Focused Ultrasound Impels the Delivery and Penetration of Model Therapeutics into Cerebral Cavernous Malformations

Introduction: Cerebral cavernous malformations (CCMs) are vascular neoplasms in the brain that can cause debilitating symptoms. Current treatments, such as surgical resection and stereotactic radiosurgery, pose significant risks to patients, motivating the development of new treatment options. Despite multiple studies screening pharmacological treatments for CCM, effective nonsurgical interventions remain elusive. The majority of tested pharmacological agents for CCM are small molecules, highlighting the unexplored potential of larger molecules such as antibodies and gene therapies. Additionally, drugs showing promise in acute CCM models often demonstrate limited efficacy in more clinically-representative chronic models, suggesting a potential need for greater local doses of these therapies. Indeed, though CCMs are known to be more permeable than healthy cerebrovasculature, delivery of systemically administered drugs to these complex lesions is poorly understood.

Recently, our group demonstrated that focused ultrasound-mediated blood-brain barrier opening (FUS), even in the absence of therapeutic delivery, arrests the formation and growth of CCMs (1). Building on this observation, the objective of this study was to establish a foundation for therapeutic delivery approaches that harness and synergize with this potent bioeffect. We have previously demonstrated that T1-contrast mapping can enable longitudinal, quantitative concentration measurements of gadolinium-based molecules in CCMs. Thus, this is an ideal method to measure FUS-induced changes for therapeutic delivery to CCMs. To this end, we employed T1-contrast mapping MRI to quantitatively evaluate the delivery of 1 kDa and 17 kDa molecules to CCMs, comparing outcomes with and without FUS.

Materials and

Methods: Quantitative T1 mapping 9.4T MRI sequences were used with 1 kDa (MultiHance; MH) and 17 kDa (GadoSpin D; GDS) contrast agents to assess the FUS-mediated delivery and penetration of model small molecule drugs and biologics, respectively, into CCMs of Krit1 mutant mice. FUS-mediated blood-brain barrier opening was achieved using a 1.1 MHz single-element transducer with a 10 ms burst length over a 2000 ms period. A total of 60 sonications were administered during a 2-min sonication duration. The feedback control system parameters were set as follows: a starting pressure of 0.2 MPa, pressure increment of 0.05 MPa, maximum pressure of 0.4 MPa, 20 sonication baselines without microbubbles, area under the curve (AUC) bandwidth of 500 Hz, AUC threshold of 10 standard deviations, pressure drop of 0.95, and frequency selection of the subharmonic, first ultraharmonic, and second ultraharmonic. Microbubbles were intravenously injected as a bolus dose of 10^5 microbubbles per gram of body weight. T1 mapping MRI sequences were used to guide sonication targeting. Six non-overlapping sonication targets were placed over one frontal hemisphere with placement optimized to target CCMs. MH and GDS were injected as a bolus intravenously at a dose of 0.01 and 0.0002 mmol, respectively, diluted in saline. Injection of contrast agent was given immediately prior to MRI acquisition for FUS- control studies and immediately following the initiation of FUS for FUS+ studies.

Results, Conclusions, and Discussions: We first tested whether FUS increases the delivery rate of a model small molecule drug (MH) to CCMs (Figure 1A). After FUS, MH accumulation rate in the lesion core was enhanced (Figure 1A, B), increasing to well-above (4.6-fold) that of contralateral FUS- CCMs (p=0.0221; Figure 1A, C). Moreover, FUS increased perilesional MH delivery rate by 6.7-fold over FUS- CCMs (p < 0.0001; Figure 1D, E). Next, we tested the ability of FUS to enhance the total delivery of a model biologic agent (GDS) to CCMs. Baseline permeability of CCMs to GDS was measured on day 1 (Figure 1F). On day 2, FUS was applied to multiple CCMs. FUS improved total GDS delivery in both the intralesional and perilesional spaces compared to baseline permeability (Figure 1G). Strikingly, FUS elicited a 22-fold increase in GDS delivery to the lesion core (p=0.0106; Figure 1H). The “area under the curve” (AUC) metric, representing the integrated exposure of tissue to drug, indicated a 4.8-fold increase (p=0.0078; Figure 1I). Meanwhile, perilesional delivery of GDS was enhanced by 3.8-fold with FUS (p= 0.0021; Figure 1J), corresponding to a 2.2-fold increase in AUC (p=0.0195; Figure 1K). Lastly, we investigated if FUS differentially affects total delivery of MH and GDS by comparing their magnitude of FUS enhancement. AUC showed an increase in the intralesion-to-perilesion ratio of model drug exposure for GDS vs. MH with FUS (p=0.045; Figure 1L). Indeed, over a 20 min post-FUS time period, this ratio was typically < 1 for MH and >2 for GDS (Figure 1M). In conclusion, FUS is capable of impelling the delivery and penetration of therapeutics into the complex and disorganized CCM microenvironment. Benefits to small molecule drug delivery are more evident in the perilesional space, while benefits to biologic delivery are more evident in CCM cores. These findings, when combined with ability of FUS alone to control CCMs (1), highlight the potential of FUS to serve as a powerful non-invasive therapeutic platform for CCM.

1. Fisher, D.G., et al. (2024) Focused Ultrasound Blood-Brain Barrier Opening Arrests the Growth and Formation of Cerebral Cavernous Malformations. bioRxiv. DOI: https://doi.org/10.1101/2024.01.31.577810.

Acknowledgements (Optional): Supported by NIH R01CA279134, R01EB030409, and R01EB030744