In recent years, with the increasing prevalence of neurological disorders such as Alzheimer's disease, depression, schizophrenia, and Parkinson's disease , the development of CNS drugs has gained significant attention. However, due to unclear disease mechanisms and challenges such as the blood-brain barrier (BBB), the success rate of CNS drug development remains low. This article will provide an overview of the relevant theoretical knowledge and research methodologies for CNS drugs from a pharmacokinetic (PK) perspective.
1. Barriers to CNS Penetration
As shown below, the BBB is composed of tightly packed brain capillary endothelial cells and neuroglial cells, which almost entirely block compounds from crossing via paracellular pathways or endocytosis. The BBB also expresses efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which pump most substrates back into the bloodstream. Therefore, most small molecules penetrate the brain primarily through passive diffusion across the lipid membranes. Additionally, the blood-cerebrospinal fluid barrier (BCSFB) is another significant barrier. Compounds in the blood can directly cross the BCSFB or, after crossing the BBB, diffuse or be carried into the cerebrospinal fluid (CSF). However, P-gp expressed at the BCSFB may pump compounds back into the blood, limiting brain exposure.
2. Key Parameters for CNS Drug Penetration
Given the difficulty in crossing the BBB, screening compounds with favorable CNS penetration in in vitro or animal studies becomes crucial. Selecting appropriate parameters to serve as evaluation criteria is key. Commonly used parameters include unbound drug concentration in the brain (Cbu), the brain-to-plasma unbound drug ratio (Kpuu or Cbu/Cpu), the brain-to-plasma total drug concentration ratio (B/P ratio), unbound drug fraction (fu), BBB permeability (Papp), and efflux ratio (ER).
2.1 Unbound Drug Concentration in the Brain
Based on the free drug hypothesis, the unbound drug concentration in the brain best reflects the effective drug concentration. However, it is difficult to directly measure the unbound drug concentration within brain cells under practical conditions. In the absence of significant transporter effects, unbound drug concentrations in plasma, CSF, or brain interstitial fluid are often used as substitute parameters.
2.2 Brain-to-Plasma Unbound Drug Ratio (Kpuu)
Kpuu is a key parameter reflecting the distribution equilibrium of compounds between blood and brain, integrating passive diffusion and transporter activity. When Kpuu approaches 1, it indicates desirable compound characteristics, such as good permeability and non-transporter substrates. In this case, the plasma unbound drug concentration can serve as a surrogate indicator. When Kpuu is significantly less than 1, the compound may be a transporter substrate or have poor permeability, necessitating structural modifications. A Kpuu greater than 1 may indicate the involvement of active transporters in the transmembrane transport process.
2.3 Brain-to-Plasma Total Drug Concentration Ratio (B/P Ratio)
Although easy to obtain, the B/P ratio can mislead understanding of a compound's brain penetration. Due to differences in the unbound fractions between plasma and brain, a high B/P ratio does not necessarily imply a high concentration of unbound drug in the brain, and vice versa. The B/P ratio is only useful for rough stratification, eliminating compounds with extremely low total brain concentrations but not for selecting superior CNS compounds.
2.4 Unbound Fraction (fu)
The unbound fraction in plasma and brain tissue is used to convert standard PK parameters into unbound drug parameters. While higher unbound fractions can indicate higher effective drug concentrations, increased plasma unbound fractions may also lead to higher metabolism or elimination rates. Similarly, higher unbound fractions in brain tissue may hinder passive diffusion into the brain. Therefore, fu cannot typically serve as a primary optimization parameter, and its value is not directly linked to efficacy.
2.5 BBB Permeability (Papp)
Papp represents the rate of passive diffusion of a compound across the BBB. However, this parameter alone is not decisive in determining brain penetration. It merely reflects the rate at which equilibrium between plasma and brain is achieved but does not imply a high absolute concentration of unbound drug in the brain. Therefore, Papp can only be used to eliminate compounds with extremely poor BBB permeability.
2.6 Efflux Ratio (ER)
ER reflects the strength of efflux transporter activity during BBB penetration. While it does not directly indicate the concentration of a compound in brain tissue, it can serve as an exclusion criterion. Ideally, CNS drugs should not be substrates of efflux transporters like P-gp or BCRP, as they hinder drug delivery to target sites and increase clinical PK variability. This is especially problematic for compounds with narrow therapeutic windows, posing risks and complicating development.
3. Evaluation Methods
3.1 Animal Studies
Animal studies are the most direct and effective means of obtaining in vivo concentrations in brain tissue, CSF, and plasma. In general, the species used for PK screening should align with those used for efficacy and toxicity studies. The route of administration is determined by the development goals. The procedure is similar to conventional PK studies, with additional attention to the collection of brain tissue or CSF. Results should be corrected using in vitro determined fu values for plasma and brain tissue, as CSF has low protein content and requires no correction. The AUCbu/AUCpu ratio represents Kpuu, which evaluates a compound's ability to cross the BBB. Comparing AUCcsf with AUCpu assesses the compound's distribution across the BCSFB, while the AUCbu/AUCcsf comparison indicates whether CSF can serve as a proxy for unbound drug concentration in brain tissue, optimizing the research process. The downside of brain PK studies is the relatively high animal use and low screening throughput.
3.2 Transporter Studies
Compared to other drugs, transporter studies play a more significant role in CNS drug research due to the broad substrate specificity of efflux transporters like P-gp and BCRP, which significantly limit brain penetration. In vitro studies often use transfected cell lines with high P-gp or BCRP expression (e.g., MDCK-MDR1 or MDCK-BCRP) for high-throughput screening, with reasonable cut-off values based on compound number and screening strategy. However, species differences in transporters can lead to discrepancies between in vitro human transporter data and in vivo animal studies, occasionally necessitating the use of knockout animal models (e.g., MDR1a/1b or BCRP KO mice), though this approach is costly and less predictive of human transporter activity.
3.3 BBB Permeability Studies
The gold standard for studying BBB permeability is in situ brain perfusion, but due to technical requirements and low screening throughput, this method is rarely used for compound screening. Commonly used methods include in vitro assays such as PAMPA-BBB or single-layer cell transport studies, which resemble conventional absorption permeability studies but are more reflective of BBB characteristics. Early-stage studies can also use computational models based on physicochemical properties to predict BBB permeability, aiding compound optimization.
3.4 Protein Binding Studies
The most common method for determining protein binding is equilibrium dialysis of plasma and brain tissue homogenates. For compounds with strong non-specific binding, it is necessary to adjust the experiment's equilibrium time to achieve true balance and avoid bias. Alternative methods include Transil and progressive equilibrium dialysis. Studies have shown that brain tissue binding is independent of species or brain regions, meaning that human brain tissue binding data can be extrapolated from single-species data. Other methods such as ultracentrifugation, microdialysis, and brain tissue slice methods are also available based on specific needs.
Conclusion
Understanding the factors influencing brain exposure of CNS drugs and applying appropriate research methodologies are critical for their development. Identifying key parameters for screening and optimization can help increase brain exposure, avoid efflux transporter substrates, and reduce systemic clearance, ensuring sufficient drug reaches target sites to exert therapeutic effects. However, the clinical translation of CNS drug PK remains challenging, and future advancements may rely on more mechanistic models based on BBB transporters and physiological structures to aid CNS drug development.
