Passive Diffusion

Beta Switch Program

The Beta Switch Program by Sue Heintze

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Trifluoroperazine

Indomethacin

Scheme 10. CNS Drugs Tend to be Basic (Clark, 2001)

Trifluoroperazine

Indomethacin

Scheme 10. CNS Drugs Tend to be Basic (Clark, 2001)

Scheme 11 shows the correlation between in vivo brain permeability and H-bonds. Permeability decreased with increasing number of H-bonds for a series of steroids (Pardridge, 1995). BBB penetration also increases with increase in lipophilicity.

Scheme 12 shows how replacement of one hydroxyl group of morphine with a methoxyl group to make codeine increased BBB penetration 10 fold. When both hydroxyl groups were replaced by acetates to make heroin, the BBB penetration increased 100 fold (Pardridge, 1995). This example suggested that BBB penetration improved with decreasing H-bonds and increasing lipophilicity.

Utilization of intra-molecular H-bonding is a very effective way to increase CNS penetration. Scheme 13 shows that, when the first compound was modified by introducing a tertiary amine and an intra-molecular H-bond, not only did the solubility increase dramatically while maintaining potency, but also , more remarkably, the brain to plasma (B/P) ratio increased 10-fold (Ashwood et al., 2001).

Progesterone N=2

Estradiol N=4

Estradiol N=4

Aldosterone N=7

Testosterone N=3 OH

Testosterone N=3 OH

Corticosterone N=6

Corticosterone N=6

o OH

o OH

Cortisol N=8

Aldosterone N=7

Cortisol N=8

Scheme 11. Effects of H-Bonding on BBB Penetration (Pardridge, 1995)

Morphine

Codeine 10 x t BBB Penetration

Heroin

Morphine

Codeine 10 x t BBB Penetration

Heroin

100 x \ BBB Penetration

Scheme 12. Effect of Lipophilicity on BBB Penetration (Pardridge, 1995)

IC50 = 5.9 nM Solubility < 2 ng/mL Vehicle = 0.76 mg/mL B/P = 0.6

ICS0 = 5.5 nM Solubility = 0.7 mg/mL Vehicle = 10 mg/mL B/P = 6.0

Scheme 13. Utilization of Intra-molecular H-bond to Increase CNS Penetration (Ashwood et al.,2001)

Cell-based Assays

Permeability plays an important role for cell-based activity assays. If a compound can not permeate through the lipid membrane and enter the cells, it will not be able to interact with the targets inside the cells. Table 5 shows two series of compounds for a particular target. The first series is very potent and the second series have moderate potency in the enzyme assay. However, the first series, with better potency, was inactive in the cell-based assay, while the second series, with weaker potency, was active. Further studies showed that the first series had low permeability in the PAMPA assay and the compounds were Pgp substrates in the Caco-2 assay. The second series showed high PAMPA permeability and no Pgp substrate specificity. Thus, because of the good permeability of the second series, they were much more active in the cell-based assay, even through the activity was weaker in the enzyme assay.

In Scheme 14, Compound A is a dicarboxylic acid and has poor permeability as indicated by Caco-2 data (< 1 x 10-7 cm/s) (Liljebris et al., 2002). A bioisoteric replacement of one of the carboxylic groups with tetrazole improved permeability of the compound, while maintaining the same potency. Even through the permeability of the compound is still low (Caco-2 Pe = 1.9 x 10-7 cm/s), it was able

Assays

Series I

Series II

Enzyme Assay

Potent

Moderate

Cell-Based Assay

Inactive

Active

PAMPA Permeability

Low

High

Pgp Efflux

Yes

No

Table 5. Correlation Between Permeability and Cell-Based Activity

Table 5. Correlation Between Permeability and Cell-Based Activity

Caco-2 = 1.9 x 107 cm/s Positive Cellular Activity

Scheme 14. Bioisosteric Replacement of Carboxylic Acid with Tetrazole to Improve Permeability (Liljebris et al., 2002)

to penetrate cell membranes better than Compound A and to show cellular activity.

The compound in Scheme 15 is a potent and selective PTP1b inhibitor (Andersen et al., 2002). The compound has acceptable oral bioavailability, despite the polar dicarboxylic acid functional groups, zwitterions at physiological pHs and low permeability in the MDCK assay. However, disappointingly, the compound was inactive in insulin-stimulated 2-deoxyglucose (2-DOG) uptake into C2C12 cells due to poor cell membrane permeability. The diethyl ester prodrug of the compound has high permeability in the MDCK assay. The insulin-stimulated 2-DOG uptake into C2C12 cells of the di-ester prodrug was 70% of maximum insulin response (Andersen et al., 2002).

These examples demonstrate that good permeability is essential in order to achieve activity in cell-based assays.

Diacids

Pi-Ethyl Ester Prodrug

In vitro (PTP1B) Oral Bioavailability (Rat) Permeability (MDCK) 2-DOG Uptake in C2C12 Cell

Potent & Selective

Inactive

Not Determined

High 70%

Scheme 15. Permeability Effects Activity in Cell-Based Assays (Andersen et al., 2002)

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