ArmaGen Technologies-Enabling Biotechnology for the Brain

What restricts the free diffusion of small molecules across the BBB?
Small molecules cross the BBB in pharmacologically significant amounts only if the molecule has the following characteristics:

The absence of uptake of a small molecule drug by the brain and spinal cord is shown in the adjacent figure; the small molecule readily crosses the porous capillaries in all organs, except for the brain and spinal cord. More than 98% of all small molecules do not cross the BBB, as illustrated in the figure. The reason that such a large number of small molecules do not cross the BBB in pharmacologically significant amounts is that few small molecules fulfull all the chemical and biological criteria listed above, which must be present for free diffusion through the BBB in vivo.

The classical use of medicinal chemistry to lipidize the small molecule drug is self-limiting

Medicinal chemistry is traditionally used to enhance BBB penetration by increasing the lipid solubility of the water soluble small molecule with poor BBB permeability. However, this approach is self-limiting, because the increase in BBB transport caused by the increased lipid solubility is neutralized by the decreased plasma area under the concentration curve (AUC) of the drug, which is also caused by the increase in lipid solubility. This can be shown by the following equation that defines the % of injected dose (I.D.) taken up per gram brain:

Increased lipidization of the drug increases the BBB permeability coefficient, but also decreases the plasma AUC, because the transport of the drug across all membranes in the body is increased. As the lipid solubility is increased, the rate of exit of the drug from blood is increased proportionately with a concomitant decrease in the plasma AUC, which offsets any increase in BBB permeability. The opposing effects of lipid solubility on the plasma AUC and the BBB permeability is the reason it is so difficult to increase brain penetration of a drug with medicinal chemistry aimed at increasing lipid solubility.

New applications of medicinal chemistry based on knowledge of endogenous BBB transporters

There are 2 fundamentally different approaches by which medicinal chemistry can be used to solve the BBB drug delivery problem: chemistry-based and biology-based approaches, as shown in the figure. The chemistry based approached seeks to increase lipid solubility by masking polar functional groups, such as hydroxyl or carboxyl groups on the lead candidate. As discussed above, this approach is made difficult by the fact that increasing lipid solubility actually works to decrease brain uptake by decreasing the plasma AUC.

ArmaGen Technologies uses the biology based approach, which requires knowledge on the endogenous BBB transporters for small molecules. There are 2 types of small molecule endogenous transporters at the BBB: carrier-mediated transporters (CMT) that cause the blood-to-brain uptake of drugs, or the active efflux transporters (AET) that cause the active efflux of drugs from brain to blood. Many of the endogenous BBB CMT and AET transporters have been cloned, and these cDNAs can be expressed to establish in vitro models of BBB transport via specific endogenous transporters. ArmaGen Technologies can guide medicinal chemists to molecular structures that favor BBB transport on endogenous transporters. The goal is to replicate the dopamine/L-DOPA model. Dopamine is a water soluble drug that does not cross the BBB. The alpha-carboxylation of dopamine converts the monamine into an amino acid, which is then able to cross the BBB on the LAT1 large neutral amino acid transporter. This model can be replicated for other drugs, based on knowledge of endogenous BBB transporters.

Carrier-mediated transport (CMT) and active efflux transport (AET) of small molecules

Whereas the RMT systems are used for BBB transport of large molecules, the BBB CMT systems can be used to deliver small molecule drugs into brain. The CMT systems include the GLUT1 glucose transporter, the LAT1 large neutral amino acid transporter, the CAT1 cationic amino acid transporter, the MCT1 monocarboxylic acid transporter, the CNT2 nucleoside transporter, and many others. The CMT systems generally mediate the transport of drug in the blood to brain direction. In contrast, the AET systems, such as p-glycoprotein, cause the active efflux of drugs from brain back to blood. However, p-glycoprotein is only one of several BBB AET systems. Moreover, energy-dependent active efflux systems, such as p-glycoprotein or the multi-drug resistance proteins (MRPs) must work in concert with energy independent anti-porters that function at the opposite pole of the brain capillary endothelial membrane. Scientists at ArmaGen Technologies can provide knowledge about the BBB CMT and AET systems to enable the Partner's medicinal chemists to modify the structure of the drug with the aim of making the lead candidate transportable across the BBB.

The traditional use of pharmaco-genomics technologies in CNS drug development is in the area of discovery of new "druggable" targets and in the evaluation of drug toxicity (see figure). However, this approach does not solve the BBB drug delivery problem for the lead drug candidate. CNS drug delivery problems can be addressed with "BBB genomics", which seeks to discover novel BBB endogenous transporters, which can be used as conduits for drug transport to the brain.

The BBB occupies a tiny volume of the brain, about 0.1% or 1:1000 parts of brain. The sensitivity of most gene micro-arrays is 0.01% or 1:10,000 parts. Therefore, only the most abundant BBB gene products will be detected in a whole brain gene micro-array. Because most BBB-specific genes will not be detected in a whole brain gene micro-array, it is necessary to establish a separate 'BBB genomics' program, if the goal is to discover novel endogenous BBB transporter genes, that could be used as conduits for drug delivery to the brain.

Scientists at ArmaGen Technologies have developed animal and human BBB genomics programs, which start with the isolation of capillaries from the brain, as shown in the nearby figure, which is a scanning electron micrograph of isolated brain capillaries. The capillaries are prepared in pure form free of brain tissue (figure). RNA is then prepared from these isolated capillaries to initiate the BBB genomics transporter discovery program. Human BBB-specific cDNA libraries are produced following the isolation of mRNA derived from freshly isolated human brain capillaries.
The BBB genomics program enables the discovery of new BBB RMT, CMT, and AET transport systems, which can be used to develop new BBB drug and gene targeting strategies in the future. It is estimated that about 15% of the genes expressed at the BBB are transporter genes and that less than half of the BBB transporter genes have been discovered to date. ArmaGen's BBB genomics programs can be used to accelerate the discovery of novel endogenous BBB CMT or AET systems.

The more information known about the structural requirements of the endogenous BBB CMT and AET systems, the more likely the success in using medicinal chemistry to modify the structure of the drug so that the drug can cross the BBB in pharmacologically significant amounts via endogenous BBB transporters. ArmaGen has developed proprietary expression systems for many of the endogenous BBB small molecule transporters, and these expression systems can be used in high throughput screening (HTS) to find drugs that can enter brain via transport on one of the endogenous BBB transporters. Typically genomics methodology is used to discover druggable targets. However, drugs that cannot be delivered to the brain cannot enter brain drug development.

ArmaGen Technologies uses BBB genomics to discover endogenous transporters to enable brain drug delivery. Novel CMT systems can be used to direct medicinal chemistry to re-formulate the drug to enable transport across the BBB on the CMT system.

		Most BBB-specific transcripts will be missed in a whole brain gene micro-array.
The volume of the capillary endothelium is 1:1000 parts of brain.	The sensitivity of most gene micro-arrays is 1:10,000 parts.		The starting point for "BBB genomics" is the purification of mRNA from isolated brain capillaries.

New AET systems can be used to develop "co-drugs', which inhibit the BBB AET system and minimize the active efflux of the CNS active drug from brain back to blood. Co-drugs, which are developed from a platform of BBB AET transporter discovery, are used to potentiate the CNS action of a drug that is normally excluded from the brain owing to active transport by the BBB active efflux transporter.

BBB genomics enables the discovery of novel BBB CMT, AET, or RMT transport systems, which provides the basis for the development of new pathways of drug delivery to the brain via the endogenous BBB transporters. With this approach, brain drug delivery can enable brain drug development programs that, otherwise, would be terminated because of the BBB problem.

Small molecule BBB drug delivery technology