8.1 Historical Background

S. Farber’s pioneering research on childhood ALL (acute lymphoblastic leukemia) treatment with the folate antagonist aminopterin laid the foundation for combinational chemotherapy as a standard treatment approach in clinical oncology (Farber et al. 1948). The discovery of methotrexate (MTX) (Fig. 8.1) was combined as emerging chemical research efforts as antifolates were first single drug entities that enabled to cure a solid tumor (Seeger et al. 1947, 1949; Farber 1949). However, two researchers, R Hertz and Min C Li from the National Institute of Health (NIH), showed that combining MTX and 6-mercaptopurine (6-MP) significantly enhanced the prognosis for fatal gestational trophoblastic disease (GTD) (Hertz et al. 1956).

Fig. 8.1
Two chemical structures of Aminopterin and M T X. The structures have bonds between atoms, and letters indicating the type of atom C, N, H, and O, and specific functional groups and atoms are labeled.

Structure of folic acid and its synthetic analog MTX

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Hence, MTX became a fundamental component in the development of combined chemotherapy. Various oncologists, including E. J. Freireich, E. Frei, J. H. Burchenal, V. DeVita, and D. Pinkel, supported the multidrug treatment approach, based on compelling rationale and empirical evidence demonstrating that combining drugs significantly increased cancer remission rates (Frei III et al. 1958, 1965).

Following their initial success, particularly in treating childhood ALL, there was a surge in drug discovery research at the federal level in the USA. This led to the establishment of the Cancer Chemotherapy National Service Center (CCNSC) in 1955 (DeVita Jr. and Chu 2008). One of the initial commercial successes in drug discovery was the introduction of a novel class of anticancer medication, vincristine, by the Eli Lilly and Company. Vincristine (Fig. 8.2) received FDA approval in 1963 (Johnson et al. 1963). Unlike many other chemotherapeutic agents, vincristine does not target DNA (Škubník et al. 2021; Sheikh-Zeineddini et al. 2020); instead, it halts mitosis by affecting the cytoskeleton (Himes et al. 1976). Its unique mode of action opened up various possibilities for drug combinations, ultimately leading to the successful development of a “quadruple” combination therapy consisting of three key drug components: vincristine, amethopterin, 6-MP, and prednisone, known as VAMP (Freireich et al. 1964).

Fig. 8.2
A molecular structure of vincristine with various atoms connected by bonds, including single, double, and dotted lines indicating different types of chemical bonds. The molecule contains multiple rings and functional groups, with atoms labeled.

Structure of antineoplastic drug, vincristine

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At present, numerous clinical trials are underway exploring frontline combination chemotherapies involving targeted medications such as the proteasome inhibitor, bortezomib, the JAK (Janus kinase) inhibitor ruxolitinib, and the PI3K/mTOR inhibitor NVP-BEZ235 (Fig. 8.3). These trials are aimed at achieving improved responses, particularly for relapsed cases of ALL, among other objectives (Terwilliger and Abdul-Hay 2017; Messinger et al. 2012; Tasian et al. 2012; Senkevitch et al. 2015; Schult et al. 2012).

Fig. 8.3
3 molecular structures. Top left. A multi-ring molecule with oxygen, nitrogen, boron, and hydrogen atoms. Top right. A multi-ring molecule with nitrogen atoms in rings and chains. Bottom. A large and complex molecule with multiple interconnected ring structures containing nitrogen and oxygen atoms.

Current targeted combinational chemotherapeutic agents

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Expanding the scope of drug combination strategies to other cancers, optimization was employed using vincristine in combination with cyclophosphamide, prednisone, and MTX for treating Hodgkin’s syndrome (Moxley III et al. 1967; Frei III et al. 1966; Devita et al. 1970). Similarly using mustard-mustargen with cyclophosphamide results in “MOPP” protocol (Fig. 8.4)—procarbazine, mustargen/mechlorethamine, prednisone, and oncovin/vincristine. MOPP achieved an 80% complete remission rate in patients with advanced Hodgkin lymphoma and served as the standard treatment protocol for two decades (Canellos et al. 1992).

Fig. 8.4
3 molecular structures. Top left. A linear arrangement of atoms with two chlorine atoms at either end and nitrogen in the center. Top right. A benzene ring has a single bond to three N H and a double bond to an O atom. Bottom. It has multiple rings of carbon atoms connected to O atoms, H atoms, and O H groups.

MOPP-combined drug protocol

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Cooper et al. showed 90% responsive rates in hormone-resistant breast cancer using a five-drug treatment regimen comprising methotrexate, cyclophosphamide, fluorouracil (5-fluorouracil [5-FU]), prednisone, and vincristine sulfate (Cooper 1969; Segaloff et al. 1985). This is noteworthy that most of the primitive drug components such as MTX, vincristine, and prednisone were carried forward and had acceptance in many international drug combination protocols and has laid the foundation for present-day adjuvant cancer therapies (Ansfield et al. 1971; Canellos et al. 1974).

8.2 Targeted Therapies

With new discoveries and significant advances in gaining knowledge of biological mechanism at molecular level, drugs designed to target particular cellular pathways have transformed Paul Ehrlich’s concept of a “magic bullet” into a practical reality (Strebhardt and Ullrich 2008). The initial successful targeted therapy, imatinib (Fig. 8.5) was discovered in 1996 (Druker et al. 1996) and gained FDA approval in 2001. This small molecule is highly specific, inhibiting the product of the reciprocal 9–22 chromosome translocation known as the “Philadelphia chromosome.” Imatinib works by binding to the ATP-binding pocket of BCR-ABL, effectively halting its kinase activity (Druker et al. 1996, 2001). It stands out as one of the most prominent examples of targeted small molecule therapeutics. Researchers observed remarkable clinical responses in chronic myeloid leukemia (CML) patients treated with imatinib and other tyrosine kinase inhibitors (TKIs) (Freireich et al. 2014). However, the main focus was on rationale design of small molecule targeting various oncogenic targets. Of the 48 approved drugs, the majority (25) target receptor protein-tyrosine kinases (Roskoski 2019). Other targets like epigenetic regulators transcriptional factors (Cheng et al. 2019; Morel et al. 2020) have captured the attention of researchers globally.

Fig. 8.5
The molecular structure of imatinib consists of multiple rings and bonds including a pyridine ring, with nitrogen and oxygen atoms.

Structure of imatinib

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8.3 CRISPR Gene-Editing Techniques

Considering the intricate layers of complexity within cancer and its capacity to adapt to changing conditions, the battle against cancer was far from finished. To overcome these challenges, CRISPR gene-editing tool revolution presented new technological niche (Shalem et al. 2014; Wang et al. 2014) for the identification of new drug combinations for cancer treatments.

Two primary methods for drug discovery integrate high-throughput CRISPR-Cas 9 technology:

  1. 1.

    Searching genetic aberration within specific genetic contexts.

  2. 2.

    Performing the pharmacological screening of known targeted therapy to reveal drug-related vulnerabilities.

Wei et al. discovered that hepatocellular carcinoma (HCC) cells, which were resistant to sorafenib, became highly responsive to treatment upon the knockout of phosphor-glycerate dehydrogenase (PHGDH) (Wei et al. 2019). Szlachta et al. conducted a CRISPR screen using an sgRNA library on trametinib in a patient-derived xenograft (PDX) model of pancreatic ductal adenocarcinoma (PDAC) to identify conditional lethality associated with MEK inhibition (Szlachta et al. 2018).

More recently, Xu et al. revealed that a group of kinases enhances the effectiveness of the standard chemotherapeutic treatment involving cisplatin and pemetrexed for malignant pleural mesothelioma (MPM). With the discovery of CRISPR-Cas gene editing tool, there was a great jump for combination drug treatment in cancers, and fruitful outcome of therapies is expected in near future (Xu et al. 2020) (Figs. 8.6 and 8.7).

Fig. 8.6
A timeline diagram of the progression of cancer treatment discoveries and developments from 1942 to 1996, including the introduction of various drugs and therapies. The timeline highlights the achievements of different researchers and the impact of their work on the field of oncology.

Combinational treatment timeline diagram in solid tumors

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Fig. 8.7
An illustration of the process of using C R I S P R whole genome library on W T cells, Mutant or K O cells, and Cancer cells, and exhibiting the outcomes with and without drug treatment.

Identifying drug vulnerabilities through CRISPR-Cas9 genome-wide screens: genetic context-derived vulnerability (1) and drug-related vulnerability (2)

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8.4 New Combination Treatment Strategies

Another intriguing treatment strategy within combination drug regimens involves “conjugation drug therapy,” which merges two drugs into a single entity or molecule. Bestrabucil (Fig. 8.8a) was among the pioneer fused molecules generated by combining an estradiol derivative with CLB amid estrogen receptor-positive (ER+) tumors (Kubota et al. 1986). Surprisingly, it displayed effectiveness against ER-negative tumors as well. A similar approach was utilized to create a single chimeric molecule integrating two distinct chemotherapeutic agents. An example of this was CM358 (illustrated in Fig. 8.8b), formed by fusing CLB with the topoisomerase II inhibitor amonafide (Gilad et al. 2017).

Fig. 8.8
2 molecular structures, labeled a and b, depict bestrabucil and C M 358, respectively. A. It contains multiple benzene rings and O, N, and C l atoms. B. It has multiple benzene rings, O, N, N H, and C l atoms.

Combining two chemotherapeutic drugs into a single compound using a chimeric approach: (a) bestrabucil and (b) CM358

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CM358 exhibited elevated cytotoxicity across various cancer models compared to its individual parent drugs, attributed to its enhanced inhibition of the topoisomerase II target by amonafide. Nevertheless, there were shortcomings of combination targeted therapeutics as listed below:

1.

Lack of preclinical and clinical in vitro and in vivo model studies. No standard protocol was carried out for screening these combination agents

2.

No preclinical evaluation was done on combination drug agents

3.

Lack of characterization methods or imaging tools to access target identification

4.

A large number of agents and targets were prepared that require target identification, a strategic synthetic plan was missing

5.

IPR rights for competing sponsor and regulatory framework for commercialization of targeted combination drug candidates were not underlined

8.5 Metal Complex-Based Combination Therapies

Metal complexes have proven to act as potent therapeutic as well as diagnostic agents to detect and treat many chronic diseases, in particular cancers. Through in vitro, in vivo, and clinical investigations, multiple metal complexes have showcased promising antitumor properties against diverse cancer cell lines. Some of these have obtained FDA approval as effective anticancer drugs. This field gained momentum after serendipitous discovery of cisplatin which was highly effective in treating many solid malignancies (Rosenberg et al. 1969). Later on, a large plethora of platinum and nonplatinum compounds were designed and prepared to mitigate the severe side effect and resistance issues of anticancer drugs (Harrap 1985; Graham et al. 2000; Kenny and Marmion 2019; Trondl et al. 2014; Ruiz-Azuara 1992; Lee et al. 2020). Nevertheless, the success cure rates of these drugs were overwhelming which brought hope to many dying patients. However, the rapid advancement of these innovative metal-based drugs introduced new challenges in pharmacology, including issues such as poor water solubility, limited bioavailability, and brief circulation periods. To address these challenges, various drug discovery methods emerged, focusing on structural enhancements involving ligand design and modification, prodrug development, and drug delivery vehicles. For example, cobalt complexes act as bioreductive prodrugs, maintaining an inert oxidized Co(III) state and a labile reduced Co(II) state (Mathuber et al. 2020). Pt(IV) metal complexes function as inert prodrugs that activate into active Pt(II) complexes upon reduction. While the efficacy of combination therapy relies on targeting multiple pathways to reduce drug resistance, drug delivery strategies aim to optimize drug concentration at the intended site and ensure that the therapeutic drug reaches the desired site by encapsulating the drug in drug delivery vehicles such as liposome or attaching it to targeting fragments. Combination therapies offer to reduce the side effects as the required dosage of drug may be lowered. Repositioning metallodrugs has demonstrated enhanced effectiveness when integrated into combination therapy, either in an additive or synergistic manner, resulting in minimal toxicity and significantly reduced drug resistance (Cirri et al. 2021).

Associating a drug with a positively charged metal complex significantly enhances its solubility in water due to the complex’s hydrophilic nature. Conversely, coupling a metal with a negatively charged group can diminish the drug’s negative charge, boosting its passive cellular uptake and absorption (Renfrew 2014). These strategies effectively improve the pharmacokinetic and pharmacodynamic properties of the original free drug. The combined action of two or more drugs or their derivatives can often be more potent than the parent organic drug, besides overcoming drug resistance.

To combat resistance to cisplatin-based chemotherapy in germ cell cancer and other chemo-sensitive tumors, aggressive therapies, such as intensifying the dose of active drugs or adding new drugs, have been introduced. Etoposide, when combined with cisplatin and bleomycin, has shown significant activity in cases of relapse (Hansen et al. 1986). Administering very high doses of etoposide (2400 mg/m2) with autologous bone marrow transplantation has led to remission in certain patients resistant to standard etoposide doses (Wolff et al. 1984). These outcomes prompted the use of etoposide in combination regimens for all types of testicular cancers. Ozols and coworkers implemented an aggressive regimen involving a doubled dose of cisplatin (40 mg/m2 daily × 5) combined with vinblastine, etoposide, and bleomycin (Ozols et al. 1988). However, it remains unclear whether the improvement in the high-dose group could be attributed to increased cisplatin doses or the inclusion of etoposide. On the other hand, Nicholos et al. suggested that doubling the dose of cisplatin increased morbidity without enhancing therapeutic efficacy (Nichols et al. 1991).

8.6 Metal Complex-Biomolecule Conjugation in Targeted Cancer Therapy

A targeted delivery system refers to a molecule (comprising ligands and metal complexes) capable of being recognized by membrane receptors on tumor cells, thereby effectively accumulating in cancerous tissues. This system employs targeting ligands coupled with metals, facilitating the transportation of drugs to specific affected regions or tissues (Singh et al. 2021; Brodyagin et al. 2021). These targeting ligands encompass folic acid, biotin, albumin, and hyaluronic acid. Folic acid, extensively used in cancer diagnosis and treatment, proves advantageous due to the increased expression of folic acid receptors (FARs) by several rapidly growing cancer cells, including those in the ovary, prostate, lung, nose, brain, and colon, while normal cells exhibit minimal FAR expression (Thoreau et al. 2018; Fig. 8.9). Leveraging these specific or overexpressed receptors allows anticancer drugs to be selectively delivered to cancerous cells and tissues.

Fig. 8.9
An illustration depicts the effects of multi-drug combination agents. It includes improving the aqueous solubility of drugs, controlled released drugs, negatively charged metal complexes, and biological activation of metal complexes. The respective effect on therapeutic potency is given alongside.

Mechanism of drug-metal bioconjugate complex and its effect on therapeutic potency

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8.6.1 Folic Acid-Conjugated Metal Complexes

Folates, also known as vitamin B9, play a critical role in fundamental metabolic processes such as RNA and DNA synthesis, crucial for the human genome (Ghosh et al. 2019). Diminished folate levels have been associated with the development of various cancers, including colorectal, lung, and breast cancer. The reduced folate carrier (RFC), an anion exchanger, transports folate through folate receptors (FARs) in an acidic environment, displaying high affinity for folic acid (FA) (Fig. 8.10). FA is transported into cells via endocytosis (Kumar et al. 2019). Due to its stability across a wide range of pH and temperature, FA can serve as a biocompatible targeting motif by being covalently linked to therapeutic agents, rendering FARs a potential target for cancer diagnosis and treatment (Consoli et al. 2015). Subsequent developments introduced lanthanide(III) compounds such as Eu-FA, Eu-MTX, fluorescent Tb-FA, and Tb-MTX probes designed for imaging folate receptors in cancer cells (Du et al. 2018).

Fig. 8.10
An illustration of two starburst shapes representing L n 3 complexes, one labeled with F A for Folic acid and the other with M T X for Methotrexate, along with a legend explaining the abbreviations and indicating L n 3 = E u, T b.

Chemical structure of Ln(III)-FA and Ln(III)-MTX

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Healthy noncancerous cells exhibit limited absorption of the folate and MTX-tethered complex, similar to their limited uptake of these compounds. In contrast, FAR-positive cancer cell lines efficiently absorbed these complexes, suggesting the safe utilization of FA and MTX in studying folate receptor targeting groups. Another intriguing concept arose when human serum albumin (HSA) was linked to FA, mitigating nonspecific damage to normal cells. Researchers developed HSA carriers for Cu(II) complexes by coupling FA with HSA (Gou et al. 2015). This approach revealed a threefold increase in cytotoxicity in cancer cells while exhibiting no toxicity in normal cells.

8.6.2 Albumin-Conjugated Metal Complexes

Cancer cells exhibit different metabolism as compared to normal cells, as they have higher intracellular ROS concentration. Metal-based drugs, due to their redox activity, elevate ROS (reactive oxygen species) levels within cancer cells, consequently inhibiting ROS-detoxifying enzymes such as glutathione. This process represents an enduring mechanism for their anticancer activity (Khan et al. 2019). HSA, being the most prevalent blood plasma protein (Matos et al. 2018; Almi et al. 2020), serves as a carrier for fatty acids, drug metal ions, and metal complexes, particularly binding at tryptophan residue which florescence intensely at 347 nm upon excitation at 295 nm (Pereira et al. 2021). Simunkova et al. reported the cytotoxicity of copper(II) NSAIDs complexes such as flufenamic, mefenamic, tolfenamic, and 1,10-penontheroline complexes (Simunkova et al. 2019). These complexes were found to interact with albumin as targeting biomolecule, sharing high binding affinity constant (K = 106) values. Copper(II) complexes also act as SOD mimics and are good intercalating agents in association with heterocyclic aromatic ligand scaffolds. Therefore, these complexes can execute the cytotoxic potency by multiple targeting methods, including ROS scavenging via hydroxyl radicals, singlet oxygen, and superoxide radicals (Ibrahim et al. 2018); π-π stacking intercalation in between the DNA bases; and other biochemical pathway known specifically for copper metal complexes such as apoptosis.

8.6.3 Biotin-Conjugated Metal Complexes

Biotin emerges as a promising targeting agent. Babak and coworkers developed ruthenium(II)-arene complexes incorporating biotin-containing ligands. Their work showcased that the coordination of ruthenium substantially increased cytotoxicity compared to biotin alone (Babak et al. 2015). In vitro screening of anticancer properties demonstrated that the half-sandwich ruthenium(II)-biotin conjugate acted as a biological vector, specifically accumulating in cancer cells, thereby limiting toxicity. Additionally, it is worth noting that biotin (vitamin H) is essential for cell proliferation, and highly proliferative cancer cells often possess elevated concentrations of biotin receptors and sodium-dependent multivitamin transporters.

Copper compounds have surfaced as a prospective group of anticancer agents (Zehra et al. 2021). Some copper complexes under trade name Casiopeínas have entered phase I clinical trial (Bravo-Gómez et al. 2009). To explain the idea of combination targeted therapy, combination of two compounds, casIII-ia with cisplatin or its analogs, was evaluated in vivo in HeLa cells with the idea of synergistic or superadditive interaction (Davila-Manzanilla et al. 2017). The two combined drugs were exhibiting different mechanisms at the molecular level: while cisplatin binds to N7 of guanine in the DNA helix, CasIII-ia interacted mainly with the phosphate group of the DNA. The observed results reflected that sum of combination effects of these two drugs to the receptor of membrane could have profound autogained and additive effect, reducing the toxicity and exhibiting 90% inhibition of proliferation in HeLa cells.

8.7 Future Prospects

Presently, targeted therapy methods are becoming more favored due to the lack of specificity of many anticancer drugs for tumor cells, leading to substantial toxicity in healthy organs during treatment.

Ru-NO@FA@CDs is known as a nanoplatform for NO delivery, where folic acid, a Ru-NO donor, and ruthenium nitrosyl molecules are attached covalently to the carbon dots (CDs) surface. Using confocal laser microscopy, the specific binding of Ru-NO@FA@CDs to the folate receptor in human cervical cancer cells was observed (Deng et al. 2016). Metal complex bioconjugates utilizing FA, albumin, and biotin are very appealing for the treatment of many cancers limiting the toxicity, providing better pharmacokinetics and pharmacodynamics features such as higher bioavailability in cancer cells.

Therefore, combining new metal-based anticancer compounds with biomolecules that focus on cancer cell metabolic pathways could pave the way for effective and safe cancer treatments.