Assessment of Brain Endothelial Permeability, Transport, and Flow

Background Introduction

The blood-brain barrier (BBB) is a critical barrier that protects the brain from harmful substances while also serving as a key obstacle for drug delivery to the brain. The permeability and transport properties of the BBB directly influence the distribution of drugs in the brain, particularly the efficacy of central nervous system (CNS) drugs. However, measuring the permeability of highly permeable drugs (e.g., lipophilic drugs) at the BBB has been a technical challenge, as traditional cerebral blood flow measurement methods struggle to accurately assess permeability for such drugs. To address this issue, researchers have developed new experimental methods aimed at overcoming the limitations of traditional approaches and directly measuring the permeability of highly permeable drugs at the BBB.

This study, led by Quentin R. Smith and colleagues, aimed to measure the BBB permeability of highly lipophilic drugs using the in situ brain perfusion technique and validate its relationship with drug lipophilicity (log Poct). The research team included members from multiple institutions, such as Texas Tech University Health Sciences Center and the University of Nebraska Medical Center. The study was published in 2024 in the journal Fluids and Barriers of the CNS.

Research Process

1. Experimental Design

The primary goal of the study was to extend the measurement range of BBB permeability using two methods: increasing cerebral blood flow rate and adding plasma proteins. These methods aimed to reduce the extraction rate of drugs, bringing it into a measurable range, thereby enabling the calculation of BBB permeability using the Crone-Renkin “diffusion-flow” equation.

2. Experimental Subjects

The study used rats and mice as experimental subjects. Specifically, adult male Sprague-Dawley rats (200-350 g) and CF-1 mice (30-40 g) were used for the in situ brain perfusion experiments. All experiments were conducted in accordance with protocols approved by the institutional animal care and use committee.

3. In Situ Brain Perfusion Technique

The study employed a modified in situ brain perfusion technique, with the following steps: - Anesthesia and Surgical Preparation: Animals were anesthetized via intraperitoneal injection (e.g., ketamine/xylazine or sodium pentobarbital), followed by carotid artery cannulation to ensure direct delivery of perfusion fluid to the brain. - Perfusion Fluid Preparation: The perfusion fluid was physiological saline containing varying concentrations of plasma proteins (e.g., serum albumin or alpha-1 acid glycoprotein) to simulate different plasma protein binding scenarios. - Perfusion Process: The perfusion fluid was infused into the carotid artery using a constant-rate pump, while perfusion pressure was monitored. The perfusion fluid contained radiolabeled test solutes (e.g., diazepam, palmitate) and flow markers (e.g., iodoantipyrine). - Brain Tissue Sampling and Analysis: After perfusion, the brain was rapidly removed and dissected into regions. Brain tissue samples were analyzed using liquid scintillation counting (LSC) to measure the content of radiolabeled substances, calculating brain uptake rate (kin) and vascular permeability (PS).

4. Data Analysis

The study used the Crone-Renkin equation to calculate BBB permeability. This equation considers the relationship between blood flow rate (F), permeability-surface area product (PS), and the free fraction of the drug (fu). Through nonlinear regression analysis, the researchers fitted the experimental data to calculate the BBB permeability of different drugs.

Key Findings

1. Measurement of BBB Permeability at High Blood Flow Rates

By increasing cerebral blood flow rates, the researchers successfully extended the measurement range of BBB permeability. The experimental results showed that the brain uptake rate (kin) of lipophilic drugs such as diazepam significantly increased at high blood flow rates and exhibited a linear relationship with blood flow rate. The BBB permeability (PS) of diazepam reached 0.94 ml/s/g, far exceeding previous study results (0.03-0.06 ml/s/g).

2. Impact of Plasma Protein Binding on BBB Permeability

The addition of plasma proteins (e.g., bovine serum albumin) reduced the free fraction (fu) of the drug, thereby decreasing the brain uptake rate. By adjusting plasma protein concentrations, the researchers were able to control the extraction rate of drugs within a measurable range and accurately calculate BBB permeability using the Crone-Renkin equation. The experimental results showed that the BBB permeability of diazepam, flunitrazepam, and palmitate was 0.81-0.93 ml/s/g, 0.14 ml/s/g, and 2.8 ml/s/g, respectively.

3. Relationship Between Drug Lipophilicity and BBB Permeability

The study analyzed the BBB permeability of 125 solutes, 78 of which showed no significant active efflux transport. The results revealed a linear relationship between BBB permeability and drug lipophilicity (log Poct), spanning 10 orders of magnitude. Notably, the BBB permeability of drugs such as diazepam, estradiol, and testosterone was significantly higher than previously reported values.

Conclusions and Significance

1. Scientific Value

This study successfully measured the BBB permeability of highly lipophilic drugs by improving experimental methods, filling a gap in traditional approaches for measuring highly permeable drugs. The results demonstrated a significant linear relationship between BBB permeability and drug lipophilicity, providing an important theoretical basis for the screening and design of future CNS drugs.

2. Practical Value

The study offers new experimental methods for drug development, particularly in assessing the BBB permeability of highly lipophilic drugs. By increasing blood flow rates and adding plasma proteins, researchers can more accurately measure drug BBB permeability, optimizing drug distribution and efficacy in the brain.

3. Research Highlights

• Innovative Experimental Methods: By increasing blood flow rates and adding plasma proteins, the researchers successfully extended the measurement range of BBB permeability.
• High-Precision Data: The study provided high-precision BBB permeability data for highly lipophilic drugs, with the permeability of drugs such as diazepam significantly exceeding previous results.
• Broad Application Prospects: The study offers new experimental methods and theoretical support for the screening and design of CNS drugs, with broad application prospects.

Additional Valuable Information

The study also found that BBB permeability is not only related to drug lipophilicity but is also influenced by molecular weight (MW). Through multiple linear regression analysis, the researchers discovered that log Poct and log MW significantly impact BBB permeability, further revealing the complex mechanisms of drug transport across the BBB.

Through innovative experimental methods and in-depth data analysis, this study provides new perspectives and approaches for measuring BBB permeability, offering significant scientific and practical value.