Avyv VAV vyyv

Scheme 6. Effect of lipophilicity on Caco-2 Permeability of Phenylalanine Dipeptide Series. Permeability values shown are Peff (apical to basolateral) x 10-6 cm/s (Goodwin et al., 2001)

Scheme 7 shows the effect of different substituents on permeability for benzyl imidazole and benzyl pyrazole core structures. Compounds with CN substitution are more permeable than NH2 or CONH2. Carboxylic acids are least permeable (Fichert, et al, 2003).


P Caco-2 X 10"












Pcaco-2 x 10"













Scheme 7. Effects of Different Substituents of Caco-2 Permeability (Fichert et al., 2003)

Enalkiren is a first generation of Renin inhibitor (Scheme 8). It is dosed in an I.V. formulation. The compound has poor oral bioavailability due to poor permeability. It was modified later by reducing H-bonds and replacing the amide group. This, not only improved permeability, but also the metabolic stability of the compound against various enzymes. The oral bioavailability was 53% in dog (Panchagnula and Thomas, 2000).

Scheme 9 shows two Factor Xa inhibitors. They have very similar potency and metabolic profile, but their oral bioavailability is 4-fold different. The tertiary amine was more permeable in the Caco-2 assay, due to fewer H-bonds than the secondary amine, which resulted in higher oral bioavailability (Quan et al., 2004).

Blood-Brain Barrier

Blood Brain Barrier and Transport Mechanisms

CNS is the second largest therapeutic area, right behind cardiovascular diseases (IMS Health, 2005). Five out of the top ten causes for disability are due to CNS disorders. Stroke is the 3rd leading cause of death. Alzheimer's disease affects 15 million people in US alone and it is the 2nd most expensive disease. However, many of the brain diseases still do not have adequate treatment (Pardridge, 2001). A major challenge for CNS therapy is the blood brain barrier (BBB). It has been estimated that only 2% of the CNS discovery compounds can cross the BBB and potentially reach the therapeutic targets.




Reduce H-bonds Improve stability 53% oral bioavailability (dog)


Scheme 8. For this Renin Inhibitor, Reduction of H-bonds Increased Permeability and Stability, Resulting in Increased Oral Bioavailability (Panchagnula and Thomas, 2000)



Caco-2 Papp


T 1/2




(xlO-6 cm/s)



















Scheme 9. Effect of Permeability on Oral Bioavailability for Factor Xa Inhibitor (Quan et al., 2004)

The BBB is the membrane that separates the blood from the interstitial fluids of the brain. There are 400 miles of blood capillaries in the brain. The BBB consists of brain endothelial cells that line the capillaries. They have very tight intercellular junctions and strong Pgp efflux activity. The function of the BBB is an interplay among four different cell types: the endoththelial cells, astrocytes, pericytes and neurons. In addition to the BBB, there is a blood-CSF barrier. The surface area of the BBB is 5000 times larger than blood-CSF barrier. The CSF flow through the arachnoid villi back to the blood is too fast to allow any significant absorption into the inner area of the brain where most of the therapeutic targets are located. Therefore, the BBB is the major barrier for CNS penetration.

The BBB has many of the transport mechanisms discussed above for oral absorption. Most CNS drugs used in the clinic pass the BBB by passive diffusion. The BBB also exhibits active processes: influx and efflux transport. There are limited pinocytosis and paracellular processes. Determination of BBB penetration is of great importance, not only for CNS drugs, but also for non-CNS therapeutic targets, where BBB penetration might cause unwanted side effects.

Methodology for BBB Penetration

There are several common approaches to predict BBB penetration, including rules, in silico prediction, physicochemical methods, cell-based models and in vivo assays. Rules are very effective for medicinal chemists. Table 4 shows Partridge's Rule of 2 and Clark's rules for predicting BBB penetration. Computational methods have gone a long way to predict BBB penetration. Both classification and QSAR models have good accuracy (Clark, 2001; Lobell et al., 2003). Computational methods are particularly useful to predict BBB penetration prior to synthesis and to guide structural modifications. Many cell-based in vitro BBB and blood-CSF barrier (BCSFB) models with different animal species have been developed to predict penetration. These cell lines include MDR1-MDCKII, Caco-2, BCEC, LLC-PK1, TM-BBB, and TR-BBB. The limitations of the cell-based assays are that many of the transporters are down regulated and variable from batch-to-batch and passage-to-passage. Furthermore, transporters are species dependent. Cell-based assays are particularly useful to diagnose Pgp efflux transport (Kerns et al., 2004), which plays a very important role in protecting the brain and is, at the same time, a major challenge for CNS drug candidates.

Pardridge's Rule of 2 (Pardridge, 1995)

Clark's Rules (Clark, 2001)

Total H-bonds < 8-10 MW < 400-500

N + O s 5 Clog P - (N + O) > 0 PSA s 60-70 MW < 450 Log D = 1-3

Table 4. Rule-Based Methods to Enhance BBB Penetration

Table 4. Rule-Based Methods to Enhance BBB Penetration

At Wyeth Research, we have developed a high throughput PAMPA-BBB assay (Di et al., 2003). The assay predicts a compound's potential for BBB penetration through passive diffusion. Passive diffusion is the major driving force to move the compounds into the brain, but there are other processes trying to prevent or slow down the compounds from entering the brain, such as Pgp efflux, protein binding and metabolism (Figure 3). A useful screening strategy is to use PAMPA-BBB for 1st -tier screening of large numbers of compounds and then validate results with in vivo studies of selected compounds. If there is a disconnect between the two assays, secondary assays can be used to diagnose transport mechanisms.

Figure 3. Complex Mechanism In Vivo for BBB Penetration

Structure-BBB Penetration Relationship

CNS drugs tend to be basic and contain basic nitrogens. Scheme 10 shows two compounds having different BBB penetration (Clark, 2001). Trifluoroperazine has a basic pKa of 7.8. It is CNS + . Indomethacin has an acidic pKa of 4.2. It is CNS-. One explanation is that the BBB is negatively charged. Acidic compounds will be negatively charged at pH 7.4 and be repulsed by the BBB. Basic amines will be positively charged at pH 7.4. The charged species will be neutralized by the negatively charged membrane, so that they can cross the BBB. Hydrogen bonds decrease the ability of a molecule to cross the BBB.

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