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About the blood-brain barrier (BBB) Effective drugs have not been developed for most brain disorders The global CNS drug market is greatly under-penetrated Why is the blood-brain barrier problem so central to CNS drug development? What is the blood-brain barrier ? Example of restricted drug transport at the BBB What are the traditional approaches for solving the BBB problem? Why most CNS drug development programs end in termination A new model for brain drug development How to study drug transport across the BBB Effective drugs have not been developed for most brain disorders
Big Pharma only develops a certain class of drug called small molecules. Although it is widely believed that any small molecule crosses the blood-brain barrier (BBB), just the opposite is true. In order to cross the BBB, the small molecule must have a molecular weight (MW) < 400 Daltons, and must also be lipid soluble. The number of small molecules that have these chemical properties is <2% of all drugs. All other drugs do not cross the BBB. Since Big Pharma does not develop BBB solutions, >98% of all drugs that could potentially treat the brain are not developed. The only drugs currently developed for the brain are lipid soluble drugs with a MW < 400 Daltons, and only a few brain disorders respond to such drugs (see Table 1). The majority of brain disorders do not respond to lipid soluble drugs with a MW < 400 Daltons, and consequently, no treatments are available (see Table 2 below). The global CNS drug market is greatly under-penetrated The global market for drugs for the central nervous system (CNS) is greatly under-penetrated, and would have to grow by >500% just to equal the cardiovascular drug market. The problem of under-penetration of the brain drug market will become even more severe in the next 20 years as the population ages and the number of people afflicted with Alzheimer's disease, Parkinson's disease, or stroke increases by 50%. Without new drug treatments and new methods for early diagnosis, the annual cost of health care for patients with Alzheimer's disease alone could approximate $0.5 trillion in 2020 in the U.S.
There are only a few diseases of the brain that are currently treated by CNS drugs:
The reason that so few CNS disorders are being treated is derived from the current industry model for neuropharmaceuticals:
Using this model, it is increasingly difficult to build a successful CNS drug development program for brain diseases other than affective disorders, pain, or epilepsy. This approach leaves un-developed very large markets of unmet needs that could be addressed if the BBB problem were not rate-limiting. The inability to treat most CNS disorders is not due to the lack of effective CNS drug discovery. Rather, it is due to the ineffective CNS drug delivery. Why is the blood-brain barrier problem so central to CNS drug development? If practical solutions to the BBB problem were available, the global CNS drug market would be the fastest growing sector of the pharmaceutical industry, because the following drug development pathways would be enabled:
What is the blood-brain barrier ?  The BBB is formed by the capillary endothelium of the vessels perfusing the brain. The brain capillary endothelium is stained red in the attached Figure. Owing to the presence of epithelial-like, high resistance tight junctions that cement together all the endothelia in brain capillaries, there is no 'para-cellular' pathway for solute or drug transport from blood to brain. In addition, the pinocytosis or 'trans-cellular' pathway of solute transport across the endothelium is reduced 100-fold in brain endothelium compared to endothelial cells in peripheral tissues. Owing to the absence in brain capillaries of the trans-cellular and para-cellular pathways that normally exist in the microvascular wall, circulating molecules may pass from blood to brain only by one of two mechanisms: (1) free diffusion owing to lipid solubility of small molecules with molecular weights < 400 Daltons. (2) Catalyzed transport using one of the different classes of BBB transport mechanisms--the BBB endogenous transport systems can be used as portals of entry into the brain for either large or small molecules. 
There are 100 billion capillaries in the human brain, and the endothelial walls of these capillaries form the BBB. The density of the capillary network in the brain is shown in the adjacent figure.
The BBB is very tight and excludes the brain uptake of all large molecule drugs and >98% of small molecule drugs. A misconception about BBB drug transport is that all small molecules readily cross the BBB. In fact, most small molecules do not cross the BBB. In order to cross the BBB in pharmacologically significant amounts, the drug must have the following characteristics: (a) lipid soluble (b) have a molecular weight < 400 Daltons (c) not be a substrate for a BBB active efflux transporter. Example of restricted drug transport at the BBB 
The nearby figure is a body scan of a mouse following the intravenous injection of a small molecule (molecular weight only about 100 Daltons). The scan shows the small molecule drug readily crosses the porous capillary barrier in all organs, but does not enter the brain or spinal cord--the drug does not cross the blood-brain barrier or the blood-spinal cord barrier. This drug cannot be further developed as a neurotherapeutic, unless the BBB problem is solved. At this point the brain drug developer has only 2 options: either terminate the drug development program, or attempt to use one of the traditional approaches for dealing with the BBB problem.
What are
the traditional approaches for solving the BBB problem?
Why most
CNS drug development programs end in termination
A new model for brain drug development ArmaGen Technologies' model to brain drug development is to develop effective BBB drug or gene targeting technologies first, and then start specific CNS drug or gene development programs. In each case, the specific CNS drug or gene is formulated to enable transport across the BBB via one of the endogenous catalyzed transport systems that normally function at the brain endothelial wall. The brain is richly perfused by a complex capillary network (see above Figure). There are about 100 billion capillaries in the human brain, and every neuron is virtually perfused by its own blood vessel. The endogenous BBB transport systems are localized to the endothelial plasma membranes that form the BBB in vivo. There is a luminal endothelial membrane (on the blood side of the capillary), and an abluminal endothelial membrane (on the brain side of the capillary). The luminal and abluminal membranes are separated by about 300 nm of endothelial cytoplasm; therefore, transport through the BBB is a process of transport through 2 membranes in series. Within the luminal and abluminal membranes are the endogenous BBB transporters, and these are classified into 3 general groups of transporters:
The 3 types of endogenous transporters are shown in the adjacent Figure. The CMT systems generally mediate the transport of small molecules in the blood to brain direction. Examples of the CMT systems include the GLUT1 glucose transporter, the LAT1 large neutral amino acid transporter, and many other CMT systems. The AET systems generally mediate the active efflux of small molecules in the brain to blood direction. The model AET system at the BBB is P-glycoprotein; however, there are many other BBB AET systems other than p-glycoprotein. The RMT systems mediate the transport in either the blood to brain or the brain to blood direction of endogenous peptides and proteins. Examples of the BBB RMT systems include the insulin receptor . BBB drug and gene targeting technology platforms are built from knowledge about the endogenous transport systems within the BBB. The endogenous transporters can be used ccessed to deliver drugs and genes to the brain via a route of administration no more invasive than an intravenous or subcutaneous administration. See Targeting Protein Drugs, Targeting Non-Viral Genes, and Targeting Small Molecules. How to measure drug transport across the blood-brain barrier (BBB) Frog oocytes and transporter cloned RNA expression Computer models are used to predict BBB transport of small molecules, based on physico-chemical properties of the drug, such as molecular weight, hydrogen bonding, lipid solubility, molecular weight, and polar surface area. These models are invariably validated by one of 3 measurements: (1) drug distribution into cerebrospinal fluid (CSF) (2) intra-cerebral dialysis fibers (3) the log BB, where BB=the brain/blood concentration ratio at some arbitrary time point after drug administration, e.g. 60 min. It can be shown that all 3 approaches have disadvantages, which limit the predictive power of the computer model. DRUG TRANSPORT INTO CSF IS NOT A MEASURE OF BBB PERMEABILITY Drug transport from blood to CSF is a function of drug transport across the choroid plexus epithelium, which forms the blood-CSF barrier in vivo. This epithelial barrier is anatomically separate from the BBB, which limits drug transport from brain into brain interstitial fluid (ISF) across the capillary endothelium. The capillary endothelium (the BBB) and the choroid plexus (the blood-CSF barrier) have different transporter gene expression profiles. Drugs may readily enter CSF, owing to rapid transport across the choroid plexus, but not undergo significant transport into brain tissue, due to limited BBB transport. This is illustrated with azidothymidine (AZT), a treatment for neuro-AIDS. AZT is readily transported into CSF, but is not transported across the BBB. INTRA-CEREBRAL DIALYSIS FIBERS MEASURE BBB TRANSPORT IN BRAIN INJURY The insertion of a dialysis fiber into brain tissue causes brain injury and BBB disruption. Thus, the permeability of the BBB is artifactually increased with this model. This is illustrated in the case of morphine and morphine 6-glucuronide (M6G). The BBB permeability of morphine is about 50-fold higher than that of M6G, when measured by standard physiological methods (see below). However, the brain uptake of morphine and M6G is comparable when measured by dialysis fibers, owing to the BBB disruption associated with this method. The disruption causes the formation of small pores within the BBB, which selectively increases the BBB permeability for molecules such as M6G, which have a very low BBB permeability coefficient. LOG BB IS NOT A DIRECT MEASUREMENT OF BBB DRUG TRANSPORT The log BB is a measure of the drug volume of distribution in brain at late time points, and this is primarily influenced by drug binding to brain cytoplasmic proteins, as well as plasma protein binding. Two drugs may have comparable BBB permeability coefficients, yet markedly different log BB values, owing to selective tissue binding of one drug relative to the other. Drug action in brain is a function of drug receptor occupancy, which is controlled by the free drug in brain, which is controlled by BBB transport of the drug, and is independent of brain cytoplasmic binding of the drug. The use of logBB to select lead candidates may not screen for drugs of high BBB permeation, and could cause CNS drug developers to choose the wrong drug lead candidate. In vitro BBB models in cell culture Capillaries can be isolated from brain, and these structures can be digested to separate brain capillary endothelial cells for growth in cell culture. The endothelial monolayer can be grown on porous membranes, which can be placed in side-by-side diffusion chambers for measurement of drug transport across the monolayer in vitro. The problem with this approach is that BBB-specific gene expression is severely down-regulated in vitro. For example, the expression of the Glut1 glucose transporter or the LAT1 large neutral amino acid transporter is down-regulated >100-fold in cultured endothelium compared to freshly isolated brain capillaries. Drug transport via carrier-mediation is under-estimated by >100-fold in the in vitro BBB model. For example, L-DOPA for Parkinsons disease is effective, because this drugs crosses the BBB on the LAT1 endogenous transporter. The CMT of L-DOPA across the BBB would most likely be missed by screening with an in vitro BBB model. In vivo methods for measurement of BBB drug transport Drug transport in the blood to brain direction: Drug transport may be measured with either carotid arterial injection/infusion methods, or by short-term intravenous injection techniques, such as: internal carotid artery perfusion (ICAP) method common carotid artery single injection or brain uptake index (BUI) method intravenous/external organ (IV/EO) method Drug transport into brain should not be measured with intra-peritoneal or subcutaneous injection methods, because it is not possible to separate transport of the parent drug from a drug metabolite. The advantage of the ICAP or BUI methods is that drug metabolism is eliminated. However, the carotid arterial injection/infusion methods are labor intensive. Drug transport into brain can measured with an intravenous injection technique, providing the time interval used to monitor drug transport (e.g. 15-60 seconds) is sufficiently short to eliminate metabolism artifacts. In addition, it is necessary to compute the area under the concentration curve (AUC) with the intravenous method. Brain drug transport should not be measured with only analyses of the terminal blood drug concentration. The plasma AUC can be conveniently measured with an external organ (EO) approach, wherein a femoral artery is cannulated and connected to a syringe pump, which withdrawls blood at a defined rate over the uptake measurement period. Drug transport in the brain to blood direction: BBB active efflux transporters (AET) such as p-glycoprotein actively export drug from brain to blood. There are many other BBB efflux systems for both small and large molecules. The efflux transporters can be measured with the brain efflux index (BEI) method, which involves the direct injection of the drug into the brain under stereotaxic guidance. The kinetics of drug loss from the brain compartment, (which is a function only of BBB efflux transport) can then be quantified. Expression of cloned BBB transporters The genes encoding many of the BBB transporters have been cloned and in vitro transcription can be used to produce cloned RNA (cRNA) encoding the functional transporter protein. The cRNA can be injected into frog oocytes, which results in expression of the cloned BBB transporter. Drug transport via the cloned BBB transporter can then be quantified. Alternatively, cell lines can be transfected with the BBB transporter cDNA, which creates a model for the high throughput screening (HTS) of drug transport via the BBB transporter. With this approach, thousands of unlabeled drugs can be rapidly screened for their affinity for one of the many BBB transporters. The discovery of new BBB transporters if accelerated with a BBB genomics program (see Small Molecules). |
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