Note: Descriptions are shown in the official language in which they were submitted.
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Chitosan-based transport system
It is one of the great goals of pharmaceutical research to
make the various barriers in the body selectively passable
for specific substances. These include the intestine-blood
barrier, the skin-blood barrier, the nasal mucosa-blood
barrier, and the blood-brain barrier (BBB).
The blood-brain barrier (BBB) is one of the most
problematic barriers as it has highly selective transport
systems and as these cells are very tightly joined. The
blood-brain barrier is formed by the endothelium of the
capillary vessels. These endothelial cells adhere by tight
junctions and prevent entry of polar substances exceeding
a specific molecular weight into the brain. However, some
nutrients (such as D-glucose) and hormones overcome the
blood-brain barrier using selective transport systems.
Tight junctions (Latin: zonulae occludentes) are strip-
shaped junctions of cell membranes that appear to be so
tight under the electron microscope as if the membranes
were fused. However, actual contact only occurs among the
proteins embedded in the outer layer of the participating
cell membranes. The protein involved is occludin, a
transmembrane protein. The tight junctions occur over
extremely short sections of a few nanometers that belong -
as becomes visible in freeze breaks only - to a network of
globular occludin molecules arranged in a chainlike order
which "weld" the epithelial cells to each other.
A particular problem is the transport of hydrophilic
substances through the BBB. Pharmaceutical researchers
therefore are looking for ways to encapsule such
hydrophilic substances in lipophilic particles or bind
them to particles with substances that permit receptor-
mediated transport across the BBB.
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In recent years, they increasingly worked on transport
systems consisting of nanoparticles. Nanoparticles mostly
consist of polymers and are about 10 to 1000 nm in size
[Kreuter, Journal of Anatomy 1996, 189, pp. 503-505]. Some
researchers managed to produce efficient nanoparticles
that ensure rapid transport of drug-charged particles
across the BBB. Nanoparticles from polybutyl cyanoacrylate
are able to transport drugs by encapsulating or binding
them to the surface of the nanoparticles [Schroeder et
al., Journal of Pharmaceutical Science 1998, 87, 11, pp.
1305-1307, Schroeder et al., Progress in Neuro-
Psychopharmacology and Biological Psychiatry 1999, 23, pp.
941-949, Alyautdin et al., Pharmaceutical Research 1997,
14, 3, pp. 325-328, Ramge et al., European Journal of
Neuroscience 2000, 12, pp. 1931-1940]. However, these
nanoparticles cannot be transported across the BBB
directly, only by coating them with polysorbate 80
[Kreuter, Advanced Drug Delivery Reviews 2001, 47, pp. 65-
81, Kreuter, Current Medicinal Chemistry-Central Nervous
System Agents 2002, 2, pp. 241-249]. Nanoparticles
consisting of polycyanoacrylate that were coated with
polyethylene glycol could only overcome the BBB if, due to
an infection of the brain, the BBB is defective and has
become less permeable [Calvo et al., European Journal of
Neuroscience 2002, 15, pp. 1317-1326]. Wang et al.
[Molecular Therapy 2001, 3, 5, pp. 658-664] found a
cationic polymer (polyethylenimine) with which you can
bypass the BBB and use an intramuscular injection in the
tongue to introduce drugs into the brain using retrograde
axonal transport. Rousselle et al. [Molecular Pharmacology
2000, 57, pp. 679-686] transported doxrubicin across the
BBB using a peptide vector. The drug to be transported is
covalently bound to D-penetrantin, a peptide, and synBl,
which facilitates transport across the BBB without causing
ejection by the P-glycoprotein. Other ways include
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transporting nanoparticles via the transferrin receptor by
binding them to ligands [Li et al., Trends in
Pharmacological Sciences 2002, 23, 5, pp. 206-209]. This
system however has the setback that you can charge the
particles with a small quantity of the substance to be
transported only.
The previous results of nanoparticle research have shown
that neither complicated manufacturing processes nor
damage to the BBB is required to transport hydrophilic
substances into the brain or that the coating substances
are insufficiently decomposed and/or decomposed into
harmful monomers.
Many prior art systems are based on coating materials that
are composed of one or several cationic and/or anionic
layers. Previous approaches were based on the assumption
that the transport systems must be physically and
chemically stable to protect their content (active agents)
and take them to their destination. This is why most
systems have very good mechanical properties and do not or
do not readily dissolve in the bloodstream.
The use of monosaccharides for overcoming the blood-brain
barrier was studied to some extent. US 6,294,520 B1
describes oral administration of monosaccharides and amino
acids, among other purposes, for supporting the treatment
of hair loss.
If an active ingredient has entered the bloodstream it can
be metabolized by the liver, discharged by the kidney, or
passed to the intestine by the gall bladder. This is why a
high dose is often needed to get the required effective
quantity to the affected tissues.
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Chitosan has been known for some years now as a drug delivery
system. Chitosan has some interesting properties and is
studied in many areas of medicine and pharmaceutics. It is
known that nanoparticles with chitosan coats or nanocapsules
can transport pharmaceuticals into the body or overcome the
skin-blood or intestine-blood barrier. These barriers are
overcome relatively easily. However the blood-brain barrier
(BBB) is one of the most problematic barriers to overcome as
it has highly selective transport systems and as the cells are
very tightly joined.
It is therefore the problem of the invention to provide a
chitosan-based transport system for overcoming the blood-brain
barrier. This transport system is to convey active agents or
markers into the brain.
According to the invention, this problem is solved by a
transport system containing at least one substance from the
group of chitin, chitosan, chitosan oligosaccharides, and
glucosamine or their derivatives, and optionally one or
several active agents and/or one or several markers and/or one
or several ligands.
Basic units of the transport system of the invention are
building blocks of chitin, chitosan, chitosan
oligosaccharides, and glucosamine or their derivatives. The
term "chitosan oligosaccharide" includes both carbohydrates
that consist of up to 10 monosaccharides and longer-chain
polysaccharides. Chitosan is obtained from natural chitin by
deacetylating the amide bond, the degree of deacetylation
(DDA) being controllable. Chain length and molecular weight of
chitosan oligosaccharides can also be accurately set during
preparation. WO 03/029297 A2 describes the respective method.
Chitin, chitosan, or chitosan oligosaccharides can have
different properties depending on chain length and degree of
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deacetylation. Both parameters, chain length and degree of
deacetylation, can be set during preparation.
Chitosan oligosaccharides, chitosan, and chitin with molecular
weights from 179 Da (glucosamine) to 400 kDa are used for the
transport system of the invention. It is preferred that the
chitosan oligosaccharides, chitosans, and chitins have
molecular weights from 179 Da to 100 kDa. Particularly
preferred are chitosan oligosaccharides, chitosans, and
chitins with molecular weights from 179 Da to 1.8 kDa and
chain lengths of 1 to 10 N-acetyl glucosamine or glucosamine
rings.
Most preferred are chitosan oligosaccharides, chitosans, and
chitins with molecular weights from 800 Da to 1.8 kDa and
chain lengths of 5 to 10 N-acetyl glucosamine or glucosamine
rings.
Chitin, the chitosans, and chitosan oligosaccarides have
degrees of deacetylation from 0 to 1000.
The preferred degree of deacetylation (DDA) is in the range
from 30 to 1000. Particularly preferred is a degree of
deacetylation of 70 to 100%.
Active agents and/or markers and/or ligands can be bound in
various ways to the basic units. A preferred way is binding
via the NHZ group of the glucosamine rings. Fig. 1 shows
diagrams of various options. Chitin, chitosan, chitosan
oligosaccharide, glucosamine or their derivative are only
referred to by the term "chitosan" in Fig. 1. These ligands
are required to dock to the receptors.
The transport system according to the invention preferably is
designed in such a way that the chitin, chitosan, chitosan
oligosaccharide, glucosamine or their derivative is bound to
one or several active agents and/or one or several markers.
One or several ligands may be bound instead of active agents
and/or markers, or, optionally, in addition to them.
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In another preferred embodiment, the transport system is
designed so that the chitin, chitosan, chitosan
oligosaccharide, glucosamine or their derivative is coated by
one or several active agents. Likewise, one or several active
agents can be coated by the chitin, chitosan, chitosan
oligosaccharide, glucosamine or their derivative. This
substance may preferably be coated by another coating
substance. Preferred coating substances are starch and/or
alginate.
It is preferred that one or several markers and/or one or
several ligands are bound to the coat of chitin, chitosan,
chitosan oligosaccharide, glucosamine or their derivative or
to the coat of active agent.
In this context, if another coating substance is present, it
is preferred that one or several markers and/or one or several
ligands are bound to the outer coat.
In another embodiment, the chitin, chitosan, chitosan
oligosaccharide, glucosamine or their derivative is present as
a chain and is bound to one or several active agents and/or
one or several markers.
It is preferred in this embodiment of the transport system
according to the invention that chain-like chitin, chitosan,
chitosan oligosaccharide, glucosamine or their derivative are
bound to the chitin, chitosan, chitosan oligosaccharide,
glucosamine or their derivative.
It is particularly preferred in the two latter embodiments
(chain) that one or several ligands are bound to the chitin,
chitosan, chitosan oligosaccharide, glucosamine or their
derivative.
The bound ligands can connect to receptors on membranes. These
substances preferably are substances from the group of
transferrin, insulin, insulin-like growth factors, and
polysorbate-80.
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The transport system according to the invention preferably
contains substances as active ingredient that develop an
effect in the brain.
The transport system according to the invention is preferably
solid, liquid, or semisolid.
It can be applied by oral, dermal, or parenteral
administration (preferably by intravenous injection).
The transport system according to the invention overcomes body
barriers (dermal, oral, etc.) and enters the vascular system.
The vascular system transports the particles, capsules, or
molecules that partially re-arrange into specific cells or
extracellular structures. Surprisingly, this also occurs
across the blood-brain barrier.
Absorption in the brain could be proven by studies in mice
that had the transport system according to the invention
containing a fluorescent marker injected intravenously. Fig. 2
shows the fluorescence microscopic picture of a section
through the brain from the area of the hippocampus. The
hippocampus is the region in the brain that is responsible for
short-term memory, forming associations, and recognition of
situations and objects. Considerable changes of this region of
the brain occur with diseases such as Alzheimer's disease.
You can see a clear accumulation of fluorescent particles in
the pyramidal cell layers (Pz) of hippocampus subregions CA1,
CA2, and CA3. (The bar at the bottom right in the figure
represents 100 um.)
Figure 3 shows a close-up shot of a neuronal cell comprising a
high content of particles of the transport system according to
the invention (black coloration) (The bar at the bottom right
in the figure represents 100 um.).
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It was found in studies that the composition can be
reconfigured in the blood if, for example, long chitin or
chitosan components disintegrate into short molecular blocks.
It was found surprisingly that the blood itself and primarily
the erythrocytes in it can assume a filtering or sorting
function causing the chitin or chitosan molecule chains to
disintegrate into molecular blocks with preferably 4 to 10
chitin or chitosan rings (N-acetyl glucosamine or glucosamine
rings). These form active agent transport mixtures that can be
transported independently and are preferably transported by
erythrocytes. Chitin or chitosan molecules having the same
structures and molecular size as glucose or glucosamine
transported by erythrocytes preferably bind to erythrocytes.
If such a rearrangement occurs in the blood, absorption
preferably takes place by glucose transport points at the
blood-brain barrier or blood-organ barrier. Depending on the
organ and configuration, transport can also be achieved via
tight junctions or endocytotic or receptor-mediated processes.
We found that the transport systems according to the invention
have great affinity for specific cells. These are cells with a
high metabolic activity (energy-consuming) cells that are
characterized by considerable glucose consumption. In the
brain, besides in microgliocytes, particles preferably
accumulate in neuronal cells, more preferably in pyramidal
neurons.
Absorption in the cells could be proven by studies in mice
that had the transport system according to the invention
containing a fluorescent marker injected intravenously. Figure
4 shows the fluorescence microscopic picture of a neuronal
cell from the brain of a mouse. The cells stained with cell-
specific and fluorescent markers (parvalbumin positive) appear
dark gray. The transport system according to the invention is
shown in black (the bar represents 50 um).
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Figures 5 and 6 also show fluorescence microscopic pictures of
tissue slices with neuronal cells. The cells are stained gray,
the transport system appears in the form of black dots (the
bar represents 50 Vim). It is clearly visible that the
transport system (black) is located within the gray area
(cell). The picture proves beyond doubt that the transport
system is absorbed in the cells.
In addition to this accumulation in and on cells, accumulation
at extracellular structures, preferably at structures rich in
protein such as ~-amyloid plaques in the Alzheimer pathology.
Figure 7 shows a fluorescence microscopic picture of a tissue
slice with a-amyloid plaque (2) where the transport system
according to the invention (1) has accumulated (the bar
represents 50um).
It can also be absorbed in inflammatory brain regions or in
tumor tissue. Absorption is similar in other body tissues.
Cells with a high energy metabolism (such as inflammations,
tumors) are addressed primarily again.
The mechanism of action of preferred absorption via the
glucose transporter in addition to endocytotic and receptor-
mediated processes also explains the special effect on active,
inflammatory, and tumor cells. These cells have a particularly
great growth-related energy demand.
Depending on the modification of the composition, the
transport system and active agent are separated in the cell or
passed on to other areas of the cell such as lipide-like
structures for absorption in or accumulation at organellas
such as mitochondria or the nucleus. If the composition or its
chitin, chitosan, chitosan oligosaccharide, or glucosamine
portion is absorbed in the nucleus, it accumulates at DNA
structures.
Accumulation sites in the cell could be proven by studies in
mice that had the transport system according to the invention
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injected intravenously. Figure 8 shows a section of electron
microscopic pictures of neuronal cell. The transport system
has accumulated at specific cell structures, here the nuclear-
investing membrane, and is indicated by a circle. The
fluorescence microscopic picture in Figure 4 also clearly
shows a concentration at specific sites in the cell.
Surprisingly, concentration (clustering) of various individual
compositions may occur at extracellular structures.
If chitin, chitosan, chitosan oligosaccharide, or glucosamine
(basic elements) and active agent are separated or if only the
basic element is introduced into the body, it can cause the
following effects:
- Accumulation at or depositing in the membranes of cells
and/or organellas and the resulting influence on signal
cascades.
- Change in absorption or discharge of substances of any
kind such as growth factors, messenger substances,
minerals, electrolytes, and others in or from the cell
These effects can also occur without separation of the basic
element from the active agent.
In addition to causing an effect in the cells, specific
structures that are marked by the basic elements can be
identified outside the cell and used for diagnostic purposes.
After the diagnosis or unfolding of the effect of the bound
substances the transport system decomposes so that the bound
substance remains in the cell or is transported as an unbound
particle through the vascular system, decomposed, or
discharged.
Chitin, chitosan, chitosan oligosaccharide, or glucosamine are
decomposed without residue in the cell or in the body.
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Thus the transport system according to the invention can, in a
cell-specific manner, dock to, or penetrate into cells that
have these features, even outside the brain.
Controlled accumulation of chitin, chitosan, chitosan
oligosaccharide, or glucosamine and active agent particles
makes it possible to introduce diagnostic or therapeutic
agents and transport them to the focus of the disease or the
action site. As the transport system according to the
invention accumulates in the metabolically active cells whose
metabolism is increased as compared to other cells low doses
of diagnostic or therapeutic agents can be administered as
these concentrate in the diseased tissues of the body.
In this respect, the transport system according to the
invention can be used to produce an agent for diagnosing
brain-specific diseases. The diagnosis of tumors and
Alzheimer's disease is preferred. In addition, the transport
system described can be used as a diagnostic or therapeutic
agent with malignant brain tumors. For example, highly
effective antitumor agents such as tamoxifen can be delivered
to the site where the effect should develop.
If you link the transport system according to the invention to
a radioactive substance, you can diagnose foci of disease
(inflammations) or tumors in vivo even if they are present at
a low concentration (metastases, tumors in their early stage).
In Alzheimer pathology, the invention enables you to bind a (3-
amyloid-affine radioactive substance to the transport system
of the invention for controlled identification of plaque foci
and concentration of diagnostics there. If the marker does not
concentrate, you can assume that there is no pathologic
change.
To diagnose beta-amyloid deposits using positron emission
tomography (PET) the chitosan transport system is labeled with
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C11 by methylating the chitosan. The target were specific
activities of more than 2000 Ci/mmol.
The transport system according to the invention can be used
for treating brain-specific diseases. The treatment of tumors
and Alzheimer's disease is preferred.
Suitable active agents for treatment can be bound to the
transport system according to the invention that concentrate
at the diseased sites and penetrate into the cells. This
allows for a relatively low dose in relation to the body,
which reduces the side effects of the drugs.
Particularly preferred are active agents selected from the
group of acetylcholine precursors, in particular, choline and
lecithin, stimulants for acetylcholine release such as
linopirdine, acetylcholine sterase inhibitors, in particular,
tacrine, donepezile, rivastigmine, metrifonate, and
galantamine, muscarine receptor agonists, in particular,
xanomeline, milameline, AF102B, Lu25-109, SB202026, and
talsaclidine, beta-sheet breakers, neutral endopeptidases such
as neprilysine, painkillers, inflammation inhibitors such as
propentofylline, ibuprofen, and indomethacin, antioxidants,
neuroprotective agents, NMDA antagonists, and antirheumatics.
The nerve growth factor (NGF) is a particularly preferred
active agent.
Preferred antioxidants are vitamins E, C; deprenyl
(selegiline; MAO-B inhibitor), and gingko biloba.
Neuroprotective agents are preferably selected from the group
of Q10, nicotin, cerebrolysin, piracetam, phosphatidyl serine,
and acetyl-L-carnitine. A particularly preferred NMDA
antagonist is memantine.
Chitin or chitosan and chitosan oligosaccharide are decomposed
without residue by the organism due to their glucose-like
structure. We observed that chitosan was surprisingly
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resorbed from the urine in the kidney and returned to the
body.
As the particles are decomposed without residue, no further
load on the organism by harmful monomers occurs, and monomer
that is formed is glucosamine.
It can be expected that the transport system of the invention,
due to its capability to be conveyed via glucose transporters
and/or the openings of tight junctions, will be able to
overcome the blood-blood barrier between mother and fetus.
This capability can be utilized at the prenatal stage for
diagnostic and therapeutic purposes.
The invention is explained in greater detail with reference to
examples.
Exemplary embodiments
Example 1
Intravenous administration of a mixture of chain-like chitosan
with a molecular weight from 1.8 kDa to 300 kDa and a degree
of deacetylation from 80 to 100% to which a peptide or
polypeptide of maritime origin was bound for treating tumor
diseases in the brain. Administration of 45 mg of active agent
per day over a period of 90 days; the transport agent/active
agent mixture is absorbed in normal saline and applied.
Example 2
Preparation of chitosan oligomer in pure form and with a low
degree of deacetylation (DDA) < 80o and a molecular weight
from 800 to 1600 Dalton for treating inflammatory diseases in
the bloodstream (phlebitis), administration of <_ 0.2 mg/100
kg body weight.
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Example 3
Preparation of chitosan oligomers with a high DDA > 80%
molecular weight 500 to 2500 Da and adding 0.2 parts of
ibuprofen or indometacin and intravenous administration of 5
0,3 mg/100 kg body weight in 2 ml NaCl solution over 14 days
to inhibit inflammations induced by local Alzheimer plaque
Example 4
Preparation of chitosan oligomers with a high DDA and mixing
with glucosamine solutions and gingko biloba extract at a
ratio of 5 . 2 . 1 for oral mucosa penetration (gel film on
palatum or lower lip area).
Example 5
Coupling of memantine to chitosan with a degree of
deacetylation of 87% and a molecular weight of 1.8 kDa. The
transport system is stabilized by another coating with
chitosan (DDA 90%) with a molecular weight of 150 kDa. The
preparation is administered orally once a day at a dose of
mg of active ingredient in the first week that is increased
at weekly increments of 2.5 mg to the maximum dose of
mg/day.
Example 6
Coupling of donepezile, rivastigmine, or galantamine to
chitosan transport system (DDA 85%,) so that the administered
dose is 2 to 5 mg of active ingredient per day; chronic
application over several weeks (40 weeks).
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Figures 1 to 8
Figure 1: Various embodiments of the transport system
according to the invention.
Figure 2: Fluorescence microscopic picture of a section
through the brain of a mouse in the hippocampus region (the
bar represents 100 um).
Figure 3: Close-up shot of a neuronal cell comprising a high
content of particles of the transport system according to the
invention (black coloration) (The bar at the bottom right in
the figure represents 100 um.).
Figure 4: Fluorescence microscopic picture of a neuronal cell
from the brain of a mouse. The stained cells are shown in dark
gray. The transport system according to the invention is shown
in black (the bar represents 50 um).
Figure 5: Fluorescence microscopic picture of a tissue slice
with neuronal cells. The cells are stained gray, the transport
system appears in the form of black dots (the bar represents
5 0 um ) .
Figure 6: Fluorescence microscopic picture of a tissue slice
with neuronal cells. The cells are stained gray, the transport
system appears in the form of black dots (the bar represents
50 um) .
Figure 7: Fluorescence microscopic picture of a tissue slice
with (3-amyloid plaque (2) where the transport system according
to the invention (1) has accumulated (the bar represents
50um) .
Figure 8: Section of a electron microscopic image of a
neuronal cell. The transport system has accumulated at the
nuclear-investing membrane and is indicated by a circle.
