**4.1.3 The rate and extent of drug penetration to the brain**

Neuropharmaceuticals should be able to permeate the BBB and enter the brain parenchyma in order to treat desired disorders whereas peripheral drugs should have limited entrance to the brain in order to decrease their neurological side effects. The drug entrance to the brain was evaluated and quantified using different methods, among them BUI, logBB, Kp,uu etc, are well studied and frequently used to measure the rate and the extent of brain drug penetration (Jeffrey & Summerfield, 2010).

The free drug is responsible for pharmacokinetic and pharmacodynamic properties of drugs and relation between dose and response is correct when free drug supplies in target tissue get into account. In this regard interstitial fluid and intra cellular fluid drug levels in brain

The traditional methods of brain homogenization destroy all compartments of brain (including brain tissue binding and plasma protein binding) and drug levels in specific compartments cannot be measured (Reichel, 2009). The plasma free fractions data cannot be used in CNS drug discovery studies, because of the different physiological properties, blood brain interstitial fluid free fractions. Some researchers used cerebrospinal drug levels (CSF sampling) as an estimate of the unbound drug levels in brain which is not so reliable because of lower tightness of cerebra-spinal blood barrier which leads to higher diffusion and overestimation of free drug concentration in brain (Read & Braggio, 2010). The microdialysis is the only *in vivo* method to provide such data directly, which is limited by its

Neuropharmaceuticals should be able to permeate the BBB and enter the brain parenchyma in order to treat desired disorders whereas peripheral drugs should have limited entrance to the brain in order to decrease their neurological side effects. The drug entrance to the brain was evaluated and quantified using different methods, among them BUI, logBB, Kp,uu etc, are well studied and frequently used to measure the rate and the extent of brain drug

Fig. 5. Different equilibria in brain.

are important data for drug discovery.

practicability.

**4.1.2 The importance of free drug measurement** 

**4.1.3 The rate and extent of drug penetration to the brain** 

penetration (Jeffrey & Summerfield, 2010).

Brain uptake index (BUI%) is one of the earliest indicators of BBB permeability of compounds and is calculated by:

$$BIII\% = 100 \frac{E}{E\_{ref}} \tag{1}$$

where E denotes the first pass extraction and the Eref referred to freely diffusible internal standard. This indicator provides information about the total concentration of the drug in the brain at early time point after administration (Lanevskij et al., 2010).

The logBB which describes the ratio between brain and blood (or plasma) concentrations and provide a measure of the extent of drug permeation is calculated using (Kerns & Di, 2008):

$$\log{BB\,\,or\,\,K\_p} = \frac{\text{AUC}\_{\,\,to\,\,brain}}{\text{AUC}\_{\,\,to\,\,hload}}\tag{2}$$

The only information provided by Kp is passive lipid partitioning of the drug which is affected by metabolism, relative binding affinity to proteins and lipid content of brain and blood or plasma and it is not a net measure of BBB permeability (Abbott, 2004; Mehdipour & Hamidi, 2009). It is highly time dependent and in order to get an overall estimation, usually is measured under steady-state conditions.

Another approach based on unbound drug fraction, for quantifying the extent of brain penetration is recommended, which is calculated by:

$$K\_{p, \text{uu}} = \frac{ALIC \\_\text{u, brain}}{ALIC \\_\text{u, blood}} \tag{3}$$

Kp,uu affected by both passive diffusion and active influx/efflux and can give information about the permeation mechanism, beyond these, it is not affected by plasma protein and brain tissue binding which interfere in logBB values (Mehdipour & Hamidi, 2009). For drugs delivered by passive diffusion, this index will be close to unity while for efflux and influx substrates it will be less than and more than unity respectively (Hammarlund- Udenaes et al., 2008).

To assess the brain drug permeability rate, the unidirectional influx constant from blood to the brain (Kin) and the product of the BBB permeability surface area (PS) which is a measure of the unidirectional clearance from blood to brain have been developed. Both parameters expressed as ml/min/g of brain (Rooy et al., 2010). PS is able to reflect the BBB permeation step more accurately (Abbott, 2004) and is valuable parameter for follow up permeation ability of drug candidates in the pharmaceutical industry and although in pathologic conditions. PS gives an estimation of unbound drug in brain but it is affected by the possible association of the drug with active influx or efflux transporters (Hammarlund- Udenaes et al., 2008).

According to the measurement method Kin and PS can be calculated from Crone-Renkin equation:

$$K\_{in} = F\left(1 - e^{\frac{-PS}{F}}\right) \tag{4}$$

Blood Brain Barrier Permeation 15

been replaced by *in situ* brain perfusion which provide higher control on experimental

The desired concentration of the studied drug was prepared using the perfusion fluid and the resulted solution is perfused directly to the brain through common artery of an anesthetized animal (commonly rat) for the suitable time and the brain sampling carry out on the predefined time intervals after stopping the perfusion (Amith & Allen, 2003). Similar to the intravenous injection method the remained intravascular perfusion fluid should be removed by brain flashing or calculated using an impermeable compound injection (Rooy et al., 2010). Direct perfusion enables scientists to study the BBB drug permeation in the absence of the first pass metabolism or drug elimination methods. Using this method, the mechanism of drug permeation can be studied using co-administered transporter inhibitors. But such as intravenous injection high resource demanding is a limitation for this method.

Another method for CNS drug partitioning study is quantitative auto radiography which can be used for regional study of total drug exposure. Using this method, the amount of radio labelled compound is measured in desired regions (e.g. stroke affected areas, brain tumours) following oral, intravenous or subcutaneous administrations to animals. Similar to previous methods after blood sampling in various time intervals, the brain is taking out and after sectioning the frozen brain to suitable sections the radioactivity is measured. Intra vascular correction is needed here too. Obtaining the regional PS values is possible using this method and the resolution of obtained data is high because of the micrometer

Positron emission tomography is a non-invasive method which is applicable in human. The suitable tracers are administered to the body and the emission is monitored using positron emission tomography scanners. The blood sampling is done in designed intervals and the brain and plasma distribution is measured using a curve fitting method. Similar to quantitative auto radiography the regional information about drug distribution is

Microdialysis is the only technique which is able to provide the concentration of CNS drug candidates in the interstitial fluid directly. A stereotaxic probe equipped with a semi permeable membrane implanted under anesthesia. The interior of the probe perfused with a physiological solution and samples are taken from freely moving animals and analyze using suitable separation techniques (commonly chromatographic systems) (Bickel, 2005; Alivajeh & Palmer, 2010). The studied compound can be administered orally, intravenously, subcutaneously or from other routes. This method is applicable for human and by implanting the probe in different regions of brain; specific data from different parts of brain (which have different properties) could be collected. The recovery of the probe is an important point in this method to get the absolute concentration data. Pharmacokinetic

The *Kin* and *PS* can be calculated using the obtained data from this method.

dimensioned studied sections (Bickel, 2005; Rooy et al., 2010).

achievable using this method (Dash & Elmquist, 2003).

condition (Kerns & Di, 2008).

**4.2.3** *In situ* **brain perfusion** 

**4.2.4 Quantitative auto radiography** 

**4.2.5 Positron emission tomography** 

**4.2.6 Intra cerebral microdialysis** 

where *F* could be considered as perfusion flow rate, or cerebral blood flow rate and *PS* is computed using:

$$PS = -F \times \ln\left(1 - \frac{K\_{in}}{F}\right) \tag{5}$$

Methods for measuring efflux of the drugs out of the brain (brain efflux index (BEI)) have been developed which represent the elimination rate constant of the drugs in brain. Using these parameters, scientists can provide information about the mechanism of BBB permeation in which for passive diffusion the efflux and influx constants will be similar. To measure all of these data, the remained drug in brain microvascular should be calculated and subtracted from total brain concentration.

#### **4.2** *In vivo*

The resulted data from *in vivo* experiments are valuable and regarded as gold standard in CNS drug discoveries. This value comprises from the experiment which uses anesthetized or cautious animals which represent full physiologic condition for study and the obtained data reflect different aspects of BBB permeation. Demanding skilled scientists and equipped laboratories are the main disadvantage of these techniques.

#### **4.2.1 Intra venous injection**

Intra venous injection methods have been developed during primary CNS studies in order to assess the BBB permeability and brain distribution of the CNS drug candidates. The radio-labelled compounds are injected intravenously and blood samples are obtained in different time intervals and a single brain tissue can be obtained at the designated time point. The measured compound concentrations in plasma and brain plotted against the time and after calculating AUC values the logBB computed using equation 2. For each time interval three animals are needed and in order to get a plot using 7 data points, 21 animals are required which is the main limitation of the method (Rooy et al., 2010). The logBB are interesting for pharmaceutical companies, because they can be easily used to rank the goals and other pharmacokinetic parameters such as Cmax and time length that the compound remains above *in vitro* determined effective concentration can be calculated. Recently these data are questioned about their ability to reflect the permeability properties of studied compounds mainly because: 1) The obtained concentrations are total, while the free fraction of the compounds are responsible for most of their pharmacokinetic properties and 2) It is a brain distribution value and the permeation rate of compounds cannot be obtained (Kerns & Di, 2008). The other parameters which can be calculated using the obtained data are rate parameters (i.e. *Kin* and *PS*).

#### **4.2.2 Single carotid injection**

Single intra carotid injection is one of the earliest BBB permeation study methods and can be done by injection of a given concentration of a labelled compound through common carotid artery of an animal along with a reference standard and experiment stopped after 5 - 15 seconds. Then the brain sampling is done and the brain uptake index (BUI%) can be calculated using the concentration of the compound and the reference standard (Pardridge, 2007). Because of the low sensitivity of the method (limited sampling time), this method has

where *F* could be considered as perfusion flow rate, or cerebral blood flow rate and *PS* is

ln 1 *Kin PS F*

Methods for measuring efflux of the drugs out of the brain (brain efflux index (BEI)) have been developed which represent the elimination rate constant of the drugs in brain. Using these parameters, scientists can provide information about the mechanism of BBB permeation in which for passive diffusion the efflux and influx constants will be similar. To measure all of these data, the remained drug in brain microvascular should be calculated

The resulted data from *in vivo* experiments are valuable and regarded as gold standard in CNS drug discoveries. This value comprises from the experiment which uses anesthetized or cautious animals which represent full physiologic condition for study and the obtained data reflect different aspects of BBB permeation. Demanding skilled scientists and equipped

Intra venous injection methods have been developed during primary CNS studies in order to assess the BBB permeability and brain distribution of the CNS drug candidates. The radio-labelled compounds are injected intravenously and blood samples are obtained in different time intervals and a single brain tissue can be obtained at the designated time point. The measured compound concentrations in plasma and brain plotted against the time and after calculating AUC values the logBB computed using equation 2. For each time interval three animals are needed and in order to get a plot using 7 data points, 21 animals are required which is the main limitation of the method (Rooy et al., 2010). The logBB are interesting for pharmaceutical companies, because they can be easily used to rank the goals and other pharmacokinetic parameters such as Cmax and time length that the compound remains above *in vitro* determined effective concentration can be calculated. Recently these data are questioned about their ability to reflect the permeability properties of studied compounds mainly because: 1) The obtained concentrations are total, while the free fraction of the compounds are responsible for most of their pharmacokinetic properties and 2) It is a brain distribution value and the permeation rate of compounds cannot be obtained (Kerns & Di, 2008). The other parameters which can be calculated using the obtained data are rate

Single intra carotid injection is one of the earliest BBB permeation study methods and can be done by injection of a given concentration of a labelled compound through common carotid artery of an animal along with a reference standard and experiment stopped after 5 - 15 seconds. Then the brain sampling is done and the brain uptake index (BUI%) can be calculated using the concentration of the compound and the reference standard (Pardridge, 2007). Because of the low sensitivity of the method (limited sampling time), this method has

*F*

(5)

computed using:

**4.2** *In vivo*

**4.2.1 Intra venous injection** 

parameters (i.e. *Kin* and *PS*).

**4.2.2 Single carotid injection** 

and subtracted from total brain concentration.

laboratories are the main disadvantage of these techniques.

been replaced by *in situ* brain perfusion which provide higher control on experimental condition (Kerns & Di, 2008).

#### **4.2.3** *In situ* **brain perfusion**

The desired concentration of the studied drug was prepared using the perfusion fluid and the resulted solution is perfused directly to the brain through common artery of an anesthetized animal (commonly rat) for the suitable time and the brain sampling carry out on the predefined time intervals after stopping the perfusion (Amith & Allen, 2003). Similar to the intravenous injection method the remained intravascular perfusion fluid should be removed by brain flashing or calculated using an impermeable compound injection (Rooy et al., 2010). Direct perfusion enables scientists to study the BBB drug permeation in the absence of the first pass metabolism or drug elimination methods. Using this method, the mechanism of drug permeation can be studied using co-administered transporter inhibitors. But such as intravenous injection high resource demanding is a limitation for this method. The *Kin* and *PS* can be calculated using the obtained data from this method.

#### **4.2.4 Quantitative auto radiography**

Another method for CNS drug partitioning study is quantitative auto radiography which can be used for regional study of total drug exposure. Using this method, the amount of radio labelled compound is measured in desired regions (e.g. stroke affected areas, brain tumours) following oral, intravenous or subcutaneous administrations to animals. Similar to previous methods after blood sampling in various time intervals, the brain is taking out and after sectioning the frozen brain to suitable sections the radioactivity is measured. Intra vascular correction is needed here too. Obtaining the regional PS values is possible using this method and the resolution of obtained data is high because of the micrometer dimensioned studied sections (Bickel, 2005; Rooy et al., 2010).

#### **4.2.5 Positron emission tomography**

Positron emission tomography is a non-invasive method which is applicable in human. The suitable tracers are administered to the body and the emission is monitored using positron emission tomography scanners. The blood sampling is done in designed intervals and the brain and plasma distribution is measured using a curve fitting method. Similar to quantitative auto radiography the regional information about drug distribution is achievable using this method (Dash & Elmquist, 2003).

#### **4.2.6 Intra cerebral microdialysis**

Microdialysis is the only technique which is able to provide the concentration of CNS drug candidates in the interstitial fluid directly. A stereotaxic probe equipped with a semi permeable membrane implanted under anesthesia. The interior of the probe perfused with a physiological solution and samples are taken from freely moving animals and analyze using suitable separation techniques (commonly chromatographic systems) (Bickel, 2005; Alivajeh & Palmer, 2010). The studied compound can be administered orally, intravenously, subcutaneously or from other routes. This method is applicable for human and by implanting the probe in different regions of brain; specific data from different parts of brain (which have different properties) could be collected. The recovery of the probe is an important point in this method to get the absolute concentration data. Pharmacokinetic

Blood Brain Barrier Permeation 17

cell based and non cell based methods. Cell based models are simplification of *in vivo* system in which the brain and non brain derived cell cultures are used to study the permeation and transport of drug candidates. The brain derived cell cultures (primary endothelial cultures) show closest phenotype to the *in vivo* brain while their preparation and handling are more difficult than non-brain derived cell lines. Primary endothelial cultures prepared by isolating animal brain micro vessels and *seeding* in culture medium where the endothelial cells grow out and make suitable mono layers for experiments. In order to mimic the *in vivo* system more closely co-cultures included astrocytes have been developed which provide more physical and physiological features in comparison with primary cell cultures (Cardoso et al., 2010). Non brain derived models use the epithelial cell cultures (e.g. Caco 2) and modified epithelial cell cultures which are used for drug absorption studies in order to rank the permeability of CNS drug candidates. Non cell based *in vitro* models include the parallel artificial membrane permeability assay (PAMPA) and immobilized artificial membranes (IAMs) which used as HPLC columns and mimic the properties of biological membrane (Abbott, 2004). PAMPA models initially developed for study passive oral absorption and successfully applied in the pharmaceutical industry. Recently, it has been modified for using in BBB permeation

studies and showed good correlation with *in vivo* findings (Mensch et al., 2010).

*In vivo*, *ex vivo* and *in vitro* methods of assessing brain drug penetration leads to high quality data resemble most of the permeation mechanisms in BBB, but they are highly cost and time demanding and are not suitable for screening of large compound libraries. As soon as BBB studies have begun, attempts to predict the BBB permeation properties of drug candidates lead to primary structure activity relationships which later accepted as essential rules of CNS drug development. These structural features later used to develop quantitative relationships to predict the pharmacokinetic properties of CNS drugs. During years and improving the knowledge about the effect of different passive and active mechanisms of brain drug penetration, the prediction models improved and specific models to predict different aspects of BBB permeation have been developed. In order to develop a model first the prediction endpoint (dependent variable or experimental value) should be measured or obtained from the literature. The quality of these data is deterministic for developed model certainty. After selection of the data set, the inclusion of each point in data set should be evaluated and possible outliers should be determined. The next step is to split data set in training and test sets and measure or calculate the desired independent descriptors. The significant descriptors should be selected and the relationship between the dependent and independent variables should be developed using appropriate modelling method. While the model has been developed, its predictive ability along with other validation parameters should be calculated and the effect of selected descriptors on the experimental value should be defined. The details of each step are provided in following sections. Some commercial software have been developed to predict the brain drug penetration which can be used to

In order to get initial information about the BBB permeation of new drug entities, studying the existing information using different methods is more interesting than experimental

**4.4 BBB permeation prediction methods (***in silico* **methods)** 

get primary estimations about the CNS activity of a compound.

**4.5 Prediction endpoints (Experimental data)** 

parameters of CNS drug candidates including half-life, Cmax, Tmax, total exposure, volume of distribution, clearance, BBB influx and efflux rates for different brain regions and most importantly the Kp,uu at steady state can be obtained and calculated using microdialysis driven data. These data can be used for pharmacodynamic studies and dosing regimens (Alivajeh & Palmer, 2010).

The methods reviewed in sections 4.2.1 to 4.2.6 give information about the overall exposure resulted from different passive or active influx and efflux systems.
