an oral formulation, which seems to mitigate the neurological and cardiac complications. Doxifluridine may have potential side effects, including gastrointestinal disturbances, myelosuppression (reduced bone marrow activity), and dermatological reactions. Close monitoring and appropriate management of these side effects are necessary during doxifluridine treatment [43].

5.6. Eniluracil

Eniluracil is a pharmacological compound that enhances the efficacy of the anti-cancer drug 5-FU while reducing its toxicity. It achieves this by acting as a potent and selective inhibitor of DPD, the enzyme responsible for the rapid degradation of 5-FU in the body. By inhibiting DPD, eniluracil increases the systemic exposure and concentration of 5-FU, thereby enhancing its anti-cancer effects. Eniluracil prolongs the 5-FU half-life and competitively inhibits DPD when administered alongside 5-FU, preventing its rapid breakdown and maintaining a sustained presence of the active drug in the body. Co-administration of eniluracil with 5-FU modifies the pharmacokinetic profile of 5-FU and inhibits the development of harmful 5-FU catabolites. Animal tests have shown that eniluracil has a significant impact on the potency of 5-FU [44]. Clinical trials are presently focusing on assessing the efficacy and safety of tegafur, carmofur, gemcitabine, capecitabine, doxifluridine and eniluracil in various cancer types and treatments [45-50].

Naturally occurring macromolecules like polysaccharides, pectin, cholesterol, etc., can substantially improve the drug release performance, and can offer controlled drug release leading to higher levels of bioavailability. Thus, a novel polysaccharide 5-FU prodrug derivative was formulated with improved specificity to treat colorectal cancer. This prodrug was obtained by joining a polysaccharide (molecular weight of 10⁵-10⁷), possessing galactose through various bridge linkages, to get therapeutic conjugates. Its unique linkage prevented premature digestion, allowing targeted delivery [51]. The prodrug’s 5-FU-galactose portion binds to galectin-3, overexpressed in colorectal cancer cells. This triggered interaction with cancer-specific ligands. Subsequently, enzymatic hydrolysis of local bacterial flora produces 5-FU from the polysaccharide to impart its specific effect on colorectal cancer cells. Reduced toxicity was seen in in vivo investigations using a mouse model of colorectal cancer, enabling dosage reduction, increased survival rates, and decreased tumor weight. This polysaccharide prodrug-5-FU also exhibited potential immunoregulatory effects, mitigating 5-FU-induced immunosuppression. This innovative approach holds promise for improved therapeutic outcomes and warrants further clinical investigation [51]. A novel drug conjugate was designed by combining carboxyalkyl-5-FU with a sulfated polysaccharide from a red alga and subsequently binding with folic acid. It exhibited a significantly sustained release of the API and increased phosphorylation in the tumor cells to promote apoptosis in HeLa and AGS cancer cells [52].

Pectin can synergistically aid in the anti-cancer potential of 5-Fu by contributing to the metastatic activity. In 2008, the dual-targeted prodrugs were reported by conjugating pectin with 5-FU to treat colon cancer. These prodrugs were designed to be guided by the carrier to the colon, and once there, they could be recognized by galactin-3, a protein highly expressed in colon cancer cells. The release of 5-FU within the cancer cells enhanced selectivity and efficacy while reducing chemotherapy side effects. Furthermore, pectin fragments potentially enhanced the anti-cancer metastatic 5-FU activity [53]. In another study, cholesterol-attached 5-FU prodrugs were prepared [54], while considering the benefits of elevated lipophilicity and targeted prodrug delivery because reduced lipoprotein receptor is overexpressed on tumor tissue. Ester links in cholesterol-conjugated 5-FU drug carriers fulfilled this need because these low-density lipoprotein receptors require more low-density lipoprotein than normal tissues do. Six compounds were developed by changing the solvents, and mouse models were used to assess each compound’s efficiency. The study revealed that synthesized prodrugs were more lethal to cells than 5-FU by itself [54].

6. Modulation strategies for 5-Fluorouracil

Modulation strategies for 5-FU involve various approaches to optimize the drug’s effectiveness and to minimize the associated toxicities. One strategy is combination therapy, where 5-FU is administered with other chemotherapeutic agents or targeted therapies to enhance the overall treatment response. Another approach is the utilization of drug delivery systems like nanoparticles, microspheres, liposomes, or viruses, which can enhance the drug’s pharmacokinetics, enhance tumor targeting, and reduce off-target effects.

6.1. Combination/co-administration strategies for 5-FU-resistant targets

Different scientists have suggested the use of biochemical modulators to enhance the anti-cancer activity of 5-FU. The biological impact of chemotherapy can be enhanced through a pharmacological agent (biochemical modulator), either by intensifying the anti-tumor action selectively or by providing a shielding effect to the host. A 5-FU modulator can operate in two ways: selectively increasing anti-tumor activity (anabolic pathways) and boosting the bioavailability of the active compound while minimizing toxic effects (catabolism) [1].

6.1.1. Non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) showed promising improvement in 5-FU treatment outcomes. NSAIDs work synergistically with 5-FU, potentially overcoming resistance, reducing inflammation, and inhibiting the formation of blood vessels. By inhibiting specific enzymes involved in inflammation and cell signaling pathways [4], NSAIDs like aspirin or indomethacin can augment the cytotoxicity of 5-FU, potentially leading to improved treatment outcomes. NSAIDs possess anti-angiogenic qualities that prevent the development of new blood arteries that oxygenate the tumor. This characteristic complements the action of 5-FU by restricting the tumor’s blood supply, potentially enhancing its anti-cancer activity and inhibiting metastasis. Inflammation plays a significant part in cancer growth and progression. NSAIDs alleviate inflammation by blocking the action of cyclooxygenase (COX) enzymes, thus reducing the production of pro-inflammatory molecules. By creating a less inflammatory environment, NSAIDs may increase the efficacy of 5-FU in targeting cells of cancer cells [55].

6.1.2. Tacalcitol

Recent research has shown that vitamin D analogues, particularly tacalcitol (Figure 9), are compounds that are currently being studied to make 5-FU treatment more effective [56]. It can boost the anti-cancer activity of both human and animal colorectal cancer (CRC) models. Tacalcitol has shown the ability to sensitize cancer cells that are resistant to 5-FU. By targeting different pathways involved in cancer progression, tacalcitol enhances treatment outcomes beyond the effects of 5-FU alone. Further research and clinical trials are necessary to validate its effectiveness, optimize dosage regimens, and ensure patient safety [56].

6.1.3. Chloroquine

Due to its demonstrated anti-cancer properties, chloroquine (CQ) has garnered significant attention as a potential antineoplastic agent and adjunct therapy in overcoming resistance to 5-FU in recent years. CQ (Figure 10) may enhance the anti-cancer effects of 5-FU by preventing CRC cells from inducing autophagy [57], which is one of the outcomes of 5-FU’s mode of action [11]. Animal models with CRC were employed to verify the results [57]. CQ has also been demonstrated to increase 5-FU sensitivity by preventing the P53 protein regulation pathway. In CRC patients, the combination of both of these techniques may be capable of overcoming 5-FU resistance.

6.1.4. Nifedipine

The expression of TS and survivin is down-regulated by the calcium ion-activated protein-coupled calcium-sensitive receptor (CaSR), which enhances the sensitivity of CRC cells to 5-FU. The combination of calcium ion (Ca²⁺) and the cardiac medication nifedipine synergistically activates CaSR, leading to increased intracellular Ca²⁺ levels. When nifedipine (Figure 11) is combined with Ca²⁺, it significantly reduces the expression of survivin and TS, rendering cancer cells more susceptible to the effects of 5-FU [58]. This combined therapy demonstrates an improved sensitivity to 5-FU compared to the use of Ca²⁺ alone, providing potential benefits for CRC patients [58].

6.1.5. Modulation by leucovorin

Leucovorin (LV), also known as folinic acid, is a reduced form of folic acid commonly used in cancer treatment as an adjuvant therapy. It enhances the effectiveness of certain chemotherapeutic agents, such as 5-FU, by modulating their effects. The modulation of 5-FU by LV (Figure 12) involves several mechanisms that enhance its cytotoxic effects while minimizing resistance [59]. LV stabilizes the binding of 5-FU’s active metabolite FdUMP to TS, disrupting DNA synthesis and promoting cell death. Additionally, LV facilitates the formation of 5,10-methylenetetrahydrofolate, which enhances the incorporation of FdUMP into DNA, thereby amplifying the 5-FU anti-tumor effects. These mechanisms not only improve the efficacy of 5-FU but also reduce its toxicity on normal tissues. A research group investigated the effect of adding LV to oral tegafur-uracil (UFT) or 5-FU prodrugs in xenografts of colorectal cancer with varying levels of TS. The combination of UFT with LV showed significant inhibition of tumor growth, especially in tumors with high TS expression. The enhanced ternary complex of TS and elevated levels of decreased folates in tumors treated with LV; this combination therapy could enhance the anti-tumor efficacy of UFT in colorectal cancer patients [59].

6.1.6. Modulation by methotrexate

Amethopterin, another name for methotrexate, is a chemotherapeutic medication and immune suppressor, widely used in cancer therapy. Methotrexate (Figure 13) [60], an inhibitor of dihydrofolate reductase and an antifolate, plays a vital role in suppressing the development of deoxythymidine monophosphate (dTMP) nucleotide, a necessary component for cell replication. Significantly surpassing the therapeutic benefits of 5-FU alone, methotrexate treatment exhibits notable advantages in cancer therapy [60].

7. Targeted delivery of 5-FU using carrier systems

When administered alone, 5-FU and its derivatives show an unsatisfactory response rate in chemotherapy. Clinical efficacy depends on maintaining high serum levels for extended periods, but only a small fraction of the administered dose is activated [5]. Unpredictable oral bioavailability due to variable enzymatic degradation further hinders effectiveness. Achieving cancer-specific delivery and incorporation of the agent into cancer cells is an ideal approach. Incorporating the agent into a vesicle can reduce side effects on normal cells. Innovative treatments are sought for improving absorption, anti-tumor effects, and reducing adverse effects. Various vectors, such as viruses, microspheres, liposomes, and nanoparticles, are being explored to achieve these objectives [6].

7.1. Nanoparticles

The advancement of nanotechnology, focusing on engineering functional systems at the nanoscale, has enabled its applications to medical challenges. Nanomedicine entails using nanoparticles for preventing and treating various diseases within the human body. Presently, nanomedicine offers potential solutions for the treatment of various tumors, particularly in targeted therapy of tumors. Nano-carriers play a crucial role in overcoming challenges linked to chemotherapy, including optimizing concentration, ensuring minimal drug exposure and improving drug selectivity [6]. The predominant approach for clinically viable strategies involving nano-carriers and macromolecules is grounded in the enhanced permeability and retention (EPR) phenomenon. The use of nano drug delivery system for 5-FU opens opportunities for combination therapies and synergistic drug combinations. By co-encapsulating 5-FU with other therapeutic agents or incorporating them into multifunctional nano-carriers, it becomes possible to achieve complementary effects, overcome drug resistance, and enhance overall treatment efficacy.

i. Chitosan-based nanoparticles

By utilizing chitosan, a biodegradable and biocompatible polymer, as the nanoparticle matrix for 5-FU delivery, the approach presents many advantages. It provides improved controlled release, drug stability, targeted delivery to tumor sites, and reduced systemic toxicity. This innovative formulation holds great possibility for improving the 5-FU’s safety and effectiveness for cancer treatment. In 2013, Chang with his fellows conducted a study on chitin nanogels loaded with 5-FU, assessing their characteristics and performance. The nanogels demonstrated pH-responsive swelling and controlled release of the drug, forming stable spherical particles when dispersed in cawater. In toxicity tests, the nanogels exhibited effective toxicity against melanoma cells while demonstrating reduced toxicity towards human dermal fibroblast cells. Confocal microscopy confirmed the uptake of the nanogels by both cell types. Skin permeation studies revealed similar flow rates compared to the 5-FU control, but with significantly higher retention of the nanogels in the inner layers of the skin, up to 4-5 times greater retention [61]. Later, another group reported chitosan nanoparticles, which have been shown to require smaller concentrations of anti-cancer medications, reducing their negative effects. After a 48 h incubation time, chitosan nanoparticles exhibited controlled and sustained release of 5-FU. The development of new chitosan Schiff base ligands using compounds related to 5-FU medicine was observed, as given in Figure 14 [62]. The carbonyl group from 5-FU is combined with the amino group of chitosan to form an intermediate (I), which subsequently eliminates a water molecule to generate the imine compound [62]. Recently, a study was conducted to develop folate-functionalized 5-FU nanoparticles using the solvent evaporation process. Folate functionalization enhanced the effectiveness of 5-FU folate nanoparticles in a xenograft mouse tumor model, resulting in significantly increased nanoparticle concentration within tumor cells in vitro. This functional network has the potential to target different drugs in future developments [63].

Formation of chitosan-based 5-FU Schiff base.

In 2024, the ionic gelation process was used to specifically target receptors of 5-FU as well as its sustained release against triple-negative breast cancer. For this purpose, thiolated chitosan nanoparticles were synthesized and decorated with hyaluronic acid to target an overexpressed receptor in most malignancies. This drug loading improved pharmacokinetic attributes as well as cell target specificity. This drug loading also resulted in improved pharmacokinetic attributes as well as cell target specificity, validated through enhanced cytotoxicity studies employing MTT assay [64]. To overcome the chemoresistance of triple-negative breast cancer, an akin gelation process was used by Fahmy et al. [65] to synthesize chitosan-based nanovectors of 5-FU and their combination with microRNA oligonucleotides to target triple-negative breast cancer. This combination synergistically showed increased tumor suppression with slow drug release.

ii. Silica-based nanoparticles

Nanoparticles, composed of silica (SiO₂), offer a promising platform for efficient drug delivery. By encapsulating 5-FU within the SiO₂ matrix, these nanoparticles provide controlled release and targeted delivery to cancer cells. The high surface area of silica nanoparticles allows enhanced drug loading capacity and improved therapeutic efficacy. Additionally, their biocompatibility and tunable surface properties make them suitable for various biomedical applications. The researchers investigated a magnetic nanocomposite with drug-loading capability and studied its behavior in loading and releasing 5-FU. They conjugated hyaluronic acid to silicon nanoparticles, which improved 5-FU uptake, resulting in significant anti-cancer efficacy. The study also explored the binding of 5-FU to the carrier and functionalized magnetic silica using different solvent systems for loading purposes [66].

iii. Poly(lactic-co-glycolic acid nanoparticles

The synthetic methods of Poly(lactic-co-glycolic acid (PLGA) nanoparticles ensure their desirable physicochemical properties, including surface morphology and particle size. The encapsulation of 5-FU within PLGA nanoparticles offers sustained release profiles, allowing for prolonged drug exposure to cancer cells. Continued research and optimization of this drug delivery system are essential for its successful translation into clinical practice, bringing us closer to more effective and personalized cancer therapies. In 2023, Dhanabalan et al. [67] developed 5-FU-encapsulated nanoparticles using PLGA and evaluated their potential in cancer treatment. The PLGA nanostructures containing 5-FU were synthesized using a modified dual emulsification procedure and exhibited two unique component pairings as illustrated in Figure 15 [67]. Tumor cell lines and glioma cell lines were utilized for biological assessment. The nanoparticles achieved a drug entrapment efficiency of less than 70%. However, in a dose- and time-dependent manner, the nanoparticles containing the PLGA 50-50 combination demonstrated superior cytotoxicity against both tumor and glioma cells compared to the drug alone.

PLGA encapsulated nanoparticles for 5-FU drug delivery.

iv. Solid lipid Nanoparticles

Solid lipid nanoparticles (SLN) drug delivery system holds great potential for overcoming drug resistance and reducing systemic toxicity, bringing us closer to more effective and targeted cancer therapies [9]. In 2016, Khallaf et al. [68] improved the degradation behavior of SLN compared to 5-FU alone, while the gel matrix aided SLN transport and release. Mice treated with SLNs exhibited higher treatment efficacy than those injected with 5-FU alone, highlighting the potential of SLNs for enhanced local administration of hydrophilic drugs. Later, a study was conducted to investigate the SLN as carriers for 5-FU in colorectal cancer treatment, aiming to enhance therapeutic efficacy. By employing hot and cold homogenization, SLNs loaded with 5-FU were developed, incorporating novel PEGylated lipids and a surfactant mixture as given in Figure 16 [69]. The efficiency of 5FU-SLN4 in HCT-116 cancer cells was evaluated through cytotoxicity tests. In vivo, anti-tumor efficacy tests on mice with HCT-116 cancer demonstrated significant inhibition of tumor growth, highlighting the high cytotoxicity of 5FU-SLN4 against HCT-116 cells.

Solid lipid nanoparticles through the high-pressure homogenization process.

v. Polyamidoamine (PAMAM) dendrimers as nanocarriers

Studies have shown that PAMAM/5-FU nanocomposites exhibit improved drug solubility, sustained release, and increased cellular uptake when compared to free 5-FU. The dendrimers protect 5-FU from degradation and allow for controlled release, leading to enhanced anti-cancer efficacy. In 2017, Rengaraj et al. [70] carried out a study to explore the use of poly(amidoamine)/5-FU as a drug delivery conjugate system targeting E7 or E6 oncoproteins prevalent in cervical malignancies, as shown in Figure 17 [70]. Molecular docking studies revealed that the 5-FU/ PAMAM combination exhibited higher affinity towards the oncoproteins compared to 5-FU alone. Both PAMAM/5-FU and PAMAM demonstrated reduced toxicity in HeLa cell lines and COS-7. E6/E7 oncoproteins and PAMAM/5-FU interaction was confirmed through enzyme P53 analysis, and hematological testing on female mice having cervical cancer further supported the safer characteristics of PAMAM/5-FU.

Enhanced drug delivery and reduced toxicity with PAMAM/5-FU conjugate.

vi. Xylan as a drug carrier

To enhance therapeutic efficacy and targeted drug delivery, Kumar et al. [71] studied and created the colon-specific prodrugs called xylan-5-fluorouracil-1-acetic acid conjugates, as given in Figure 18 [71]. Human cancer cell lines (HT-29 and HTC-15) were used to evaluate the toxicity investigations of the prodrugs in vitro, which revealed that the conjugates were more lethal than the free drug. As a result, the findings showed the Xyl-5-FUAC conjugates as promising candidates for colon-specific delivery of drugs in the treatment of colon cancer with little adverse side effects.

Simplified proposed reaction mechanism for Xyl-5-FUAC conjugate synthesis.

In another study, a novel zwitterionic 5-FU co-crystal with L-proline (PL) was integrated into poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) carriers to produce co-crystal micelles. Yu et al. [72] used solvent-assisted grinding and solution evaporation crystallisation to accelerate the co-crystal formation. Using various analytical techniques, the shape of the co-crystal micelles and the structure of the 5-FU-PL co-crystal were characterised. Theoretical and experimental methodologies were consistently used to undertake comparative investigations of in vivo/vitro characteristics. The outcomes highlighted the improved solubility and permeability of co-crystals. Enhanced relative bioavailability of 5-FU-PL co-crystal micelles compared to 5-FU, facilitated improved in-vivo absorption. Notably, the 5-FU-PL co-crystal micelles exhibited more effective sustained-release properties than 5-FU, resulting in an extended biological half-life and therapy duration.

vii. CaCO₃ nanoparticles as drug carrier

In 2024, Deng et al. [73] used cockle shells to synthesize CaCO₃ nanoparticles, and 5-FU was made using a homogenizer and combined with thymoquinone to evaluate the cytotoxicity against colon cancer. The synthesized 5-FU-CaCO₃ nanoparticles not only reduce the side effects of 5-FU on NIH₃T₃ normal cells but also improve the cytotoxic potential against the CT26 cell line of colon cancer. It was attributed to improved sustained drug release as well as biocompatibility, affirmed through a molecular docking study.

viii. COF and MOF as drug carriers

With the advent of nanotechnology, covalent organic frameworks (COFs) and metal organic frameworks (MOFs) have garnered substantial attention due to their exceptional tunable properties, like controlled pore size and huge surface area, offering immobilization support for controlled drug release as well as sensing applications. Our research group published a review article pertaining to the use of COF for drug sensing as well as drug delivery, a section of which reports updated literature regarding the use of COF as drug carriers for 5-FU [41]. Methyl imidazole and zinc-based MOF, named as ZIF-8, being pH-sensitive, was synthesized and studied for drug loading and release in the acidic environment of oral tumors. Along with the slow release of 5-FU, the cytotoxicity assay showed a synergistic inhibitory effect [74].

7.2. Microspheres

In recent years, there has been a growing interest in employing microspheres for the controlled delivery of 5-FU. Microspheres, a type of multiparticulate delivery system, are formulated to achieve sustained drug release, thereby enhancing stability, bioavailability, and targeted delivery to specific sites. The use of microspheres can provide several advantages, including minimizing fluctuations within the therapeutic range, reducing side effects, lowering dosing frequency, and improving patient compliance. Faisant et al. [75] introduced a groundbreaking approach employing biodegradable microspheres for the targeted delivery of an anti-cancer agent in the treatment of glioblastoma. This innovative strategy involved the use of implantable microspheres that released 5-FU, combined synergistically with radiotherapy and surgery. This integrated therapeutic approach holds great promise for improving treatment outcomes and enhancing patient survival rates [75]. A novel approach for targeted drug delivery to the colon using colon-specific microspheres encapsulating 5-FU was developed by Rahman et al. [76]. Alginate core microspheres were created employing a modified emulsification method to facilitate sustained drug release. The results demonstrated a remarkable drug release duration of up to 10 h in core formulations and up to 20 h in coated formulations, ensuring prolonged therapeutic efficacy. Moreover, the microspheres retained their stability even after 6 months of storage, underscoring their potential as a promising strategy for colon cancer treatment.

7.3. Liposomes

TS overexpression has been found to correlate with 5-FU resistance in colorectal cancer patients. To optimize the anti-cancer effectiveness of 5-FU, it is crucial to control the activation pattern of the drug, specifically targeting the inhibition of TS FdUMP synthesis. The key enzyme involved in generating intratumor FdUMP, which is crucial for the activation of 5-FU through the DNA pathway, is TP. The potential of 2’-deoxyinosine as a modulator of 5-FU is hindered by two factors. Firstly, its pharmacokinetic profile has shown significant improvement. Additionally, it undergoes extensive metabolism within erythrocytes [77].

A first pegylated liposomal formulation of 2′-deoxyinosine (d-Ino), a 5-FU modulator, was designed to spare it from erythrocytic clearance. The liposomal d-Ino exhibited a potent effect as a modulator of fluoropyrimidines, both in vitro and in xenografted mice, due to its optimized pharmacokinetic profile [78]. In 2009, Fanciullino and Ciccolini [79] reported a two-fold liposomal formulation that encapsulated both 5-FU and its modulator, providing an improved therapeutic index for 5-FU. This innovative medicament offers promising potential for patients experiencing chemoresistance to 5-FU or other fluoropyrimidine drugs. HA-modified liposomes encapsulating 5-FU were tested against colorectal and hepatoma cell lines, showing reduced colony formation. Hyaluronic acid, known to be overexpressed in many cancer cells, offers targeted delivery potential. This optimized targeted delivery of 5-FU into colorectal cancer cells induced apoptosis, cell-cycle arrest, and decreased colony formation, suggesting a promising approach for cancer treatment [80].

7.4. Viruses

Chemotherapy using the Hemagglutinating Virus of Japan Envelope (HVJ-E) vector, incorporating an anti-cancer agent, can be employed as a standalone treatment for humans. Alternatively, it can be locally administered to patients with advanced cancer who may not be suitable for direct administration of an anti-cancer agent, this approach aims to induce cancer regression. Simultaneously, combining it with radiotherapy and/or surgical interventions can enhance the overall anti-cancer effects. Shibata et al. [81] successfully developed a patented medicinal preparation utilizing 5-FU-encapsulated viruses as a gene delivery vector for transferring chemotherapeutic agents, such as 5-FU or its derivatives, into cells or living organisms, with a focus on enhancing therapeutic efficacy in cancer treatment.

8. Derivatization at N₁ and N₃ of 5-FU

Alkylated metabolites can extend the plasma half-life of 5-FU, although they are not directly responsible for the increase in cytotoxicity in tumor tissues. The pharmacological effects of 5-FU on different alkylating groups, such as methyl and activated methylene groups, can vary. In the case of anti-cancer drugs like 5-FU, alkyl groups can be added to the N₁ or N₃ positions to form dialkyl derivatives or to the N₁ site alone to form mono-alkyl derivatives. By understanding these principles, N₁, N₃-dialkyl or N₁-monoalkyl derivatives can be synthesized, as shown in Figure 19 [82].

A series of novel N-acyl and N-(alkoxycarbonyl)-5-FU derivatives were synthesized, with substituents such as benzoyl, o-toluyl, acetyl, propionyl, heptanoyl, ethoxycarbonyl, phenoxycarbonyl, and benzyloxycarbonyl groups at N₁ and/or N₃ positions. The anti-tumor activities of these derivatives were evaluated, with N₃-benzoyl and N₃-o-toluyl derivatives showing significant activity against experimental tumors. Notably, N1-acetyl-N₃-o-toluyl-5-fluorouracil exhibited the most promising results among them, retaining higher activity toward various tumors with lower toxicity and favorable blood levels, even for oral administration [83]. In 2008, Zheng et al. [84] synthesized a series of N₁-acetylamino-(5-alkyl/aryl-1,3,4-thiadiazole-2-yl)-5-fluorouracil derivatives, as shown in Figure 20 [84]. To evaluate their potential as anti-cancer agents, the team performed MTT assays against Bcap-37 (human breast cancer cells) and A-549 (human lung cancer cells). Remarkably, the results revealed significantly higher anti-cancer activity of these derivatives compared to 5-FU, demonstrating their enhanced potency in combating cancer [84]. A stable chemical linker was used to create several complexes between 5-FU with alepterolic acid, as well as the benzyl group on FU’s 3-N position was then removed. Due to piperazine’s excellent water solubility, low toxicity, and great stability, it is frequently employed in the manufacturing of pharmaceuticals. The outcomes showed the in vitro cytotoxicity of the MCF-7 and A549 cell lines [85].

Synthesis of 5-FU Alkyl derivatives (2-amino-aryl/alkyl-1,3,4-thiadiazoles).

9. Efficacy in animals

Being a cornerstone chemotherapeutic drug, 5-FU has been extensively studied in animal models to unfold underlying operative mechanisms and therapeutic efficacy. 5-FU and Kaempferol were administered in combination to the Wister rat model to evaluate the hematological indices. Kaempferol possesses immunomodulatory and hepatoprotective features by protecting various organs, countering the adverse effects of 5-FU [86]. Although 5-FU is the most prescribed antineoplastic agent, its administration is found to cause intestinal injury. So, to access the dose-response relationship, an animal model of mice was used. It showed severe weight loss and mortality at higher administered dosages. 25 mg kg^-1 was found to be the optimum dose without any intestinal damage or associated side effects [87]. 5-FU has also been found to increase cell response and apoptosis in various animal models.

As the limited bioavailability of 5-FU has restricted its oral administration, 5-FU loaded on different nonpolar nanoemulsions was topically applied to the skin of goat, cow, and rat models. Thermodynamically stable optimized 5-FU-nanoemulsions with improved permeability were selected following a cytotoxicity study. Animal models are also front-line experimental species in order to explore the mechanism of cardiotoxicity associated with antineoplastic therapy. Nevertheless, mechanisms of drug resistance, permeability, and bioavailability studies, histopathology studies, and optimization of dose-response relationship are amply studied in animal models owing to the higher efficacy of 5-FU.

10. Conclusions

Over several years, efforts have been made to make 5-FU more effective and more target-specific; this comprehensive review article explores the various aspects of 5-FU modulation. The article provides insights into the molecular mechanisms of 5-FU’s anti-cancer effects, its formulation strategies using co-crystals, the potential of prodrugs to improve drug delivery and selectivity, and also discusses modulation strategies to overcome resistance to the drug and reduce side effects, alongside the importance of alkyl derivatization in optimizing 5-FU’s performance. The review paper examines how these approaches can increase the 5-FU therapeutic effect and overcome its limitations. It also provides a compilation of clinically approved prodrugs, highlighting their role in improving the therapeutic performance of 5-FU. Overall, this review contributes to the field of cancer chemotherapy and guides the advancement of more effective treatment approaches.

11. Future directions for 5-FU research

The horizon of 5-FU research continues to expand, with numerous promising strategies being explored to enhance its therapeutic efficacy and treatment response. This section outlines the emerging advancements and potential future directions in this evolving field, i.e., i) Personalized medicine: In an attempt to improve the efficacy of the drug and to minimize the possible side effects, personalized medicines can employ modifications employing pharmacogenetics. Particularly for 5-FU, dose adjustments and personalized treatment plans can be conducted, which will also help predict the patient’s response to 5-FU. Subsequently, biomarkers can be designed or researched to monitor therapeutic response as well as disease progression. ii) Novel drug administration: Targeted administration, improving the availability of 5-FU, can be done with innovative delivery systems. For sustained and localized drug release, hydrogels and implantable devices are a promising future direction. Highly biocompatible nanoparticles based composite materials like COF-metal oxides, organic metal oxides, COF-MOF, MOF-Metal-CF, porous organic polymeric materials (POPs capable of encapsulation or integration with 5-FU can improve its stability and drug targeting specificity. Furthermore, designing pH and temperature-responsive hydrogels and implantable devices can significantly contribute towards the targeted delivery of 5-FU. iii) Combination therapies: Combining 5-FU with other synergistically functionalizing chemotherapeutic agents improves its anti-cancer effects and helps cope with drug resistance. Development of effective immunotherapies, like potential integrations with targeted bioreceptors like monoclonal antibodies and enzyme inhibitors, can lead to improved drug response.

Moreover, emerging endeavors such as the Internet of Things (IoT), machine learning, artificial intelligence, gene-editing techniques, organoids, and multidimensional cell cultures can be strategically integrated to enhance drug efficacy and improve patient outcomes.

Acknowledgements

The authors are thankful to the Deanship of Graduate Studies and Scientific Research at University of Bisha for supporting this work through the Fast-Track Research Support Program. We are thankful to the reviewers for their insightful comments and constructive suggestions, which greatly contributed to the improvement of the paper’s content.

CRediT authorship contribution statement

Farhat Jubeen: Conceptualization; Project administration; Supervision; Misbah Sultan: Formal analysis; Aqsa Aslam: Investigation; Writing - original draft; Kald M. A. Algarni: Data curation: Methodology; Muhmmad K. Saleemi: Resources; Visualization; Wissem Mnif: Software; Funding acquisition; Farooq Anwar: Resources; Validation; Munawar Iqbal and Naveed Ahmad: Validation; Visualization; Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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Recent advances in 5-fluorouracil: Co-crystal synthesis, prodrug derivatives and modulation strategies to enhance anti-cancer activity

Abstract

Keywords

5-Fluorouracil

Modulation strategies

Nanoparticles/drug carriers

Pharmaceutical co-crystals

Prodrugs

1. Introduction

2. 5-Fluorouracil

2.1. 5-FU anti-cancer: Mechanism of action

3. Pharmaceutical co-crystallization

3.1. Co-crystal preparation techniques

3.1.1. Solution-based method

3.1.2. Solid-based method

4. Co-crystallization of 5-FU

4.1. Two-component synergy in co-crystals

5. 5-FU prodrug derivatives

5.1. Tegafur

5.2. Carmofur

5.3. Gemcitabine

5.4. Capecitabine

5.5. Doxifluridine

5.6. Eniluracil

6. Modulation strategies for 5-Fluorouracil

6.1. Combination/co-administration strategies for 5-FU-resistant targets

6.1.1. Non-steroidal anti-inflammatory drugs

6.1.2. Tacalcitol

6.1.3. Chloroquine

6.1.4. Nifedipine

6.1.5. Modulation by leucovorin

6.1.6. Modulation by methotrexate

7. Targeted delivery of 5-FU using carrier systems

7.1. Nanoparticles

7.2. Microspheres

7.3. Liposomes

7.4. Viruses

8. Derivatization at N1 and N3 of 5-FU

9. Efficacy in animals

10. Conclusions

11. Future directions for 5-FU research

Acknowledgements

CRediT authorship contribution statement

Declaration of competing interest

Declaration of generative AI and AI-assisted technologies in the writing process

References

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