*Review* **Modulation of the Blood–Brain Barrier for Drug Delivery to Brain**

**Liang Han**

Jiangsu Key Laboratory of Neuropsychiatric Diseases Research, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China; hanliang@suda.edu.cn

**Abstract:** The blood–brain barrier (BBB) precisely controls brain microenvironment and neural activity by regulating substance transport into and out of the brain. However, it severely hinders drug entry into the brain, and the efficiency of various systemic therapies against brain diseases. Modulation of the BBB via opening tight junctions, inhibiting active efflux and/or enhancing transcytosis, possesses the potential to increase BBB permeability and improve intracranial drug concentrations and systemic therapeutic efficiency. Various strategies of BBB modulation have been reported and investigated preclinically and/or clinically. This review describes conventional and emerging BBB modulation strategies and related mechanisms, and safety issues according to BBB structures and functions, to try to give more promising directions for designing more reasonable preclinical and clinical studies.

**Keywords:** blood–brain barrier modulation; tight junction; active efflux; transcytosis; drug delivery

#### **1. Introduction**

The blood–brain barrier (BBB) plays a crucial protective role in maintaining a highly precise brain microenvironment for neuronal activity by regulating material transport into and out of the brain. The structural bases of the BBB (Figure 1) are brain capillary endothelial cells with tight junctions, active efflux transporters, and major facilitator superfamily domain-containing protein 2a (Mfsd2a), which jointly endow the BBB with extremely low both paracellular permeability and transcellular permeability [1]. Tight junctions seal endothelial paracellular gaps, leading to high trans-endothelial electrical resistance and limited paracellular transport. Transmembrane tight junction proteins include claudins, occludin, and junctional adhesion molecules, which all attach to intracellular actin cytoskeleton by membrane-associated proteins (e.g., zonula occludins-1). Highly expressed active efflux transporters include P-glycoprotein (Pgp), breast cancer resistant protein (BCRP), and multidrug-resistance proteins (MRPs). Mfsd2a mediates unique BBB endothelial lipid composition via transporting lysophosphatidylcholine esterified docosahexaenoic acid to BBB endothelial cells, to limit formation of caveolae-mediated transcytotic vesicles [2–4]. In addition, endothelial cells, pericytes, and astrocytes jointly form the neurovascular unit (Figure 1), and regulate the development and function of the BBB microcirculation by interacting with each other via secreting several factors [5–7]. These above properties cause the BBB to be constantly and dynamically modulated by both physiological and pathological factors [8,9].

Despite its protective function, the BBB blocks the entry of therapeutic substances into the brain. Although various brain diseases can lead to BBB breakdown with impaired structure and increased permeability [8], BBB around lesion margins or after repairing (e.g., Pgp upregulation in epilepsy and brain tumor) can still block drug delivery to the brain [9–12]. Therefore, systemic drug therapy for brain diseases is severely limited by the BBB. BBB modulation contributes to an increased drug concentration in the brain, and thus increases the efficiency of various systemic therapies [13]. Crucial proteins and structures in

**Citation:** Han, L. Modulation of the Blood–Brain Barrier for Drug Delivery to Brain. *Pharmaceutics* **2021**, *13*, 2024. https://doi.org/10.3390/ pharmaceutics13122024

Academic Editors: Yuan Huang, Jingyuan Wen and Ruggero Bettini

Received: 23 October 2021 Accepted: 25 November 2021 Published: 27 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

formation and regulation of BBB and their changes in brain diseases have been selectively regulated to improve drug delivery for systemic therapies against various brain diseases. tures in formation and regulation of BBB and their changes in brain diseases have been selectively regulated to improve drug delivery for systemic therapies against various brain diseases.

BBB. BBB modulation contributes to an increased drug concentration in the brain, and thus increases the efficiency of various systemic therapies [13]. Crucial proteins and struc-

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 2 of 19

**Figure 1.** The neurovascular unit (**left**) and the mechanisms of transport inhibition by the BBB (**right**). **Figure 1.** The neurovascular unit (**left**) and the mechanisms of transport inhibition by the BBB (**right**).

This review describes various conventional and emerging strategies for BBB modulation that increase both paracellular permeability and transcellular permeability of the BBB, and classifies these strategies according to BBB structures and functions including tight junctions, active efflux, and low transcytosis (Table 1). Furthermore, mechanisms responsible for increased BBB permeability and safe issues related to various strategies are also discussed, to try to give more promising directions for designing more reasonable preclinical and clinical studies. This review describes various conventional and emerging strategies for BBB modulation that increase both paracellular permeability and transcellular permeability of the BBB, and classifies these strategies according to BBB structures and functions including tight junctions, active efflux, and low transcytosis (Table 1). Furthermore, mechanisms responsible for increased BBB permeability and safe issues related to various strategies are also discussed, to try to give more promising directions for designing more reasonable preclinical and clinical studies.


**Table 1.** BBB regulation strategies and related advantages and disadvantages. **Table 1.** BBB regulation strategies and related advantages and disadvantages.

#### **2. Modulation of Tight Junctions** This strategy has been translated into the clinic to increase chemotherapy efficiency for

*2.1. Osmotic BBB Disruption* 

Opening BBB tight junctions is supposed to increase paracellular BBB permeability and facilitate paracellular drug transport into the brain [14]. Ideally, tight junction opening should be transient and selective in a controlled manner to prevent unwanted accumulation (and toxicity) in the brain, and also avoid any short- or long-term peripheral side effects [15]. Various tight junction opening strategies have been reported with robust both preclinical and clinical performance (Figure 2). However, concerns of causing severe toxicity constantly exist, because the non-specific accumulation of neurotoxic blood components may induce neuronal degenerative changes and even cognitive impairments [16–18]. Various reported strategies are discussed here, which may help to promote the emergence of highly efficient approaches with minimal side effects. brain tumors, and the tight junction opening window by osmotic BBB disruption can last for hours in humans [21]. Other hyperosmotic agents that transiently open tight junctions also include arabinose, lactamide, saline, urea, and radiographic contrast agents [15]. Osmotic BBB disruption is generally nonselective with uncontrolled flow into whole brain regions, such as neurotoxic blood components (e.g., albumin), leading to edema, neurological toxicity, epilepsy, aphasia, and hemiparesis [15,22–24]. In addition, the invasive nature and general anesthesia render the technique impractical for drug therapy against chronic brain diseases [14]. Therefore, the use of osmotic BBB disruption is confined to only clinical management of brain tumors.

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 3 of 19

Upregulation of

gence of highly efficient approaches with minimal side effects.

**2. Modulation of Tight Junctions** 

Transcytosis Upregulation of LRP1 Drug-specific Slow and possible LRP1-asso-

Opening BBB tight junctions is supposed to increase paracellular BBB permeability

Intra-arterial infusion of 25% hyperosmotic mannitol into the carotid or vertebral artery can induce vasodilation, endothelial cell shrinkage, and subsequent tight junction loosening and separation, leading to transient and reversible BBB disruption [15,19]. While conventional intra-arterial administration increases drug exposure of brain tumors 10-fold, osmotic BBB disruption can further increase drug exposure by up to 100-fold [20].

and facilitate paracellular drug transport into the brain [14]. Ideally, tight junction opening should be transient and selective in a controlled manner to prevent unwanted accumulation (and toxicity) in the brain, and also avoid any short- or long-term peripheral side effects [15]. Various tight junction opening strategies have been reported with robust both preclinical and clinical performance (Figure 2). However, concerns of causing severe toxicity constantly exist, because the non-specific accumulation of neurotoxic blood components may induce neuronal degenerative changes and even cognitive impairments [16– 18]. Various reported strategies are discussed here, which may help to promote the emer-

versible

GLUT1 Efficient Fasting-associated poor com-

Inhibition of Mfsd2a Transient and re-

ciated side effects

Possible Mfsd2a-associated side effects

pliance

**Figure 2.** The BBB tight junctions and typical modulation strategies. **Figure 2.** The BBB tight junctions and typical modulation strategies.

#### *2.1. Osmotic BBB Disruption*

 Intra-arterial infusion of 25% hyperosmotic mannitol into the carotid or vertebral artery can induce vasodilation, endothelial cell shrinkage, and subsequent tight junction loosening and separation, leading to transient and reversible BBB disruption [15,19]. While conventional intra-arterial administration increases drug exposure of brain tumors 10-fold, osmotic BBB disruption can further increase drug exposure by up to 100-fold [20]. This strategy has been translated into the clinic to increase chemotherapy efficiency for brain tumors, and the tight junction opening window by osmotic BBB disruption can last for hours in humans [21]. Other hyperosmotic agents that transiently open tight junctions also include arabinose, lactamide, saline, urea, and radiographic contrast agents [15]. Osmotic BBB disruption is generally nonselective with uncontrolled flow into whole brain regions, such as neurotoxic blood components (e.g., albumin), leading to edema, neurological toxicity, epilepsy, aphasia, and hemiparesis [15,22–24]. In addition, the invasive nature and general anesthesia render the technique impractical for drug therapy against chronic brain diseases [14]. Therefore, the use of osmotic BBB disruption is confined to only clinical management of brain tumors.

#### *2.2. Radiation-Mediated BBB Disruption*

Radiation cannot only induce tumor cell apoptosis, but also disrupt the BBB [7,18,25–31]. Although the underlying mechanisms are still uncertain, BBB disruption induced by radiation leads to enhanced paracellular diffusion and transcellular transport [7]. Radiation therapy has been combined with systemic therapies to treat brain tumors. Although some study suggests that radiation failed to increase intracranial drug concentrations, increased gefitinib concentration in cerebrospinal fluid was shown with escalating radiation dose in patients with brain metastases in clinical trials [32,33]. Therefore, further research is needed to verify whether enhanced drug delivery to the brain can indeed occur after

radiation and whether it is based on the effects on the BBB [34]. It has been reported that the disrupted BBB by radiation needs hours to years to recover [27]. Therefore, irradiation involves acute, subacute, and chronic dose-dependent toxicity [26,27]. For example, vasogenic edema from vascular damage causes early radiation toxicity syndrome including headache, nausea, or neurologic deficits [18]. Subacute side effects may appear around six months post radiation and progress into chronic dysfunction. Chronic side effects include radiation-induced necrosis, demyelination, leukoencephalopathy, cerebral atrophy, and neurocognitive deficits, and so on [35,36]. Stereotaxic radiosurgery may be an alternative approach to reduce radiation-related intracranial side effects and simultaneously maintain the BBB disrupting effects.

#### *2.3. Activating Bradykinin B2 Receptor*

Bradykinin B2 receptor is constitutively expressed on BBB endothelial cells. Its stimulation can rapidly and transiently disengage tight junctions and increase BBB permeability [37]. The expression of bradykinin B2 receptor is upregulated in the blood–tumor barrier (BTB) in brain tumors [38,39]. Therefore, activating the bradykinin B2 receptor may selectively modulate the BTB permeability and increase drug delivery to brain tumors. This strategy may be able to avoid side effects of osmotic BBB disruption towards the normal brain, owing to targeting effects on the BTB. Nonapeptide RMP-7 can selectively stimulate bradykinin B2 receptor and possesses longer blood circulation than endogenous bradykinin [37]. RMP-7 has been shown to be effective in opening BBB tight junctions and increasing intracranial drug concentrations in normal animal and in brain tumor animal models after intravenous infusion or intra-arterial injection [40–42].

Bradykinin B2 receptor is also expressed at numerous additional sites, and its activation at these sites can induce a wide variety of physiological responses including smooth muscle relaxation (e.g., vasculature) and contraction (e.g., intestine and uterus), inflammation modulation, pain mediation, and dose-limiting side effects (e.g., hypotension) [37]. The major side effects of intravenously administered tolerable RMP (up to 300 ng/kg over 10 min) were immediate and transient and included flushing, nausea, headache, and tachycardia [43–45]. At clinically approved dosage, the effects of intravenously infused RMP-7 weren't shown in Phase II clinical trials in patients with brain tumors [38,44–46]. Intracarotid injection rather intravenous infusion has the potential of concentrating RMP-7 to the brain and reducing effects on peripheral tissues. Except for transient decreases in arterial blood pressure, intra-arterial administration of RMP-7 wasn't shown to produce any other side effects, such as apparent cerebrovascular abnormalities and neurologic deficits in swine [47]. It is to be noted that bradykinin-increased BBB permeability may also be related with increased vesicular transport [48]. Considering the specific effect of RMP-7 on the BTB and the evidence demonstrated with the U87 glioma model that 7~100 nm pores in BTB are sufficient to allow the translocation of certain nanoparticles [49], the possibility of combining RMP-7 with targeting macromolecules or nanomedicine should be further evaluated.

#### *2.4. Direct Interference of Tight Junctions*

Claudins are major components of tight junctions, and claudin-5 dominates the BBB tight junctions by limiting paracellular penetration of small molecules [50–52]. Knockdown of BBB endothelial claudin-5 using specific siRNA was also shown to be able to transiently and reversibly increase BBB permeability to small molecules (MW up to 742) in mice [53]. The BBB opening and increased permeability after claudin-5 siRNA treatment were found to be size-selective and last for 72 h for small molecules with MW 443 and for 48 h for small molecules with MWs 562 and 742. It is also noteworthy that BBB opening after claudin-5 siRNA treatment also contributed to the clearance of water from the brain with cognitive improvement in mice with focal cerebral edema [54]. Anti-claudin-5 antibody can specifically recognize and bind with the extracellular loop domain of claudin-5, leading to impaired BBB tight junctions and increased BBB permeability to small molecules (e.g.,

sodium fluorescein with MW 376) [55–57]. The 3 mg/kg antibody didn't induce any liver and kidney injury, change of plasma biomarkers of inflammation, and behavior change in cynomolgus monkeys while vasodilation in liver, lung, and kidney, lung hemorrhage, and brain edema were shown with 6 mg/kg antibody [55]. The side effects of high dose of anti-claudin-5 antibody can be ascribed to the wide expression of claudin-5 in the vascular endothelium of peripheral tissues [52]. The narrow window between the tight junction opening and peripheral side effects should be considered and local delivery of anti-claudin-5 antibody may be able to prevent the above side effects. Peptide C5C2 can bind with the first extracellular loop of claudin-5 and was shown to internalize and downregulate claudin-5 [58]. However, in contrary to anti-claudin-5 antibody and claudin-5 siRNA, the transient and reversible BBB opening mediated by C5C2 was found to allow brain entry of molecules up to 40 kDa.

Angulin-1 and tricellulin constitute the functional BBB tricellular tight junctions, which blocking brain entry of macromolecules only [50,59,60]. Angubindin-1 is derived from the receptor-binding domain of *Clostridium perfringens* iota-toxin and can bind with angulin-1 of tricellular tight junctions and remove angulin-1 and tricellulin from tricellular tight junctions, leading to enhanced BBB permeability to macromolecules [61]. Intravenously injected angubindin-1 disrupted tricellular tight junctions without any overt adverse effect and increased BBB permeability for transient brain entry of macromolecules [60].

#### *2.5. Other Potential Strategies*

There also reported numerous other strategies for opening BBB tight junctions with enormous potential. For example, as a G protein-coupled receptor, sphingosine 1-phosphate receptor-1 (S1PR1) plays an important role in the barrier function of the BBB and peripheral vessels [17]. Knockout or downregulation of endothelial S1PR1 transiently and reversibly altered distribution of BBB tight junction proteins and allowed increased brain penetration of small molecules with MW less than 3000 in mice. The opening of BBB tight junctions by S1PR1 inhibition via FTY720 didn't show any signs of brain inflammation or injury. Controversially, FTY720 was also reported to reverse downregulation of S1P1 and S1P3 in retinas of diabetic rats and repair BBB by upregulating claudin-5 and downregulating VCAM-1 [62,63]. Therefore, further research is needed to verify whether FTY720 can indeed open BBB tight junctions and enhance paracellular drug delivery to the brain. The upregulation of astrocytic S1PR3 was linked to high permeability of brain metastases from breast cancer [64–66], suggesting contrary pathophysiological effects of S1PR3 to those of S1PR1. Further studies are also needed to elucidate the respective roles of S1PR1 and S1PR3 in the BBB.

Intracarotid injection of alkylglycerols was shown to transiently increase paracellular BBB permeability to small molecules and macromolecules with an efficiency comparable to that of osmotic BBB disruption and higher than that of intracarotid infusion of bradykinin [67–71]. Although intracarotid administration is an invasive procedure and the effects of alkylglycerols haven't been proven clinically, the strategy of alkylglycerolmediated BBB opening didn't reveal any sign of toxicity at the animal level after long-term in vivo analyses [71]. In addition, intracarotid infusion of oleic acid or linoleic acid was also found to cause reversible BBB disruption and increase BBB permeability, but with brain edema, necrosis, and demyelination [72,73].

In theory, selectively disrupting the diseased BBB is more advantageous than nonspecific BBB disruption when systemic therapy of brain diseases is considered, owing to the absence of unwanted side effects to normal brain regions, e.g., the strategy of activating the bradykinin B2 receptor in 2.3. Pericytes derived from glioblastoma were reported to be directly associated with the BTB tight junctions and poor response of glioblastoma to chemotherapy [74]. Reducing pericyte coverage of the BTB was found to successfully increase paracellular BTB permeability and then improve chemotherapy efficiency against glioblastoma [75]. Ibrutinib with the ability of eliminating glioblastoma-derived pericytes

by inhibiting BMX kinase was proven to be able to selectively impair the BTB tight junctions to enhance the therapeutic efficacy of drugs with poor BTB penetration [74].

Substance P is an important proinflammatory neuropeptide that functions as an immunoneuromodulator in the brain. Notably, substance P is also expressed by breast cancer and involves in chemoresistance and BBB crossing of breast cancer cells to form brain metastases [76]. Substance P secreted by breast cancer cells induces BBB endothelial cells to successively secret tumor necrosis factor alpha (TNFα) and angiopoietin-2 (Ang-2), which further activate their receptors to reorganize endothelial cytoskeleton and destabilize inter-endothelial adhesion complexes to alter distribution of tight junction proteins such as claudin-5 [76–79]. In addition, increased BBB permeability by secreted Ang-2 is also correlated with upregulated caveolin-1 and intensified caveolae-mediated vesicular transport [80]. Considering the substance P-mediated effects and corresponding specific expression of TNF receptors in the BTB (brain metastases), substance P, TNFα, Ang-2 and their derivatives can be used to open tight junctions in the BBB and tumor-associated BTB, respectively.

#### **3. Modulation of Active Efflux**

Active efflux transporters are selective gatekeepers at the BBB and cooperate with tight junctions to regulate substance transport into and out of the brain. Pathophysiological processes and pharmacological intervention further aggravate the efflux effect by intensifying expression and activity of these efflux transporters. Therefore, targeting regulatory pathways of BBB efflux transporters is supposed to be a feasible approach for efficient drug delivery to the brain [81,82]. BBB efflux transporters include Pgp, BCRP and MRPs. Although the role of other efflux transporters may be underestimated, Pgp with multiple binding domains for broad substrate spectrum is thought to be a predominant BBB efflux transporter [81,82]. Therefore, this section is focused on the modulation of Pgp. Typical strategies including direct inhibition and inhibiting transcriptional activation are introduced here. Notably, preserving and restoring their normal expression and activity after treatment is of specific importance, owing to the protective roles of active efflux. Many other modulating mechanisms of BBB Pgp expression and activity, such as posttranscriptional mechanisms, posttranslational mechanisms, and intracellular and intercellular trafficking, were not reviewed here, owing to the absence of reported pharmacological intervention [81].

#### *3.1. Direct Inhibition of Efflux Transporters*

Pgp activity can be directly inhibited using specific competitive inhibitors, such as verapamil (Figure 3) [83,84]. In vivo cerebral microdialysis can be used to directly measure the concentration of free drug in the brain to study possible drug–Pgp interactions [85]. For example, through brain microdialysis in rats, it has been shown that Pgp inhibition enhanced the brain concentration of Pgp substrates ceftriaxone and seliciclib [86,87]. Evaluated by intracerebral microdialysis on mice, topotecan penetration in gliomas was enhanced by modulating Pgp using gefitinib [88]. However, high dosed inhibitors are often used, owing to their low Pgp binding affinity and greater resistant Pgp inhibition at the BBB than peripheral Pgp [89], which may lead to tolerability concerns and side effects. In addition, Pgp inhibition at the BBB can enhance brain concentrations of unwanted substrates, which could lead to serious intracranial side effects from the unwanted compounds [85]. The second-generation inhibitors with improved tolerability, including valspodar, possess the shortcomings of inhibiting cytochrome P450 enzymes, leading to delayed drug clearance and prolonged systemic exposure of co-administered therapeutic drugs [82]. Thus, the effects on drug metabolism and pharmacodynamics limit the application of these two generation inhibitors. Third-generation inhibitors (e.g., elacridar) affect BBB efflux efficacy by inducing Pgp conformation changes.

fect BBB efflux efficacy by inducing Pgp conformation changes.

enhanced the brain concentration of Pgp substrates ceftriaxone and seliciclib [86,87]. Evaluated by intracerebral microdialysis on mice, topotecan penetration in gliomas was enhanced by modulating Pgp using gefitinib [88]. However, high dosed inhibitors are often used, owing to their low Pgp binding affinity and greater resistant Pgp inhibition at the BBB than peripheral Pgp [89], which may lead to tolerability concerns and side effects. In addition, Pgp inhibition at the BBB can enhance brain concentrations of unwanted substrates, which could lead to serious intracranial side effects from the unwanted compounds [85]. The second-generation inhibitors with improved tolerability, including valspodar, possess the shortcomings of inhibiting cytochrome P450 enzymes, leading to delayed drug clearance and prolonged systemic exposure of co-administered therapeutic drugs [82]. Thus, the effects on drug metabolism and pharmacodynamics limit the application of these two generation inhibitors. Third-generation inhibitors (e.g., elacridar) af-

**Figure 3.** The strategy of directly inhibiting efflux transporters. **Figure 3.** The strategy of directly inhibiting efflux transporters.

## *3.2. Targeting Regulatory Pathways of Efflux Transporters*

*3.2. Targeting Regulatory Pathways of Efflux Transporters*  Inhibiting the signal pathways intensifying Pgp expression and activity is supposed to overcome Pgp-mediated efflux and chemoresistance [90]. A number of "orphan" nuclear receptors are key transcriptional regulators and their expression at the BBB can upregulate Pgp, BCRP, and MRPs to respond to potentially harmful compounds. For example, pregnane X receptor (PXR) directly participates in Pgp upregulation by anticancer drugs [91–93]. Antagonists of these orphan nuclear receptors, such as ketoconazole, were shown to effectively inhibit rifampicin- and paclitaxel-induced Pgp upregulation, and sensitize brain cancers to anticancer drugs [94]. It is to be noted that these Pgp regulating Inhibiting the signal pathways intensifying Pgp expression and activity is supposed to overcome Pgp-mediated efflux and chemoresistance [90]. A number of "orphan" nuclear receptors are key transcriptional regulators and their expression at the BBB can upregulate Pgp, BCRP, and MRPs to respond to potentially harmful compounds. For example, pregnane X receptor (PXR) directly participates in Pgp upregulation by anticancer drugs [91–93]. Antagonists of these orphan nuclear receptors, such as ketoconazole, were shown to effectively inhibit rifampicin- and paclitaxel-induced Pgp upregulation, and sensitize brain cancers to anticancer drugs [94]. It is to be noted that these Pgp regulating mechanisms at the BBB likely exist in peripheral tissues. Strategies reversing Pgp upregulation might also reduce Pgp in other tissues and thereby cause unintended side effects.

mechanisms at the BBB likely exist in peripheral tissues. Strategies reversing Pgp upregulation might also reduce Pgp in other tissues and thereby cause unintended side effects. The signaling pathway of glutamate/NMDA-R/COX-2/prostaglandin E2 EP1 receptor induces Pgp and BCRP overexpression at the BBB in epileptic brains (Figure 4). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDA-R), was proven to effectively prevent glutamate-induced Pgp upregulation [95]. However, the side effects severely restrict the development of this approach [96]. COX inhibition using indomethacin and celecoxib was proven to prevent seizure-induced Pgp overexpression and enhance delivery of antiepileptic drugs to the brain in epilepsy model with negligible effect on basal Pgp expression and transport activity [97–99]. Unfortunately, COX-2 inhibitors are also associated with an enhanced risk of cardiovascular and cerebrovascular events and the controversial impact on seizure thresholds and seizure severity [100]. Inhibiting the prostaglandin E2 EP1 receptor by SC-51089 was further demonstrated to abolish glutamateinduced Pgp increases at the BBB, and enhance antiepileptic drug efficacy [82,101]. Neurodegeneration aggravation after COX-2 inhibition can be attributed to the blocking of EP2, EP3, and EP4 downstream of prostaglandin E2 [102–104]. Therefore, antagonism of The signaling pathway of glutamate/NMDA-R/COX-2/prostaglandin E2 EP1 receptor induces Pgp and BCRP overexpression at the BBB in epileptic brains (Figure 4). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDA-R), was proven to effectively prevent glutamate-induced Pgp upregulation [95]. However, the side effects severely restrict the development of this approach [96]. COX inhibition using indomethacin and celecoxib was proven to prevent seizure-induced Pgp overexpression and enhance delivery of antiepileptic drugs to the brain in epilepsy model with negligible effect on basal Pgp expression and transport activity [97–99]. Unfortunately, COX-2 inhibitors are also associated with an enhanced risk of cardiovascular and cerebrovascular events and the controversial impact on seizure thresholds and seizure severity [100]. Inhibiting the prostaglandin E2 EP1 receptor by SC-51089 was further demonstrated to abolish glutamate-induced Pgp increases at the BBB, and enhance antiepileptic drug efficacy [82,101]. Neurodegeneration aggravation after COX-2 inhibition can be attributed to the blocking of EP2, EP3, and EP4 downstream of prostaglandin E2 [102–104]. Therefore, antagonism of the prostaglandin E2 EP1 receptor may be the most promising approach to control Pgp expression and enhance entry of antiepileptic drugs to epileptic brains. Strategies of reversing Pgp upregulation in epilepsy can be extended to the application in treating brain ischemia, because the glutamate release and similar Pgp upregulation mechanisms also exists in brain ischemia [105]. In contrary, as a critical factor for intracranial clearance of amyloid β-protein (Aβ), Pgp expression at the BBB is often downregulated to promote intracranial Aβ accumulation in Alzheimer's disease [106–109]. Signaling pathways inducing Pgp upregulation may be carefully harnessed to treat Alzheimer's disease. For example, PXR ligands (e.g., hyperforin) and EP1 receptor agonists hold the potential for upregulating Pgp to interfere with Alzheimer's disease. In addition, strengthening the Wnt/β-catenin signaling may also be able to increase Pgp to reduce Aβ burden in Alzheimer's disease [110].

the prostaglandin E2 EP1 receptor may be the most promising approach to control Pgp expression and enhance entry of antiepileptic drugs to epileptic brains. Strategies of reversing Pgp upregulation in epilepsy can be extended to the application in treating brain ischemia, because the glutamate release and similar Pgp upregulation mechanisms also exists in brain ischemia [105]. In contrary, as a critical factor for intracranial clearance of amyloid β-protein (Aβ), Pgp expression at the BBB is often downregulated to promote intracranial Aβ accumulation in Alzheimer's disease [106–109]. Signaling pathways inducing Pgp upregulation may be carefully harnessed to treat Alzheimer's disease. For example, PXR ligands (e.g., hyperforin) and EP1 receptor agonists hold the potential for upregulating Pgp to interfere with Alzheimer's disease. In addition, strengthening the Wnt/β-catenin signaling may also be able to increase Pgp to reduce Aβ burden in Alz-

#### **4. Modulation of Transcytosis 4. Modulation of Transcytosis**

heimer's disease [110].

Receptor-mediated transcytosis is often used to mediate transcellular BBB crossing, owing to the extremely low paracellular BBB permeability controlled by the tight junctions and active efflux transporters. Receptor-specific ligands can be used to decorate drug delivery systems (e.g., multifunctional nanoparticles) to initiate transcellular transport across the BBB [8,49,111–117]. However, the density of these target receptors at the BBB is much lower than that of nutrient transporters (e.g., glucose transporter) [118]. More importantly, exclusively expressed Mfsd2a limits formation of caveolae-mediated transcytotic vesicles and the transcytosis rate at the BBB by regulating BBB endothelial lipid composition [1–6,119–121]. Therefore, the efficiency of transcellular transport at the BBB should be modulated to improve brain accumulation of ligand-modified drug deliv-Receptor-mediated transcytosis is often used to mediate transcellular BBB crossing, owing to the extremely low paracellular BBB permeability controlled by the tight junctions and active efflux transporters. Receptor-specific ligands can be used to decorate drug delivery systems (e.g., multifunctional nanoparticles) to initiate transcellular transport across the BBB [8,49,111–117]. However, the density of these target receptors at the BBB is much lower than that of nutrient transporters (e.g., glucose transporter) [118]. More importantly, exclusively expressed Mfsd2a limits formation of caveolae-mediated transcytotic vesicles and the transcytosis rate at the BBB by regulating BBB endothelial lipid composition [1–6,119–121]. Therefore, the efficiency of transcellular transport at the BBB should be modulated to improve brain accumulation of ligand-modified drug delivery systems.

#### ery systems. *4.1. Upregulation of LRP1*

*4.1. Upregulation of LRP1*  Low-density lipoprotein receptor-related protein 1 (LRP1) is expressed at both luminal and abluminal sides of the BBB. While abluminal LRP1 is primarily responsible for clearing Aβ from the brain to blood [122], luminal LRP1 has been extensively studied to mediate drug delivery to the brain. Inspired by the fact that statins can suppress cholesterol synthesis and then induce compensatory expression of LRP1 [118,123–126], simvastatin-loaded nanoparticles were reported in our previous work to upregulate LRP1 at the Low-density lipoprotein receptor-related protein 1 (LRP1) is expressed at both luminal and abluminal sides of the BBB. While abluminal LRP1 is primarily responsible for clearing Aβ from the brain to blood [122], luminal LRP1 has been extensively studied to mediate drug delivery to the brain. Inspired by the fact that statins can suppress cholesterol synthesis and then induce compensatory expression of LRP1 [118,123–126], simvastatinloaded nanoparticles were reported in our previous work to upregulate LRP1 at the BBB and boost LRP1-targeting chemotherapy efficiency against brain metastases from breast cancer [114]. In addition, LRP1 can respond to astrocytic apolipoprotein E to maintain the BBB integrity by suppressing the BBB-degrading pathway of activation of cyclophilin A-matrix metalloproteinase 9 [127,128]. More importantly, the diminishment of abluminal LRP1 is closely related to intracranial Aβ accumulation in Alzheimer's disease, and also to the aggregation of α-synuclein into Lewy bodies in Parkinson's disease [127,128]. Therefore, the strategy of upregulating LRP1, a potentially important therapeutic target of BBB breakdown-related diseases, holds the potential to be used to treat both Alzheimer's disease and Parkinson's disease. In fact, delivery of LRP1 gene to the BBB has been reported to facilitate Aβ clearance via upregulating LRP1 [127,129].

#### *4.2. Inhibition of Mfsd2a*

Reversible inhibition of Mfsd2a holds the potential to temporarily liberate the limited transcytosis at the BBB [2]. In our previous work, Mfsd2a inhibitor tunicamycin was

delivered to the BBB and shown to be able to enhance brain accumulation of subsequent therapeutic nanoparticles and the efficiency in treating brain metastases from breast cancer in mice [117]. Owing to the crucial role of Mfsd2a in transporting essential fatty acids and promoting BBB formation and brain development, Mfsd2a knockout induces microcephaly, Allan-Herndon-Dudley syndrome, and other severe side effects (e.g., BBB breakdown, neuronal loss, cognitive impairment, intellectual disability, behavioral deficits, spasticity, and absent speech and so on) [4,121,127,128,130–132]. In clinical practice, the loss of BBB Mfsd2a is often found in Alzheimer's disease, traumatic brain injury, stroke, and brain tumor. Mfsd2a may be a potential therapeutic target for these diseases and remains to be explored further [130,131]. However, tunicamycin-mediated Mfsd2a inhibition is likely to be reversible, because the inhibition mechanism is supposed to be just physical binding and the inhibitor would dissociate from Mfsd2a after entering the brain [2]. Therefore, side effects associated with Mfsd2a deficiency could be avoided.

#### *4.3. Upregulation of GLUT1*

Glucose transporter 1 (GLUT1) at the BBB maintains the continuous high glucose and energy demands of the brain. Based on its essential role in transporting glucose and its participation in pathological processes of various brain diseases such as Alzheimer's disease, ischemia, and brain tumors, upregulation of GLUT1 has been proposed to treat hypoglycemic conditions, while its downregulation or inhibition could be used to cope with hyperglycemic conditions [133]. In addition to being direct therapeutic targets, wide expression of GLUT1 at the BBB has been extensively used to mediate drug delivery to the brain. GLUT1 upregulation at the luminal side of the BBB via hypoglycemia and its migration to the abluminal side were implemented via rapid glycemic increase after fasting [134]. Then, the brain accumulation of properly configured glucose nanoparticles was shown to reach 6% dose/g-brain in normal mice with glycemic control. Because Alzheimer's disease is characterized by reduced GLUT1 at the BBB and a reduction of glucose transport [129], this strategy of rapid glycemic increase after fasting holds the potential to treat Alzheimer's disease via upregulating GLUT1.

#### **5. Multifunctional Strategies by Multiple BBB Modulation**

All the above strategies increase BBB permeability via separately modulating tight junctions, active efflux, or transcytosis. In fact, there many other multifunctional strategies were also reported, which can simultaneously modulate multiple controlling factors and achieve theoretically higher BBB permeability for efficient drug delivery to the brain.

#### *5.1. Focused Ultrasound*

Low intensity focused ultrasound is a noninvasive technique that is combined with intravenously injected gaseous perfluorocarbon-filled microbubbles to transiently and focally modulate the BBB [135,136]. With the help of stable oscillation of microbubbles, the BBB is transiently and reversibly disrupted and characterized by (1) disintegration of tight junctions including claudin-5; (2) fenestration and channel formation; (3) Pgp suppression; and (4) upregulation of caveolin-1 and caveolae-mediated transcytotic vesicles, which jointly facilitate both paracellular transport and transcellular passage through the BBB [137–141]. Under the guidance by magnetic resonance imaging, microbubbleenhanced focused ultrasound can act on specific intracranial areas with negligible toxicity to adjacent normal brain cells [142–146]. Further, ultrasmall superparamagnetic iron oxide nanoparticles can be encapsulated into microbubbles and nanobubbles to increase the BBB disruption efficiency and monitor post-sonication BBB opening and drug delivery across the BBB [147,148]. Generally, microbubble-enhanced focused ultrasound is less invasive than BBB disruption induced by osmotic agents with minimal neurotoxicity, inflammation, and stroke occurrences in clinical settings [135,149–153]. However, increasing acoustic energy is associated with increasing risk of side effects including vascular damage, edema, parenchymal damage, microhemorrhage, and over-activation of the immune

system (e.g., autoimmunity) [137,154,155]. Therefore, adjusting ultrasound parameters is necessary for reducing risks, especially for repeated treatments and the application of mediating drug delivery to Alzheimer's disease owing to Aβ-mediated resistance of BBB disruption [156,157].

#### *5.2. Activating A2A Adenosine Receptor*

A2A adenosine receptor interacts with G<sup>s</sup> protein to activate adenylate cyclase and further increase intracellular cAMP [154]. It is located on platelets, leukocytes, blood vessels and intracranial regions such as striatum [158]. Its activation can inhibit platelet aggregation and regulate blood pressure through vasodilation [159]. Its expression can be altered by pathological conditions, e.g., upregulation on glial cells by hypoxia and inflammation and at the BBB by brain tumors [10,160], to protect against damage via reducing inflammation [161]. The activation of A2A adenosine receptor at the BBB can increase BBB permeability by rapid and reversible decrease of tight junction proteins (e.g., claudin-5), Pgp and BCRP [154,162]. Intravenous injection of clinically used regadenoson was shown to be able to increase intracranial concentrations of small molecules and macromolecules in preclinical studies [163–167]. However, regadenoson treatment at FDA-approved doses in humans (bolus injection of 0.4 mg) was found without increased intracranial concentrations of temozolomide in patients with recurrent glioblastoma [168,169], which may be attributed to the insufficient dose of this strategy for effective BBB modulation, and warrants the necessity of studies on whether higher dose or different agonists would be effective [154]. The alternative option of nanomedicine-mediated targeted agonist delivery holds the potential of not only enhancing selectivity, intensifying the BBB opening effect, and prolonging the BBB opening time window from up to 50 min to up to 2 h [170–174], but also avoiding affecting peripheral A2A adenosine receptors to minimize systemic side effects, e.g., excessive vasodilatation of the peripheral vascular bed, dizziness, and headaches [154].

#### *5.3. Activating Potassium Channels*

Blood vessel endothelium widely expresses potassium channels, especially ATPdependent potassium channels (*K*ATP) [175,176]. Activation of *K*ATP can regulate vascular hyperpolarization, relaxation, dilatation and vessel permeability [175–178], making *K*ATP a therapeutic target for hypertension. *K*ATP is often upregulated in hypoxic environments including brain tumors and ischemia [178,179]. The regulatory effects on BTB permeability by activating the *K*ATP are expected to be more significant than those of the BBB [176,180]. These effects include intensified paracellular diffusion and transcellular transport, which involve in downregulated tight junction proteins and upregulated caveolin-1 and caveolaemediated transcytotic vesicles [176,181,182]. BTB modulation by strengthening the activation of *K*ATP can be tightly controlled by inhibitors and has been used via minoxidil to increase Herceptin delivery to primary or metastatic brain tumors [183–186]. Although minoxidil was found to be nontoxic in both mice and rats [175], nonselective activation of *K*ATP may induce pericardial effusion, cardiac tamponade, reflex tachycardia, myocardial necrosis, coronary arteriopathy, degeneration of renal tubules, hypotension, dermatologic reactions, and hypertrichosis [154,187]. Intracarotid injection rather intravenous infusion holds the potential of concentrating minoxidil to the brain and reducing effects on peripheral tissues. As an alternative approach, in our previous work, minoxidil was delivered by hyaluronic acid modified nanoparticles to specially intensify the activation of BTB *K*ATP to enhance specific accumulation of subsequently injected therapeutic nanoparticles in brain metastases in mice [188].

#### *5.4. Other Potential Multifunctional Strategies*

As a key factor in hypertension, diabetes and aging, angiotensin-II can increase BBB permeability in both paracellular and transcellular manner by altering the distribution of tight junction proteins, decreasing Mfsd2a, and increasing caveolin-1 [189]. Thus, angiotensin-II can be used to open the BBB for increased drug delivery into the brain to treat various brain diseases. As a surgical technique, laser interstitial thermal therapy has been widely used to ablate brain tumors [190,191]. Interestingly, increasing data indicate that thermal therapy can disrupt the BBB via temporarily altering tight junctions and increasing transcytosis [190]. Although this technique is invasive and requires general anesthesia, combination of laser interstitial thermal therapy with other systemic therapies still holds the potential for synergistic therapeutic effect.

#### **6. Conclusions and Future Perspectives**

Modulation of the BBB, including tight junctions, active efflux transporters, and transcytotic vesicles, has been extensively studied to increase drug delivery to the brain. Although improved intracranial drug concentrations were often shown for almost all approaches, most of these studies were conducted preclinically and focused on brain tumors with very few exceptions on epilepsy. Side effects associated with these modulating strategies need to be carefully handled to extend these technologies to various brain diseases, including neurodegenerative diseases. First, although any delivery route can be used including intravenous, intracarotid or stereotactic administration, these BBB modulation approaches by themselves (e.g., radiation and various modulators) can be severely toxic. Second, besides the BBB's protective roles, BBB modulations are likely to impair the intracranial physiological functions of related targets, e.g., normal physiological actions of bradykinin B2 receptor, S1PR1, Pgp, Mfsd2a, LRP1, GLUT1, A2A adenosine receptor, and *K*ATP. Third, increased drug concentrations in normal brain and peripheral tissues resulting from efflux inhibition or tight junction opening may worsen side effects of subsequent therapeutic drugs. Fourth, unwanted accumulation of endogenous neurotoxic blood components and xenobiotics in normal brain regions (even specific accumulation in diseased regions) may lead to severe neurological complications. Therefore, the modulation window of various modulation strategies should be carefully investigated for safe clinical translation, especially those multifunctional strategies that combine multiple BBB modulations.

**Funding:** This work was funded by the National Natural Science Foundation of China (81973254 and 32171381), the Natural Science Foundation of Jiangsu Province (BK20191421), the Suzhou Science and Technology Development Project (SYS2019033) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).

**Conflicts of Interest:** The author declares that there are no conflict of interest.

#### **References**


*Review*

**Julian S. Rechberger 1,2,\*, Frederic Thiele <sup>3</sup> and David J. Daniels 1,4**


**Abstract:** Intra-arterial drug delivery circumvents the first-pass effect and is believed to increase both efficacy and tolerability of primary and metastatic brain tumor therapy. The aim of this update is to report on pertinent articles and clinical trials to better understand the research landscape to date and future directions. Elsevier's Scopus and ClinicalTrials.gov databases were reviewed in August 2021 for all possible articles and clinical trials of intra-arterial drug injection as a treatment strategy for brain tumors. Entries were screened against predefined selection criteria and various parameters were summarized. Twenty clinical trials and 271 articles satisfied all inclusion criteria. In terms of articles, 201 (74%) were primarily clinical and 70 (26%) were basic science, published in a total of 120 different journals. Median values were: publication year, 1986 (range, 1962–2021); citation count, 15 (range, 0–607); number of authors, 5 (range, 1–18). Pertaining to clinical trials, 9 (45%) were phase 1 trials, with median expected start and completion years in 2011 (range, 1998–2019) and 2022 (range, 2008–2025), respectively. Only one (5%) trial has reported results to date. Glioma was the most common tumor indication reported in both articles (68%) and trials (75%). There were 215 (79%) articles investigating chemotherapy, while 13 (65%) trials evaluated targeted therapy. Transient blood–brain barrier disruption was the commonest strategy for articles (27%) and trials (60%) to optimize intra-arterial therapy. Articles and trials predominately originated in the United States (50% and 90%, respectively). In this bibliometric and clinical trials analysis, we discuss the current state and trends of intra-arterial therapy for brain tumors. Most articles were clinical, and traditional anti-cancer agents and drug delivery strategies were commonly studied. This was reflected in clinical trials, of which only a single study had reported outcomes. We anticipate future efforts to involve novel therapeutic and procedural strategies based on recent advances in the field.

**Keywords:** brain tumor; glioma; drug delivery; injection; intra-arterial; chemotherapy; targeted therapy; immunotherapy; nanoparticles; treatment

## **1. Introduction**

Conventional treatment options for brain tumors rely on surgery, radiotherapy, and systemic pharmacotherapy. Oral and intravenous drug administration is often associated with poor brain distribution and bioavailability, limiting therapeutic effect, and contributing to unsatisfactory clinical outcomes [1–6]. High-grade gliomas, including glioblastoma and H3K27-altered diffuse midline glioma, with a median survival of approximately 12–15 months after diagnosis, stand a grim example of this failure to develop effective treatments [7–11]. In this multiomics era of biomedical research, insights into biological aspects of cancer have allowed us to identify potential targets that could improve the clinical course of these devastating diseases [12–15]. The first-pass effect and the blood– brain barrier (BBB), however, remain significant obstacles for therapeutic access to the brain and hinder novel therapies from unfolding pharmacologic potential [16–20].

**Citation:** Rechberger, J.S.; Thiele, F.; Daniels, D.J. Status Quo and Trends of Intra-Arterial Therapy for Brain Tumors: A Bibliometric and Clinical Trials Analysis. *Pharmaceutics* **2021**, *13*, 1885. https://doi.org/ 10.3390/pharmaceutics13111885

Academic Editors: Jingyuan Wen and Yuan Huang

Received: 20 September 2021 Accepted: 4 November 2021 Published: 6 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

One proposed solution to overcome these hurdles comprises strategies to minimize systemic drug exposure and modulate the BBB, which could expand the spectrum of usable drugs and potentially improve therapeutic efficacy and tolerability. Intra-arterial injection into intracranial vessels is one such strategy, with the potential to increase drug responses to primary and metastatic brain tumors [21–30]. Intra-arterial infusion of anti-cancer therapies can be combined with concurrent administration of a variety of agents, including chemical reagents, penetration drug carriers, or microbubbles for focused ultrasound, to selectively open the BBB in areas of interest [17,25,31,32]. By accessing intracranial vessels through peripheral arteries and directly administering BBB-disrupting and therapeutic agents into the arterial supply to the brain, intra-arterial injection facilitates greatly improved local drug delivery, increased intra-tumoral concentration, and lowered systemic exposure [33–37].

Since it was first described more than half a century ago, there have been considerable efforts not only to explore the biological mechanisms behind intra-arterial therapy but also to evaluate its applicability to a wide range of diseases. To date, multiple research studies are quoted to have investigated intra-arterial drug administration, yet there has been little, if any, translational impact observed for brain tumors [31,34,38–43]. Therefore, it is important to characterize how impactful the literature and previous clinical trials have been to predict where this drug delivery approach is heading. The aim of this study was to analyze the bibliometric parameters of available articles and evaluate registered clinical trials that have incorporated intra-arterial drug injection as a treatment strategy for brain tumors. This will provide a profile of the most impactful articles and trials to better inform clinicians of the current research landscape of intra-arterial drug delivery. Furthermore, this will enable future clinical trials to optimize and justify their design based on previous experiences to maximize trial discoveries and outcomes.

#### **2. Methodology**

The search strategy was designed to capture all possible Scopus-indexed articles and ClinicalTrials.gov-registered clinical trials referring to intra-arterial therapies for the treatment of brain tumors. Elsevier's Scopus facilitates access to peer-reviewed articles from approximately 22,000 journals. It offers one of the largest scientific literature capture reaches of biomedical electronic research databases [44]. ClinicalTrials.gov is a database provided by the US National Library of Medicine that contains referenced clinical trials on a wide range of conditions and diseases conducted around the world. It has been shown to have entries on 388,133 research studies from all 50 states of the USA and 219 countries worldwide [45,46]. Both databases were searched and screened independently by two investigators (J.S.R. and F.T.). We searched Scopus for referenced articles from its date of inception to August 2021 using the following string of search terms: (intra-arterial) AND (therapy OR treatment) AND (brain tumor OR glioma). The ClinicalTrials.gov portal was searched in August 2021 using "brain tumor", "glioma", and "intra-arterial injection" search terms for Condition or disease and Intervention/treatment, respectively. Any discrepancies were resolved by discussion until consensus was reached. Publications were limited to the English language.

To be included in our subsequent analyses, articles and clinical trials were required to investigate (1) intra-arterial administration of (2) therapeutics as (3) a treatment strategy for (4) tumors related to (5) the brain. In the case of articles and research studies that explored intra-arterial injection as a purely diagnostic tool, focused on diseases other than primary or secondary brain tumors, or investigated tumors of other organ systems, these were not included due to lack of specificity. Assessment of articles and trials to satisfy these criteria was performed independently by two investigators (J.S.R and F.T.), with any discrepancies resolved by discussion. There was no location restriction for eligible database entries.

The following validated article variables were then extracted from the Scopus database: article title, year, authors, number of authors, country of correspondence of the senior author, journal, Scopus citations, document type, study type, tumor type, therapy type, and type of treatment strategy for optimizing intra-arterial administration. Regarding the

latter variable, 5 categories were defined: (1) nanoparticles, (2) transient BBB disruption, (3) transient cerebral hypoperfusion or flow arrest, (4) superselective intra-arterial cerebral infusion, and (5) the combination of imaging techniques with intra-arterial infusion of contrast agents or labeled therapeutic agents. With respect to study type, articles were dichotomized to be either basic science (BSc) or clinical (CL). BSc articles were ones primarily describing nonpatient investigations, such as in vitro and in vivo models, whereas CL articles were ones focusing on patient outcomes, including feasibility, safety, and survival. Clinical trial outcomes extracted from ClinicalTrials.gov included National Clinical Trial (NCT) number, title, sponsor, institution of correspondence, country of origin of the corresponding institution, number of institutions involved, involvement of outside countries, status, availability of results, type of condition, type of primary intervention, primary and secondary outcome measurements, gender enrollment, age of enrollment, number of patients enrolled, study phases, study type, start year, completion year, year of the first release of results, and last updated year [47]. Missing data were denoted as "not reported". All data analyses, including the generation of figures and tables, were performed using Pandas 1.3.2 (i.e., Python Data Analysis Library), an open-source data analysis and manipulation tool that is built on top of the Python programming language [48]. No statistical comparisons were conducted.

#### **3. Results**

ble S3).

articles [34,36,37,51,55–66] (Table S4).

## *3.1. Article Characteristics*

A total of 546 articles were retrieved from Scopus after the initial database search. We screened titles and abstracts to obtain 357 articles not meeting any exclusion criteria. Full-text evaluation yielded 271 articles that were finally included in our study (Figure 1). A summary of the whole article cohort is provided in Table 1, and detailed results can be found in Tables S1–S13, Supplementary Materials. We identified 227 (84%) as original articles and 44 (16%) as review articles. There were 70 BSc articles (26%) and 201 CL articles (74%). Fifty-four (20%) were published open access, and therefore freely accessible online (Table S1). The most common articles for intra-arterial drug delivery in brain tumors were for gliomas (*n*= 184, 68%), including glioblastoma, gliosarcoma, diffuse intrinsic pontine glioma and glioma without further specification, brain metastasis (*n* = 12, 4%), and lymphoma (*n* = 5, 2%). Sixty-six articles reported inclusion of multiple tumor types (24%) (Table S2). *Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 5 of 21

**Figure 1.** Methodological approach to identify articles and clinical trials on intra-arterial brain tumor therapy via databases and registers. articles published in 1986. Original articles and reviews peaked with respect to their **Figure 1.** Methodological approach to identify articles and clinical trials on intra-arterial brain tumor therapy via databases and registers.

The median citation count was 15 (range, 0–607), with the most-cited article to date a review by Hochberg et al. [49], published in 1988 with 607 citations ("Primary central

With regard to contributing authors, the median number of authors for original and review articles was five (range, 1–18). The most authored article was the original, CL study by Angelov et al. [51], published in 2009 with 18 authors ("Blood–brain barrier disruption and intra-arterial methotrexate-based therapy for newly diagnosed primary CNS lymphoma: A multi-institutional experience" in the *Journal of Clinical Oncology*). The highest number of authors for BSc articles was 12: Liu et al. [52] published their manuscript in 1991 ("Effects of intracarotid and intravenous infusion of human TNF and LT on established intracerebral rat gliomas" in Lymphokine and Cytokine Research) whereas the article by Mao et al. [53] was published in 2020 ("Peritumoral administration of IFNβ upregulated mesenchymal stem cells inhibits tumor growth in an orthotopic, immunocompetent rat glioma model" in Journal for ImmunoTherapy of Cancer). The most authored review article was by Aoki et al. [54], published in 1993 with 13 authors ("Supraophthalmic chemotherapy with long tapered catheter: Distribution evaluated with intraarterial and intravenous Tc-99m HMPAO" in Radiology). The authors with the most senior-authored articles overall were E.A. Neuwelt and J.A. Boockvar, who both contributed eight

All articles were published between 1962 and 2021 (Figure 2), with a median of 5 publications per year. The peak year (median) with most-published articles was 17 (6%)

efficacy of a multicenter study using intraarterial chemotherapy in conjunction with osmotic opening of the blood–brain barrier for the treatment of patients with malignant brain tumors" in Cancer). Matsukado et al. [50] published in 1996 the most-cited BSc article with 151 citations ("Enhanced tumor uptake of carboplatin and survival in gliomabearing rats by intracarotid infusion of bradykinin analog, RMP-7" in Neurosurgery) (Ta-


**Table 1.** Summary of article characteristics.

\* Categorical data reported as *n* (% total). # Does not sum to 271 as studies could report more than one tumor type and therapeutic approach.

The median citation count was 15 (range, 0–607), with the most-cited article to date a review by Hochberg et al. [49], published in 1988 with 607 citations ("Primary central nervous system lymphoma" in the *Journal of Neurosurgery*). The most cited original article was the CL study by Doolittle et al. [34], published in 2000 with 300 citations ("Safety and efficacy of a multicenter study using intraarterial chemotherapy in conjunction with osmotic opening of the blood–brain barrier for the treatment of patients with malignant brain tumors" in Cancer). Matsukado et al. [50] published in 1996 the most-cited BSc article with 151 citations ("Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7" in Neurosurgery) (Table S3).

With regard to contributing authors, the median number of authors for original and review articles was five (range, 1–18). The most authored article was the original, CL study by Angelov et al. [51], published in 2009 with 18 authors ("Blood–brain barrier disruption and intra-arterial methotrexate-based therapy for newly diagnosed primary CNS lymphoma: A multi-institutional experience" in the *Journal of Clinical Oncology*). The highest number of authors for BSc articles was 12: Liu et al. [52] published their manuscript in 1991 ("Effects of intracarotid and intravenous infusion of human TNF and LT on established intracerebral rat gliomas" in Lymphokine and Cytokine Research) whereas the article by Mao et al. [53] was published in 2020 ("Peritumoral administration of IFNβ upregulated mesenchymal stem cells inhibits tumor growth in an orthotopic, immunocompetent rat glioma model" in Journal for ImmunoTherapy of Cancer). The most authored review article was by Aoki et al. [54], published in 1993 with 13 authors ("Supraophthalmic chemotherapy with long tapered catheter: Distribution evaluated with intraarterial and intravenous Tc-99m HMPAO" in Radiology). The authors with the most senior-authored articles overall were E.A. Neuwelt and J.A. Boockvar, who both contributed eight articles [34,36,37,51,55–66] (Table S4).

All articles were published between 1962 and 2021 (Figure 2), with a median of 5 publications per year. The peak year (median) with most-published articles was 17 (6%) articles published in 1986. Original articles and reviews peaked with respect to their annual publication number in 1986 and 2020, respectively. Most BSc articles were published in 1999, while CL articles had their peak year in 1986 (Table S5).

A total of 20 countries were denoted as the location for correspondence of all articles (Figure 3). The USA was the country with the highest contribution, with 135 articles (50%), followed by Japan and Canada, with 46 (17%) and 19 (7%), respectively. The USA was the most common country of correspondence for all document and study types (Table S6).

One hundred and twenty journals contributed to articles of intra-arterial therapy for the treatment of brain tumors. The most common ones were the *Journal of Neuro-Oncology*, with 48 (18%) articles, the *Japanese Journal of Cancer and Chemotherapy* (*n* = 14, 5%), and *Neurosurgery* (*n* = 13, 5%). The journal publishing most original studies, review articles, BSc articles, and CL articles was the *Journal of Neuro-Oncology* (Table S7).

In terms of therapy types used with intra-arterial delivery, general chemotherapy was the most common, with 215 (79%) articles, followed by targeted therapy (*n* = 40, 15%), and radiosensitizing or neutron capture therapy (*n* = 17, 6%). The number of articles per therapy type per year of publication is illustrated in Figure 4. Chemotherapy was the top therapeutic strategy in all but three years (1973, 2014, and 2017). The most commonly studied chemotherapeutic agents included i.a. carmustine (*n* = 29, 13%), i.a. nimustine (*n* = 20, 9%), and i.a. cisplatin (*n* = 23, 11%). Twenty-three articles (11%) mentioned the general concept of intra-arterial chemotherapy without further specification (Tables S8–S12).

At least 1 additional treatment strategy for optimizing intra-arterial drug delivery was evaluated in 104 articles (Figure 5). The most common strategy was transient BBB disruption, mentioned in 74 articles (27%). Transient BBB disruption was followed by superselective intra-arterial cerebral infusion (*n* = 27, 10%), and nanoparticles (*n* = 17, 6%). Among BBB-opening modalities, mannitol was the most common one, referenced in 48 articles (65%), followed by bradykinin/RMP-7 (*n* = 16, 22%) (Table S13).

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 6 of 21

lished in 1999, while CL articles had their peak year in 1986 (Table S5).

lished in 1999, while CL articles had their peak year in 1986 (Table S5).

annual publication number in 1986 and 2020, respectively. Most BSc articles were pub-

annual publication number in 1986 and 2020, respectively. Most BSc articles were pub-

**Figure 2.** Distribution of articles about intra-arterial drug delivery for the treatment of brain tumors based on the country of correspondence. **Figure 2.** Distribution of articles about intra-arterial drug delivery for the treatment of brain tumors based on the country of correspondence. followed by Japan and Canada, with 46 (17%) and 19 (7%), respectively. The USA was the most common country of correspondence for all document and study types (Table S6).

**Figure 3.** Distribution of articles about intra-arterial drug delivery for the treatment of brain tumors based on the year of publication. **Figure 3.** Distribution of articles about intra-arterial drug delivery for the treatment of brain tumors based on the year of publication.

One hundred and twenty journals contributed to articles of intra-arterial therapy for

the treatment of brain tumors. The most common ones were the *Journal of Neuro-Oncology*, with 48 (18%) articles, the *Japanese Journal of Cancer and Chemotherapy* (*n* = 14, 5%), and

**Figure 3.** Distribution of articles about intra-arterial drug delivery for the treatment of brain tumors

the treatment of brain tumors. The most common ones were the *Journal of Neuro-Oncology*, with 48 (18%) articles, the *Japanese Journal of Cancer and Chemotherapy* (*n* = 14, 5%), and *Neurosurgery* (*n* = 13, 5%). The journal publishing most original studies, review articles,

BSc articles, and CL articles was the *Journal of Neuro-Oncology* (Table S7).

One hundred and twenty journals contributed to articles of intra-arterial therapy for

In terms of therapy types used with intra-arterial delivery, general chemotherapy

In terms of therapy types used with intra-arterial delivery, general chemotherapy

At least 1 additional treatment strategy for optimizing intra-arterial drug delivery

At least 1 additional treatment strategy for optimizing intra-arterial drug delivery

was evaluated in 104 articles (Figure 5). The most common strategy was transient BBB disruption, mentioned in 74 articles (27%). Transient BBB disruption was followed by superselective intra-arterial cerebral infusion (*n* = 27, 10%), and nanoparticles (*n* = 17, 6%).

was evaluated in 104 articles (Figure 5). The most common strategy was transient BBB disruption, mentioned in 74 articles (27%). Transient BBB disruption was followed by su-

articles (65%), followed by bradykinin/RMP-7 (*n* = 16, 22%) (Table S13).

articles (65%), followed by bradykinin/RMP-7 (*n* = 16, 22%) (Table S13).

*Pharmaceutics* **2021**, *13*, x FOR PEER REVIEW 7 of 21

was the most common, with 215 (79%) articles, followed by targeted therapy (*n* = 40, 15%), and radiosensitizing or neutron capture therapy (*n* = 17, 6%). The number of articles per therapy type per year of publication is illustrated in Figure 4. Chemotherapy was the top therapeutic strategy in all but three years (1973, 2014, and 2017). The most commonly studied chemotherapeutic agents included i.a. carmustine (*n* = 29, 13%), i.a. nimustine (*n* = 20, 9%), and i.a. cisplatin (*n* = 23, 11%). Twenty-three articles (11%) mentioned the general concept of intra-arterial chemotherapy without further specification (Tables S8–S12).

was the most common, with 215 (79%) articles, followed by targeted therapy (*n* = 40, 15%), and radiosensitizing or neutron capture therapy (*n* = 17, 6%). The number of articles per therapy type per year of publication is illustrated in Figure 4. Chemotherapy was the top therapeutic strategy in all but three years (1973, 2014, and 2017). The most commonly studied chemotherapeutic agents included i.a. carmustine (*n* = 29, 13%), i.a. nimustine (*n* = 20, 9%), and i.a. cisplatin (*n* = 23, 11%). Twenty-three articles (11%) mentioned the general concept of intra-arterial chemotherapy without further specification (Tables S8–S12).

**Figure 4.** Different therapy types investigated in articles of intra-arterial drug delivery for the treatment of brain tumors based on the year of publication. **Figure 4.** Different therapy types investigated in articles of intra-arterial drug delivery for the treatment of brain tumors based on the year of publication. **Figure 4.** Different therapy types investigated in articles of intra-arterial drug delivery for the treatment of brain tumors based on the year of publication.

**Figure 5.** Treatment strategies to optimize intra-arterial drug delivery for the treatment of brain tumors in pertinent articles based on the year of publication. **Figure 5.** Treatment strategies to optimize intra-arterial drug delivery for the treatment of brain tumors in pertinent articles based on the year of publication. **Figure 5.** Treatment strategies to optimize intra-arterial drug delivery for the treatment of brain tumors in pertinent articles based on the year of publication.

#### *3.2. Clinical Trial Characteristics 3.2. Clinical Trial Characteristics 3.2. Clinical Trial Characteristics*

The initial search of the ClinicalTrials.gov portal yielded 21 clinical trials for screening. One trial was excluded because it did not investigate intra-arterial drug injection, but rather looked at cerebral blood perfusion changes during emergence from general anesthesia for craniotomy using an intra-arterial pressure line [67]. Consequently, 20 trials The initial search of the ClinicalTrials.gov portal yielded 21 clinical trials for screening. One trial was excluded because it did not investigate intra-arterial drug injection, but rather looked at cerebral blood perfusion changes during emergence from general anesthesia for craniotomy using an intra-arterial pressure line [67]. Consequently, 20 trials The initial search of the ClinicalTrials.gov portal yielded 21 clinical trials for screening. One trial was excluded because it did not investigate intra-arterial drug injection, but rather looked at cerebral blood perfusion changes during emergence from general anesthesia for craniotomy using an intra-arterial pressure line [67]. Consequently, 20 trials were included in our study, all of which were interventional in nature. Included trials have been summarized in Table 2, with individual details listed in Tables S14–S24.

> Glioblastoma was the most common brain tumor indications for trials involving intra-arterial drug delivery (*n*= 13, 65%), followed by anaplastic astrocytoma (*n* = 8, 40%) (Table S14). The median commencement year was 2011, with trials reporting start dates between 1998 and 2019. As for expected completion year, the median was 2022 (range, 2008–2025) (Table S15). As of August 2021, 6 (30%) trials are reported to have completed

recruiting patients, 8 (40%) are still recruiting, and 2 (10%) are active but not recruiting. Two (10%) trials are declared as suspended (Table S16).

All trials reported target enrollment sizes between 3 and 60 patients, with the Portlandbased trial "NCT00075387: Combination Chemotherapy With or Without Sodium Thiosulfate in Preventing Low Platelet Count While Treating Patients With Malignant Brain Tumors" [68] targeting the most (*n* = 60). This trial has an estimated study completion date in spring 2023 (Table S17). With regard to age of enrollment, the median minimum patient age was 18 years (range, 1 month–18 years), with 18 (90%) of trials using this threshold. The median maximum patient age was 99 years (range 17–120 years), with 2 (10%) trials focusing solely on the pediatric demographic while 18 (90%) also included adult patients (Table S18).

**Table 2.** Summary of clinical trial characteristics.


**Table 2.** *Cont.*


PFS, progression-free survival; OS, overall survival; QOL, quality of life. \* Continuous data reported as the median (range) and categorical data reported as *n* (% total). ˆ Only 1 trial. # Does not sum to 20 as trials could report more than one condition, outcome, and therapeutic approach. <sup>1</sup> Superselective intra-arterial cerebral infusion into a major tumor feeding artery was performed using neurovascular microcatheter systems under fluoroscopic guidance to increase the concentration of drug delivered to the tumor while sparing the patient of systemic side effects. <sup>2</sup> Temporary opening of the blood–brain barrier was achieved by treating patients with an intra-arterial infusion of the osmotic agent mannitol followed by intra-arterial administration of therapeutic agents (mannitol 20–25%; 3–12.5 mL over 2 min).

Phase category was reported for all clinical trials included in this study. With 9 (45%) trials, phase 1 was the most common phase design. Eight (40%) trials were registered as both phase 1 and 2. A total of 3 (15%) trials were exclusively phase 2. Two phase 2 studies were randomized. Allocation was non-randomized in 4 phase 1 trials, while allocation type was not available for all other trials (Table S19).

In terms of the different types of primary intervention, therapeutic drug alone was the most common, with 14 (70%) trials. Combinations of radiation and drug therapy as well as therapeutic drug and psychological assessments were applied in 2 (10%) trials each. Investigations of biological agents alone (*n* = 1, 5%) and in combination with drug therapy (*n* = 1, 5%) were also reported (Table S20). Overall, there were 20 different types of primary intervention combinations evaluated in clinical trials of intra-arterial therapy for brain tumors (Table S21). Based on our original classification, targeted therapy was the therapeutic strategy most commonly investigated (*n* = 13, 65%), followed by chemotherapy (*n* = 8, 40%). Of these, i.a. bevacizumab (*n* = 5, 25%), i.a. cetuximab (*n* = 3, 15%), and i.a. melphalan (*n* = 2, 10%) were most common (Table S22). Transient BBB disruption was used in 12 (60%) trials. Thirteen (65%) trials explored superselective intra-arterial cerebral infusion as a strategy to optimize intra-arterial administration (Table S23).

Feasibility, safety, and toxicity of a treatment or intervention was the most common primary outcome reported (*n* = 11, 55%). With respect to other primary outcomes, 6 (35%) trials reported progression-free survival and 5 (25%) reported overall survival. One study (5%) reported tumor response and intracellular carboplatin accumulation as primary outcome measurement. The most common secondary outcome reported was progressionfree survival 11 (55%), followed by feasibility, safety, and toxicity (*n* = 9, 45%) and overall survival (*n* = 8, 40%) (Table S24).

As of August 2021, only 1 (5%) trial has posted results (NCT00362817: Carboplatin and Temozolomide (Temodar) for Recurrent and Symptomatic Residual Brain Metastases) [69]. This study started recruiting patients in 2004 and reported results in 2015. Seventeen patients older than 18 years, who had all received prior systemic chemotherapy for primary cancers in parts of the body other than the brain, were enrolled to investigate the use of intra-arterial carboplatin and oral temozolomide for the treatment of recurrent and symptomatic residual brain metastases. In terms of primary outcome, the reported response rate, evaluated by MRI criteria (MacDonald criteria), was approximately 43%. Secondary outcome measures included 25 weeks overall survival, 23 weeks progression-free survival,

and no incidence of CNS toxicities or CNS tumor-related deaths. In 7 (41%) cases, systemic disease progression was determined as cause of death (Table 3).


**Table 3.** Summary of clinical trial with reported results.

A total of 10 different institutions coordinated all 20 clinical trials on intra-arterial therapeutic delivery to brain tumors. Three different countries were listed as the location for correspondence of all trials, with the USA contributing 18 (90%) trials. The Lenox Hill Brain Tumor Center, located in New York City, coordinated the most trials (*n* = 10, 50%) [70–79]. The only other institution coordinating more than one trial was the OHSU Knight Cancer Institute in Portland, OR (*n* = 3, 15%) [68,80,81]. Only two trials had corresponding institutions outside the USA, with the Beijing YouAn Hospital (China) and the Centre hospitalier universitaire de Sherbrooke (Canada) both coordinating one (5%) trial [82,83] (Table S25). The median number of institutions involved was one (range, 1–3), with 18 (95%) studies involving a single institution. All trials were conducted in a single country.

#### **4. Discussion**

The intention of this study was to identify and characterize the published literature and registered clinical trials on intra-arterial drug administration for brain tumor treatment. We identified 271 articles and 20 trials to meet our inclusion criteria. These numbers are reflected in the quoted numbers reported by recently published reviews [31,84], even though this is the first study to offer a precise number of clinical trials involving intraarterial brain tumor therapy, highlighting a previously unreported area in the field as to how many distinct trials have officially been registered and conducted since the technique was first described in 1950 [85]. The complexity of successfully translating intra-arterial drug delivery into the clinic is demonstrated by the fact that in the last 20 years, only 6 trials eligible for this review have completed recruitment [69,74,76,77,82,86], and results of just a single study are publicly available at the ClinicalTrials.gov portal as of August 2021 [69]. Despite these findings, given the discovery of novel biological and molecular features of brain tumors potentially amenable to therapy [12,13,87,88], we posit that more tumor-specific intra-arterial interventions will emerge in future trials to add to the current body of research studies.

The development of intra-arterial technologies was historically driven by the need to minimize the systemic toxicity of traditional anti-cancer agents and propelled by advances in endovascular techniques; however, very few studies took into consideration the pharmacokinetic characteristics underlying intra-arterial drug delivery [89]. Although the neuro-oncological application of intra-arterial technology has been established by impactful CL articles [34,38,85], accurate and reliable pharmacological models to optimize

the method, rate, and duration of drug injection for high local extraction and systemic clearance may be lacking to date [90]. Furthermore, biological hurdles to intra-arterial therapy of brain tumors, including the vascular heterogeneity within the tumor microenvironment, have to be considered in ongoing research efforts and future refinements [91]. The lack of effective therapies to be delivered by the intra-arterial route and reported in BSc articles could in part explain why CL articles and clinical trials remain without definitive success. Consequently, we expect to observe an increase in BSc articles in the future as our understanding of this technology and our ability to modify relevant drug properties continue to grow.

For those within the field of intra-arterial therapy for brain tumor treatment, it is not surprising that E.A. Neuwelt and J.A. Boockvar were identified as particularly impactful authors who pioneered the field and spearheaded recent advances of this drug delivery technique in terms of preclinical and clinical research. The portfolio of both these authors was predominantly focused on CL articles and clinical trials based in the USA. When considering all articles included in this study, E.A. Neuwelt was the author of most with 14 articles overall, of which he senior-authored 8 CL articles [34,51,55–60,92–94]. Through the Neuro-Oncology Blood-Brain Barrier Program at OHSU, he was also involved in initiating 3 clinical trials that are currently being conducted at the OHSU Knight Cancer Institute [68,80,81]. J.A. Boockvar authored 10 articles overall, serving as the corresponding author of 5 original and 3 review CL articles [35–37,61–66,95]. In his role as vice chair of neurosurgery at Lenox Hill Hospital, he has also been in charge of 10 clinical trials that were registered to evaluate intra-arterial brain tumor therapies, including bevacizumab, cetuximab, trastuzumab, and temozolomide alone or in combination with carboplatin, radiotherapy, and/or mannitol, for conditions such as glioblastoma, anaplastic astrocytoma, vestibular schwannoma, and brain metastasis [70–79]. Collectively, the clinical trials overseen by J.A. Boockvar and E.A. Neuwelt account for more than half of all trials included in this study. The general focus of both authors on translational research is largely reflected in the current research landscape of intra-arterial drug delivery for brain tumors, highlighting their significant impact on the field.

When Perese et al. [96] first proposed the intra-arterial route as a drug delivery strategy for treating patients with malignant brain tumors, they posited that this technology could be used to deliver large concentrations of a variety of anti-cancer agents to the brain without causing much systemic reaction. More than a half-century later, based on this study of pertinent articles and clinical trials, it appears their vision has, in part, been realized, but new hurdles have emerged. The largest indication for intra-arterial administration was chemotherapy, with 215 of 271 articles describing its BSc or CL use. Indeed, intra-arterial chemotherapy allowed relatively high dosing while minimizing systemic toxicity [41,59,63,64]. However, articles on chemotherapeutic drugs peaked over three decades ago, possibly indicating that these therapies were lacking efficacy with intraarterial use. Furthermore, several articles alluded to chemotherapy-related safety concerns, some of which were unique to the intra-arterial delivery route [38,58,60,93]. This could explain why only a few traditional chemotherapeutics, all of which had previously demonstrated safety and efficacy in preclinical models and via other drug delivery strategies, have found their way to clinical trials. The fact that targeted therapy was the most commonly used therapy type in clinical trials could suggest a paradigm shift towards novel therapeutic strategies. Based on exciting developments in modern neuro-oncology, involving precision medicine [97], immunotherapy [98], and stem cell technology [99], we anticipate an increased number of articles and clinical trials investigating these therapies with intra-arterial injection in the future [100].

As effective treatment modalities for different types of brain tumors are desperately needed, it is not surprising that a number of technologies to improve the therapeutic effect of intra-arterial drug delivery have been proposed [24,30]. BBB penetrance, targeting, and accuracy of intra-arterial administration are of major interest, which is underscored by the high number of articles and clinical trials exploring strategies to account for these parameters. We found chemical reagents, and mannitol in particular, to be the oldest and most common approach to open the BBB. However, the capability of mannitol and newer agents, such as the bradykinin agonist RMP-7, to permeabilize the BBB is limited [50,63,101]. Focused ultrasound is a non-invasive strategy for disrupting BBB tight junctions in a reversible and controlled fashion and has the potential to be used with intra-arterial technologies [32,102,103]. The scientific complexity of this concept is underlined by the fact that of all 271 articles included in this study, only 2 review articles described focused ultrasound in combination with intra-arterial drug delivery [20,104]. Although mentioned by only a small proportion of articles, superselective intra-arterial cerebral infusion into the tumor-feeding arteries was reported in 13 of 20 clinical trials. This possibly indicates a trend towards using this advanced endovascular procedure to reduce neurotoxic side effects and ensure targeted intra-arterial therapy [35–37,61,63–65,95]. It is interesting to observe that several articles published in recent years investigated nanoparticles. The attraction of nanotechnology for intra-arterial drug delivery is multifold. Various authors noted these small particles could not only be loaded with different therapies, including small-molecule inhibitors, gene therapies or siRNAs, but could also be modified to cross the BBB through a variety of transport mechanisms and remain at the target site for longer periods of time to allow for a gradual release of loaded therapeutics [23,105–113].

We speculate that future refinements of intra-arterial brain tumor therapy will come from multiple angles. From a therapeutic perspective, studies to date are using mostly approved agents, as they are easier to translate; however, certain novel compounds may have superior pharmacokinetics and be ultimately more useful for intra-arterial drug delivery. Disease-specific drugs will enhance efficacy and minimize systemic and CNS-related side effects [87]. Drug modification or loading into nanoparticles can allow for improved BBB penetration, tumor targeting, and drug-tumor contact time of active compounds [114–116]. Labeled therapeutic agents as well as theragnostic nanoparticles have the potential to be used with advanced imaging techniques [50,106,109]. From a procedural point of view, optimizing currently available endovascular technologies and combining them with innovative strategies, such as superselective intra-arterial cerebral infusion or transient cerebral hypoperfusion/flow arrest, is crucial to ensure procedural safety and effect [31,61,89]. With regard to clinical trials, it will be important to move from phase 1 and 2 to phase 3 trials and to evaluate novel drugs and procedures in the context of randomized patient allocations. This will help to ensure the validity of conclusions not only from a feasibility and safety perspective but also in terms of efficacy. The inability of the vast majority of clinical trials to report results suggests that a collaborative approach may be necessary to improve the fate of future trials. Therefore, we stress the importance of intra- and inter-institutional collaboration in preclinical and clinical research for progress within the field of intra-arterial brain tumor therapy.

The intra-arterial therapeutic concept constitutes only one of many technologies to facilitate drug delivery to the brain and brain tumors. An enhanced understanding of the BBB physiology has led to the development of a multitude of noninvasive and invasive strategies to target tumor cells present beyond this barrier. Locoregional invasive technologies, in particular, are a remarkably fast-evolving segment within the neurooncology field. These technologies are based on local delivery of therapeutics directly to the brain, thereby bypassing the BBB entirely and facilitating smaller initial drug dosage and minimal systemic absorption. They include drug delivery to the cerebrospinal fluid via intrathecal or intraventricular injections and interstitial delivery via biodegradable polymers or catheters [17]. Diffusion-based approaches such as intracavitary wafers placed at the time of tumor resection, intrathecal injection using the Ommaya reservoir, and intraventricular injection via lumbar puncture are limited by the restricted tissue penetrance of most therapeutic agents into structures not immediately adjacent to the brain surface, hindering them from reaching deep and infiltrative tumor cells [21,27,117]. However, molecularly engineered cells, especially chimeric antigen receptor (CAR) T-cells and natural killer cells, feature enhanced tumor-homing abilities and have shown promise in preclinical and

clinical investigations of these strategies in various brain tumors [118–126]. In contrast to locoregional therapies, intra-arterial administration takes advantage of the branched blood vessel system that is feeding into brain tumors, thereby reaching even distant, infiltrative tumor cells, and enabling site-directed infiltration of cell-based therapies or diffusion of macromolecules [127].

Direct interstitial drug infusion to brain tumors is achieved by placing one or multiple catheters under stereotactic guidance into the bulk tumor. These cannulas can be connected to osmotic or mechanical pumps and allow for direct, targeted delivery [28,29]. The former can provide continuous drug delivery at a set infusion rate, whereby the infusion is driven by an osmotic pressure gradient [128]. Convection-enhanced delivery (CED), which describes direct interstitial infusion under a mechanical pressure gradient, has further advantages, including a larger, more homogeneous volume of drug distribution [129]. This technology is currently being used in multiple clinical trials for brain tumors like glioblastoma and diffuse intrinsic pontine glioma (DIPG) [130–138]. In addition to technical and procedural challenges, such as catheter design and placement, tracking of infusate distribution, prevention of reflux along the canula tract, and reduction in edema and mechanical tissue damage, the success of CED is hampered by the potential requirement of repetitive infusions [139,140]. Almost 100 clinical trials for DIPG have failed, and it has recently been shown that the brain half-life of panobinostat, a small molecule inhibitor, after CED is only 2.9 h [141,142]. Intra-arterial technologies address some of these pitfalls, allowing for repeated and prolonged therapeutic administration to ensure pharmacologic effect, but are accompanied by their own constraints, as discussed above. Consequently, a "one size fits all" approach may not be effective, and a rational combination of therapeutics and their delivery strategies should be considered. In this way, a comprehensive treatment regime to attain optimal concentrations in brain tumors over a prolonged period of time while minimizing off-target effects could be established.

There are certain limitations that are inherent to bibliometric and clinical trials analyses. First, Elsevier's Scopus and ClinicalTrials.gov are only two of many databases for articles and clinical trials, respectively. With both of them being US-based, this may have influenced the predominance of US-based literature and trials in this study. There is validity to the argument that our representation of the research landscape of intra-arterial brain tumor therapy would have been more comprehensive if additional registries would have been included. Even though both Scopus and ClinicalTrials.gov constitute major databases in their respective field, holding registrations from journals and institutions around the world, there is a distinct possibility that this study is an underestimate of all articles and trials evaluating intra-arterial brain tumor treatments. Second, since our therapy type and treatment strategy classifications were solely based on the literature and our own experience, it is unclear whether the presented categories and proportions optimally reflect the current status and trends of the field. It is possible that more refined categorizations would have led to a better representation. Finally, article and clinical trial parameters, e.g., citation count and number of institutions involved, are regularly updated to rigorously reflect the current state of affairs. As it is difficult to continue updating these parameters once they have been extracted, our study represents the status quo and trends of articles and clinical trials on intra-arterial drug delivery for brain tumor treatment as of August 2021. We anticipate that analyses in the future will confirm whether these were accurate.

#### **5. Conclusions**

In this bibliometric and clinical trials analysis, we identified, characterized, and analyzed available parameters of preclinical and clinical research on intra-arterial therapy for brain tumors. Overall, 271 articles and 20 clinical trials were sufficiently specific for inclusion. Among articles, most were CL and chemotherapy was the most common therapeutic modality. With respect to treatment strategies for optimizing intra-arterial drug delivery, transient blood–brain barrier disruption using mannitol was the most frequently studied. These trends were reflected in clinical trials, but unfortunately only a single phase 1/phase 2 study has reported outcomes to date. Given the longstanding history of intra-arterial brain tumor therapy research, our results mandate the consideration of novel therapeutic and procedural strategies, including precision medicine, nanoparticles, and superselective intra-arterial cerebral infusion, to foster the preclinical research basis and set the stage for more robust, systematic clinical trials in the future.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/pharmaceutics13111885/s1, Table S1. Articles on intra-arterial therapy for brain tumors based on publication type; Table S2. Classification of tumors investigated in articles on intra-arterial therapy for brain tumors; Table S3. Citations for articles on intra-arterial therapy for brain tumors; Table S4. Authors of articles on intra-arterial therapy for brain tumors; Table S5. Years of publications of intraarterial therapy for brain tumors differentiated by article type; Table S6. Involvement of countries in articles on intra-arterial therapy for brain tumors; Table S7. Involvement of journals in articles on intra-arterial therapy for brain tumors; Table S8. Investigated chemotherapies and application ways in articles on intra-arterial therapy for brain tumors; Table S9. Investigated targeted therapies in articles on intra-arterial therapy for brain tumors; Table S10. Investigated immunotherapies in articles on intra-arterial therapy for brain tumors; Table S11. Investigated radiosensitizing/neutron capture therapies in articles on intra-arterial therapy for brain tumors; Table S12. Investigated stem cell therapies in articles on intra-arterial therapy for brain tumors; Table S13. Investigated treatment strategies in articles on intra-arterial therapy for brain tumors; Table S14. Tumor types investigated in clinical trials on intra-arterial therapy for brain tumors; Table S15. Timeline of clinical trials on intra-arterial therapy for brain tumors, including start year, completion year, year of the first release of results, and last updated year; Table S16. Recruitment status of clinical trials on intra-arterial therapy for brain tumors; Table S17. Number of patients enrolled in clinical trials intra-arterial therapy for brain tumors; Table S18. Minimum and maximum age of enrollment in clinical trials on intra-arterial therapy for brain tumors; Table S19. Distribution of the different study phases of clinical trials on intra-arterial therapy for brain tumors; Table S20. Type of primary intervention investigated by the clinical trials on intra-arterial therapy for brain tumors; Table S21. Detailed primary interventions applied by the clinical trials on intra-arterial therapy for brain tumors; Table S22. Therapies investigated in clinical trials on intra-arterial therapy for brain tumors; Table S23. Treatment strategies investigated in clinical trials on intra-arterial therapy for brain tumors; Table S24. Different outcome measurements of clinical trials on intra-arterial therapy for brain tumors with respect to primary and secondary outcome, overall survival, progression-free survival, safety aspects, and quality of life; Table S25. Sponsors, institutions and countries involved in clinical trials on intra-arterial therapy for brain tumors.

**Author Contributions:** Conceptualization, J.S.R. and D.J.D.; methodology, J.S.R.; software, F.T.; validation, J.S.R. and D.J.D.; formal analysis, J.S.R. and F.T.; investigation, J.S.R.; resources, J.S.R.; data curation, J.S.R. and F.T.; writing—original draft preparation, J.S.R.; writing—review and editing, F.T. and D.J.D.; visualization, F.T.; supervision, D.J.D.; project administration, J.S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data supporting reported results can be found in the manuscript and supplementary materials to the manuscript.

**Acknowledgments:** No individuals other than the listed co-authors contributed to this publication.

**Conflicts of Interest:** The authors declare no conflict of interest.

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