Therapeutic potential of colchicine-binding site inhibitors in breast cancer brain metastasis

Abstract

Breast cancer brain metastasis (BCBM) represents one of the most aggressive and lethal complications of breast cancer, arising when malignant cells colonize the brain. Patients diagnosed with BCBM face a poor prognosis, with median survival times ranging from 2 to 25.3 months [1], and experience significantly reduced quality of life compared with those patients with extracranial metastases. Current therapeutic strategies, including surgical resection, whole-brain radiation therapy, and stereotactic radiosurgery, offer limited benefit and are rarely curative. Despite advances in breast cancer management, effective treatment for BCBM remains a major clinical challenge, primarily due to the restrictive nature of the blood-brain barrier (BBB), which hinders the delivery and accumulation of most chemotherapeutics within brain tissue. For example, paclitaxel, a widely used microtubule-targeting agent in breast cancer therapy, has limited efficacy against BCBM because it is a substrate of ATP-dependent efflux transporters, particularly P-glycoprotein, at the blood-brain barrier, with its physicochemical properties, such as high molecular weight, further restricting effective intracranial drug exposure. In contrast, emerging evidence suggests that colchicine-binding site inhibitors (CBSIs), a novel class of microtubule-targeting agents, possess the ability to penetrate the BBB, making them promising candidates for the treatment of BCBM. This review highlights recent advances in the development of CBSIs for BCBM and extends the discussion to their evaluation in other brain cancers, such as glioma. Furthermore, we examine strategies to enhance drug delivery across the BBB and to improve therapeutic selectivity toward cancer cells while sparing healthy brain tissue.

1. Introduction

Breast cancer remains the most commonly diagnosed cancer and the leading cause of cancer-related mortality among women worldwide. According to the GLOBOCAN 2022 estimates, breast cancer accounted for approximately 2.3 million new cases and 665,000 deaths globally, surpassing lung cancer as the most frequently diagnosed malignancy in women [2]. In the United States alone, the American Cancer Society projected that in 2025, over 316,000 new cases of invasive breast cancer would be diagnosed in women, with nearly 43,000 deaths [3]. Despite substantial progress in early detection and therapeutic intervention, breast cancer continues to impose a significant global health burden due to its heterogeneity, propensity for metastasis, and endogenous or acquired therapeutic resistance [4].

Breast cancer is not a single disease, but is a biologically and clinically heterogeneous group of malignancies [5]. Classification based on gene expression profiling and immunohistochemical (IHC) markers has led to the identification of at least four intrinsic molecular subtypes based on the presence or absence of the hormone receptors (HR), estrogen receptor (ER), progesterone receptor (PR), and the human epithelial growth factor receptor 2 (HER2): luminal A (ER+/PR+/HER2−), luminal B (ER+/PR+/HER2+), HER2-enriched (± ER or PR; HER2+ or amplified), and triple-negative/basal-like breast cancer (TNBC) (ER−/PR−/HER2−) [6]. Each subtype exhibits distinct molecular characteristics, clinical behaviors, and responses to therapy. While all subtypes are surgically resected when able, they can be treated with targeted therapies depending on their receptors. Luminal A tumors are associated with a more favorable prognosis and responsiveness to endocrine therapy. While luminal B tumors expressing ER ± PR are treated with endocrine therapies, they are also treated with targeted therapy to HER2+ [7]. HER2-enriched tumors are treated with radiation therapy, chemotherapy, and targeted anti-HER2 therapies [8]. In contrast, TNBC lacks expression of all three receptors, and is characterized by aggressive behavior, high rates of recurrence, and limited treatment options [9]. TNBC patients have extremely high metastatic potential, are subject to developing resistance to chemotherapy, and have an overall poor prognosis [4, 10, 11]. TNBC patients are treated with a combination of chemotherapies consisting of taxanes, anthracyclines, and platinum agents along with radiation and surgery [12]. There are currently no first-line targeted treatment options for TNBC due to the lack of hormone receptors. In 2020, an antibody-drug conjugate (ADC) targeting the trophoblast cell-surface antigen 2 (TROP-2) was approved as a second-line therapy for heavily pre-treated TNBC patients [13].

1.1. Chemoresistance in breast cancer

Chemoresistance remains a significant barrier to the effective treatment of breast cancer, particularly in the metastatic setting, where it contributes to disease progression and therapeutic failure. Resistance to chemotherapy can be broadly categorized as intrinsic, where tumor cells possess pre-existing features that confer insensitivity to drugs, or acquired, which arises after repeated exposure to chemotherapeutic agents [14]. Mechanistically, chemoresistance in breast cancer involves a complex interplay of cellular, molecular, and microenvironmental factors. Key contributors include the upregulation of drug efflux transporters such as P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), which actively pump chemotherapeutic agents out of cells such as paclitaxel (Taxol) and vincristine (P-gp) or doxorubicin and SN-38 (BCRP) [15]; alterations in drug targets or DNA repair pathways, such as increased expression of βIII-tubulin or enhanced homologous recombination repair [16, 17]; epithelial-to-mesenchymal transition (EMT), which facilitates phenotypic plasticity and survival [18, 19]; and the presence of cancer stem-like cells (CSCs) that exhibit intrinsic resistance to cytotoxic agents [20, 21]. Furthermore, the tumor microenvironment, including hypoxia, acidic pH, stromal cell interactions, and immune evasion mechanisms, can support a chemoprotective niche [4, 22]. The emergence of multi-drug resistance (MDR) phenotypes not only complicates therapeutic decision-making but also underscores the need for novel agents that can bypass common resistance mechanisms. Current research is focused on overcoming chemoresistance through strategies such as combination therapies, inhibition of resistance-associated pathways, and nanoparticle-mediated drug delivery systems that enhance intratumoral drug accumulation [15].

1.2. Metastatic breast cancer

Metastatic breast cancer (MBC) is defined by the dissemination of malignant cells beyond the primary tumor site to distant organs, including the bones, lungs, liver, and brain [23]. Approximately 20–30% of women diagnosed with early-stage disease will ultimately develop stage IV metastatic disease, which accounts for 90% of cancer-related deaths in breast cancer patients [24, 25]. The metastatic cascade is a complex, multistep process involving local invasion, intravasation into the vasculature or lymphatics, survival in circulation, extravasation into distant tissues, and colonization of secondary sites [26]. Successful metastasis requires cancer cells to acquire invasive properties, evade immune surveillance, and adapt to the microenvironment of distant organs. Epithelial-to-mesenchymal transition (EMT), loss of cell-cell adhesion, increased motility, and remodeling of the extracellular matrix are critical for initial invasion [27–29]. Tumor cells must also overcome anoikis, shear stress, and evade immune cells during hematogenous or lymphatic spread [30, 31]. While MBC is generally considered incurable, systemic therapies can significantly prolong survival and maintain quality of life [32]. Treatment strategies are typically subtype-specific and include endocrine therapy for hormone receptor-positive tumors, HER2-targeted agents for HER2-amplified cancers, and cytotoxic chemotherapy for triple-negative disease [33]. Nevertheless, the prognosis for MBC remains poor, with a 2–4-year median survival time [34].

1.2.1. Bone metastasis

Bone is the most common site of distant metastasis in breast cancer, occurs at higher frequency in women with ER+ tumors than TNBC, and occurs in approximately 70% of total metastatic cases [35, 36]. The bone microenvironment is highly dynamic and characterized by continuous remodeling through the coordinated actions of osteoclasts and osteoblasts [37]. Breast cancer cells exploit this microenvironment by secreting paracrine factors such as parathyroid hormone-related protein (PTHrP), interleukin-11 (IL-11), and transforming growth factor-beta (TGF-β), which promote osteoclast differentiation and bone resorption [35, 38]. This process releases growth factors embedded in the bone matrix, such as insulin-like growth factors (IGFs), that further stimulate tumor growth in a feed-forward loop known as the “vicious cycle” of bone metastasis [38]. Bone-targeted agents such as bisphosphonates (e.g., zoledronic acid) or a RANK ligand inhibitor (e.g., denosumab) are routinely used as anti-resorptive agents to reduce the progression of bone metastasis [39].

1.2.2. Lung metastasis

The lungs are another frequent site of breast cancer metastasis, accounting for approximately 30% of MBC patients [36, 40, 41]. BC patients with lung metastasis have a poor prognosis and a median survival time of 25 months [42]. The premetastatic lung microenvironment is conditioned by tumor-derived exosomes, cytokines, and myeloid-derived suppressor cells (MDSCs) that facilitate the extravasation, survival, and colonization of disseminated tumor cells [41]. In particular, tumor-secreted lysyl oxidase (LOX) has been shown to support lung metastasis development [43]. Lung metastasis is diagnosed mostly frequently in TNBC patients and is associated with high expression of epidermal growth factor receptor (EGFR) [44]. Therapeutic management of lung metastases typically relies on systemic treatment with chemotherapy, targeted agents, or immunotherapy, depending on the molecular subtype [45].

1.2.3. Liver metastasis

The liver is the third most common site of breast cancer metastasis and accounts for approximately 25% of metastatic cases [35, 36, 40]. Liver metastases often indicate an aggressive disease course and are associated with poor prognosis, with median survival ranging from 3 to 15 months depending on response to therapy [46]. The hepatic microenvironment poses unique challenges to metastatic colonization. Kupffer cells (liver-resident macrophages), hepatic stellate cells, and sinusoidal endothelial cells contribute to immune tolerance and may enable immune evasion by disseminated tumor cells [47, 48]. Systemic treatment remains the cornerstone of management. HER2-targeted therapies (e.g., trastuzumab, pertuzumab, T-DXd) have shown efficacy in patients with HER2-positive liver metastases, while a combination of chemotherapy and PARP inhibitors is most efficacious in TNBC [49].

1.2.4. Brain metastasis

Central nervous system (CNS) metastases represent one of the most devastating complications of metastatic breast cancer. BCBM occur in approximately 10–30% of patients with advanced disease and are particularly prevalent in the HER2-positive and TNBC subtypes [50]. The incidence is rising due in part to improved systemic control of extracranial disease and longer patient survival [51]. The brain represents a unique sanctuary site due to the protective function of the BBB, which restricts the entry of most chemotherapeutic and monoclonal antibody therapies, and this challenge is further compounded by the complex, multistep process through which breast cancer cells disseminate and ultimately colonize the central nervous system [52]. After a primary tumor emerges and expands within the breast, malignant cells acquire the capacity to infiltrate surrounding structures, penetrate the vasculature, circulate systemically, and extravasate across the BBB [35]. Upon arrival, these disseminated cells must acclimate to the distinct metabolic and immunological conditions of the neural niche, enabling the establishment of early micrometastases that have the potential to develop into overt brain lesions (Figure 1) [35]. Breast cancer cells that reach the CNS can disrupt the BBB through the secretion of proteolytic enzymes such as matrix metalloproteinases (MMPs), activation of vascular endothelial growth factor (VEGF) pathways, and interactions with astrocytes and microglia that remodel the local microenvironment to facilitate tumor cell survival and proliferation [53, 54]. Once established, brain metastases are often associated with profound neurological symptoms, including headaches, seizures, cognitive dysfunction, and focal neurological deficits, depending on the location of lesions [55]. BCBM treatment usually relies on whole brain radiotherapy, stereotactic radiosurgery, HER2-targeted therapies for HER2-overexpressing brain metastases, and a combination of chemotherapies for TNBC that have adequate BBB penetration, such as capecitabine, platinum compounds, and temozolomide [55].

Figure 1. Breast cancer cells form brain metastasis by invading through the blood-brain barrier.

This schematic illustrates the sequential stages underlying the metastatic cascade from the primary breast tumor to the formation of micrometastatic lesions in the brain. (1) A primary breast tumor arises within the breast tissue and undergoes malignant expansion. (2) Tumor cells acquire invasive properties that allow them to breach the basement membrane, intravasate into nearby blood vessels, and enter the systemic circulation. (3) Circulating tumor cells subsequently invade into the brain through the blood-brain barrier. (4) Once within the brain, disseminated breast cancer cells adapt to the neural microenvironment and establish micrometastases that may later progress to clinically detectable brain metastatic lesions. Created in BioRender.

1.3. Microtubules

Microtubules (MT) are dynamic, cylindrical polymers composed of α- and β-tubulin heterodimers that play a vital role in numerous cellular processes, including intracellular transport, maintenance of cell shape, and mitotic spindle formation during cell division [56]. Their inherent dynamic instability (Figure 2), characterized by alternating phases of polymerization and depolymerization, is tightly regulated during the cell cycle, particularly during mitosis [57]. During the polymerization phase, guanosine triphosphate (GTP) subunits are added to the end of the MT and subsequently hydrolyzed to guanosine diphosphate (GDP) [58]. The leading “GTP cap” is maintained while GTP-bound tubulin dimers are added to the growing microtubule [58]. This cap stabilizes the microtubule by promoting lateral interactions between protofilaments and maintaining a straight conformation conducive to further polymerization [59]. MT depolymerization, or “catastrophe,” occurs when the GDP-tubulin subunits are released from the end of the MT at a very rapid rate [60]. Conversely, during MT shrinkage, it can undergo a “rescue” event in which the MT transitions back to the growth phase [60]. Given their essential role in chromosomal segregation and various cellular processes, microtubules are a well-validated therapeutic target in cancer, which is characterized by the hallmarks of aberrant mitotic activity and genomic instability [61].

Figure 2. Microtubule dynamics.

This schematic depicts the fundamental processes that govern microtubule growth and shrinkage. During polymerization, GTP-bound tubulin dimers are added to the plus end of the microtubule, forming a stabilizing GTP cap that supports continued elongation. Over time, incorporated GTP is hydrolyzed to GDP, generating a GDP-tubulin core that is intrinsically less stable. Loss of the GTP cap triggers catastrophe, initiating rapid depolymerization in which GDP-tubulin dimers peel away from the microtubule lattice. Conversely, the transition from shrinkage back to growth, termed rescue, occurs when conditions favor re-establishment of the GTP cap and renewed polymerization. Together, these alternating phases of growth and shrinkage characterize microtubule dynamic instability, a process essential for cytoskeletal function, mitotic spindle assembly, and intracellular trafficking. Created in BioRender.

1.3.1. Microtubule Inhibitors

Tubulin inhibitors exert their antitumor activity by disrupting microtubule dynamics, thereby interfering with mitotic spindle assembly and inducing mitotic arrest, which ultimately leads to apoptosis [61]. These agents can be broadly classified into microtubule-stabilizing agents (e.g., taxanes) and microtubule-destabilizing agents (e.g., vinca alkaloids and colchicine site-binding agents) [62]. There are seven known binding sites on the microtubule structure, taxane site, vinca site, colchicine site, maytansine site, laulimalide site, pironetin site, and gatorbulin site [63].

Taxanes, such as paclitaxel and docetaxel, bind to the taxane-binding site on β-tubulin and promote microtubule polymerization while preventing depolymerization, resulting in hyperstabilized, nonfunctional microtubules [64]. This stabilization impairs the dynamic reorganization of the microtubule network required for spindle formation and chromosome congression, which is critical for the metaphase-to-anaphase transition in the cell cycle [65]. Cells treated with taxanes typically exhibit prolonged mitotic arrest in the G₂/M phase of the cell cycle, followed by apoptosis [66]. In addition to their antimitotic effects, taxanes exert cytotoxic effects through additional pathways, such as the disruption of intracellular trafficking and cell migration [67, 68]. These additional functions of tubulin also contribute to the therapeutic efficacy and adverse outcomes of taxanes, including peripheral neuropathy, which is a major dose-limiting toxicity for this class of tubulin inhibitors [69].

In contrast, microtubule-destabilizing agents inhibit polymerization by binding to distinct sites on tubulin, including the vinca domain and the colchicine-binding site [70–72]. These agents disrupt mitotic spindle formation, trigger the spindle assembly checkpoint, and lead to mitotic catastrophe [73]. Vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine) bind to the β-tubulin subunit on the vinca domain at the interface of the two tubulin dimers [72, 74, 75]. This interaction disrupts the contact required for tubulin elongation, thereby promoting microtubule disassembly [72]. Vinca alkaloids cause metaphase arrest and are widely used in the treatment of hematological malignancies and solid tumors [75].

Colchicine-binding site inhibitors (CBSIs) represent a versatile group of agents that bind to the colchicine-binding site located at the interface of α- and β-tubulin [74]. Classical CBSIs such as colchicine, combretastatin A-4 (CA4), and podophyllotoxin, induce depolymerization by inducing a curved conformation in tubulin dimers, thereby preventing their incorporation into growing microtubules [74, 76]. More recently, a variety of synthetic small-molecule CBSIs, including chalcones, stilbenes, benzimidazoles, and indole derivatives, have been developed to exploit this binding site for anti-cancer and anti-angiogenic purposes [77]. Unlike taxanes and vinca alkaloids, CBSIs often exhibit favorable pharmacological profiles such as lower susceptibility to MDR transporters, and a comparatively lower incidence and severity of peripheral neuropathy, although neurotoxicity can still occur depending on the specific compound and dosing regimen [72, 78–80]. Despite their clinical efficacy, the therapeutic potential of classical tubulin inhibitors is often limited by resistance mechanisms, dose-limiting toxicities (notably neurotoxicity), and poor blood-brain barrier (BBB) penetration [62, 81].

1.4. CBSIs in Overcoming Chemoresistance

CBSIs represent a promising class of tubulin-targeting agents with unique structural and functional properties that may circumvent some limitations of traditional agents. Unlike taxanes and vinca alkaloids, CBSIs bind at the interface between α- and β-tubulin heterodimers and prevent microtubule polymerization at an early stage, leading to depolymerization and mitotic arrest [76]. CBSIs are highly potent against taxane-resistant cancer models, in part due to their reduced susceptibility to common resistance mechanisms such as P-glycoprotein-mediated efflux and alterations in β-tubulin isotypes, including βIII-tubulin, which is commonly overexpressed in aggressive breast cancers [82, 83]. Furthermore, CBSIs may disrupt tumor vasculature through anti-vascular mechanisms, leading to ischemic necrosis within the tumor core [84, 85]. These characteristics make CBSIs attractive candidates for overcoming MDR, particularly in TNBC, where limited therapeutic options and high recurrence rates remain clinical challenges [86]. The ability of CBSIs to act through mechanisms distinct from other microtubule inhibitors may provide a therapeutic advantage.

In addition to direct tubulin-targeting agents, recent therapeutic strategies have explored dual-targeting approaches that simultaneously disrupt microtubule dynamics and inhibit complementary oncogenic pathways. These bifunctional or multitarget compounds aim to enhance antitumor activity by addressing mechanisms that contribute to therapeutic resistance. For example, tubulin/histone deacetylase (HDAC) inhibitors combine microtubule disruption with epigenetic modulation, potentially enhancing apoptosis and impairing tumor cell survival [87, 88]. Similarly, tubulin/Src inhibitors target both cytoskeletal regulation and Src-mediated signaling pathways involved in tumor proliferation, invasion, and metastasis [89, 90]. Tubulin/poly(ADP-ribose) polymerase (PARP) inhibitors represent another approach that integrates mitotic disruption with impaired DNA damage repair, providing potential opportunities for improved efficacy in tumors with defective DNA repair pathways [91, 92].

1.5. CBSIs Evaluated in Preclinical Breast Cancer Brain Metastasis Models

One of the major limitations of conventional tubulin-targeting agents is their poor ability to penetrate the BBB, restricting their use in the treatment of CNS metastases, which are increasingly common in HER2-positive and TNBC breast cancer subtypes [70]. The BBB is a selectively permeable interface composed of tightly joined endothelial cells joined by tight junctions, pericytes, astrocytes, and a basal lamina [93, 94]. This complex structure restricts the translocation of therapeutic agents into the brain parenchyma, particularly those that are hydrophilic or substrates of efflux transporters, such as P-gp and breast cancer resistance protein (BCRP) [95, 96]. Paclitaxel, although a well-known and effective chemotherapy in metastatic breast cancer, is largely ineffective in CNS metastases due to poor BBB permeability and active efflux by P-gp [97]. However, in late-stage disease, where the BBB becomes structurally compromised and evolves into a heterogeneous blood-tumor barrier (BTB), paclitaxel may exert limited efficacy in a subset of brain metastases. In such cases, sufficient drug accumulation and apoptotic response have been observed in approximately 10% of lesions with the highest permeability, suggesting that paclitaxel’s therapeutic impact is restricted to metastases with significantly disrupted BBB integrity [98]. Notably, several CBSIs have been identified that are capable of crossing the BBB (Figure 3). These structural changes have focused on enhancing lipophilicity, reducing molecular weight, and reducing substrate affinity for efflux transporters [99]. Preclinical pharmacokinetic studies have identified CBSIs with promising brain-to-plasma (B/P) ratios that reach therapeutic concentrations, thus supporting their potential as therapeutic agents for brain metastases [100, 101].

Figure 3.

Chemical structures of representative CBSIs investigated for central nervous system delivery, blood-brain barrier penetration, or intracranial tumor therapy.

Although numerous CBSIs have demonstrated the ability to penetrate the BBB, only a limited number have been evaluated in bona fide preclinical BCBM models. To date, SB-216 and SP-1-39 are the only CBSIs with published in vivo efficacy data in experimental BCBM models. The remaining compounds discussed below have primarily been investigated in glioblastoma multiforme (GBM), other primary brain tumor models, or pharmacokinetic studies demonstrating BBB penetration. These agents are included because they provide valuable mechanistic and medicinal chemistry precedents for the development of next-generation brain-penetrant CBSIs for metastatic breast cancer.

Throughout this review, the reported B/P ratios represent total drug concentrations measured in brain tissue and plasma, as reported in the original studies. Although total B/P is a useful indicator of CNS distribution, it may overestimate pharmacologically relevant brain exposure because it includes drug that is nonspecifically bound to brain tissue. Consequently, high total B/P values do not necessarily indicate that therapeutically active unbound drug concentrations are achieved within the brain. The unbound brain-to-plasma partition coefficient (K_p,_uu) is increasingly recognized as the preferred pharmacokinetic metric for assessing BBB penetration because it reflects the equilibrium distribution of free drug across the BBB [102]. Unfortunately, K_p,_uu measurements were not reported for the CBSIs discussed in this review; therefore, comparisons are necessarily based on total B/P ratios together with demonstrated in vivo efficacy in intracranial tumor models.

1.5.1. SB-216

SB-216 is a novel CBSI derived from the structural scaffold of Azixa (discussed in section 1.6.1) and demonstrates robust anti-cancer activity, including efficacy in a paclitaxel-resistant melanoma in vivo model (compound 5m) [103]. Notably, SB-216 exhibits strong BBB permeability, achieving total B/P ratios of 20, 42, 67, and 20 at 0.5, 1, 2, and 8 hours post-administration, respectively [101]. In an intracardiac injection model using MDA-MB-231 BrM2 cells, intravenous (IV) administration of SB-216 at a low dose of 1 mg/kg twice weekly reduced brain metastatic burden by 2.88-fold and extended median overall survival from 28 to 35.5 days relative to vehicle-treated mice in a separate survival study [101]. In a pre-existing metastatic model in which treatment began five days after intracardiac injection, SB-216 similarly decreased brain metastasis by 3.1-fold, with additional reductions observed across major organs including the lungs, liver, spleen, kidneys, and bones [101]. A minimal effective dose study further demonstrated that twice-weekly IV administration of 0.8 mg/kg, but not 0.64 mg/kg, significantly inhibited brain metastatic colonization, establishing 0.8 mg/kg as the lowest effective therapeutic dose for this compound.

1.5.2. SP-1-39

SP-1-39 is a CBSI developed from the structural scaffold of SB-216 and has demonstrated strong activity in a paclitaxel-resistant prostate cancer model (compound 12k) [104]. Although the structural modifications incorporated into SP-1-39 did not enhance BBB permeability relative to SB-216, the compound nevertheless exhibits substantial BBB penetration, achieving total B/P ratios of 9, 23, 24, and 38 at 0.5, 1, 2, and 8 hours post-injection, respectively [101]. To evaluate its therapeutic potential in metastatic disease, mice were inoculated via intracardiac injection with the paclitaxel-refractory patient-derived xenograft (PDX) model HCI-10-Luc2. Due to the slow-growing nature of this PDX line, dosing commenced five weeks after intracardiac injection. Treatment with SP-1-39 at 5 mg/kg administered intraperitoneally three times per week resulted in a 6.11-fold reduction in brain metastatic burden relative to vehicle-treated controls, with minimal associated body weight loss. Additionally, SP-1-39 markedly diminished metastatic dissemination to major organs, including the lungs, bones, liver, spleen, and kidneys, underscoring its broad anti-metastatic efficacy [101].

1.6. CBSIs Evaluated in Glioma and Other CNS Tumor Models

The following CBSIs have demonstrated BBB penetration or antitumor activity in glioma or other CNS tumor models but have not yet been evaluated in published BCBM models. Although these studies provide valuable medicinal chemistry and pharmacokinetic precedents, their interpretation should be tempered by the fact that many orthotopic glioma models develop a partially disrupted BBB, which may permit greater drug penetration than occurs during the early stages of breast cancer brain metastasis. Consequently, efficacy observed in glioma models should not be considered equivalent to efficacy in clinically relevant BCBM models with a relatively intact BBB.

1.6.1. Azixa

Azixa (MPC-6827, verubulin) has emerged as a promising CBSI and vascular-disrupting agent with the notable ability to effectively penetrate the BBB, exhibiting a B/P ratio of approximately 14 in murine models [105, 106]. In preclinical studies across a range of solid tumor types, including melanoma, breast, and pancreatic cancers, Azixa demonstrated significant antitumor efficacy by reducing primary tumor size and growth rate [107]. However, despite its favorable pharmacokinetic (PK) profile and CNS penetration, clinical outcomes were less encouraging. A Phase II clinical trial (NCT00892931) investigating Azixa in patients with recurrent GBM, either as a monotherapy or in combination with bevacizumab, revealed limited overall response rates. These results ultimately led to the discontinuation of Azixa’s clinical development for oncology [108]. More recently, Azixa has been repurposed as a BBB-permeable radiotracer for positron emission tomography (PET) imaging in the brain, highlighting its potential utility in neuroimaging applications [105, 109].

1.6.2. RGN3067

RGN3067 is a CBSI developed as a brain-penetrant microtubule targeting agent (MTA) for GBM by optimizing physiochemical properties of parent molecules by reducing molecular weight and polar surface area and improving multiparametric optimization (MPO) score [110]. In preclinical PK studies, RGN3067 demonstrated equivalent distribution between the plasma and brain at 1, 4, and 8 hours following oral administration of 100 mg/kg, indicating favorable brain exposure. Moreover, RGN3067 is not a substrate for P-gp, as evidenced by its potency in a MDR1-MDCK cell permeability model. In vitro, RGN3067 exhibited potent anti-proliferative effects across multiple GBM cell lines, including U87 and the temozolomide (TMZ)-resistant LN-18 cells. In vivo efficacy studies using a PDX model further demonstrated that RGN3067 significantly inhibited tumor growth without inducing observable toxicity, highlighting both its therapeutic promise and favorable safety profile [110]. However, it is important to note that efficacy studies to date have been conducted in subcutaneous tumor models. Thus, despite its favorable PK profile, it remains to be definitively demonstrated whether RGN3067 achieves sufficient brain penetration to exert therapeutic effects in orthotopic brain tumor models.

1.6.3. ST-401

ST-401 is a brain-penetrant CBSI that has been investigated in glioma models [100]. Preclinical PK studies in mice demonstrated a half-life of approximately 6 hours and a B/P ratio of 2.2 following a single intraperitoneal dose of 150 mg/kg in an RCAS/tv-a-driven PDGFB expressing glioma mouse model. In this model, combination treatment with ST-401 and either TMZ or radiation therapy significantly enhanced therapeutic efficacy, increasing survival by 60 and 66%, respectively. In contrast, ST-401 monotherapy did not confer a statistically significant survival benefit [100].

1.6.4. AG119

AG119 is a rationally designed small molecule with dual functionality, targeting both angiogenesis and microtubule dynamics [111, 112]. In an orthotopic high-grade glioma mouse model, intraperitoneal administration of AG119 at 30 mg/kg twice weekly for two weeks significantly prolonged survival, with a median of 29 days compared to 20 days in vehicle-treated controls [113]. However, the survival benefit observed with AG119 was less pronounced than that achieved with TMZ, which extended median survival to 34 days. Importantly, the authors note that glioma models frequently exhibit a compromised or “leaky” BBB, which may enhance the penetration of therapeutic agents that otherwise display limited permeability in models with an intact BBB [113, 114]. In the absence of a proper PK study to determine the BBB permeability of AG119, it remains speculative to definitively state that AG119 crosses a fully intact BBB; therefore, further studies are necessary to establish it as a brain-penetrant CBSI.

1.6.5. ST-11

ST-11 was developed as a candidate for the treatment of human GBM through the screening of a library of analogs. In vitro, ST-11 exhibited an EC₅₀ of 2.4–8.6 μM across multiple GBM cell lines, without affecting the viability of normal mouse astrocytes or neurons, suggesting selective cytotoxicity toward cancer cells [115]. PK studies using a 40 mg/kg dose given intraperitoneally revealed a high amount of ST-11 in the brain tissue with a C_max of 8.7 ± 0.9 μM after 60 minutes. ST-11 remained above its EC₅₀ for up to 8 hours post-injection. For in vivo evaluation, mice were orthotopically injected with DBT glioblastoma cells and, one week later, received daily intraperitoneal injections of ST-11 at doses of 5, 15, or 40 mg/kg for two weeks. The results demonstrated a dose-dependent reduction in tumor volume, with statistical significance observed only at the 40 mg/kg dose.

1.6.6. 6-MOMIPP

6-MOMIPP is a novel indole-based chalcone CBSI that has been shown to penetrate the BBB for the treatment of glioblastoma. A preliminary PK study in mice, using a single intraperitoneal dose of 20 mg/kg, demonstrated that 6-MOMIPP crosses the BBB and accumulates in brain tissue, achieving a B/P ratio of approximately 1.0 [116]. For in vivo efficacy, mice were orthotopically inoculated with U251-Luc glioblastoma cells and treated with intraperitoneal injections of 20 mg/kg 6-MOMIPP twice daily for 12 days. Treatment with 6-MOMIPP resulted in significant reduction in tumor size compared to vehicle-treated controls.

1.6.7. 6t

Compound 6t is part of a series of (E)-N-aryl-2-arylethenesulfonamides developed to generate novel microtubule inhibitors with potential BBB penetration [117]. Although in vivo studies were not conducted to directly assess BBB permeability, an in vitro assay using MDCK-MDR1 cells, a widely accepted model for evaluating BBB transport, was performed [118]. The results indicated that compound 6t is not a subject to active efflux, suggesting that it may have favorable properties for brain penetration and could be a promising candidate for the treatment of brain tumors and metastases.

1.6.8. Plinabulin

Plinabulin (BPI-2358) is a synthetic analog derived from an Aspergillus metabolite and functions as a potent CBSI. It is currently under investigation in phase III clinical trials [89]. In a PK study, Sprague-Dawley rats were administered 5 mg/kg plinabulin intravenously, and concentrations were measured in plasma, cerebral cortex, cerebellum, and spinal cord at multiple time points. These findings confirmed that plinabulin crosses the BBB [119]. Building on this observation, investigators evaluated plinabulin in a glioma model, where mice were treated intraperitoneally with 7.5 mg/kg daily for five consecutive days. Treatment significantly prolonged survival compared with vehicle-treated controls [119].

1.6.9. PTC596

In an orthotopic U-87 human GBM model, PTC596 was administered orally at 20 mg/kg once weekly. Treatment extended the median survival of tumor-bearing mice to 20 days compared with 15 days in vehicle-treated controls, representing a significant 33% increase in lifespan [120]. Although PTC596 has been reported to cross the BBB, a definitive study directly confirming this could not be identified. Nonetheless, in vivo efficacy studies suggest that PTC596 achieves sufficient brain penetration to exert therapeutic activity [120].

1.6.10. 4AZA2891

A library of compounds was screened for anti-proliferative and clonogenic activity, leading to the identification of a 3-nitropyridine derivative with potent activity against HT-29 colorectal adenocarcinoma cells. 4AZA2891 was subsequently evaluated in CD1 mice, where intravenous administration at 20 mg/kg demonstrated effective distribution to the brain, achieving a B/P ratio of 2.7 [121]. Although these pharmacokinetic findings are encouraging for potential application in CNS malignancies, no published studies to date have reported on the therapeutic efficacy of 4AZA2891 within the brain.

Overall, BBB-penetrant CBSIs represent a promising class of therapeutics for addressing CNS-resident tumor cells and preventing metastatic colonization in the brain. By combining their ability to disrupt microtubule dynamics with demonstrated brain distribution, these agents have the potential to overcome the sanctuary site limitations imposed by the BBB. Preclinical models, including those of glioblastoma and breast cancer brain metastases, suggest that CBSIs can induce mitotic arrest, reduce tumor burden, and extend survival. However, while these findings underscore their translational promise, additional pharmacokinetic and efficacy studies, particularly in the context of more clinically relevant brain metastasis pre-clinical models, are essential to fully establish their therapeutic utility. Table 1 highlights that, despite encouraging brain exposure for many CBSIs, only SB-216 and SP-1-39 have been evaluated in bona fide breast cancer brain metastasis models. Most remaining compounds have been tested exclusively in glioma models or have only pharmacokinetic evidence of brain penetration, emphasizing the need for validation in clinically relevant BCBM systems.

Table 1.

Summary of brain-penetrating CBSIs

Compound	Brain-exposure metric	Primary in vivo model	Key efficacy finding	Evidence from BCBM model?
SB-216	B/P ratios 20, 42, 67, 20 (0.5, 1, 2, 8 h)	Intracardiac MDA-MB-231 BrM2 BCBM	Reduced brain metastatic burden 2.9–3.1-fold and prolonged survival	Yes
SP-1-39	B/P ratios 9, 23, 24, 38 (0.5, 1, 2, 8 h)	Intracardiac HCI-10-Luc2 PDX BCBM model	6.1-fold reduction in brain metastases with decreased systemic metastasis	Yes
Azixa	B/P ratio ~14	Multiple solid tumor models; glioblastoma clinical studies	Antitumor activity preclinically; limited efficacy in GBM clinical trial	No
RGN3067	B/P ratio ~1	Subcutaneous glioblastoma PDX	Significant tumor growth inhibition	No
ST-401	B/P ratio 2.2	Orthotopic glioma	Combination with TMZ or radiation increased survival; monotherapy inactive	No
AG119	No definitive BBB PK study	Orthotopic glioma	Prolonged survival versus vehicle	No
ST-11	Brain Cmax 8.7 μM; maintained above EC50 for ~8 h	Orthotopic glioblastoma	Dose-dependent reduction in tumor volume	No
6-MOMIPP	B/P ratio ~1.0	Orthotopic glioblastoma	Significant reduction in tumor size	No
6t	No in vivo PK; MDR1-MDCK suggests low efflux	None	No in vivo efficacy reported	No
Plinabulin	BBB penetration in rats	Orthotopic glioma	Prolonged survival	No
PTC596	BBB penetration inferred; no definitive PK study	Orthotopic glioblastoma	33% increase in median survival	No
4AZA2891	B/P ratio 2.7	PK study only	Brain distribution demonstrated; no intracranial efficacy reported	No

1.7. Future directions and limitations

While this review primarily focused on CBSIs investigated in the context of BCBM, several novel BBB-penetrant CBSIs were also highlighted due to their promising potential in future therapeutic options for brain tumors. Continued progress in this field will require medicinal chemistry-based optimization of CBSI scaffolds to enhance target selectivity, improve pharmacokinetic profiles, and minimize off-target toxicities. An additional strategy involves the incorporation of cellular targeting moieties, such as Angiopep-2, to facilitate selective delivery to tumors and metastatic sites. Angiopep-2, a peptide for the low-density lipoprotein receptor-related protein-1 (LRP1), is abundantly expressed at the BBB and in multiple tumor types, including brain metastases [122]. Conjugating CBSIs with Angiopep-2 could leverage receptor-mediated transcytosis to increase drug accumulation in intracranial tumors. This approach has already shown translational promise in other CNS-directed therapies (e.g., ANG1005) [122] and may substantially improve the therapeutic index of CBSIs for BCBM and other CNS malignancies.

Despite these encouraging advances, important limitations remain. Many studies evaluating BBB-penetrant CBSIs have been conducted in glioblastoma models, which are limited by the methodology of localization to the site of injection, rather than development of micrometastasis through the brain. Because they quickly generate a large tumor mass, causing edema, intracranial GBM models often exhibit a disrupted or “leaky” BBB, potentially overestimating drug penetration compared to metastatic models with an intact barrier [123, 124]. For this reason, intracardiac injection models that seed micrometastases to the brain from brainenriched tumor types, including lung cancer, melanomas and breast cancers, may be more useful for testing metastatic progression in the brain over a longer period [125, 126]. Furthermore, while pharmacokinetic data demonstrate brain distribution for several CBSIs, definitive evidence linking intracranial drug concentrations to therapeutic efficacy in BCBM remains lacking for most reported compounds. Additional challenges include the potential for neurotoxicity with long-term microtubule inhibition, an incomplete understanding of CBSI interactions with efflux transporters, and the scarcity of clinically relevant preclinical models of BCBM that metastasize to the brain from the mammary gland (the orthotopic site).

Future research should prioritize rigorous pharmacokinetic and pharmacodynamic studies, including orthotopic BCBM models, to better define the therapeutic window and minimal effective doses of CBSIs. Integration of rational drug design, targeted delivery strategies, and advanced pre-clinical model systems will be critical to translating BBB-penetrant CBSIs into effective clinical options for patients with brain metastases.

Significance.

Breast cancer brain metastasis (BCBM) remains a major clinical challenge due to the restrictive blood-brain barrier (BBB), which limits the efficacy of most chemotherapies. Novel colchicine-binding site inhibitors (CBSIs) show promise by penetrating the brain and targeting microtubules, offering new therapeutic potential. This review highlights recent advances in CBSIs for BCBM and related brain cancers, while also discussing strategies to enhance drug delivery and improve treatment selectivity for cancer cells over healthy brain tissue.

1. Current chemotherapies are limited by their efficacy in treating BCBM.

2. Examines CBSIs as promising brain-penetrant microtubule-targeting therapeutic.

3. Highlights strategies to enhance drug delivery across the blood-brain barrier.