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Workshop Reports
Hereditary Disease Foundation
In conjunction with:
The Jennifer Jones Simon Foundation
Delivering Therapeutic Molecules to the Brain
May 31 and June 1, 1997
Santa Monica, California
Prepared by Kerry Thompson
Delivering Therapeutic Molecules to the Brain
May 31 - June 1, 1997
Santa Monica, California
These multidisciplinary workshops are designed to stimulate research into causes and cures
for Huntington's disease. The Foundation's creator, Milton Wexler, a psychoanalyst by
profession, felt that free association is the key to creativity. The workshops have therefore
dispensed with formal presentations, in order to have scientists talk with each other rather than
at each other. The inspiration for this particular meeting came from the Jennifer Jones Simon
Foundation's interest in cancer and cancer therapies and the Hereditary Disease Foundation's
(HDF) interest in Huntington's disease (HD). The discovery of the gene that causes HD has
increased hope for novel therapeutics and prophylaxis. Experiments with growth factors, the
HD-gene product (as well as HD-protein-associated molecules), and gene therapy suggest a
number of therapeutic strategies for HD. However, the issue of how to get therapeutic agents
to the brain (across the blood brain barrier) remains critical. The workshop participants have
brought their expertise from several scientific arenas to discuss various strategies, invasive
and noninvasive, for delivering neuroactive agents to the brain.
Participants included:
Keith Black
University of California Los Angeles
Ulrike Bl”mer
The Salk Institute of Biological Studies
Ruben J. Boado
University of California Los Angeles
Eric Crumpler
Massachusetts Institute of Technology
Robert L. Dedrick
National Institutes of Health
Matthew During
University of Auckland
Dwaine Emerich
Alkermes, Inc.
William M. Gelbart
University of California Los Angeles
M. Frederick Hawthorne
University of California Los Angeles
David R. Jacoby
Massachusetts General Hospital Kam Leong
Johns Hopkins University
Ed Neuwelt
Oregon Health Sciences University
William Pardridge
University of California Los Angeles
Manuel Penichet
University of California Los Angeles
Terry Reisine
University of Pennsylvania
Joel A. Saydoff
CytoTherapeutics
Kerry Thompson (Recorder)
University of California Los Angeles
Allan J. Tobin
University of California Los Angeles
Patrick A. Tresco
University of Utah
Nancy S. Wexler
Columbia University Huntington's disease:
A brief discussion of the research findings over the last few years is provided
below. This information is the product of previous meetings, clinical and scientific
reports over the past several years. Although not comprehensive, this information
is a framework with which to consider therapeutic strategies discussed at this
workshop.
Huntingtin, the protein encoded by the HD gene, is about 3000 amino acid
residues long, with 67 exons. In the first exon there is a tract of glutamines that
extends up to 34 in unaffected people. When there are more than 35 glutamines,
the protein somehow triggers the onset of the disease. There is some debate on
how many glutamines are actually required to cause HD, but the current consensus
seems to be above 36 (between 35 and 40 CAG's lead to variable penetrance --
sometimes the disease is expressed and sometimes not. The CAG repeat size can
expand above 40 in the next generation). This mutant protein can be visualized on
western blots because it migrates anomalously. The protocol for identifying this
protein using western blots has only recently been refined. Using a very low
percentage acrylamide gel with long running times clearly demonstrates
differences between the two alleles. The larger the number of glutamines the more
anomalous the migration.
Patients with more than 60 glutamines or more can present with symptoms as
children. The expansion of this CAG repeat may occur during meiosis but this is
subject to research investigation. These repeated glutamines occur near the N-
terminal. Knocking out the gene in mice causes embryonic death. An obvious
question is whether the normal function of the gene has anything to do with the
abnormal function seen in the disease state. Nancy Wexler has looked at patient
populations in Venezuela and shown that there is no apparent difference between
homozygotes and heterozygotes. However, homozygotes may have a slightly
earlier age of onset compared to their heterozygote siblings. Other factors seem to
be comparable in this patient population.
The knock-out mice have not provided much information about the protein other
than that it is essential for normal mouse development. The initial studies
suggested that wild-type huntingtin prevents apoptotic death. Simple knock-outs,
however, do not reveal whether huntingtin is required in adults. Allan Tobin
suggested that HD may be a true gain-of-function mutation, where some acquired
function of the mutated protein causes the disease. There may be phase transition
in the structure of polyglutamine, as the number increases from 35 to 36. There
are correlations between the number of repeats and the age of onset, but these
data are not conclusive.
HD-associated proteins have been found. A two-hybrid screen, using exon 1 as
"bait" , allowed the identification of Huntingtin-Associated Protein (HAP-1). Other
partners have been found but all of these associations are relatively weak. Affinity
purification with immobilized polyglutamine has pulled out glucose-6-phosphate
dehydrogenase (G6PD), a very abundant cytosolic protein. This is interesting
because spinocerebellar ataxia, another expanded repeat disease, also showed an
association between the mutated protein and G6PD.
Attempts to produce huntingtin in bacterial or eukaryotic expression systems
has not been very successful. David Jacoby has been looking to see if the
expression of polyglutamine is itself toxic to cells. A group at Duke University has
reported polyglutamine toxicity in E. coli, but other laboratories have not been
successful at replicating these results. One problem is that polyglutamines are not
soluble, suggesting that over-expressing any protein with a strong promoter, to the
point of precipitation, may have limitations as a disease state model. This area of
research requires development.
Some transcription factors have glutamine tracts and some investigators have
argued that huntingtin is nuclear, others however, have suggested that it is
cytoplasmic or synaptic. Since this workshop took place, new data suggest that
wheras huntingtin is normally a cytoplasmic protein, the pathological expanded-
repeat form, and probably only the N-terminal fragment thereof, is found in the
nucleus. Site-directed mutagenesis experiments have suggested that
polyglutamine in transcription factors bind DNA. This fact implies that therapies
designed to enhance or disrupt binding at these polyglutamines may be possible.
Currently there is no cellular assay for the action of this protein.
Transgenic mice made with only the N-terminal exon 1 portion of the gene have
severe behavioral abnormalities and some pathology, suggesting that this small
part of the corresponding protein is enough to cause disease. These animals will
soon be available and seem to model the severely affected juveniles. The mice
begin getting sick at around 6 weeks. They then start showing feeding problems,
seizures, and subsequently die. Neuropathology did not reveal much until jost
prior to death. At these longer survival times a small amount of cell death has now
been shown, but markers such as GFAP staining are increased particularly in the
caudate. Other groups are exploring additional transgenic mice, and also "knock-
in" mice where altered genes replace the native ones rather than, as in
transgenics, being present as additional copies.
Blood Brain Barrier:
The blood-brain-barrier (BBB) is a selectively permeable barrier which regulates
the flow of ions and proteins entering the brain from the bloodstream. This barrier
allows exquisite control of the neuronal microenvironment under normal
physiological conditions, but presents an obstacle for clinicians seeking to
introduce therapeutic agents to the brain.
The endothelial cells comprising the vascular tree of the brain are specialized in
that they form tight junctions. Tight junctions are the junctions between endothelial
cells which are " zippered" shut. The junctions between brain endothelial cells do
not show fenestrations which are seen in endothelial cells from other parts of the
body, and thus prohibit paracellular movement. These junctions produce a
prohibitive pore for larger molecules (above 500 daltons) that are not lipophilic.
The pore size is somewhere in the neighborhood of 5-10 nm but may be up to 40
nm.
The fusion of the plasma membrane during development of the BBB occurs
in the rat at about E13 and is associated with junctional strands, which are protein-
lipid structures specific to brain endothelia. It is widely believed that these
junctional strands play an important role in the adhesion of brain endothelial cells,
but little is known about their molecular structure and function.
Brain endothelial cells are also associated with astrocytic endfeet. Trophic
factors secreted by these astrocytes may induce the formation of tight junctions.
Type I astrocytes implanted into non-neuronal tissue induce a permeability barrier
in invading endothelial cells. This finding is interesting because tumors may not
have this type of trophic signaling and subsequently do not form tight junctions. It
is well known that tumor vasculature carries a higher permeability than
nonpathological brain vasculature.
The BBB can be disrupted by a number of mechanisms: Introduction of
agents, such as bradykinin and RMP-7 which bind receptors (possibly B2
receptors) on the endothelial cells, causes Ca2+ shifts that are correlated with
disruption of the barrier. Stress or severe emotionality may also cause transient
openings of the barrier in specific regions of the brain, presumably due to an
increase in circulating stress-related hormones. There are also methods that
transiently change the permiability of tight junctions with osmotic disruption using
agents such as mannitol.
Technologies:
The following section will cover the experimental strategies presented by the
participants during discussions over the course of two days. This section is divided
into two broad categories: invasive and noninvasive. From the discussion at the
workshop, a dichotomy could be drawn between those investigators who favor
invasive strategies and those who favor noninvasive strategies.
The former group, generally speaking, supports aggressive surgical
techniques that are currently being used by neurosurgeons around the world. The
group argues that these strategies are currently the best way to ensure delivery of
therapeutic agents to the brain in clinically useful concentrations. Patients with
severe neurodegenerative diseases show willingness to undergo such treatments.
The latter group argues that these strategies are inherently dangerous for
the patient and carry heavy costs for health care providers which make them
clinically impractical. More practical approaches include (1) the use of carriers
within the BBB endothelium and (2) disruption of the tight junctions.
Invasive techiniques.
Dwaine Emerich has participated in the development of a technology that
involves encapsulation within a semi-permeable porous membrane of genetically
modified cells. The capsule is a rod-shaped copolymer of polyvinylchloride. These
capsules begin as a polymer dope that is precipitated into a tubular form within a
water bath. Studies at Cytotherapeutics used encapsulated fibroblasts genetically
engineered to produce neurotrophic factors to screen such factors for effects on
GABAergic neurons (the principal neurons lost in Huntington's patients). One
molecule, CNTF, worked remarkably well. Capsules implanted into the lateral
ventricle or directly into the striatum produced protection from neurodegeneration
as well as cognitive and behavioral deficits seen after excitotoxic lesions of the
striatum in rodents and primates (one model of HD). The mechanism of protection
is unknown because there is no evidence of neuronal receptors for CNTF within
the striatum. The positive effects could possibly be achieved through effects on
astrocytes which have been shown to express CNTF receptors. CNTF has an
effect on a wide range of cell types and is the only factor that has been shown to
protect GABAergic cells within the striatal circuitry.
Viability of encapsulated cells has been documented out to 13.5 months.
Retrieval from the brain, with subsequent ELISA analysis for growth factor levels
and genetic analysis for gene copy numbers in engineered cells, show no
significant differences between pre- and post-implanted cells. This study also
looked at long-term effects on basal forebrain cholinergic cell growth to check for
hypertrophy of these cells toward the site of diffusion in response to prolonged
exposure to trophic agents. The results suggest that pathological growth did not
occur.
Joel Saydoff is involved in the clinical application of the encapsulated
fibroblasts. The fibroblasts are transfected with a dihydrofolate reductase based
plasmid vector with the human gene of interest under a metallothionein promoter.
Data suggests that behavioral deficits are reduced and sometimes totally reversed
with this technique which suggests that these are clinically useful strategies. This
strategy can avoid problems with toxicity of the therapeutic agents by providing
local delivery in a specific area of the brain. In the case where multiple factors
need to be delivered over a long period of time, this technique is particularly
advantageous.
In order to justify these invasive strategies which carry inherent risks, there
are many issues that need to be weighed against the benefit to the patients. We
do not know for example, how much tissue must be exposed to growth factors, so
the number of implanted devices per patient cannot be assessed at this time.
There are also issues of bystander effects on nearby cell populations, as well as
the actual mechanism of the positive effect. In this system continuous diffusion
occurs over the course of hours to days, which could potentially cause
desensitization. Better constructs to control gene production are currently being
sought, for example using inducible promoters (such as the RU486-sensitive
promoter) can overcome issues of continuous diffusion causing desensitization.
Patrick Tresco, in collaboration with laboratories at the University of
Washington, has been developing specialized catheters for neuroimplantation. He
views this technology as a mechanism to explore ex vivo gene therapy in a
reasonably safe manner. These systems can be viewed as sustained delivery
devices for neurotrophic agents in a site-specific manner. These devices provide
the significant advantage of intraparenchymal infusion. Capsules provide the
additional advantage of being retrievable for subsequent analysis of the
engineered cells. Long-term expression of engineered cells, as well as the host
response to secreted factors, can be studied from a basic science perspective.
Encapsulation technology is evolving rapidly out of a relatively old
technology (i.e. the systems used for dialysis cartridges in water filtration system).
Thermoplastics can be manufactured to specification, developed and analyzed
using conventional physical chemistry techniques. Thirty thousand synthetic
polymers are available for the synthesis of these capsules. A coupling of polymer
technology, and data on the effects of growth factors, and the use of regulatable
gene cassettes would unquestionably lead to progress in this field. Significant
clinical advances could be achieved if this technology were focused on one of
these [neurodegenerative] diseases with the appropriate financial and technical
support (perhaps on the order of the resources devoted to space exploration).
Human studies are necessary to provided biocompatibility data on
materials that will be of clinical use. Among the issues to be addressed are, how
the CNS reacts to different materials with glial scarring. Currently, nonadherent
surfaces are being investigated in primate studies at Cytotherapeutics. Engineers
have discovered ways of modifying the surface to reduce glial adhesion and
facilitate retrieval without significant hemorrhaging. These problems are being
addressed are similar to the those associated with shunt technology currently
being used by neurosurgeons. With state-of-the-art neurosurgical techniques,
these types of implants should effectively influence electrical activity of neuronal
pools and become valuable assets to neurosurgeons.
Kam Leong is also working on delivery of therapeutic agents from implanted
microspheres. These studies have been addressing the issues of drug perfusion
after implantation. Studies using encapsulated radiolabeled molecules suggest
that drug delivery is dominated by convection in the first 3-5 days following
implantation. In these experiments, the biodistribution of labeled IL-2 includes the
entire hemisphere on the implanted side. This convection may be related to the
effects of brain edema resulting from surgical trauma. This widespread distribution
was seen despite a rapid drop in concentration over the distribution area.
Investigation into the time course of drug delivery has revealed that the use of
biodegradable polymers to deliver lipophilic compounds, such as taxol, presents no
problem with getting local sustained release for months. However, hydrophilic
compounds are much more difficult to deliver in a controlled manner. Small
hydrophilic compounds are pulled by a huge chemical gradient and leach out
immediately. There is also an issue of drugs becoming biologically inactive due to
the process of encapsulation. Studies have shown that there is always some loss
of activity of the protein. Theoretically, the cellular system would deliver the native
protein and eliminate this problem.
Eric Crumpler has been working with a group which is trying to redesign the
chemical composition of some of these polymers to prevent loss in bioactivity of
encapsulated therapeutic agents. These biodegradable polyanhydride wafers are
designed for patients with recurring tumors, with the goal of slowing tumor
progression over periods of months. Both small and large growth-inhibiting
molecules are being studied for delivery from these polymers. Dopamine, for
example, is released for up to 6 months, but other molecules are more problematic,
because of the poor control over delivery.
Matt During is using 60 nm microsomes containing condensed DNA. These
microsomes do not aggregate, which is a problem that has been reported with
other cationic liposomes. Avoiding aggregation depends on having the appropriate
combination of the condensing reagent with the DNA. When the microsomes
form, DNA is apparently bound to the surface of the liposomes. These microsomes
are delivered directly to the brain and have shown expression as long as 12
months. In these experiments During used a nuclear targeting signal to enhance
nuclear distribution but the problem of getting integration and long-term expression
remains. Changing the DNA structure and sequences to get integration is now the
major focus. Most of this work is done in vivo because it has been difficult to
extrapolate and apply in vitro data.
M. Fredrick Hawthorne has been experimenting with liposomes as carriers for
boron, which then serves as a target for neutron-capture and tumor destruction.
His studies have been successful in getting high levels of boron into target tissue.
For example, with an injected dose of 10 mg/kg body weight (i.v.) the tumor
concentrations of boron produced exceed 30 mg of boron / gram of tumor, when
borine is in the aqueous core of the liposomes. Thirty hours after injection, the
tumor cells have taken up the boron, which is distributed within the cytoplasm.
These results have been achieved in carcinoma studies using mice, but there is an
obvious potential for clinical application. Additionally, using boron-rich phosphate
diesters have shown that this is a potential application for DNA and RNA delivery.
There are numerous criticisms of the liposome mediated delivery of
therapeutic agents like CNTF. The most serious include: (1) In the studies which
show neuronal protection from encapsulated CNTF-producing cells there seems to
be a diffusional limit for protection to (1-2 mm from the capsule site). The
parameters that limit the diffusion and convection are not well understood.
Capsule implantation may therefore be impractical as a neurosurgical intervention
because of the requirement for multiple implantations. This requirement increases
the risk of complications due to surgery (bleeding in 1-3% of implantations) and
increases surgical expense which reduces the likelihood of managed health care
backing of such procedures, although Parkinson's patients with multiple implants
have shown some clinical benefit. In addition, the mechanisms of CNTF efficacy
are unknown; this lack of knowledge is especially troubling since the striatum does
not contain CNTF recptors.
Viral vectors for gene delivery
David Jacoby has been working with virus-based technologies. The herpes
virus has been used in many applications for long term, non-toxic gene delivery.
These experiments show that this procedure may have clinical utility. Engineering
a plasmid with the herpes packaging sequence on it produces a sequence that can
be packaged as a herpes virion. In the recent generations of this vector, all of the
herpes virus genes have been deleted. This produces a system in which there is
no contaminating virus and substantial space for subcloning a gene of interest
(plasmids in the 25 kb range have not presented any problems). This vector
provides flexibility for combinations of gene inserts and for investigation of
sequence interactions.
These virions infect neurons as does the herpes virus. They are uncoated at
the cell surface when the capsid binds microtubules and are subsequently
retrogradely transported into the nucleus. The capsid fuses with the nuclear
membrane and the plasmid is thereby introduced into the nucleus of the target
neuron. These constructs have been producing 108 transducing units/ml which can
be used in any application for infecting neurons. These viruses can cross
synapses and therefore many neuroanatomic pathways can be targeted, with
limited toxicity. Currently the virus is being engineered for enhanced long-term
expression. Promoter manipulations, study of matrix attachment regions, and
altering chromatin for increased access to transcriptional may also enhance
expression.
Ulrike Bl”mer uses a lentivirus system. This virus has the ability to infect
terminally differentiated cells. This is currently a three-plasmid system. The HIV
backbone, and the packaging system are provided during transfections. The viral
coat has been modified to broaden the range of cell types which can be infected.
Stable cell lines are currently being produced by this group. Current methods have
generated titers of 109 after two ultracentrifugation steps. With this particular
vector however, there is the real fear of generating wild-type virus. Manipulation
requires special handling facilities and stringent controls to check for the
generation of wild-type viruses. This requirement has prevented extensive
behavioral analysis.
After injection of virus particles into the striatum and the hippocampus,
expression has continued as long as 11 months. A 2 ml solution of 108 viral
particles produces an infection area of about 9 mm3, and no toxicity has been
reported. Comparisons between the lentivirus system, adenovirus (AV), and AAV
for expression, duration of expression, and immune response using reporter
genes have been performed. The results show that, unlike the lentovirus, AV
demonstrates comparable expression and efficiency but shows high infiltration.
AAV showed long-term expression, but low efficiency of infection and no
integration.
The NGF gene has been packaged into this construct and expression has
been demonstrated out to 6 months in experimentally lesioned animals. In vitro the
observation has been made that nondividing cells are more easily infected than
dividing cells with this virus. This may explain why neurons are preferentially
infected, which has been shown by cell counts in 45 animals. The reason for this
selectivity is unknown.
Matt During has also worked extensively with the AAV system. He reported that
the toxicity issue is a large one. Even after knocking out the viral genes in these
systems, there is still an immunological response. This may be true of all viral
vectors. Also, with AAV the difference between particle titers, and transducing
units is a problem. The number of transducing units is 3 orders of magnitude less
than the particle titers so the best concentration achieved in his hands is 109 , even
in the most permissive cells. A reason to choose this system over others is
because it is small and diffuses better than any of the others. It has the simplest
protein structure, and engineering may improve integration and packaging. Getting
the virus into the nucleus of cells has proven a problem. This may be related to its
single stranded DNA. Regulation of second-strand synthesis may be part of the
problem.
AAV has been used in a large number of monkeys, and the immunological
response and toxicity has been minimal (although there may be strain differences,
as suggested by other studies). In comparison, other studies using reporter assays
with AV showed that repeated injections over the course of 4 months caused an
immunological response. Any previous injection with the virus reduced the ability
of the virus to infect upon subsequent exposure with another reporter gene.
Similar studies are ongoing with AAV. There is also a critical issue of where the
virus is introduced into the brain. AAV-packaged genes were differentially
expressed under the CMV promoter depending on the brain region being
evaluated. This may suggest additional differences between AAV and lentivirus.
Progress in the expression of tyrosine hydroxylase using AAV has been
achieved by coexpressing the enzymes needed for dopamine (DA) synthesis
(tyrosine hydroxylase and aromatic amino acid decarboxylase (AADC)), in either a
two plasmid system or by using bicistronic constructs. The infected cells produce
and release DA under depolarizing conditions. AADC also increases after
infection. Desensitization has not been reported. It is suspected that there is not
enough DA produced in these models to produce desensitization.
Commentary: Issues surrounding drug diffusion
Regardless of the method used to introduce therapeutic agents to the brain,
the size and shape of the molecule of interest is an important issue, for example,
viruses diffuse differently from globular proteins. Each molecule to be developed
therefore requires separate study. The area over which a molecule will diffuse
depends upon a number of parameters including the diffusivity constant, but
special attention must be given to the clearance or consumption of that agent in
tissue. This parameter sets an absolute limit of diffusion in brain. However,
perivascular transport changes the classic distribution.
Dr. Dedrick also suggests that convection (bulk flow) rather than diffusion is
important for delivery of large molecules that exit an implanted catheter with a
semipermeable membrane. With a steady diffusion rate of a finite volume within a
catheter, the delivery of the therapeutic agent will be more extensively distributed
as well as more uniformly distributed by means of convection. This is supported by
the report of Kam Leong.
Additionally, arterial infusion at a slow rate has been shown in several
species to result in gross maldistribution of the agents in tissue. Dr. Dedricks'
group has experimented with pulsatile delivery techniques which improve the
distribution. These data will provide better predictive ability and a more complete
understanding of how various molecules will behave in brain tissue.
Neurosurgical Considerations
Ed Neuwelt believes that transvascular delivery of therapeutic agents can be
accomplished with the appropriate neurosurgical techniques. He suggested that
optimum dose calculations be based on the amount of drug per gram of tissue, but
this parameter was contested by the pharmacologists because it can not be
normalized experimentally between species. To explore drug administration
experimentally, Dr. Neuwelt has been looking at a number of techniques to deliver
growth factors in models requiring repeated administration of therapeutic agents
and BBB disruption. The models he used included the 6OHDA-model of
Parkinson's disease and a cat model of Tay Sachs disease, in which cats have a
single nucleotide deletion of the 5' end of the gene coding for b-hexosamidase.
A number of methods have been explored for BBB disruption. One method
is transient opening using osmotic changes produced by the administration of
mannitol. Another method that has been used capitalizes on the ability of brain
endothelial cells to internalize molecules conjugated to mannose-6-phosphate,
taking advantage of endothelial-receptor-mediated transcytosis. Enzyme delivery
after BBB disruption in Dr. Neuwelt's models produces positive effects for as long
as 28 days. To avoid repeated disruptions of the BBB, one-time delivery of the
gene in a viral vector would be better therapeutically. The available vectors,
however, all show toxicity.
Dr. Neuwelt suggests that iron particle conjugate delivery may be another
experimental possibility. Dextran coated iron oxide crystals (22 nm) have a high
affinity for neurons. This method has been combined with transient BBB opening
with the introduction of a hypertonic solution (1500 mosmols, 20 s (Rappaport
method)) and has been used in both animals and humans (1/1000 stroke rate) to
deliver drugs and particles into the brain. "Stealth" liposomes (200 nm and smaller)
are another possibility. Stealth liposomes have polyethylene glycol barriers on the
outer surface which allows the material to persist in circulation much longer than it
would normally.
Clinically, Dr. Neuwelt and his colleagues at the Oregon Health Sciences
University have been successful at repeated BBB disruptions in patients
undergoing treatment for brain lymphomas (up to 50 times a year, with no cognitive
dysfunction out to 15 years). These brain tumors are whole brain diseases. A
large fraction of these tumors are leaky (infiltrating cells spreading to other areas of
the brain) which is why BBB disruption is a better clinical strategy than regional
therapy. To improve the treatment paradigm filtered mannitol is used. This
filtration step has eliminated the detrimental effects seen in a Swedish study
showing pathological sequelae of BBB disruption in rats. No magnetic resonance
imaging (MRI) abnormalities or deficits in psychological health have been seen.
The practice of very short duration BBB disruptions may be key in preventing side
effects. These techniques are derived form an earlier study by Rappaport which
showed that arabinose loosens the tight junctions which are normally present
between the brain endothelial cells (see above). Insertion through the disrupted
tight junctions after hypertonic solution administration has been shown with EM
visualizing lanthanumm or iron particles.
Keith Black suggested that global disruption of BBB with osmotic disruption may
be necessary for large-scale drug delivery to the brain, but other techniques for
local delivery are also effective. Using vasoactive compounds like bradykinin, the
bradykinin analogue RMP7, or leucotrines, his group has been able to show
differential effects between the capillaries within the tumor and normal brain
capillaries. There is a 2-10-fold increase in permeability in abnormal (tumor) vs.
normal capillaries. High molecular weight compounds, such as Dextran, show a
10-fold increase of delivery into the targeted tumors. Bradykinin is clearly the most
potent but other factors (histamine, vascular permeability factor) have been used.
Animal and clinical data show that this is a viable method, especially in the case
where the therapeutic drug is neurotoxic. The use of MRI has greatly improved this
technique but this enhanced permeability is true even for microscopic foci less than
1 mm in diameter, which is less than the resolution of an MRI scan. This method
could also be used to deliver compounds like boron.
BBB physiology:
Two criteria generally need to be met to get agents through the BBB without
intervention: (1) the molecules have to be small (molecular weight under 500
daltons) and (2) the molecules have to be lipid soluble. These criteria are the
driving force behind the research and development of drugs that have clinical
utility. These criteria also provide insight into the biology of the BBB. Permeability
of the membrane is based on variables such as thickness and fluidity of the lipid
bilayer which can be investigated and possibly manipulated. The size requirement
for drug entry provides information about the width of junctional pores and
suggests that protein mass may be important.
Information provided by research into the basic molecular structure and
function of BBB may be necessary for the development of better strategies for
getting agents into the brain. For example one study exposed ivermectin (a
neurotoxin which has low BBB permeability) to P-glycoprotein mutants (MDR
knockouts). These mice are marketed as BBB-less mice. P-glycoprotein is
colocalized with GFAP. The ivermectin was highly enriched in the MDR knockouts
suggesting that P-glycoprotein may play a role in the BBB at the end foot process.
One interpretation suggests that an active efflux system in the astrocytic endfoot
necessary for getting molecules out of the brain is involved. Inhibiting this process
(as in the MDR knockouts) would permit drugs that all ready have a finite
permeability at the endothelial cell to enter the brain.
Protein vector based systems have been used by Ruben Boado to take
advantage of transporter-mediated entry into the brain. A fusion protein is created
with a monoclonal antibody to the transferrin receptor conjugated to streptavidin.
These vectors are actually too big to undergo glomerular filtration and have a much
longer plasma life than smaller molecules. Any biotinylated protein can be
combined with this "non-invasive" vector for entry into the brain. Currently
antisense therapy is also being pursued. Several generations have produced
modified antisense RNA molecules which are resistant to endonuclease activity.
Using this strategy it is possible to deliver 0.1% of the injected dose into the brain.
These oligonucleotides are 14mers-18mers, with molecular weight of about 6000
daltons. Cell specificity can be accomplished with a second conjugated antibody
directed toward a cell specific antigen.
William Partridge has developed an endothelial-specific antibody for
development as potential protein carriers into the brain. Other carriers are anti-
transferrin receptor for rats and anti-insulin for humans and old-world primates.
Currently fusion proteins are being developed with these antibodies. These
conjugates provide an advantage when streptavidin is added which allows the
subsequent addition of any biotinylated protein. One antibody can serve as the
carrier and the other can be targeted for neurons. Large molecules (>4 Kd) can
get across the BBB with this method.
When these antibody-conjugate systems are used for antisense therapy a
third barrier has to be crossed, that is, the endosomal barrier. Experimentation has
found that phosphothioate nucleotides are very neurotoxic so peptide-nucleic acids
are a better strategy. These are polypeptide backbones with high hybridization
affinities for the target nucleic acids. These types of molecules could potentially be
directed toward the polyglutamines of the HD protein and be designed to bind
those with 36 or more glutamine residues. Therapeutic use is licensed exclusively
to Isis Technologies.
Suggestions for future research
Bill Partridge suggests that resources be invested in the basic biology of the
blood brain barrier. The invasive technology will be hard to sell to patients and
health care management organizations. He suggests that too much attention has
been paid to brain drug development and not enough to brain drug delivery. One
directed approach would be to look for brain specific delivery systems with basic
research techniques. Experiments such as subtraction cloning of brain vs. non-
brain capillaries to look for brain specific genes. Another approach is using pro-
drug chemistry with drugs that do cross the BBB. Still another approach is to study
how viruses enter the brain.
Comments: David Jacoby mentions that viruses actually do not get into the brain
on their own with ease. A high titer (a viremia) needs to be built up in order to get
brain infection. This is why stereotaxy is used to get these agents into the brain.
HIV uses macrophages to cross the blood brain barrier.
Robert Dedrick suggests that diffusivity is an important issue that needs to be
carefully studied for the agent to be delivered. The diffusion tensor imaging
technique (weighted MRI imaging) gives us power to predict how diffusion will
occur in the brain. He warns that the computational requirements are enormous
but the are interpretable for the purpose of predicting the best therapeutic
approach. This is an alternative to the "brute force" method of delivery.
Joel Saydoff suggests that since we are not at a point where we can selectively
deliver drugs to specific areas of the brain in a safe manner noninvasively, then
use of encapsulated cell-based delivery system is the best method presently
available. Dwaine Emerich echoes this idea and states that these are strategies
that are practiced currently clinical settings around the world. These techniques
therefore should be developed to their fullest extent until something better is
available such as transgenes and engineered cell delivery. Dr. Emerich adds that
special attention should be paid to the diffusion and bioavailability of agents like
CNTF so that the number and duration of implant can be optimized.
Ed Neuwelt suggests that since none of the current vectors have clinical utility
presently, delivery should be carried out using neurosurgical approaches. For
example, if the head of the caudate needs to see a drug, then it can be bathed via
catheter through one of the supplying vessels. Larger brain areas can be perfused
through the internal carotid or the vertebral artery and this can be done repeatedly
with very little surgical risk. In terms of future research he suggests that interstitial
infusion of the HD protein using a viral vector (i.e. HSV) unilaterally will probably
produce the best animal model of the disease.
Comment: David Jacoby suggests that the construct which goes into the model is
very important to consider. Over expressing the HD protein is certainly possible
but other approaches may be tried. Generating a cell line to express HD in a
sustained manner and then transplanting them is another route. Researchers
need to replicate the chronic disease and investigate the prevention of cell death
as well as the rescue neuronal function. These vectors [viral] will serve as tools to
generate models with which to approach these problems.
William Gelbart suggests that more research should be devoted to the nature of
tight junctions, because so little is known about what the tight junctions are
composed of at the molecular and protein level. An understanding of what these
proteins are and what happens to them during osmotic disruptions is necessary to
formulate an effective approach to selectively disrupt the BBB.
Ruben Boado suggests that an antisense strategy for binding the abnormal HD
protein glutamine expansions could be designed and delivered through the BBB
with the antibody (i.e. transferrin receptor) vectors that he and others have been
using. New technologies such as DNA or polyamide ("peptide") nucleic acid (PNA)
clamping can be used to block or arrest target genes in diseases such as HD.
These constructs can be delivered with antibody conjugate vectors. This antibody
based technology may also be used to generate animal models of HD.
Matt During suggests that more needs to be learned about the disease using
animal models. The development of transgene delivery techniques using viral
vectors is probably the best strategy to pursue. The CRE-LOX system could be
used to knock out the HD gene and investigate more about the function of the HD
gene product. Reservations were expressed with the strategy of long-term
exposure to trophic factors like CNTF.
Robert Dedrick suggests that more attention should be paid to
pharmacodynamics of any drug that would be used for treatment of brain diseases
such as HD. Whatever method that is used for the introduction of therapeutic
agents, treatment paradigms need to consider such factors as clearance of the
drug and diffusivity of the drug. These are answerable questions which will
ultimately produce better treatment strategies. These questions should be
addressed before we attempt to start therapies like CNTF containing implants.
David Jacoby suggests that the viral vectors are the most promising avenue of
research. By going to humans as quickly as possible researchers will learn a lot
about how these techniques can be used to treat disease. Invasive techniques are
currently being used to treat these disease states. Intervention at the genetic level
using viral vectors will allow us to develop interventions at the level of the gene, the
protein and the cell. Development of cell lines which express HD will provide
platforms to test treatments for the disease. Dr. Jacoby is currently working with
human neuronal surrogates and getting them to overexpress disease genes.
Xenographic transplants will also contribute to treatment development. Porcine
cells are one way to go. Additionally, HSV vectors have room for subcloning the
entire HD gene and gives the power to manipulate the gene of interest. For
example, the number of CAG repeats expressed in the system can be controlled
by straightforward gene engineering strategies.
Patrick Tresco suggests that we use the implant technology that is available
now as simple site specific delivery devices of therapeutic agents. More
sophisticated strategies for drug delivery will be developed with time. Human data
is also necessary to advance our understanding of these treatment strategies.
However, Dr. Tresco suggests that the molecule to be delivered will dictate what
mode of delivery will be best. For this reason, the focus should be on finding the
best therapeutic agent and deciding what technology is best for delivering that
agent. Once an agent is found there will be a surge of research by pharmaceutical
companies to get it into the brain. |
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