HK1178909A - Aprotinin and analogs as carriers across the blood-brain barrier - Google Patents

Description

Aprotinin and analogs as carriers across the blood-brain barrier

The present application is a divisional application of patent application No. 200480003544.8, filed on 2004, 1/5, entitled "aprotinin and analog as a vehicle crossing the blood brain barrier".

Technical Field

The present invention relates to improvements in the field of drug delivery. More particularly, the present invention relates to non-invasive and flexible methods and carriers for transporting a compound or drug across the blood-brain barrier of an individual.

Background

In the development of new therapies for brain pathologies, the Blood Brain Barrier (BBB) is considered to be a major obstacle to the potential use of drugs for the treatment of Central Nervous System (CNS) diseases. In 1998 the global market for CNS drugs was $330 billion dollars, which is roughly half of the global market for cardiovascular drugs, and even in the united states, CNS-disordered patients are almost twice as many as cardiovascular disease patients. The reason for this imbalance is that more than 98% of potential CNS drugs cannot cross the blood-brain barrier. Furthermore, more than 99% of CNS drug development worldwide is directed only to CNS drug discovery, and less than 1% is directed to CNS drug delivery. This ratio may prove why no effective treatment is currently available for major neurological diseases such as brain tumors, alzheimer's disease and stroke.

The brain is protected against potentially toxic substances by two barrier systems: the Blood Brain Barrier (BBB) and the blood cerebrospinal fluid barrier (BCSFB). The BBB is thought to be the major route of uptake of serum ligands, as its surface area is about 5000-fold greater than that of BCSFB. The brain endothelium, which constitutes the BBB, represents a major obstacle to the use of potential drugs against various diseases of the CNS. As a general rule, only lipophilic molecules smaller than about 500 daltons are able to cross the BBB, i.e., from the blood to the brain. However, the size of many drugs that show promising results in animal studies for the treatment of CNS diseases is considerably larger.Thus, peptide and protein therapeutics are generally excluded from transport from the blood to the brain because the brain capillary endothelial wall is almost impermeable to the drug. Brain Capillary Endothelial Cells (BCEC)_s) Sealed by tight junctions, with few openings and intracellular vesicles as compared to capillaries of other organs. BCECs are surrounded by extracellular matrix, astrocytes, pericytes and microglia. The tight association of endothelial cells and the astrocyte foot processes with the basement membrane of capillaries is important for the formation and maintenance of the properties of the BBB, which allows tight control of blood-brain exchange.

There is no effective drug delivery method available to the brain. The methods under investigation for the delivery of peptide and protein drugs to the brain can be divided into three main strategies. First, invasive methods include direct intraventricular administration of drugs by surgical methods, and transient disruption of the BBB by intra-carotid infusion of high osmolarity solutions. Second, the pharmacologically-based strategy appears to force peptides or proteins across the BBB by increasing their lipid solubility. Third, physiologically-based strategies exploit the various vector mechanisms of the BBB, which have been characterized in recent years. In this approach, the drug is attached to a protein carrier that behaves like a receptor-targeted delivery vehicle on the BBB. The method is very specific, shows high efficacy and is very flexible for clinical indications with indefinite targets. In the present invention, the last method is studied.

It would be highly desirable to provide improvements in the field of drug delivery.

It would also be highly desirable to provide a non-invasive and flexible method and vector for transporting a compound or drug across the BBB of an individual.

Disclosure of Invention

It is an object of the present invention to provide an improvement in the field of drug delivery.

It is another object of the present invention to provide a non-invasive and flexible method and carrier for transporting a compound or drug across the blood-brain barrier of an individual.

According to one embodiment of the present invention, there is provided a method for transporting an agent across a blood-brain barrier of a patient, the method comprising the steps of: administering to the patient a compound comprising said agent linked to aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin, or a pharmaceutically acceptable salt of a fragment of aprotinin.

According to a further embodiment of the present invention, there is provided the use of aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin or a pharmaceutically acceptable salt of a fragment of aprotinin for transporting a compound attached thereto across the blood-brain barrier of a patient.

According to another embodiment of the present invention, there is provided a use of aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin or a pharmaceutically acceptable salt of a fragment of aprotinin for the manufacture of a medicament for treating a neurological disease across the blood-brain barrier of a patient.

According to yet another embodiment of the present invention, there is provided a use of aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin or a pharmaceutically acceptable salt of a fragment of aprotinin for the manufacture of a medicament for treating a central nervous system disorder across the blood-brain barrier of a patient.

According to another embodiment of the present invention, there is provided a compound of the formula R-L-M or a pharmaceutically acceptable salt thereof, wherein R is aprotinin or a fragment thereof, L is a linker or a bond, and M is an agent or drug selected from the group consisting of small molecule drugs, proteins, peptides and enzymes.

In accordance with another embodiment of the present invention, there is provided a method of treating a neurological disease in a patient comprising administering to the patient a medicament comprising aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin or a pharmaceutically acceptable salt of a fragment of aprotinin, and a compound suitable for treating the disease, said compound being linked to aprotinin.

According to a further embodiment of the present invention there is provided a method of treating a central nervous system disorder in a patient, the method comprising administering to the patient an agent comprising aprotinin, a pharmaceutically acceptable salt of aprotinin, a fragment of aprotinin or a pharmaceutically acceptable salt of a fragment of aprotinin, and a compound suitable for treating the disease, said compound being linked to aprotinin.

According to one embodiment of the present invention, there is provided a carrier for transporting an agent attached thereto across a blood-brain barrier, wherein the carrier is capable of crossing the blood-brain barrier after attachment to the agent, and thereby transporting the agent across the blood-brain barrier.

In a preferred embodiment of the invention, said transporting does not affect the integrity of the blood-brain barrier.

In a preferred embodiment of the invention, the carrier is selected from the group consisting of aprotinin, a functional derivative of aprotinin, angiopep 1 (Angio-pep 1) and a functional derivative of angiopep 1.

In a preferred embodiment of the invention, the agent is selected from the group consisting of a drug, a medicament, a protein, a peptide, an enzyme, an antibiotic, an anti-cancer agent, a molecule active at the central nervous system level, a radioimaging agent, an antibody, a cytotoxin, a detectable label and an anti-angiogenic compound.

In a preferred embodiment of the invention, the anti-cancer agent is paclitaxel.

In a preferred embodiment of the invention, the detectable label is selected from the group consisting of a radioactive label, a green fluorescent protein, a histidine tag (histag) protein and β -galactosidase.

In a preferred embodiment of the invention, the agent has a maximum molecular weight of 160,000 daltons.

In a preferred embodiment of the invention, the transport is effected by receptor-mediated transcytosis or adsorptive-mediated transcytosis.

In a preferred embodiment of the invention, the medicament is for the treatment of a neurological disease.

In a preferred embodiment of the invention, the neurological disease is selected from the group consisting of brain tumors, brain metastases, schizophrenia, epilepsy, alzheimer's disease, parkinson's disease, huntington's disease, stroke and blood-brain barrier related dysfunctions.

In a preferred embodiment of the present invention, the blood-brain barrier related malfunction disease is obesity.

In a preferred embodiment of the invention, the transporting results in delivery of the agent to the Central Nervous System (CNS) of the individual.

In a preferred embodiment of the invention, the agent is releasable from the carrier after transport across the blood-brain barrier.

In a preferred embodiment of the invention, the agent is released from the carrier after transport across the blood-brain barrier.

In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition for transporting an agent across the blood-brain barrier, the composition comprising a carrier according to an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for treating a neurological disease, comprising a carrier according to an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for delivery of an agent to the CNS of an individual, the composition comprising a carrier according to an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to another embodiment of the present invention, there is provided a conjugate for transporting an agent across a blood-brain barrier, the conjugate comprising: (a) a carrier; and (b) an agent attached to the carrier, wherein the conjugate is capable of crossing the blood-brain barrier and thereby transporting the agent across the blood-brain barrier.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for transporting an agent across a blood-brain barrier, the composition comprising a conjugate according to an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to an embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of neurological diseases, comprising a conjugate according to an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to another embodiment of the invention, there is provided a pharmaceutical composition for delivery of an agent to the CNS of an individual, the composition comprising a conjugate according to an embodiment of the invention in association with a pharmaceutically acceptable excipient.

In accordance with another embodiment of the present invention, there is provided a use of a carrier for transporting an agent attached thereto across a blood-brain barrier in the manufacture of a medicament for transporting an agent across the blood-brain barrier.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for transporting an agent across a blood-brain barrier, the composition comprising a medicament prepared as defined in an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

In accordance with another embodiment of the present invention, there is provided a use of a carrier for transporting an agent attached thereto across a blood-brain barrier in the manufacture of a medicament for treating a neurological disease in an individual.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of neurological diseases, said composition comprising a medicament prepared as defined in an embodiment of the invention in association with a pharmaceutically acceptable excipient.

In accordance with another embodiment of the present invention, there is provided a use of a carrier for transporting an agent attached thereto across a blood-brain barrier in the manufacture of a medicament for treating a central nervous system disorder in an individual.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of central nervous system disorders, comprising a medicament prepared as defined in an embodiment of the present invention in association with a pharmaceutically acceptable excipient.

According to another embodiment of the present invention, there is provided a conjugate of the formula R-L-M or a pharmaceutically acceptable salt thereof, wherein R is a carrier capable of crossing the blood-brain barrier after attachment to L-M, thereby enabling transport of M across the blood-brain barrier, L is a linker or a chemical bond, and M is selected from the group consisting of drugs, proteins, peptides, enzymes, antibiotics, anti-cancer agents, molecules active at the level of the central nervous system, radioimaging agents, antibodies, cytotoxins, detectable labels and anti-angiogenic compounds.

According to another embodiment of the present invention, there is provided the use of a conjugate of an embodiment of the present invention for transporting an agent attached thereto across a blood-brain barrier.

According to another embodiment of the invention, there is provided a use of a conjugate of an embodiment of the invention for treating a neurological disease in an individual.

According to another embodiment of the invention, there is provided a use of a conjugate according to an embodiment of the invention for treating a central nervous system disorder in an individual.

According to another embodiment of the present invention, there is provided a method of transporting an agent across the blood-brain barrier, the method comprising the step of administering to an individual a pharmaceutical composition according to an embodiment of the present invention.

In a preferred method of the invention, the pharmaceutical composition is administered to the individual intra-arterially, intra-nasally, intra-peritoneally, intravenously, intramuscularly, sub-cutaneously, transdermally or orally.

According to another embodiment of the present invention, there is provided a method of treating a neurological disease in an individual, comprising administering to the individual in need thereof a therapeutically effective amount of a pharmaceutical composition according to an embodiment of the present invention.

According to another embodiment of the present invention, there is provided a method of treating a central nervous system disorder in an individual, comprising administering to the individual in need thereof a therapeutically effective amount of a pharmaceutical composition according to an embodiment of the present invention.

For the purposes of the present invention, the following terms are defined as follows.

The term "carrier" refers to a compound or molecule that is capable of crossing the blood-brain barrier and is capable of being attached to or conjugated with another compound or agent and thereby capable of transporting the other compound or agent across the blood-brain barrier. For example, such a vector may bind to receptors present on brain endothelial cells and thereby be transported across the blood-brain barrier by transcytosis. Preferably, the carrier is a protein or molecule that achieves a high level of transendothelial transport without any effect on the integrity of the blood-brain barrier. The vector may be, but is not limited to, a protein, peptide or peptide mimetic, and may be naturally occurring or obtained by chemical synthesis or recombinant genetic technology (genetic engineering).

The term "carrier-agent conjugate" refers to a conjugate of a carrier and another compound or agent. Conjugation may be chemical in nature, for example with a bond; or of genetic nature, for example by recombinant genetic techniques, such as in the form of fusion proteins with, for example, green fluorescent protein, beta-galactosidase or histidine-tag proteins.

The expression "small molecule drug" refers to a drug having a molecular weight of 1000g/mol or less.

The terms "treat," "treating," and the like, refer to obtaining a desired pharmacological and/or physiological effect, e.g., inhibiting the growth of cancer cells, death of cancer cells, or amelioration of a neurological disease or condition. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or therapeutic in terms of complete or partial cure for the disease and/or adverse effects resulting from the disease. "treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, including: (a) preventing the occurrence of a disease or condition (e.g., preventing cancer) in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease (e.g., arresting disease progression); or, (c) alleviating the disease (e.g., alleviating symptoms associated with the disease). As used herein, "treatment" encompasses any administration of a drug substance or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a condition in the individual, including but not limited to the administration of a carrier-agent conjugate to the individual.

The term "cancer" refers to any cellular malignancy, which is characterized exclusively by loss of normal control (resulting in unregulated growth), lack of differentiation, and the ability to invade local tissues and migrate. Cancer can occur in any tissue of any organ. More particularly, cancer is meant to include, but is not limited to, cancer of the brain.

The terms "administration" and "administering" refer to a means of delivery, which includes, but is not limited to, intra-arterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, or oral. Oral administration is preferred. The daily dose may be divided into one, two or more doses of a suitable form for administration in one, two or more doses over the entire period of time.

The term "therapeutically effective" refers to an amount of a compound sufficient to substantially ameliorate some symptom associated with a disease or medical condition. For example, in the treatment of cancer or a psychiatric condition or neurological or CNS disease, an agent or compound that reduces, prevents, retards, inhibits or arrests the symptoms of any disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition, but will provide treatment to a disease or condition so as to delay, block, prevent the onset of, or alleviate symptoms of, a disease or condition, or alter the duration of a disease or condition or, for example, be less severe, or accelerate recovery in an individual.

The carriers and carrier-agent conjugates of the invention can be used in conjunction with conventional methods of treatment and/or therapy, or can be used separately from conventional methods of treatment and/or therapy.

When the carrier-agent conjugates of the present invention are used in combination with therapy with other agents, they may be administered to the individual sequentially or simultaneously. Alternatively, as described herein, the pharmaceutical compositions of the present invention may be comprised of a combination of a carrier-agent conjugate of the present invention in combination with a pharmaceutically acceptable excipient and another therapeutic or prophylactic agent known in the art.

It will be understood that the particular "effective amount" for any particular individual will depend upon a variety of factors including: the activity of the particular agent employed, the age, body weight, general health, sex, and/or diet of the individual, the time of administration, the route of administration, the rate of excretion, drug combination, and the severity of the particular disease undergoing prophylaxis or treatment.

Pharmaceutically acceptable acid addition salts may be prepared by methods well known in the art.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents (such as phosphate buffered saline buffers, water, saline), dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional vehicle or agent in the therapeutic compositions is contemplated except insofar as it is incompatible with the active ingredient. Additional active ingredients may also be incorporated into the composition.

The term "functional derivative" refers to a "chemical derivative", "fragment" or "variant" biologically active sequence or portion of a carrier or agent or carrier-agent conjugate of the invention or a salt thereof. The carrier functional derivative is capable of being attached to or conjugated to another compound or agent and crossing the blood-brain barrier, thereby enabling transport of the other compound or agent across the blood-brain barrier.

The term "chemical derivative" refers to a carrier, agent, or carrier-agent conjugate of the invention that contains an additional chemical moiety that is not part of the carrier, agent, or carrier-agent conjugate. Covalent modifications are also included within the scope of the invention. Chemical derivatives may be conveniently prepared by direct chemical synthesis using methods well known in the art. Such modifications can be, for example, introduced into a protein or peptide carrier, agent, or carrier-agent conjugate by reacting the target amino acid residue with an organic derivatizing agent capable of reacting with a selected side chain or terminal residue. The carrier chemical derivative is capable of crossing the blood-brain barrier and is capable of being attached to or conjugated with another compound or agent and thereby transporting the other compound or agent across the blood-brain barrier. In a preferred embodiment, very high levels of transendothelial transport across the blood-brain barrier are obtained without any effect on the integrity of the blood-brain barrier.

The term "fragment" refers to any fragment or portion of a carrier, agent, or carrier-agent conjugate. Fragments of a protein or peptide, for example, may be a subset of amino acids that make up the entire protein or peptide. The carrier fragment is capable of being attached to or conjugated to another compound or agent and crossing the blood-brain barrier, and thereby capable of transporting the other compound or agent across the blood-brain barrier.

The term "variant" refers to a carrier, agent, or carrier-agent conjugate that is substantially similar to any of the structures of the carrier, agent, or carrier-agent conjugate of the invention, or any fragment thereof. The carrier variant is capable of being attached to or conjugated to another compound or agent and crossing the blood-brain barrier, thereby transporting the other compound or agent across the blood-brain barrier. Variant proteins, peptides, peptidomimetics, and chemical structures of the vectors of the invention are contemplated.

The term "aprotinin fragment" refers to the portion of aprotinin that still transports a compound across the BBB. Such fragments may contain at least 12 amino acids, preferably at least 25 amino acids, more preferably at least 35 amino acids. Studies to determine The minimum sequence of effective aprotinin that interacts with megalin were performed in The mammalian low-sensitivity protein receptor family (No. rev. nutr.1999,19, 141-172). For example, the minimum sequence of the interaction of aprotinin with the megalin receptor was identified as CRAKRNNFKSA (SEQ ID NO: 1). Thus, it is meant that fragments containing the minimal sequence are also included in the term.

The term "agent" refers indiscriminately to a drug or compound, such as a therapeutic agent or compound, a label, a tracer or an imaging compound.

The term "therapeutic agent" or "agent" refers to an agent and/or drug for treating the symptoms of a disease, physical or mental condition, trauma, or infection, including but not limited to antibiotics, anti-cancer agents, anti-angiogenic drugs, and molecules active at the central nervous system level, such as paclitaxel, that can be administered intravenously to treat brain cancer.

The term "patient" or "treated individual" refers to any patient or individual receiving a medical treatment, including the administration of a carrier-agent or compound conjugate for the purpose of detecting, tracking, labeling or visualizing a condition, such as a tumor. Preferably, the patient or subject to be treated is a mammal, more preferably a human.

The term "condition" refers to any condition that causes pain, discomfort, nausea, illness or disability (mental or physical) to or in an individual, including neurological disease, injury, infection, or chronic or acute pain. Neurological diseases that can be treated with the present invention include, but are not limited to, brain tumors, brain metastases, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease and stroke.

Detailed description of the preferred embodiments

The present invention relates to novel vectors for delivery of agents, drugs or other molecules to the brain and/or Central Nervous System (CNS). The carrier allows passage of agents, drugs or other molecules which are linked or coupled (conjugated) to the carrier and which themselves are unable to cross the blood-brain barrier, to be transported across the blood-brain barrier. The carrier-conjugate can be a carrier-therapeutic agent conjugate. Such conjugates can be in the form of a composition, such as a pharmaceutical composition, for treating a disease or condition. The present invention is based on the discovery that aprotinin binds to and crosses the endothelial wall of brain capillaries in a very effective manner. Aprotinin is known in the art as an alkaline polypeptide that effectively inhibits various serine proteases including trypsin, chymotrypsin, kallikrein and pepsin. The transendothelial transport of aprotinin is about 10-50 times higher than that of other proteins including transferrin or ceruloplasmin. This high rate of passage is not caused by disruption of the integrity of the blood brain barrier, since the permeability coefficient of sucrose is not affected by aprotinin.

This approach is versatile because it allows conjugation of small as well as large molecules with a very wide variety of therapeutic targets.

According to the present invention, a method of transporting an agent across the blood-brain barrier comprises administering to an individual an agent comprising an active ingredient or drug substance linked to a carrier, such as aprotinin or a functional derivative thereof.

According to the present invention, the compounds can be administered to a patient intra-arterially, intra-nasally, intra-peritoneally, intravenously, intramuscularly, sub-cutaneously, transdermally or orally. The agent is preferably an anti-angiogenic drug. The agent may have a maximum molecular weight of 160,000 daltons. Preferably, the agent is a label or drug, such as a small molecule drug, protein, peptide or enzyme. The medicament is preferably suitable for treating a neurological disease or a central nervous system disorder in a patient. The drug may be a cytotoxic drug and the label may be a detectable label, such as a radioactive label, green fluorescent protein, histidine-tagged protein or beta-galactosidase. The agent is preferably delivered into the central nervous system of the patient.

According to yet another preferred embodiment of the present invention, the use, method, compound, medicament or medicament of the present invention does not alter the integrity of the blood-brain barrier of a patient.

According to a further preferred embodiment of the invention, aprotinin may be linked to an agent or compound for transporting the agent or compound across the blood-brain barrier of a patient, said agent or compound being suitable for the treatment of a neurological disease or for the treatment of a central nervous system disorder.

The carrier of the invention or a functional derivative thereof or a mixture thereof may be linked to or labelled with a detectable label such as a radioimaging agent, such as those emitting radiation, for detecting a disease or condition, for example, by using a radioimaging agent-antibody-carrier conjugate in which the antibody binds to a disease or condition specific antigen. Other binding molecules besides antibodies are also contemplated by the present invention, as are known and used in the art. Alternatively, the vectors of the invention or functional derivatives thereof or mixtures thereof may be linked to a therapeutic agent to treat a disease or condition, or may be linked to or labelled with a mixture thereof. Treatment is accomplished by administering one of the carrier-agent conjugates of the invention to an individual under conditions that permit transport of the agent across the blood-brain barrier.

The therapeutic agent of the present invention may be a drug, radiation-emitting substance, cytotoxin (e.g., chemotherapeutic agent) and/or biologically active fragments thereof, and/or mixtures thereof that allows for cell killing, or it may be an agent that treats, cures, alleviates, ameliorates, reduces or inhibits a disease or condition in the treated individual. The therapeutic agent may be a synthetic product or a product of fungal, bacterial or other microorganism such as mycoplasma, viruses, etc., animal, such as reptile, or plant origin. The therapeutic agents and/or biologically active fragments thereof may be enzymatically active substances and/or fragments thereof, or may act by inhibiting or blocking important and/or essential cellular pathways, or by competing with important and/or essential naturally occurring components.

Radiation-emitting radioimaging agents (detectable radiolabels) useful in the invention are exemplified by indium-111, technetium-99, or low dose iodine-131.

Detectable labels for use in the present invention may be radiolabels, fluorescent labels, nuclear magnetic resonance active labels, luminescent labels, chromophore labels, positron emitting isotopes for PET scanners, chemiluminescent labels or enzyme labels. Fluorescent labels include, but are not limited to, Green Fluorescent Protein (GFP), fluorescein, and rhodamine. Chemiluminescent labels include, but are not limited to, luciferase and beta-galactosidase. Enzyme labels include, but are not limited to, peroxidase and phosphatase. The histidine tag may also be a detectable label.

It is contemplated that the agent may be released from the carrier after crossing the blood-brain barrier, for example by enzymatic cleavage or breaking of a chemical bond between the carrier and the agent. Subsequent release of the agent may exert its intended ability in the absence of the carrier.

Brief description of the drawings

FIG. 1 is a graph showing the results of transcytosis assays of aprotinin (●), p97 (. diamond-solid.), and ceruloplasmin (■) across Bovine Brain Capillary Endothelial Cells (BBCECs);

FIG. 2 is a summary of results showing the transcytosis assay of aprotinin (●) and transferrin (o) across Bovine Brain Capillary Endothelial Cells (BBCECs);

FIG. 3 is a bar graph illustrating that the transcytosis capacity of aprotinin is higher than transferrin in the blood brain barrier model;

FIG. 4 is an SDS-PAGE analysis demonstrating that aprotinin integrity is not affected by its transcytosis across the BBECE monolayer;

FIG. 5 is [ 2 ] expressed as a function of time¹⁴C]Graph of sucrose clearance. (ii) measuring clearance of sucrose with and without 250nM aprotinin;

FIG. 6 is a graph showing the results of sucrose permeability tests for Bovine Brain Capillary Endothelial Cells (BBCECs).

FIG. 7 is [ 2 ] expressed as a function of time¹⁴C]-a plot of sucrose clearance, demonstrating that aprotinin does not affect blood brain barrier integrity. (ii) determining clearance of sucrose with and without 5 μ M aprotinin;

FIG. 8 is an explanatory view of [ 2 ]¹²⁵I]Bars of accumulation of aprotinin in human and rat capillaries.

FIG. 9 is a graph illustrating the time course of aprotinin uptake in human and rat capillaries.

FIG. 10 is a bar graph illustrating that aprotinin-biotin conjugate and aprotinin have the same transcytosis capacity;

FIG. 11 is a graph illustrating that aprotinin and aprotinin-biotin conjugate transcytosis is temperature-dependent and conformation-dependent;

FIGS. 12A and 12B are a set of graphs illustrating the effect of temperature and heat on (A) aprotinin and (B) aprotinin-biotin conjugate transcytosis in BBCEC cells;

FIG. 13 is a bar graph illustrating the increase in streptavidin transcytosis in the presence of aprotinin-biotin conjugate;

FIG. 14 is a bar graph illustrating LRP antagonist, receptor-related protein (RAP), inhibits aprotinin transcytosis;

FIG. 15 is a bar graph illustrating aprotinin uptake in an in situ brain perfusion assay;

FIG. 16 illustrates a synthetic aprotinin sequence;

FIG. 17 illustrates a sequence comparison between aprotinin and three human proteins with similar domains;

FIG. 18 is a bar graph illustrating in situ brain perfusion for transferrin, aprotinin and angiopep 1;

FIG. 19 is a transcytosis diagram illustrating the comparison of angiopeptin 1 with aprotinin; and

fig. 20 is a transcytosis diagram of angiopep 1 across an in vitro blood brain barrier model.

Detailed Description

The invention will be more readily understood by reference to the following examples, which are given to illustrate the invention, but not to limit the scope thereof.

Test section

Determination of suitable vectors

Reproducible blood brain barrier in vitro models demonstrating in vivo characteristics have been used for screening assays and mechanistic studies of drug delivery to the brain. This effective blood-brain barrier in vitro model is prepared from CELLIAL^TMDeveloped by Technologies, which is most important for reliably assessing the ability of different vectors to reach the brain. The model consisted of a co-culture of bovine brain capillary endothelial cells and rat glial cells. It represents the ultrastructural features of the brain endothelium including tight junctions, lack of membrane pores, lack of transendothelial channels, low permeability to hydrophilic molecules and high electrical resistance. Furthermore, the model shows a good correlation coefficient between in vitro and in vivo analysis of a wide range of tested molecules. All data obtained so far show that this BBB model reproduces a certain data The complexity of the cellular environment present in vivo closely mimics the in vivo situation while retaining the experimental advantages associated with tissue culture. Thus, many studies have demonstrated that the cell co-culture is one of the most reproducible in vitro models of the BBB.

The in vitro model of BBB was established by using co-cultures of BBCECs and astrocytes. The upper side of the plate insert (Millicell-PC 3.0. mu.M; diameter 30-mm) was coated with rat tail collagen prior to cell culture. They were then placed in six-well microtitre plates containing astrocytes and the BBCECs placed on top of the filter in 2mL of co-culture medium. This BBCEC medium was changed three times per week. Under these conditions, after 7 days, the differentiated BBCECs formed a confluent monolayer. The test was performed between day 5 and 7 after reaching confluence. The permeability coefficient of sucrose was determined to verify the permeability of the endothelium.

Primary cultures of mixed astrocytes were prepared from neonatal rat cerebral cortex (Dehouck M.P., Meress S., Delorme P., Fruchart J.C., Cecchelli, R.an Easier, reproduction, and Mass-Production Method to StudyBlood-Brain Barrier In vitro, J.neurochem,54, 1798-. Briefly, after removal of the meninges, brain tissue was gently passed through an 82 μm nylon mesh. Astrocytes were plated at 1.2X10 in 2mL optimal Medium (DMEM) ⁵cells/mL concentration were placed on six-well microplates, the medium was supplemented with 10% heat-inactivated fetal bovine serum. Medium was changed twice a week.

Bovine Brain Capillary Endothelial Cells (BBCECs) were obtained from Cellial Technologies. Cells were cultured in the presence of DMEM medium supplemented with 10% (v/v) horse serum and 10% heat-inactivated calf serum, 2mM glutamine, 50. mu.g/mL gentamicin and 1ng/mL basic fibroblast growth factor, added every other day.

To determine suitable vectors of the invention, tests were performed using the BBB in vitro model. As shown in FIG. 1, different proteins (aprotinin (●), p97 (. diamond-solid.) and blood were performedCeruloplasmin (■)) passed through bovine brain capillary endothelial cells (transcytosis assays for BBCECs). FIGS. 2 and 3 show the results of a transcytosis assay performed with aprotinin (●) and transferrin (o) and using the same method as in the assay of FIG. 1. One insert covered by BBCECs was placed in a six-well microplate with 2mL Ringer-Hepes and preincubated at 37 ℃ for 2 hours. Will 2¹²⁵I]Aprotinin, (-) enzyme¹²⁵I]-p97、[¹²⁵I]-ceruloplasmin or [ solution ]¹²⁵I]Transferrin (final concentration 250nM) was added to the upper side of the filter covered by cells. At a different time, the insert is transferred to another hole to avoid the possibility of [ 2 ] ¹²⁵I]Protein is re-endocytosed by the proximal (basal) side of the BBECEs. At the end of the test, by the action of TCA precipitation¹²⁵I]Proteins were tested in the lower chamber of a 500 μ L well. The results obtained show that aprotinin has a higher transcytosis capacity than transferrin, p97 or ceruloplasmin in the blood-brain barrier model.

Aprotinin, p97 and bovine holo-transferrin were applied using iodine-beads (obtained from Sigma)^TM) Iodination was performed using standard procedures. Bovine all-transferrin was diluted in 0.1M phosphate buffer pH6.5 (PB). P97 from Synapse Technologies neutralized citrate at pH7.0 was dialyzed against this PB. Two beads of iodine were used for each protein. These beads are available in Whatman^TMThe filter was washed twice with 3mL PB and resuspended in 60 μ L PB. Will be obtained from Amersham-Pharmacia Biotech¹²⁵I (1mCi) was added to the suspension of beads at room temperature for 5 minutes. Iodination of each protein was initiated by the addition of 100. mu.g (80-100. mu.L). After incubation at room temperature for 10 min, the supernatant was applied to a desalting column pre-loaded with 5mL of Sephadex (from Pierce) and eluted with 10mL of PBS¹²⁵I-protein. Fractions of 0.5mL were collected and radioactivity was determined in each 5 μ L fraction. Mixing and blending ¹²⁵The corresponding fraction of protein I was dialyzed against Ringer-Hepes pH 7.4. The efficiency of radiolabelling is between 0.6 and 1X 10⁸cpm/100. mu.g protein.

From FIGS. 1-3, it is clear that the transcytosis capacity of aprotinin is much higher than that of the other test proteins. The data of FIGS. 1-3 are summarized in Table 1, where comparisons were made for different proteins.

TABLE 1

¹²⁵Comparison of the transcytosis of I-protein (250nM) across the BBCEC monolayer

Table 2 summarizes another experiment in which different proteins were added for comparison.

TABLE 2

Efficiency of aprotinin crossing the blood brain barrier

In view of tables 1 and 2, it can be seen that, with respect to aprotinin, superior transendothelial transport was obtained compared to the other test proteins, and that aprotinin transcytosis was about 10 to 50-fold higher than these other proteins.

Its transcytosis across the BBCEC monolayer does not affect aprotinin integrity

The final concentration of the protein is 250nM¹²⁵I]Proteins (0.5-1.5. mu. Ci/assay) were added on the top side of the filters, with or without BBCEC cells in 6-well plates. At each time point, the filter was placed into the next well of the 6-well plate. At the end of the experiment, aliquots were taken from each well and subjected to SDS-PAGE. The gels were then assayed by autoradiography. The results, shown in figure 4, show that its transcytosis across the BBCEC monolayer does not affect aprotinin integrity.

Aprotinin does not affect blood brain barrier integrity

By measuring the value of "BBB" in the model¹⁴C]Permeability of sucrose on BBCEC monolayers grown on filters (in the presence of astrocytes) and a further experiment was performed to determine the effect of aprotinin on BBB integrity at 250nM, to achieve this, brain endothelial cell monolayers grown on inserts were transferred to Ringer-Hepes 6-well plates (basolateral compartment) containing 2mL per well and held at 37 ℃ for 2 hours. Ringer-Hepes solution was prepared from 150mM NaCl, 5.2mM KCl, 2.2mM Cal₂、0.2mM MgCl₂、6mM NaHCO₃5mM Hepes, 2.8mM Hepes, pH 7.4. In each apical chamber, the medium is replaced with 1mL of a solution containing the tag [ 2 ]¹⁴C]Ringer-Hepes of sucrose. At a different time, the insert was placed in another well. The assay was performed at 37 ℃ on filters without cells (□) or with BBCEC cells coated, without 5. mu.M aprotinin (. DELTA.) or with 5. mu.M aprotinin (. smallcircle.) (FIG. 6)¹⁴C]And (3) passing the sucrose. The results obtained are plotted as sucrose clearance (μ l) as a function of time (min). The sucrose permeability coefficient is then determined. The permeability coefficient (Pe) is calculated as follows:

1)

wherein: [C] a = near tracer trace concentration

VA is the volume of the proximal cavity

[C] L = luminal tracer concentration

2)1/Pe = (1/PSt-1/PSf)/area of filter (4.2 cm)²)

At the end of the test, the amount of radiotracer in the compartment outside the substrate was determined in a liquid scintillation counter. As previously described, permeability coefficient (Pe) of sucrose was calculated using EC-coated or non-EC-coated filters (dehauck, m., p., Jolliet-Riant, p., Bree, f., Fruchart, j.c., Cecchelli, r.,tillemen, J.P., J.neurohem.58: 1790-. The results of the two tests are respectively based on¹⁴C]Sucrose (. mu.L) was plotted as a function of time (min) (FIGS. 5 and 6). In fig. 5 and 6, PSt represents the permeability x surface area of the filter of the co-culture and PSf represents the permeability of the filter coated with collagen and astrocytes placed on the bottom side of filter B. The permeability coefficient (Pe) was calculated, which confirmed that the integrity of BBB was not affected by aprotinin (Pe in fig. 6 was calculated from fig. 5, and Pe in table 3 was calculated from fig. 7).

TABLE 3

The permeability coefficient of aprotinin proves that the aprotinin does not influence the integrity of the blood brain barrier

[ ¹²⁵ I]Accumulation of aprotinin in human and rat capillaries

Accumulation was measured at 37 ℃ for 1 hour. The incubation medium contained a Ringer/Hepes solution of aprotinin at a final concentration of 100 nM. Accumulation was stopped by adding ice cold stop solution and passing through a 0.45 μ M filter in vacuo. Nonspecific binding of aprotinin to the capillary surface was assessed by adding an ice-cold solution prior to addition of the incubation medium. This value is subtracted from the accumulation value to obtain a true accumulation value. The results of this test are shown in fig. 8.

Time course of aprotinin uptake in human and rat capillaries

The uptake of aprotinin was determined for variable times at 37 ℃. The incubation medium contained a Ringer/Hepes solution of aprotinin at a final concentration of 100 nM. At each time point, the solution was stopped by adding ice cold and stopped from accumulating under vacuum through a 0.45 μ M filter. At each time point, nonspecific binding of aprotinin to the capillary surface was assessed by adding an ice-cold solution prior to addition of the incubation medium. The results of this test are shown in fig. 9.

Aprotinin-biotin conjugate: biotinylation step

The water-soluble biotin analogue Sulfo-NHS-LC-biotin (Pierce) was used for conjugation. The analog is reacted with a primary amine under conditions that are free of organic solvents and at a neutral pH. To a 10mg/ml aprotinin solution was added a 12-fold excess of biotin analogue. The biotin analogue and aprotinin mixture was incubated at 4 ℃ for 2 hours. Unreacted biotin agent was removed and dialysis was performed overnight in slide-a-lyzer dialysis cassette (Pierce) with a 3500Da cut-off value. The binding of biotin was then determined using the dye HABA (2- (4' -hydroxyazobenzene) -benzoic acid) which binds to avidin, which absorbs at 500 nm. This binding can be replaced by either free biotin or biotinylated protein, allowing quantification of biotin binding. The conjugate was obtained in a ratio of three biotins per aprotinin.

Aprotinin-biotin conjugate and aprotinin have the same transcytosis capacity

Evaluation at 37 ℃¹²⁵I]Aprotinin and¹²⁵I]-aprotinin-biotinylation. The final concentration of the protein is 250nM¹²⁵I]Protein (0.5-1.5. mu. Ci/assay) was added to the upper side of the cell-covered filter for transcytosis assays. At the end of the test, the [ 2 ] is determined directly by TCA precipitation¹²⁵I]-transcytosis of protein cells. The results of this experiment are shown in figure 10.

Aprotinin and aprotinin-biotin conjugate transcytosis is temperature-dependent and conformation-dependent

The assessment [ 2 ] at 37 ℃ and 4 ℃ or at 37 ℃ after boiling the protein at 100 ℃ for 10 minutes¹²⁵I]Aprotinin and¹²⁵I]accumulation of aprotinin-biotin. The final concentration of the protein is 250nM¹²⁵I]-eggsWhite light (0.5-1.5. mu. Ci/assay) was added to the upper side of the cell-covered filter for transcytosis assays. At the end of the test, the cell-coated filter is cut off and the value of [ 2 ] is determined directly by TCA precipitation¹²⁵I]-accumulation of protein cells. The results of this experiment are shown in fig. 11.

Effect of temperature and Heat on the transcytosis of aprotinin and aprotinin-biotin conjugates in BBCEC cells

The assessment [ 2 ] at 37 ℃ and 4 ℃ or at 37 ℃ after boiling the protein at 100 ℃ for 10 minutes¹²⁵I]Aprotinin (FIG. 12A) and [ 2 ] ¹²⁵I]-transcytosis of aprotinin-biotin (fig. 12B). The final concentration of the protein is 250nM¹²⁵I]Protein (0.5-1.5. mu. Ci/assay) was added to the upper side of the cell-covered filter for transcytosis assays. At each time point, the filter was transferred to the next well in a 6-well plate. At the end of the test, the [ 2 ] is evaluated by TCA precipitation in a porous lower compartment¹²⁵I]-a protein.

Increase of streptavidin transcytosis in the presence of aprotinin-biotin conjugate

Evaluation alone or in the presence of an aprotinin-biotin conjugate¹²⁵I]-transcytosis of streptavidin. The final concentration of the protein is 250nM¹²⁵I]Protein (0.5-1.5. mu. Ci/assay) was added to the upper side of the cell-covered filter for transcytosis assays. At each time point, the filter was transferred to the next well in a 6-well plate. At the end of the test, the [ 2 ] is evaluated by TCA precipitation in a porous lower compartment¹²⁵I]-a protein. The results of this experiment are shown in fig. 13.

Inhibition of aprotinin transcytosis by LRP antagonists, receptor-related proteins (RAPs)

Protein transcytosis was assessed at 37 ℃. The final concentration of the protein is 250nM¹²⁵I]Aprotinin (0.5-1.5. mu. Ci/assay) was added to the upper side of the cell-covered filter with or without rap. At the end of the experiment, the precipitate was precipitated in a porous lower compartment by TCA Evaluation [ 2 ]¹²⁵I]Aprotinin. The results of this experiment are shown in fig. 14.

Aprotinin uptake in situ mouse brain perfusion

Procedure for surgery

Measured using the in situ brain perfusion method¹²⁵I]Uptake of aprotinin into the capillary luminal side of the mouse brain, which was adapted in our laboratory for studying drug uptake in the mouse brain (Dagenais et al, 2000, J.Cereb.blood Flow Metab.20(2): 381-386). Briefly, mice anesthetized with right common carotid ketamine/seralazine (140/8mg/kg i.p.) were exposed and ligated at the level of the common carotid bifurcation, at the level of the occipital artery beak. The common carotid beak was then cannulated with a polyethylene tube (0.30mm i.d.x0.70mm o.d.) filled with heparin (25U/ml) and mounted on a 26 gauge needle. A solution containing a perfusion fluid (10 nM in Krebs/bicarbonate buffer, pH 7.4)¹²⁵I]Aprotinin, gas filled 95% O₂And 5% CO₂) The syringe(s) was placed in an infusion pump (Harvard pump PHD 2000; Harvard Apparatus) and connected to a catheter. Just before perfusion, the heart is stopped by cutting off the chamber to eliminate the contralateral blood flow contribution. Brain perfusion was carried out for 10 minutes at a flow rate of 2.5 ml/min. After perfusing for 10 min, the brain was again perfused with Ringer/HEPES solution (150 mM NaCl, 5.2mM KCl, 2.2mM Cal) ₂、0.2m MMgCl₂、6mM NaHCO₃5mM HEPES, 2.8mM glucose, pH7.4) was perfused for 30 seconds to wash off the excess [ 2 ], [ solution ]¹²⁵I]Aprotinin. Mice were then sacrificed to terminate perfusion and the right hemisphere was separated on ice before capillary flow reduction was performed (Triguero et al, 1990, J neurohem.54 (6): 1882-8). Aliquots of the homogenate, supernatant, pellet and perfusate were removed and determined by TCA precipitation¹²⁵I]-the content of aprotinin, and the apparent volume of distribution is evaluated.

Determination of BBB transport constant

Briefly, the calculation was performed as previously described by Smith (1996, pharm. Biotechnol.8: 285-. Aprotinin uptake is expressed as volume of distribution (Vd), which is obtained from the following equation:

Vd=Q^* _br/C^* _pf

wherein Q^* _brIs calculated per gram of the right hemisphere¹²⁵I]Amount of aprotinin, C^* _pfIs the labeled tracer concentration measured in the perfusion fluid.

The results of this assay are shown in figure 15, which shows that uptake of aprotinin is higher than transferrin and that conjugation with biotin does not alter the brain uptake of aprotinin.

In view of the results obtained from the tests described above, aprotinin is a promising vector for transporting agents or compounds across the BBB because it has higher transcytosis across the BBCEC monolayer than other proteins and does not alter the integrity of the blood brain barrier. Furthermore, aprotinin is not degraded during transcytosis, and the conjugation of aprotinin to biotin does not affect its transcytosis. Furthermore, aprotinin is a versatile and flexible carrier, since many molecules such as small drug molecules, proteins, peptides and enzymes can be easily linked to aprotinin protein, thereby facilitating their passage across the BBB. It is envisaged that these molecules may be linked to aprotinin via a linker.

It has also been determined that aprotinin has a higher brain volume of distribution than transferrin. It was further established that transcytosis is temperature sensitive and conformation dependent, suggesting that the LDL-R family of receptors, and possibly LRP, are involved in aprotinin transcytosis.

Thus, aprotinin is an effective and efficient vehicle for delivery of agents into the brain through the blood-brain barrier.

Design of peptides as drug carriers for the brain

Using BLAST on the National Center for Biotechnology Information (NCBI) website^TMProgram pairThe N-terminal sequence (MRPDFCLEPPYTGPCVARIIR) (FIG. 16) (SEQ ID NO:2) of aprotinin was subjected to sequence comparison. This sequence comparison resulted in four identified sequences. None of these identified sequences corresponds to a human protein.

The sequence comparison of the C-terminal sequence (GLCQTFVYGGCRAKRNNFKSAE) (FIG. 16) (SEQ ID NO:3) of aprotinin was also performed at the NCBI website. This sequence comparison produced 27 identified sequences, some of which correspond to human proteins. The highest scoring sequence was then aligned with the aprotinin sequence (FIG. 17). From the alignment, the following angio-peptide 1 peptides were generated: TFFYGGCRGKRNNFKTEEY (net charge +2) (SEQ ID NO: 4).

In situ brain perfusion of transferrin, aprotinin and angiopep 1

Measure [ 2 ]¹²⁵I]-transferrin [ alpha ], [ alpha ]¹²⁵I]Aprotinin and¹²⁵I]brain apparent distribution volume of angiopep 1. The mouse brain was perfused for 10 min. Brain capillary drainage was performed to assess apparent volume distribution in brain parenchyma. The results of this experiment are shown in fig. 18.

Transcytosis of angiopep 1 in contrast to aprotinin

Comparison of the transcytosis of angiopep 1 with aprotinin. As described above, the determination [ 2 ]¹²⁵I]Angiopep 1 and [ [ alpha ] ]¹²⁵I]Transport of aprotinin from apical-to-basolateral side of the endothelial cell monolayer. The final concentration of angiopeptin 1 and aprotinin used in this experiment was 2.5. mu.M. The results of this experiment are shown in fig. 19.

Transcytosis of angiopep 1 across an in vitro blood brain barrier model

The apical-to-basolateral transport of angiopep 1 from the insert covered or not covered with an endothelial monolayer was determined. The results are expressed as clearance of angiopep 1 as a function of time. The slope corresponds to the permeation of the peptide alone through the filter (Psf) and the total permeability of the endothelial cell monolayer (Pst). Permeability of angiopep 1Coefficient (Pe) of 1.2x10^-3cm/min. The results of this experiment are shown in figure 20.

Permeability coefficients for angiopep 1, aprotinin, leptin, and transferrin were determined using an in vitro blood brain barrier model. The permeability coefficient (Pe) is calculated as described above. A comparison of permeability coefficients is shown in table 4.

TABLE 4

Permeability coefficients for angiopep 1, aprotinin, leptin and transferrin

The above experiments show that the brain penetration of angiopeptin 1 is higher than that of aprotinin and transferrin. The experiment also showed that the transcytosis of angiopep 1 was higher than other peptides, including aprotinin, leptin, and transferrin, as determined using the in vitro blood brain barrier model.

While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the certain features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (19)

1. A vector selected from the group consisting of angiopep 1 of sequence TFFYGGCRGKRNNFKTEEY, and functional derivatives thereof.

2. The vector of claim 1, wherein the functional derivative comprises a fragment of the peptide.

3. The vector of claim 1, wherein the fragment of the peptide is a subset of amino acids of the peptide.

4. The carrier according to any one of claims 1 to 3, wherein the brain penetration or transcytosis of the carrier across the blood-brain barrier is greater than the brain penetration or transcytosis of aprotinin and transferrin.

5. The vector of any one of claims 1 to 4, wherein the vector comprises a domain that binds to megalin.

6. The vector of claim 1, wherein the vector is angiopep 1.

7. A conjugate, comprising:

(a) the vector of any one of claims 1 to 6; and

(b) an agent linked to the carrier, wherein the conjugate is capable of crossing the blood-brain barrier.

8. The conjugate of claim 7, wherein the agent has a maximum molecular weight of 160,000 daltons.

9. The conjugate of claim 8, wherein the agent is a small molecule drug having a molecular weight of 1000g/mol or less.

10. The conjugate of claim 7, wherein the agent is selected from the group consisting of a drug, a protein, a peptide, an enzyme, an antibiotic, an anti-cancer agent, a molecule active at the central nervous system level, a radioimaging agent, an antibody, a cytotoxin, a detectable label, and an anti-angiogenic compound.

11. The conjugate of claim 10, wherein the agent is an anti-cancer agent.

12. The conjugate of claim 11, wherein the anti-cancer agent is paclitaxel.

13. The conjugate of claim 10, wherein the agent is an antibody.

14. The conjugate of any one of claims 7 to 13, wherein transport of the conjugate across the blood-brain barrier does not affect the integrity of the blood-brain barrier.

15. A pharmaceutical composition comprising a conjugate according to any one of claims 7 to 14, together with a pharmaceutically acceptable excipient.

16. The pharmaceutical composition of claim 15, wherein the composition is administered intra-arterially, intra-nasally, intra-peritoneally, intravenously, intramuscularly, sub-cutaneously, transdermally or orally.

17. Use of a conjugate or composition according to any one of claims 7 to 16 in the manufacture of a medicament for the treatment of a neurological disease in a patient.

18. The use of claim 17, wherein the neurological disease is selected from the group consisting of a brain tumor, a brain metastasis, schizophrenia, epilepsy, alzheimer's disease, parkinson's disease, huntington's disease, stroke, and blood-brain barrier related malfunction disease.

19. The use of claim 18, wherein the neurological disease is a brain tumor or brain metastasis.