PL2360258T3

Aprotinin polypeptides for transporting a compound across the blood-brain barrier

Abstract

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Term

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Projected expiry passed 21 July 2025, 1.2 years ago.

1 claim: 1 independent, 0 dependent

1
Patent Claim Zastrzeżenie patentowe 1. TFFYGGSRGKRNNFKTEEYC peptide. 1. Peptyd TFFYGGSRGKRNNFKTEEYC. Prepared and verified Sporządziła i zweryfikowała Mirosława Ważyńska Patent attorney Mirosława Ważyńska Rzecznik patentowy Fig. 1 Fig. 1 - 46 Ο "· - 46 Ο "· IZ IZ - 49 Cross-linking agent * BS - 49 Czynnik sieciujący * BS 40:1 ratio Stosunek 40:1 Vector (Aprotinin or angiopep) + - B, labeled alleys and °dem-123 (IgG, Fab, Fab '2) Wektor (Aprotynina lub angiopep) +- B,ałka znakowane i°dem-123 (IgG, Fab, Fab’2) BS addition 3 (in excess) hours, 4 ° C Dodanie BS 3 (w nadmiarze) godz., 4°C AND I Dializa - bufor Ringera Odłączenie 100 000 daltonów (Da) Dialysis - Ringer buffer Disconnection of 100,000 daltons (Da) Fig. 5 Cross-linking agent: sulfo-EMCS Fig. 5 Czynnik sieciujący: sulfo-EMCS IgG IgG AND I Exposure of free SH groups, partial IgG reduction with low DTT concentration Odsłonięcie wolnych grup SH częściowa redukcja IgG niskim stężeniem DTT Gel filtration column p Żelowa kolumna filtracyjna s Aprotinin-EMCS + SH-IgG (4: 1) hours, 4 ° C Aprotynina-EMCS + SH-lgG (4:1) godz., 4°C Dializa: odłączenie 100 000 daltonów (Da) Dialysis: disconnection of 100,000 daltons (Da) AND I Iodination of conjugate Jodynacja koniugatu Fig. 6 Fig. 6 - 51 Increased brain entry of IgG-aprotinin conjugates ("= *> {" = 3) - 51 Zwiększone wnikanie do mózgu koniugatów IgG-aprotynina ("=*> {„=3) Ratios: IgG / IgG-aprotinin (BS3) = 17.4 Stosunki: IgG/lgG-aprotynina (BS3) = 17,4 IgG / IgG-aprotinin (S-EMCS) = 16 IgG/lgG-aprotynina (S-EMCS) = 16 · Fig. 7 Fig. 7 Charge +: lysine (K), arginine (R) Ładunek + :lizyna (K), arginina (R) Charge -: glutamic acid (E), aspartic acid (D) Ładunek -: kwas glutaminowy (E), kwas asparaginowy (D) Fig. 9 Fig. 9

523 paragraphs in 11 sections, as filed

[0001] The invention relates to improvements in the field of drug delivery, in particular the invention relates to a peptide.

BACKGROUND OF THE INVENTION [0002] The main obstacle to the potential use of drugs for the treatment of central nervous system disorders arising in the development of new therapies in the treatment of brain pathology is the blood-brain barrier (BBB). In 1998, the global central nervous system (CNS) drug market brought $ 33 billion, which is only half the profit from selling cardiovascular drugs, despite the fact that in the United States alone, twice as many people suffer from CNS disorders than for cardiovascular disease. This is due to the fact that over 98% of all potential CNS drugs do not cross the blood-brain barrier. Furthermore, more than 99% of the world's laboratories developing CNS drugs focus only on discovering new CNS drugs, while less than 1% on their delivery. This relationship may be the reason for the current lack of effective treatment for most neurological diseases such as brain tumors, Alzheimer's disease and stroke.

[0003] There are two barrier systems that protect the brain from toxic substances: the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). The BBB barrier is considered to be the main pathway for the capture of serum ligands, as its surface is approximately 5000 times larger than the surface of the BCSFB barrier. The BBB barrier endothelium of the brain's blood vessels is a major obstacle to the use of potential drugs used to treat numerous CNS disorders.

[0004] As a general rule, only lipophilic particles smaller than 400 Daltons can pass through the BBB barrier, i.e. from the blood to the brain. However, the particle sizes of many drugs that give promising results in animal studies devoted to the treatment of CNS disorders are much larger. Thus, peptide and protein therapeutic agents are excluded from transport from the blood to the brain because they are not allowed by the minimal permeability of the endothelial wall of the brain capillaries. Brain capillary endothelial cells (BCECs) are tightly sealed with sealed connections, have few holes and few endocytic vesicles compared to capillaries of other organs. BCEC cells

- 2 surrounds the extracellular matrix, astrocytes, pericytes and microglia cells. The close association of endothelial cells with processes in astrocyte spikes and the capillary membrane is important in the development and preservation of BBB barrier properties that allow close control of blood exchange.

[0005] International publication WO2004 / 060403 describes the invention associated with molecules intended for the transport of drugs across the blood-brain barrier. The document describes angio-pep 1 (TFFYGGCRGKRNNFKTEEY) used in transporting compounds across the blood-brain barrier. Said peptide is a humanized peptide obtained from the C-terminus of bovine aprotinin peptide and achieves better results in crossing the blood-brain barrier than aprotinin. In addition, no effective method of delivering drugs to the brain has yet been developed. Among the studied methods of delivering peptide and protein drugs to the brain, three main strategies can be distinguished. The first of these is invasive methods, which include direct intraventricular administration of the drug by surgery and temporary disturbance of the BBB barrier by intraventricular infusion of hyperosmolar solutions (with elevated osmotic pressure). The second strategy, based on pharmacology, is to facilitate the passage through the BBB barrier by increasing the solubility of peptides and proteins in fat. The third strategy, based on physiology, uses various BBB barrier carrier mechanisms that have been described in recent years. In the latter method, the drugs are attached to a protein carrier that acts on the BBB barrier like a receptor-directed carrier. This method is extremely specific, it brings great efficiency and exceptional flexibility in clinical indications, without any restrictions on therapeutic goals. The latter method has been and still is studied by the inventors who developed the molecules described in the above-mentioned publication, as well as by the inventors.

[0006] US 5,807,980 describes the bovine pancreatic inhibitor trypsin (aprotinin) derivative inhibitors, methods of their preparation and their therapeutic use. These peptides find use in the treatment of a condition characterized by an abnormal appearance or abnormal amount of tissue factor and / or factor VIIIa, such as in thrombosis.

[0007] US 5,780,265 describes serine protease inhibitors capable of inhibiting plasma kallikrein.

[0007] US 5,118,668 describes variants of the bovine pancreatic trypsin inhibitor.

[0009] It would be extremely beneficial to obtain molecules acting as carriers or vectors transporting compounds or drugs across the patient's BBB barrier.

SUMMARY OF THE INVENTION [0010] One of the objectives of the invention is to improve the field of drug delivery.

[0011] Another object of the present invention is to provide a non-invasive and flexible carrier for transporting compounds or drugs across the patient's blood-brain barrier.

The invention relates to a peptide with the sequence TFFYGGSRGKRNNFKTEEYC. .

The document shows new molecules capable of, e.g., transporting the indicated compounds across the blood-brain barrier.

[0012] A biologically active polypeptide is disclosed that may be capable of passing (ie, crossing) a layer of mimicking cells (which mimics) the blood-brain barrier in mammals in an in vitro assay, wherein the polypeptide may be selected from, for example, from the group of:

- aprotinin (SEQ ID NO .: 98),

- aprotinin analogue,

- a fragment of aprotinin which may contain the amino acid sequence specified (or may consist mainly of the amino acid sequence specified) as SEQ ID NO .: 1,

- biologically active analog SEQ ID NO .: 1,

- a biologically active fragment of SEQ ID NO .: 1, and;

- biologically active fragment of SEQ ID NO .: 1.

[0013] A biologically active polypeptide is disclosed that can pass (i.e., exceeds) a cell layer mimicking (which mimics) a blood-brain barrier in a mammalian in vitro assay, wherein the polypeptide can be selected from, e.g.

- an aprotinin fragment that may contain the amino acid sequence set out in SEQ ID NO .: 1,

- biologically active analog SEQ ID NO .: 1,

- a biologically active fragment of SEQ ID NO .: 1, and;

- biologically active fragment of SEQ ID NO .: 1.

[0014] It is disclosed that the aprotinin fragment may consist of the sequence set out in SEQ ID NO .: 1. In addition, according to the invention, the aprotinin fragment may comprise SEQ ID NO .: 1 and may be from about 19 amino acids to about 54 amino acids, e.g. 10 to 50 amino acids in length, 10 to 30 amino acids in length, etc.

[0015] It has been disclosed that the biologically active analog of the SEQ ID NO .: 1 sequence may be from about 19 amino acids to about 54 amino acids in length (e.g. containing 21 to 23, 25 to 34, 36 to 50 and 52 to 54) or from about 19 amino acids to about 50 amino acids, or from about 19 amino acids to about 34 amino acids (e.g. 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 ) or from 19 amino acids to about 23 amino acids or about 19, 20, 21, 22, 23, 24, 35, 51 amino acids.

[0016] The biologically active polypeptide fragment (e.g., about 19 amino acids) described herein may include, for example, a polypeptide of about 7, 8, 9 or 10-18 amino acids. Therefore, the biological active fragment of SEQ ID NO .: 1 or the SEQ analogue

ID NO .: 1 may be from about 7 to about 18 amino acids in length or from about 10 to about 18 amino acids.

[0017] US 5,807,980 describes a polypeptide which is referred to as SEQ ID NO .: 102.

[0018] US Patent 5,780,265 describes a polypeptide which is referred to as SEQ ID NO .: 103.

[0019] The amino acid sequence of aprotinin (SEQ ID NO .: 98), the amino acid sequence of angiopep-1 (SEQ ID NO.:67), as well as some biologically active analog sequences can be found, for example, in international publication PCT / CA2004 / 000011, published July 22, 2004 as international publication WO2004 / 060403. Additionally, international publication No. WO04 / 060403 describes the polypeptide identified in this work as SEQ ID NO .: 104.

[0020] US 5,118,668 describes polypeptides having the sequence set forth in SEQ ID NO: 105.

[0021] Examples of aprotinin analogues can be found by performing a protein Blast (Gene Bank: <a href="http://www.ncbi.nlm.nih.gov/">www.ncbi.nlm.nih.gov/</a> BLAST /) for the synthetic aprotinin sequence (or part thereof) disclosed in PCT / CA2004 / 000011. Exemplary aprotinin analogues can be found, for example, under catalog numbers CAA37967 (GI: 58005), 1405218C (GI: 3604747) etc.

[0022] Next, a biologically active polypeptide is disclosed that has the ability to pass (i.e. cross) a cell layer that mimics (which mimics) the blood-brain barrier in a mammalian in vitro assay, which polypeptide can be selected e.g. from the group:

- a fragment of aprotinin from 19 to 54 amino acids in length (e.g. 19-50), which may include SEQ ID NO .: 1.

- a biologically active fragment consisting of SEQ ID NO .: 1,

- the biologically active SEQ ID NO .: 1 analogue of about 19 to 50 amino acids in length, and the biologically active SEQ ID NO .: 1 fragment (10-8 amino acids) or the biologically active fragment of SEQ ID NO .: 1 (about 10-18 amino acids).

[0023] Next, the biologically active analog SEQ ID NO .: 1 is disclosed, which can be selected e.g. from the group consisting of:

- an SEQ ID NO .: 1 analogue that may have at least 35% identity with the amino acid sequence of SEQ ID NO .: 1.,

- an SEQ ID NO .: 1 analogue that may have at least 40% identity with the amino acid sequence of SEQ ID NO .: 1.,

- 5 - of the SEQ ID NO .: 1 analogue, which may have at least 50% identity with the amino acid sequence of SEQ ID NO .: 1.,

- an SEQ ID NO .: 1 analogue that may have at least 60% identity with the amino acid sequence of SEQ ID NO .: 1.,

- an SEQ ID NO .: 1 analogue that may have at least 70% identity with the amino acid sequence of SEQ ID NO .: 1.,

- an SEQ ID NO .: 1 analogue that may have at least 80% identity with the amino acid sequence of SEQ ID NO .: 1.,

- an SEQ ID NO .: 1 analogue that may have at least 90% identity with the amino acid sequence of SEQ ID NO .: 1., and

- an SEQ ID NO .: 1 analogue that may have at least 95% (i.e. 96%, 97%, 98%, 99% and 100%) identity with the amino acid sequence of SEQ ID NO .: 1.

[0024] For example, the biologically active analog of the sequence SEQ ID NO .: 1 may include an amino acid sequence selected from the group consisting of the amino acid sequence defined in any one of SEQ ID NO.:2 to SEQ ID NO.:62, SEQ ID NO .:

to SEQ ID NO .: 93 and SEQ ID NO .: 97, as well as 99, 100 and 101. If the polypeptide comprises, for example, SEQ ID NO.:99, 100 or 101, it may have an amino acid sequence from about 10 up to 50 amino acids, e.g., from 10 to 30 amino acids in length.

[0025] Next, it was disclosed that the biologically active analog of SEQ ID NO .: 1 may include the amino acid sequence specified in SEQ ID NO .: 67 (i.e., polypeptide No. 67, which is the amidated version of SEQ ID NO .: 67 (angiopep-1)) .

[0026] The disclosed polypeptides may be amidated, i.e. they may have an amidated amino acid sequence. For example, polypeptides with the sequence SEQ ID NO .: 67 may be amidated (polypeptide No. 67).

[0027] The disclosed molecule is a biologically active polypeptide that may have the ability to pass (i.e. exceed) the cell mimicking (which mimics) blood-brain barrier in mammals in an in vitro assay, the polypeptide may be selected from, e.g.

- an aprotinin fragment of 19 to 54 amino acids in length (e.g. 19-50), which may include SEQ ID NO .: 1.

- a biologically active fragment consisting of SEQ ID NO .: 1,

- a biologically active analog of the SEQ ID NO .: 1 sequence from about 19 to 50 amino acids in length, provided that the analog does not include the SEQ ID NO .: sequence numbers 102, 103, 104 and 105 and provided that that the analog consists of the sequence SEQ ID NO .: 67, this analogue is amidated,

- a biologically active fragment of the sequence SEQ ID NO .: 1 with 10-18 amino acids and;

- a biologically active fragment of SEQ ID NO .: 1 with approximately 10-18 amino acids.

In addition, the biologically active fragment of the SEQ ID NO .: 1 sequence or the biologically active fragment of the SEQ ID NO .: 1 sequence analog may contain at least 9 or 10 (consecutive or adjacent) amino acids of the sequence SEQ ID NO .: 1 or the sequence analogue SEQ ID NO .: 1.

[0029] The disclosed polypeptides may have an amino acid sequence which may contain between 1 and 12 amino acid substitutions (i.e. SEQ ID NO .: 91). For example, amino acid substitution may range from 1 to 10 amino acid substitutions or 1 to 5 amino acid substitutions.

[0030] The amino acid substitution may be a non-conservative or conservative substitution.

[0031] For example, if the disclosed polypeptide contains amino acids that are identical to those found in SEQ ID NO .: 1 and other amino acids that are not identical (non-identical), then the non-identical may be a conservative amino acid substitution. Comparing identical and non-identical amino acids can be done by looking at analogous sites.

[0032] Examples of the SEQ ID NO .: 1 sequence analogue that may have at least 35% identity include, for example, polypeptides comprising (composed of) the amino acid sequence specified in SEQ ID NO .: 91 (about 36.8% identity, i.e. 7 out of 19 amino acids of SEQ ID NO .: 91 are identical to SEQ ID NO .: 1), a polypeptide containing (consisting of) the amino acid sequence specified in SEQ ID NO .: 98 (about 68.4% identity, i.e. 13 of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), a polypeptide containing (composed of) the amino acid sequence specified in SEQ ID NO .: 67 (about 73.7% identity, i.e. 14 of the 19 amino acids are identical to the sequence of SEQ ID NO .: 1), a polypeptide containing (composed of) the amino acid sequence set out in SEQ ID NO .: 76 (about 73.7% identity, i.e. 14 of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), and a polypeptide containing (composed of) the amino acid sequence of SEQ ID NO .: 5 (about 79% identity, i.e. 15 of the 19 amino acids are identical to the sequence of SEQ ID NO .: 1).

[0033] Examples of the SEQ ID NO .: 1 sequence analog that may have at least 60% identity include, for example, a polypeptide comprising (composed of) the amino acid sequence specified in SEQ ID NO .: 98 (about 68.4% identity, i.e. 13 amino acids of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), a polypeptide containing (consisting of) the amino acid sequence specified in SEQ ID NO .: 67 (about 73.7% identity, i.e. 14 of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), a polypeptide containing (composed of) the amino acid sequence of SEQ ID NO .: 76 (about 73.7% identity, i.e. 14 of the 19 amino acids are identical to the sequence of SEQ ID NO .: 1) and a polypeptide containing (composed of) the amino acid sequence set out in SEQ ID NO: 5 (about 79% identity, i.e. 15 amino acids out of 19 amino acids are identical to the sequence SEQ ID NO .: 1).

[0034] Examples of the SEQ ID NO .: 1 sequence analogue that may have at least 70% identity include, for example, a polypeptide having (composed of) the sequence of the amine additions specified in SEQ ID NO .: 67 (about 73.7% identity , i.e. 14 of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), SEQ ID NO .: 76 (about 73.7 identities, i.e. 14 of 19 amino acids are identical to the sequence of SEQ ID NO .: 1), SEQ ID NO .: 5 (approximately 79%, i.e. 15 amino acids from 19 amino acids identical to the sequence of SEQ ID NO .: 1).

[0035] Accordingly, it has been disclosed that the carrier may in particular be selected from the group consisting of peptide Nos. 5, 67, 76, 91 and peptide 97 (i.e. SEQ ID NOs: 5, 67, 76, 91 and 97 (angiopep-2)). This carrier can be used, for example, to transport the attached agent across the blood-brain barrier. The carrier may be able to cross the blood-brain barrier after the agent is attached to it, and therefore it may be able to carry this factor across the blood-brain barrier.

[0036] According to the invention, the peptide may be in isolated or substantially purified form.

[0037] More specifically, the invention provides a carrier for transferring an agent associated thereto through the blood-brain barrier, wherein the carrier may be able to cross the blood-brain barrier after the agent is attached thereto and thereby transfer the agent across the blood-brain barrier.

[0038] The carrier is selected from the peptide of the invention.

[0039] Carrier transfer activity does not affect the blood brain barrier integrity. For example, the result of a factor transfer may be its delivery to the patient's central nervous system (CNS).

[0040] It is to be understood that the peptide of the invention may be obtained by chemical synthesis (e.g. solid phase synthesis) or produced by recombinant DNA technology. Codons encoding specific amino acids are well known to those skilled in the art and are discussed, for example, in Biochemistry (third edition; 1988, Lubert Stryer, Stanford University, WH Freeman and Company, New York). Therefore, a nucleotide sequence encoding a carrier of the invention is disclosed herein.

[0041] Next disclosed is a conjugate which may be a carrier consisting of a peptide of the Invention and an agent selected from the group consisting of, for example, a drug (e.g. a small drug molecule, e.g. an antibiotic), a drug, a detectable label, a protein (e.g. an enzyme) , a protein-based compound (e.g., a single chain or multiple peptide chain protein complex) and a polypeptide (peptide). More particularly, the agent may be an active molecule at the central nervous system level. The agent may be any agent intended for the treatment or detection of neurological diseases.

[0042] It is disclosed that the carrier, as part of the conjugate, can be selected, for example, from the group:

- an aprotinin fragment of 10 to 54 amino acids in length (e.g., 19-50), which may include SEQ ID NO .: 1.

• the aprotinin fragment consisting of SEQ ID NO. : 1 • a biologically active analog of the SEQ ID NO .: 1 sequence (e.g., from about 19 to 50 amino acids in length) provided that if the analog consists of the SEQ ID NO .: 67 sequence, this analogue is amidated , • biologically active fragment of the sequence SEQ ID NO .: 1 with a length of from

- 18 amino acids, and;

• biologically active fragment of SEQ ID NO .: 1 with approximately 10-18 amino acids.

[0043] According to the invention, the maximum molecular weight of the agent may be about 180,000 daltons.

[0044] In addition, receptor-induced transcytosis or adsorption-induced transcytosis may affect transport activity. The factor can be transported by such a mechanism.

[0045] Furthermore, the conjugate may be in the form of a fusion protein, the first part of which may essentially consist of the carrier of the invention and the second part may consist essentially of a protein or protein-based agent.

[0046] Exemplary neurological diseases that can be treated or detected with a carrier and / or conjugate may be selected from the group consisting of, e.g., brain tumor, brain metastasis, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke and malfunction associated with the blood-brain barrier (e.g. obesity).

[0047] It has been disclosed that obesity associated with the blood-brain barrier is obesity. Furthermore, it has been disclosed that leptin is a factor that can be conjugated to a carrier of the invention. The leptin conjugate and carrier can be used, e.g., in the treatment of obesity.

[0048] At the same time, it has been disclosed that the detectable label may be a radio imaging agent. Examples of labels that can be conjugated to a carrier of the invention include, for example, an isotope, fluorescent label (e.g., rhodamine), reporter molecule (e.g., biotin), etc. Other examples of detectable labels include, e.g., green fluorescent protein, biotin, His protein -Tag and β-galactosidase.

[0049] Examples of the protein or protein-based compound that can be conjugated to a carrier of the invention and are described herein include an antibody, an antibody fragment, (e.g., an antibody binding fragment, such as an Fv, F (ab) 2, F fragment (ab) 2 'and Fab and the like), a peptide or protein based drug (e.g., positive pharmacological modulator (agonist) or pharmacological inhibitor (antagonist)), etc. Other examples of factors described herein include cellular toxins (e.g.

- 9 monomethyl derivative of auristatin E (MMAE)), endotoxins and bacterial exotoxins; diphtheria toxins, botulinum toxin, tetanus toxin, pertussis toxin, staphylococcal enterotoxins, TSST-1 toxic shock toxin, adenyl cyclase toxin, Shiga toxin, cholera enterotoxin and others) and anti-angiogenic compounds (endostatin, catechin) , matrix metalloproteinase (MMPI) inhibitors, anastelin, vitronectin, antithrombin, tyrosine kinase inhibitors, VEGF inhibitors, anti-receptor antibody, herceptin, avastin, panitumumab and others).

[0050] It has been disclosed that the agent may be a small molecule of a drug such as an anti-cancer drug (e.g. for the treatment of a brain tumor). Anticancer drugs may include, for example, a drug having a group that allows it to attach to the carrier of the invention. Examples of anti-cancer drugs include, for example, drugs that can be selected from the group consisting of paclitaxel (taxol), vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, taxotere, melphalan, chlorambucil and any combination thereof.

[0051] In particular, the conjugate may have the formula RLM or pharmaceutically acceptable salts thereof, wherein R is a class of molecules associated with aprotinin (e.g., aprotinin, aprotinin fragment, Angiopep-1, Angiopep-2, analogs, derivatives or fragments). For example, R may be a carrier selected from the class of aprotinin-related molecules that can cross the blood-brain barrier after attachment to the LM, and thus transfer M through the blood-brain barrier. L may be a linker or a bond (chemical bond). M may be a factor selected from the group consisting of drugs (e.g. small molecule drugs), drugs, (detectable) label, protein or protein-based compound (e.g. antibody, antibody fragment), antibiotic, anti-cancer agent, anti-anglogenic compound and polypeptide or any active molecule at the central nervous system level. According to the description, it should be understood that the RLM formula is not intended to be limited to a particular order or ratio. As illustrated in this paper, M may occur in several proportions relative to R.

[0052] For example, conjugates of formula RLM or their pharmaceutically acceptable salts can be used to transport M across the blood-brain barrier, where R can, for example, be a carrier selected from the group consisting of peptides numbered: 5, 67, 76, 91 and 97, as specified in this work. The carrier may be able to cross the blood-brain barrier when attached to the LM, and thus may transfer M across the blood-brain barrier.

[0053] M may be a helpful factor in the treatment or diagnosis of neurological diseases.

[0054] It should be understood here that if more than one attachment site is available, then more than one drug or drug molecule can be attached to the carrier of the invention. Therefore, such a conjugate may contain one or more drug molecules. The conjugate knives may be active as such, i.e. the drug may be active after attachment to the carrier.

In addition, the compound may or may not be released from the carrier, i.e. generally after passage across the blood-brain barrier. Thus, the compound may be released from the conjugate (or carrier) and become activated after that release. The agent can be released from the carrier after crossing the blood-brain barrier.

[0055] A conjugate is disclosed for transporting agents across the blood-brain barrier, the conjugate may be: (a) a carrier and (b) a factor attached to the carrier, wherein the conjugate is able to cross the blood-brain barrier and thus may pass the agent through this barrier.

[0056] In addition, the use of the carrier of the invention is disclosed for carrying a factor across the blood-brain barrier in mammals in need of treatment.

[0057] Further disclosed is the use of a class of aprotinin-related molecules for transporting compounds attached thereto through the patient's blood-brain barrier.

[0058] In addition, the use of a carrier or conjugate, as defined herein, for diagnosing a neurological disease or central nervous system disease is disclosed. For example, the carrier or conjugate can be used to detect a neurological disease in vivo. The carrier may be selected, for example, from a (biologically active) group:

• aprotinin (SEQ ID NO: 28) • a fragment of aprotinin which may include the sequence of the amine additions specified in SEQ ID NO. : 1 • a fragment of aprotinin consisting of SEQ ID NO. : 1 • the biologically active analog of SEQ ID NO .: 1, and • the biologically active fragment of SEQ ID NO .: 1 or the biologically active fragment of SEQ ID NO .: 1.

[0060] In particular, the carrier may be selected e.g. from the (biologically active) group:

• a fragment of aprotinin which may include the amino acid sequence set out in

SEQ ID NO. : 1 • a fragment of aprotinin consisting of SEQ ID NO. : 1 • the biologically active analog of SEQ ID NO .: 1, and • the biologically active fragment of SEQ ID NO .: 1 or the biologically active fragment of SEQ ID NO .: 1.

Furthermore, it has been disclosed that the carrier can be selected, for example from the group:

• a 10 to 54 amino acid fragment of aprotinin which may include SEQ ID NO .: 1.

• an aprotinin fragment consisting of SEQ ID NO. 1

- a biologically active analogue of the sequence SEQ ID NO .: 1 (e.g. from a length of about 19 to 50 amino additions) provided that if the analog consists of the sequence SEQ ID NO .: 67, amidated, • biologically active fragment of the sequence SEQ ID NO .: 1 with a length of from

- 18 amino acids, and;

• a biologically active fragment of SEQ ID NO .: 1 analogue of about 10-18 amino acids in length.

[0061] The use of a class of molecules associated with aprotinin in the manufacture of a drug is disclosed.

[0062] The use of a class of molecules associated with aprotinin is disclosed in the manufacture of a medicament intended for the treatment of neurological diseases or for the treatment of central nervous system disorders.

[0063] Disclosed is the use of the carrier or conjugate described in this work in the manufacture of a medicament intended for the treatment of brain diseases (brain-related diseases) or neurological diseases, for the diagnosis of brain diseases or neurological diseases, or for transporting agents across the blood-brain barrier.

[0064] Disclosed is the use of a carrier according to the invention in the treatment of mammals suffering for example from neurological diseases or for the diagnosis of neurological diseases in mammals which require it.

[0065] Neurological diseases include, for example, brain tumor, brain metastasis, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke and blood brain barrier dysfunction.

[0066] A method of transferring factors across the blood-brain barrier of a mammal (human, animal) is disclosed, which may comprise the step of administering to the mammal a compound which is an agent attached to the class of aprotinin-related molecules.

[0067] A method of treating a patient's neurological diseases is disclosed, comprising administering to the patient a drug comprising a class of aprotinin-related molecules and a compound adapted to treat those diseases, wherein the compound is attached to a class of aprotinin-related molecules.

[0068] Disclosed is a method of treating a patient's central nervous system disorder, comprising administering to the patient a drug comprising a class of aprotinin-related molecules and a compound adapted to treat these diseases, a compound attached to aprotinin.

[0069] In an additional aspect, a method of transmitting an agent across the blood-brain barrier is disclosed, comprising the step of administering to a patient a pharmaceutical composition disclosed herein.

[0070] The invention also discloses a method of treating a mammal (e.g., a patient) in need of drug administration (e.g., a patient with a neurological disease). The method may comprise administering to the mammal a carrier, conjugate and / or pharmaceutical composition as described herein.

[0071] Also disclosed is a method (for) the diagnosis (i.e. diagnostic method) of a neurological disease in a mammal (e.g. a patient) in need of treatment. the method may comprise administering to the mammal (subject, patient, animal) a carrier, conjugate and / or pharmaceutical composition described herein.

[0072] Administration can be by intraarterial, intranasal, intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal or oral methods.

[0073] The pharmaceutical composition may be administered to a mammal in a therapeutically effective amount.

[0074] The mammal in need of treatment (the person in need of treatment) may be, for example, a mammal suffering from or at risk of a neurological disease, central nervous system disease, brain cancer, brain metastasis, etc.

[0075] A pharmaceutical composition is disclosed, which may be, for example:

- a carrier according to the invention; and

a pharmaceutically acceptable carrier, e.g. a pharmaceutically acceptable excipient.

[0076] The pharmaceutical composition may be used, for example, in the treatment of a neurological disease.

[0077] The pharmaceutical composition may be used, for example, in the diagnosis of neurological diseases.

[0078] The pharmaceutical composition may be used, for example, to carry agents across the blood-brain barrier.

[0079] The pharmaceutical composition may be used, for example, to deliver agents to the patient's CNS.

[0080] The pharmaceutical composition may be used, for example, in the treatment of central nervous system disorders in mammals requiring treatment.

[0081] The pharmaceutical composition may be used to deliver an agent to a patient's CNS.

[0082] It is to be understood herein that a pharmaceutically acceptable salt of a carrier (polypeptide) or conjugate is disclosed.

[0083] Thus, the composition (pharmaceutical composition) may therefore include a drug produced according to the present disclosure, associated with a pharmaceutically acceptable excipient.

[0084] For the purposes of the invention, the following terms are specified below.

[0085] The term "carrier" or "vector" is intended to mean a compound or molecule, such as a polypeptide, capable of transporting the compound. Transport can take place, for example, across the blood-brain barrier. The carrier may be combined (covalently or not), or conjugated with another compound or agent, and thereby transfer that compound or agent across the blood-brain barrier. For example, the carrier can bind to receptors present on brain endothelial cells and thus can be transferred by transcytosis across the blood-brain barriers. The carrier may be a molecule for which high levels of endothelial transport can be obtained, without compromising the integrity of the blood-brain barrier. The carrier may or may not be a protein, peptide or peptidomimetic and may be naturally occurring or produced by chemical synthesis or recombinant genetic material (genetic engineering) technology.

[0086] The term "conjugate" is intended to mean association of the carrier with another compound or agent. This coupling may be of a chemical nature, such as in the case of linker (linker) or genetic linking, for example, by genetic recombination, such as in the case of fusion proteins with e.g. a reporter molecule (e.g. green fluorescent protein, β-galactosidase, His -Tag etc.).

[0087] The expression "small molecule drug" is intended to mean a drug with a molecular weight of 1000g / mol or less.

[0088] The terms "treatment", "treat" and the like mean the intended pharmacological and / or physiological effect, e.g., inhibition of tumor cell growth, death of cancer cells, or improvement of health in a neurological disease or neurological disorder. The effect may be prophylactic in terms of preventing the disease or its symptoms completely or partially and / or therapeutic in terms of curing it in full or in part and / or of the adverse effects attributed to the disease. The term "treatment" as used herein refers to any form of treatment for a mammalian disease, particularly in humans, and includes: (a) preventing a disease or condition (e.g. cancer prevention) in a person who may be susceptible to the disease, even if the disease has not yet been diagnosed; (b) inhibiting the disease, (e.g., arresting the development of the disease); or alleviating the disease (e.g., attenuation of symptoms associated with the disease). The term "treatment" as used herein refers to any administration of a pharmaceutical agent or compound to treat, cure, alleviate, weaken or inhibit a disease or improve a person's health, including, without limitation, administering a carrier-agent conjugate to the individual.

[0089] The term "cancer" is intended to mean any cancerous nature of cells that is characterized by a loss of proper control and, as a result, uncontrolled growth, lack of differentiation and the ability to invade adjacent tissues as well as metastases. Cancer can occur in any tissue and organ. Specifically, the cancer includes, without limitation, brain cancer.

[0090] The terms "administration" and "administration" are intended to mean a method of delivering a drug, including without limitation the arterial, intranasal, intraperitoneal,

- 14 Intravenous, intramuscular, subcutaneous, transdermal or oral. The daily dose may be given as a single dose or divided into two or more doses in a form suitable for being administered once, twice or more times over a given period.

[0091] The term "therapeutically effective" or "effective amount" is intended to mean an amount of compound sufficient to significantly improve symptoms associated with the disease or condition. For example, when treating a tumor, mental, neurological or CNS disease, a therapeutically effective agent would be one that reduces, prevents, suppresses or inhibits any symptoms of the disease or condition. A therapeutically effective amount of a factor or compound is not required to cure the disease or condition, but to provide treatment for the disease or condition so as to delay or hinder its onset, prevent the onset of the disease or condition, or alleviate the symptoms or change the duration disease or condition or, e.g., that their course is less severe and to accelerate the patient's recovery.

[0092] The carrier and conjugates may be used in conjunction with conventional methods of treatment and / or therapy or may be used alone.

[0093] If conjugates are administered together with therapies or other agents, they may be administered to the patient sequentially or simultaneously. Optionally, the pharmaceutical compositions may consist of a carrier-agent conjugate combination with a pharmaceutically acceptable excipient as defined and other therapeutic or prophylactic agents known to those skilled in the art.

[0094] Pharmaceutically acceptable acid addition salts may be prepared by methods known and used by those skilled in the art.

[0095] The term "functional derivative" is intended to mean a "chemical derivative," "fragment," or "variation," of a biologically active sequence or portion of a carrier, agent or conjugate, and salts thereof. The functional derivative of the carrier may be able to attach to or bind to another compound or agent and cross the blood-brain barrier so that it may be able to carry another compound or agent across the blood-brain barrier.

[0096] The term "chemical derivative" is intended to mean a carrier, agent or disclosed conjugate that contains additional chemical residues that are not part of the carrier, agent, or carrier-agent conjugate. Covalent modifications are enabled. The chemical derivative may conveniently be prepared by direct chemical synthesis using methods known to those skilled in the art. For example, such modifications can be made to a protein or peptide carrier, carrier or agent-factor conjugate, by carrying out a reaction between target amino acid residues and organic derivatizing agents capable of reacting with selected side chains or terminal chain residues. The chemical derivative of the carrier is able to cross the blood-brain barrier and to attach to or associate with another compound or agent and thereby to carry that compound or agent across the blood-brain barrier. In a preferred embodiment, they are achieved

- very high levels of endothelial transport across the blood-brain barrier without any effect on the blood-brain barrier integrity.

[0097] The term "agent" is intended to mean without distinction an antibody, drug (such as a therapeutic agent) or compound such as a therapeutic agent or compound, marker, label or imaging compound.

[0098] The term "therapeutic agent" or "agent" is intended to mean an agent and / or medication and / or medicament used to treat the symptoms of a disease, mental or physical disorder, injury or infection, and includes antibiotics, anti-cancer agents, anti-angiogenic agents and molecules active at the level of the central nervous system, without being limited to them. For example, paclitaxel may be administered intravenously to treat brain cancer.

[0099] The term "medical condition" is intended to mean any situation that causes pain, discomfort, illness or loss of function (mental or physical) in a person, including neurological diseases, injuries, infections, and acute or chronic pain. Neurological diseases that can be treated according to the invention include, but are not limited to, brain tumors, brain metastases, schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease and stroke.

[0100] The term "pharmaceutical composition" as used herein means therapeutically effective amounts of an agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. The term "therapeutically effective amount" as used refers to that amount that provides a therapeutic effect in a given disease state and in a given drug administration regimen. Such compositions are liquids or lyophilisates, or dry forms of the drug, and they contain diluents with different buffer content (e.g. TrisHCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent surface absorption, detergents (e.g. Tween 20, Tween 80, Pluronic F68, bile salts). Factors affecting solubility (e.g. glycerol, poly (ethyl oxide), antioxidants (e.g. ascorbic acid, sodium bisulphite), preservatives (e.g. thimerosal, benzyl alcohol, parabens), swelling agents or osmotic pressure modifiers (e.g. lactose, mannitol), covalent attachment of polymers such as poly (ethyl oxide) with protein, complexation with metal ions, or incorporation of mass / material in or into molecular preparations of polymer compounds such as polylactic acid, polyglycolic acid, hydrogels etc., or liposomes, microemulsions, micelles, single-lamellar and multi-lamellar carriers, shadows of erythrocytes or spheroplasts. Such compositions affect the physical state, solubility, stability, release rate in vivo and purification rate in vivo. Controlled or sustained release compositions include drug forms in lipophilic depots (e.g., fatty acids, waxes, oils). In addition, polymer-coated molecular compositions (e.g., poloxamers, poloxamines) are disclosed.

[0101] Also disclosed are protective coatings of molecule forms, protease inhibitors and penetration enhancers for various routes of administration, including parenteral, pulmonary, intranasal, oral, vaginal and rectal administration.

[0102] The pharmaceutical compositions may be administered parenterally, extra-neoplastically, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneous, intraperitoneal, intraventricular, intracranial and intratumoral.

[0103] Furthermore, the terms "pharmaceutically acceptable carrier" and "pharmaceutical carrier" are known to those skilled in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer and 0.8% brine. In addition, such pharmaceutically acceptable carriers can be aqueous or anhydrous solutions, suspensions and emulsions. Examples of anhydrous solvents include propylene glycol, polyethylene oxide, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, aqueous / alcoholic solutions, emulsions and suspensions, including brine and buffering agents. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or solid oils. Intravenous vehicles include fluid and nutritive complement, electrolyte complement such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antibacterial agents, antioxidants, consolidating agents, inert gases and the like may also be present.

[0104] The term "analog" should be understood as a polypeptide derived from the original sequence or from part of the original sequence which may contain one or more modifications; for example one or more modifications in the amino acid sequence (e.g., addition, deletion, insertion, substitution, etc. of an amino acid); one or more modifications in the main or side chain of one or more amino acids (side chains or main chain). The term "analog" is therefore understood to mean a molecule with biological activity and chemical structure (or part of a structure) similar to that of the described polypeptide. An analog contains a polypeptide in which, for example, one or more amino acid insertions occur, at one or both ends of the polypeptide and / or within the amino acid sequence of the polypeptide.

[0105] "Analog" may exhibit sequence similarity and / or identity to the original sequence or part thereof, and may have a modification of its structure as mentioned. The degree of similarity between the two sequences is based on the percentage of identity (identical amino acids) and the percentage of conservative substitutions (substitutions).

[0106] Similarity or identity can be compared, for example, in the region of 2, 3, 4, 5, 10, 19, 20 amino acids or more (and any number between the above). Identity may relate to amino acids that are identical to the original peptide and that may occupy the same or similar position as compared to the original polypeptide. An analogue showing 50% identity with the original polypeptide may be

An example is an analogue containing 50% of the amino acid sequence of the original polypeptide, similarly for the remaining percentages. It should be understood that there are gaps between the amino acids of analogs that are identical or similar to the amino acids of the original peptide. These gaps can mean a lack of amino acids, one or more amino acids that are not identical or similar to the original peptide.

[0107] The same percentage can be determined, for example, using the GAP, BESTFIT or FASTA algorithm in Wisconsin Genetics Software Package Release 7.0, using the so-called default gap weights.

[0108] For example, an analogue may have 50% identity with the original amino acid sequence, and a portion of the remaining amino acids in a similar position may, for example, be a non-conservative or conservative amino acid substitution.

[0109] Therefore, the disclosed analogs include those that may have at least 90% sequence similarity to the original sequence or part of the original sequence. For example, an analogue may exhibit at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) of sequence similarity to the original sequence or parts of the original sequence. The analogue may also show, e.g. at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) of sequence similarity to the original sequence with a combination of one or more modification within the main or side amino acid chains, or with the addition of a group or other molecule, etc. Exemplary amino acids that are intended to be similar (conservative amino acids) to others are known to those skilled in the art and include, for example, those listed in Table 1.

[0110] Analogs: the disclosed analog also includes those that exhibit at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) sequence identity to the original sequence or part of the original sequence. The analogue may also exhibit e.g. at least 35%, 50%, 60%, 70%, 80%, 90% or 95% identity (sequence) relative to the original sequence (i.e. an analogue that is at least 35%, 50%, 60%, 70%, 80%, 90% or 95% identical to the original peptide) with a combination of one or more modifications within the main or side amino acid chains, or with addition group or other molecule, etc.

[0111] The term "fragment" should be understood as a polypeptide derived from a portion of the original or parent sequence or an analog of the parent sequence. Fragments include polypeptides with one or more amino acids truncated, where the truncation may be at the amino terminus (N-terminus), carboxy terminus (C-terminus), or inside the protein. The fragment may contain the same sequence as the corresponding part of the original sequence. Disclosed herein are biologically active fragments of the carrier (polypeptide).

[0112] Thus, biologically active polypeptides in the form of original polypeptides, fragments (modified or unmodified), analogues are disclosed herein

- 18 (modified or unmodified), derivatives (modified or unmodified), homologues (modified or unmodified) of the vehicle described herein.

[0113] Therefore, any polypeptide disclosed herein having a modification compared to the original polypeptide whose biological activity has not been significantly disturbed is disclosed. Those skilled in the art know that the polypeptides of the invention can undergo many modifications without adversely affecting their biological function. On the contrary, these modifications may support or increase the biological activity of the original polypeptide or may optimize one or more properties (e.g. polypeptide stability, bioavailability, etc.).

[0114] The disclosed polypeptides include, for example, those containing amino acid sequences modified either by natural processes such as post-translational processing or by chemical modification techniques known to those skilled in the art. Modifications can occur anywhere in the polypeptide, including the core of the polypeptide, in side chains, and at the amino and carboxy termini. It will be beneficial if the same type of modification can occur at the same or different degrees in several places of a given polypeptide. A given polypeptide may contain numerous modifications. Polypeptides may be branched by ubiquitination or cyclic, with or without branching. Cyclic, branched and cyclic branched peptides may arise from natural post-translational processes or may be produced by synthesis. Modifications include, for example and without limitation, pegylation, acetylation, acylation, acetamide methyl addition (Acm), ADPribosylation, alkylation, amidation, biotinylation, carbamylation, carboxyethylation, esterification, covalent flavin attachment, covalent attachment to a heme residue, covalent covalent or a nucleotide derivative, covalent attachment of a drug, covalent attachment of a marker (e.g. fluorescent, radioactive etc.), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent network formation, cystine, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, formation of hydroxylation , iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoization, sulfation, addition of amino acids to proteins mediated by RNA transfer, e.g. arginylation and ubiquitination, etc.

[0115] As discussed above, modification of the polypeptide may include, for example, insertion (i.e., addition), deletion and substitution (i.e., replacement) of an amino acid, conservative or non-conservative (e.g., D-amino acids, non-amino acids) in the polypeptide sequence, where such changes do not significantly affect the overall biological activity of the polypeptide.

[0016] Examples of substitution may be conservative (i.e. where the residue is replaced with another residue of the same general type or from the same group) or non-conservative (i.e. where the amino acid residue is replaced by a different type of amino acid). In addition, a naturally occurring amino acid can be

- a substituted amino acid that is not naturally occurring (i.e., unnatural conservative amino acid substitution or unnatural non-conservative amino acid substitution).

[0117] It is understood that naturally occurring amino acids can be divided into subclasses as acidic, basic, neutral and polar or neutral and non-polar. Furthermore, three of the encoded amino acids are aromatic. It may be helpful that encoded polypeptides that differ from the specific disclosed polypeptide contain substituted codons for amino acids of the same type or from the same group as the amino acids to be replaced. Thus, in some cases, the basic amino acids Lys, Arg and His may be interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln and Asn can be interchangeable; the non-polar aliphatic amino acids Gly, Ala, Val, Ile and Leu are interchangeable, but due to their size, Gly and Ala are more closely related, whereas the aromatic amino acids Phe, Trp and Tyr can be interchangeable.

[0118] It should also be noted that if the polypeptides are synthetically produced, it is also possible to substitute for amino acids that are not naturally encoded by DNA (an unnaturally occurring or unnatural amino acid).

[0019] An unnaturally ascending amine additive is to be understood as an amino acid that is not naturally produced or found in mammals. A non-naturally occurring amino acid is a D-amino acid, an amino acid whose acetylaminomethyl group is attached to a sulfur atom of a cysteine, a pegylated amino acid, etc. The incorporation of a non-naturally occurring amino acid into a specific polypeptide sequence will give rise to a derivative of the original polypeptide. Unnaturally ascending amino acids (residues) also include omega amino acids of the formula NH2 (CH2) nCOOH, where n is 2-6, such as neutral non-polar amino acids, sarcosine, t-butylalanine, t-butylglycine, N-methylisoleucine, norleucine, etc. Phenylglycine can replace Trp, Tyr or Phe; citrulline and methionine sulfoxide are neutral, nonpolar, cysteic acid is acidic and ornithine is basic. Proline can be substituted with hydroxyproline, maintaining conformational properties.

[0120] Those skilled in the art know that analogs can be prepared by substitution mutagenesis and the biological activity of the disclosed polypeptides can be preserved.

[0121] In these analogues, at least one amino acid residue in the protein molecule is removed and another residue is introduced in its place. Table 1 provides examples of substitutions referred to as "conservative substitutions." If such substitution results in a change that is not desired, then other substitution types are introduced, which are set out in the "exemplary substitutions" in Table 1 or described later in the amino acid classes.

[0122] In some cases, it may be of interest to modify the biological activity of a polypeptide by substitution, insertion or deletion of an amino acid. For example, modification of a polypeptide can lead to an increase in its biological activity, it can also modulate its toxicity, change its biological

- availability or stability, or modulate its immunological activity or identity. By choosing substitutions (substitutions) that differ significantly in their effect on the maintenance (a) of the polypeptide core structure in the substitution region, for example as β-cards or helix conformation (b) the charge or hydrophobicity of the target or (c) the extended side chain, significant modifications in the activity or immunological identity. Based on the common properties of the side chain, naturally occurring residues are divided into groups:

(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe ), (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) (3) acid / negatively charged: aspartic acid (Asp), glutamic acid (Glu) (4) basic: asparagine (Asn) , glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg) (5) residues that affect chain orientation: glycine (Gly), proline (Pro);

(6) aromatic: tryptophan (Trp), tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), (7) polar: Ser, Thr, Asn, Gln (8) basic positively charged: Arg, Lys, His, and (9) charged: Asp, Glu, Arg, Lys, His [0123] Non-conservative substitutions require the exchange of a member of one of these classes for another. Conservative substitution requires the exchange of a member of one of these groups for another from those groups. Table 1 lists the remaining possible amino acid substitutions.

Table 1. Amino acid substitutions.

<td>The rest is original</td><td>Example substitution</td><td>Substitution conservative</td>

<td>Cys (C)</td><td>Cheese</td><td>Cheese</td>

<td>How many (i)</td><td>Leu, Val, Met, Ala, Phe, norleucine</td><td>Leu</td>

<td>Leu (L)</td><td>norleucine, Ile, Val, Met, Ala, Phe</td><td>How much</td>

<td>Thr (t)</td><td>Cheese</td><td>Cheese</td>

<td>Val (V)</td><td>Ile, Leu, Met, Phe, Ala, norleucine</td><td>Leu</td>

[0124] The biologically active analog may be, for example, an analog having at least one (i.e. non-conservative or conservative) amino acid substitution in the original sequence. The biologically active analog may also be, for example, an analog having the insertion of one or more amino acids.

[0125] Other exemplary analogs include, for example:

- Analog SEQ ID NO: 1, which may have the formula I X1- SEQ ID NO: 1 -X2

- Analog angiopep-1, which may have formula X1-angiopep-1-X2 and

- An angiopep-2 analogue that may have the formula X1-angiopep-2-X2 [0126] X1 and X2 may independently be an amino acid sequence of 0 to about 100 (e.g., between 0 and 30 to 50) amino acids. X1 and X2 can be obtained from subsequent amino acids of aprotinin or aprotinin analogues (homologous amino acid sequence) or they can be any other amino acid sequence (heterologous amino acid sequence). Compounds of formulas I, II, III may also contain amino acid substitution, deletion or insertion within the amino acid sequence of angiopep-1, angiopep-2 or SEQ ID NO: 1. It is preferred, however, that the analog is biologically active as defined in one of tests described or in other similar or identical tests.

[0127] The biologically active polypeptide (e.g., carrier) can be identified by one of the tests or methods described. For example, the carrier may be made by conventional peptide synthesis, linked to a taxol as shown and

22 were tested in an in vivo model as well as described. The biologically active carrier can be identified, for example, based on its efficacy in increasing the survival of an animal that has been injected with tumor cells and which has been treated with a conjugate compared to a control that did not receive the conjugate. The biologically active carrier can also be identified based on its location in the parenchyma (parenchyma) in in situ cerebral perfusion testing.

BRIEF DESCRIPTION OF THE DRAWINGS [0128]

Fig. 1 shows an example of analysis using tricine gels;

Fig. 2 shows a method of attaching a vector or carrier to paclitaxel;

Fig. 3 shows the effect of treating a glioma model in Lewis rats with pactitaxel attached to aprotinin;

Fig. 4 shows the effect of treating a glioblastoma model in nude strain mice with paclitaxel attached to AgioPep-1;

Fig. 5 shows the protocol used to combine aprotinin with IgG using the agent 3 crosslinking BS;

Fig. 6 shows the protocol used to connect aprotinin to IgG using the sulfo-EMCS crosslinker;

Fig. 7 shows the penetration of IgG-aprotinin conjugates into the brain;

Fig. 9 Shows the effect of taxol-angiopep-2 conjugate treatment on the survival of glioma-implanted mice (nude thymus-bearing mice) and;

Fig. 9 shows the structure of exemplary polypeptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION [0129] The invention relates to a new molecule that can act as vectors or carriers transporting an agent, drug or other molecule to the brain and / or central nervous system (CNS). Factors, drugs and other molecules that are not themselves able to cross the blood-brain barrier can be transferred through it after attachment or attachment (coupling) to a vector or carrier. Also, a factor that is not able to pass through the blood-brain barrier alone can improve its transport when combined with the carrier of the invention. To treat a disease state or disease, such conjugates may take the form of a composition such as a pharmaceutical composition.

Designing particles as vector carriers [0130] In international publication WO 2004/060403, the inventors disclosed that angiopep-1 (SEQ ID NO .: 67) and aprotinin (SEQ ID NO .: 98) are effective transfer vectors of interest molecules across the blood-brain barrier. The inventors have shown that other molecules can also be used as carriers for transporting agents across the blood-brain barrier. In a similar way, potential carriers are peptides having similar domains as in the case of aprotinin and angiopep-1 and modified forms of angiopep-t (amidated, peptide No. 67). These peptides resemble aprotinin and angiopep-1, but contain different amino acid insertions and carry different changes. So far, the 96 peptides listed in Table 2 as well as the additional peptides listed in the sequence listing have been tested for their potential role as carriers.

[0131] It should be understood that the peptides for subsequent experiments were selected based on their higher activity compared to other peptides. Those that have not been selected for further research should not be regarded as non-functional. These peptides have significant activity and are used as (biologically active) carriers.

- Table 2. 96 peptides designed on the basis of aprotinin-like and angiopep-1-like domain with different amino acid charges and insertions

ORGANIZED PEPTIDES AT SYNPEP (California, USA)

<td colspan="2">-1 Characteristic features of proteins</td><td>#Pep</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td> 13</td><td> 14</td><td> 15</td><td> 16</td><td> 17</td><td> 18</td><td> 19</td><td> 20</td><td> 21</td><td> 22</td>

<td colspan="2">Aprot-synth</td><td> 1</td><td>T</td><td>F</td><td>V</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>AND</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>S</td><td>AND</td><td>E</td><td>D</td><td></td><td></td><td></td>

<td colspan="2">Bikunin HI-30</td><td> 2</td><td>T</td><td>F</td><td>Q</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>M</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>V</td><td>T</td><td>E</td><td>K</td><td>E</td><td></td><td></td><td></td>

<td colspan="2">amyloid</td><td> 3</td><td>p</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>G</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>F</td><td>D</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td colspan="2">Kunitz type 1 inhibitor</td><td> 4</td><td> 5</td><td>F</td><td>Y</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>E</td><td>E</td><td>E</td><td></td><td></td><td></td>

<td>peptides</td><td>LOAD (+6)</td><td> 5</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>AND</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>R</td><td>AND</td><td>K</td><td>Y</td><td></td><td></td><td></td>

- 25 II

<td></td><td>HUMAN</td><td> 38</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>G</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 39</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>M</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 40</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>S</td><td>M</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td> —</td><td>HUMAN</td><td> 41</td><td>P</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>Y</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 42</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 43</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>Y</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 50</td><td>P</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>G</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 51</td><td>S</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>M</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 52</td><td>P</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>G</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>L</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 53</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 54</td><td>S</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 55</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>S</td><td>L</td><td>G</td><td>N</td><td>G</td><td>N</td><td>N</td><td>F</td><td>V</td><td>R</td><td>E</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 59</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>F</td><td>L</td><td>R</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 60</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 61</td><td>P</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>G</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>HUMAN</td><td> 62</td><td>P</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>S</td><td>G</td><td>G</td><td>N</td><td>R</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td> —</td>

<td>- aprotinin</td><td>vs M-terminal APROTININ</td><td> 63</td><td>M</td><td>R</td><td>P</td><td>D</td><td>F</td><td>C</td><td>L</td><td>E</td><td>P</td><td>P</td><td>Y</td><td>T</td><td>G</td><td>P</td><td>C</td><td>V</td><td>AND</td><td>R</td><td>AND</td><td></td><td></td><td></td>

<td></td><td>(1 α helix, Aterminal)</td><td> 64</td><td>AND</td><td>R</td><td>AND</td><td>AND</td><td>R</td><td>Y</td><td>F</td><td>Y</td><td>N</td><td>AND</td><td>K</td><td>AND</td><td>G</td><td>L</td><td>C</td><td>Q</td><td>T</td><td>F</td><td>V</td><td>Y</td><td>G</td><td></td>

<td></td><td>(2 β sheets, Yterminal)</td><td> 65</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>AND</td><td>K</td><td>R</td><td>N</td><td>N</td><td>Y</td><td>K</td><td>S</td><td>AND</td><td>E</td><td>D</td><td>C</td><td>M</td><td>R</td><td>T</td><td>C</td><td>G</td>

<td>AngioPep</td><td>AnglaPep-1</td><td> 67</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>AngioPEPI (lysine)</td><td> 68</td><td>K</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>AngioPEP1 (4Y)</td><td> 69</td><td>T</td><td>F</td><td>Y</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>Y</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>cysteine bridge</td><td> 70</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>S</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>N-terminal cysteine</td><td> 71</td><td>C</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>C</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td>

<td></td><td>C-terminal cysteine</td><td> 72</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td>C</td><td></td><td></td>

<td></td><td>N-terminal cysteine</td><td> 73</td><td>C</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>S</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td>

<td></td><td>C-terminal cysteine</td><td> 74</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>S</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td>C</td><td></td><td></td>

<td></td><td>permuted cys (-)</td><td> 84</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>C</td><td>G</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>permuted cys (+)</td><td> 85</td><td>T</td><td>F</td><td>F</td><td>Y</td><td>G</td><td>G</td><td>R</td><td>C</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>Q instead of F</td><td> 87</td><td>T</td><td>F</td><td>Q</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>R</td><td>G</td><td>K</td><td>R</td><td>N</td><td>N</td><td>F</td><td>K</td><td>T</td><td>E</td><td>E</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>blend ANGIOPEP</td><td> 88</td><td>Y</td><td>N</td><td>K</td><td>E</td><td>F</td><td>G</td><td>T</td><td>F</td><td>N</td><td>T</td><td>K</td><td>G</td><td>C</td><td>E</td><td>R</td><td>G</td><td>Y</td><td>R</td><td>F</td><td></td><td></td><td></td>

<td>TFPI</td><td>TFPI (similar domain)</td><td> 89</td><td>R</td><td>F</td><td>K</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>M</td><td>N</td><td>N</td><td>F</td><td>E</td><td>T</td><td>L</td><td>E</td><td>E</td><td></td><td></td><td></td>

<td></td><td>LOAD + 5 (Human)</td><td> 90</td><td>R</td><td>F</td><td>K</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>F</td><td>L</td><td>R</td><td>L</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>LOAD + 5 (Human)</td><td> 91</td><td>R</td><td>F</td><td>K</td><td>Y</td><td>G</td><td>G</td><td>C</td><td>L</td><td>G</td><td>N</td><td>K</td><td>N</td><td>N</td><td>Y</td><td>L</td><td>R</td><td>L</td><td>K</td><td>Y</td><td></td><td></td><td></td>

<td></td><td>TFPI (c-terminal) (2Y)</td><td> 92</td><td>K</td><td>T</td><td>K</td><td>R</td><td>K</td><td>R</td><td>K</td><td>K</td><td>Q</td><td>R</td><td>V</td><td>K</td><td>AND</td><td>AND</td><td>Y</td><td>E</td><td>E</td><td>AND</td><td>F</td><td>K</td><td>N</td><td>Y</td>

<td></td><td>TFPI (c-terminal clipped)</td><td> 93</td><td>K</td><td>T</td><td>K</td><td>R</td><td>K</td><td>R</td><td>K</td><td>K</td><td>Q</td><td>R</td><td>V</td><td>K</td><td>AND</td><td>AND</td><td>Y</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td rowspan="2">peptides alkaline</td><td>SynB1</td><td> 94</td><td>R</td><td>G</td><td>G</td><td>R</td><td>L</td><td>S</td><td>Y</td><td>S</td><td>R</td><td>R</td><td>F</td><td>S</td><td>T</td><td>S</td><td>T</td><td>G</td><td>R</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Penotratin (PAntp43-68)</td><td> 96</td><td>R</td><td>Q</td><td>AND</td><td>K</td><td>AND</td><td>IN</td><td>F</td><td>Q</td><td>N</td><td>R</td><td>R</td><td>M</td><td>K</td><td>IN</td><td>K</td><td>K</td><td></td><td></td><td></td><td></td><td></td><td></td>

In vitro model selection [0132] An in vitro model was used in the screening test and mechanistic studies of drug transport to the brain. An effective in vitro blood brain barrier model was developed by CELLIAL ™ Technologies. The ability of various carriers to reach the brain was assessed using an in vitro reproducible model. This model consists of a co-culture of bovine capillary endothelial cells and glial rat cells. It has ultrastructural features characteristic of the brain's endothelium, including tight junctions, no holes, no endothelial channels, poor permeability to hydrophilic molecules, and high electrical resistance. In addition, this model showed a good correlation coefficient between in vitro and in vivo analysis of a wide range of tested molecules. All data collected to date show that this BBB barrier model accurately mimics the in vivo situation by recreating some of the complexity of the cellular environment existing in vivo, while maintaining the benefits of tissue culture. Many studies have confirmed that this co-cell culture is one of the most reproducible in vitro BBB barrier models.

[0133] An in vivo BBB model was created using co-culture of bovine brain capillary endothelial cells (BBCEC) and astrocytes. Before establishing the culture, the upper side of the cartridges (MillIcell-PC 3.0 gM; 30-mm diameter) were covered with rat tail collagen. The cartridges were then placed in 6-well microplates containing astrocytes, and BBCEC cells were applied to the top of the filters in 2 ml culture medium. BBCEC medium was changed three times a week. Under these conditions, 7 days later, differentiated BBCECs formed a confluent layer on the surface of the medium. The experiments were carried out between 5 and 7 days after the cell layer merged. To verify endothelial permeability, the sucrose permeability coefficient was measured.

[0134] Primary [directly from tissue] mixed cultures of astrocytes were made from the cortex of newly born rats (Dehouck MP, Meresse S., Delorme P., Fruchart JC, Cecchelli, R. An Easier, Reproductible, and Mass-Production Method to Study the Blood-Brain Barrier In Vitro. J. Neurochem, 54, 1798-1801, 1990). Briefly, after removing the meninges, brain tissue was gently forced through a nylon sieve. Astrocytes were placed on 6-well microplates at a concentration of 1.2 x 105 cells / ml in 2 ml optimal medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum. The medium was changed twice a week.

[0135] Bovine brain capillary endothelial cells (BBCEC) were obtained from Cellial Technologies. Cells were cultured in the presence of DMEM medium supplemented with 10% by volume horse serum and 10% heat inactivated calf serum, 2 mM glutamine, 50 gg / ml gentamicin and 1 ng / ml basic fibroblast growth factor, added every other day.

[0136] Initially, at the first level of selection, 96 peptides described in Table 2 were tested as a vehicle in the in vitro BBB barrier model. Each peptide was applied to the top of cartridges coated or uncoated with endothelial cells for 30 minutes at 37 ° C. After incubation, the peptides located in the lower part of the chamber were broken down by electrophoresis. Electrophoresis gels were stained with Coomassie blue to visualize the peptides as shown (without limitation) in Fig 1. Angiopep-1 (or SEQ ID NO .: 67 or peptide No. 67 (amidated form)) is often used as a reference or in comparative purposes. In Fig 1, each first peptide applied to the upper side of the filters was applied to an electrophoretic gel (ini) as a control. After 90 minutes of transcytosis, a volume of 50 gl was also applied to the trinid gels from the side-basal side of the filters coated with endothelial cells (+) or uncoated endothelial cells (-). To visualize the peptides, gels were stained with Coomassie blue.

[0137] After the first screening step, peptides detected with Coomassie blue staining in the lower part of the chambers (5, 8, 45, 67, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 90 and 91) were selected for further study with iodinated peptides. Briefly, selected peptides were iodized by standard procedures using the iodo-beads reagent from Sigma. There were two iodo-beads for each protein. Iodo-beads were washed twice with 3 ml phosphate buffer (PB) on Whatman ™ filter and re-suspended in 60 g PB. Iodo-beads are added to the suspension25I (1 mCi) from AmershamPharmacia biotech, for 5 minutes and at room temperature. Iodination of each peptide was initiated by the addition of 100 gg (80-100 gg) suspension. After incubation at room temperature for 10 minutes, the supernatants were applied to a desalting column previously coated with 5 ml of Pierce cross-linked dextran ™ and proteins125And 10 ml of PBS were eluted. Fractions of 0.5 ml were removed and radioactivity measured in 5 g of each fraction. Fractions corresponding to proteins125They were pooled and dialyzed in Ringer / Hepes buffer at pH 7.4. The radiolabeling efficiency was between 0.6-1.0x108 cpm / 100gg protein.

[0138] Iodized peptides were also tested using an in vitro BBB model. Each peptide was added to the upper surface of plates coated or uncoated with endothelial cells for 90 minutes and at 37 ° C. After the incubation period, peptides from the lower part of the chamber were precipitated using TCA. Results are expressed as the ratio of the number of cells per milliliter (cpm). For each peptide [125I] the number of cells per milliliter in the lower part of the chamber was divided by the total number of cells per milliliter added to the filter covered with endothelial cells (+ cells / initial) or uncovered (-cells / initial). The ratio between the amount of peptide was also calculated [125I] occurring in the lower part of the chamber of filters coated and not covered with endothelial cells (+ cells / cells). The very low -cells / initial ratio indicates that the filters may interact with the peptides (peptide 5 and 8). A high ratio + cells / initial and + cells / -cells indicates better passage of peptides through brain endothelial cells. Table 3 shows the results of previously selected 18 peptides.

- 31 Table 3

Peptide screening results after the second screening level

<td rowspan="2"># Peptides</td><td colspan="3">relations</td>

<td>cells / Initial</td><td>+ Cells / initial</td><td>+ Cells / -komórki</td>

<td>aprotinin</td><td> 0.260</td><td> 0.022</td><td> 0.08</td>

[0139] From the above results, 12 peptides were selected with ratios + cells / -cells generally higher than 0.35, namely: 5, 8, 67, 75, 76, 77, 78, 79, 81, 82, 90 and 91. Peptides # 91 and # 77 were also selected for further studies because of their + cell / cell ratio (> 0.2).

[0140] 12 selected peptides were tested for their permeability coefficients using an in vitro BBB barrier model. Impact of each selected peptide at

- 32 250 nM on BBB barrier integrity was determined by measuring sucrose permeability [14C] in the BBB model on BBCEC cell layers cultured on filters in the presence of astrocytes. To this end, layers of brain endothelial cells grown on cartridges were transferred to 6-well plates containing 2 ml of Ringer-Hepes buffer per hole (lateral basal compartment) for 2 hours at 37 ° C. The Ringer-Hepes solution was composed of 150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2, 6 mM NaHCO3, 5 mM Hepes, pH 7.4. In each apical chamber, the culture medium was replaced with 1 ml of Ringer-Hepes solution containing labeled sucrose [14C]. At different times, the cartridges were placed in subsequent wells. Transition of sucrose [14C] was measured at 37 ° C, on filters without cells or on filters coated with BBCEC cells. Peptides were added at the beginning of the experiment, in zero time. Results are presented as sucrose purification (gl) as a function of time (min).

Purification fun = fCIA x VA [C] L [C] A = concentration of abluminal tracer

VA = volume of the abluminal chamber [C] L = concentration of the luminal tracer [0141] The slope of the linear variation (gl / min) is the sucrose permeability coefficient for the cellless filter (Psf) and for the filter with BBCEC (PSt) in the presence of peptide. [0142] The permeability coefficient Pe was calculated as:

1 / Pe = (1 / PSt - 1 / PSf) / Filter surface (4.2 cm 2) [0143] Peptides with the highest permeability coefficient (Pe) were selected: 67, 76, 90, 91.5.79, 8, and 78.

[0144] In situ cerebral perfusion (in mice) was used as the fourth selection level to isolate the best peptides. This method also makes it possible to distinguish between compounds remaining in the blood vessels of the brain from those that crossed the abluminal endothelium to penetrate the brain parenchyma. Moreover, the method of post-perfusion capillary depletion allows you to assess whether the molecule actually crossed the endothelium and penetrated into the brain parenchyma. The use of this method showed that certain peptides generally accumulate in the brain parenchyma (see Table 4).

- 33 Table 4

<td rowspan="3">peptides</td><td colspan="5">Distribution volume (perfusion 5 min)</td>

<td>The homogenate</td><td colspan="2">Capillaries (capillaries)</td><td colspan="2">Flesh (parenchyma)</td>

[0145] Four peptides, namely 5, 67, 76 and 91 showed the highest level of distribution in the parenchyma, reaching a volume above 20 ml / 100g, which is at least 25% of the volume for the entire brain (homogenate), thus showing the highest potential as carriers used as transporting vectors. Peptide 79 was eliminated due to the low volume of distribution in the brain parenchyma (18 ml / 100g). Peptide 67 represents the amidated form of angiopep-1 described in the previous application of the inventors. Peptide amidation affects the overall peptide load. As can be seen in Tables 2 and 3, two peptides with different charge do not necessarily show the same activity.

[0146] The vector or carrier of the present invention may therefore be used in a method of transferring agents across the blood-brain barrier and includes administering to the patient an agent containing the active ingredient or a pharmaceutical agent attached to the peptide of the invention.

[0147] The carrier or conjugate may be administered to the patient intra-arterially, intranasally, intraperitoneally, intravenously, intramuscularly, subcutaneously, transdermally or orally. For example, the agent may be an anti-angiogenic compound. The maximum mass of the medium can be 160,000 daltons. As discussed herein, the agent may be a marker or a drug, such as a small molecule drug, protein, peptide, or enzyme. Such a drug may be adapted, for example, for the treatment of a patient's neurological diseases or central nervous system disorders. Such a drug may be a cytotoxic drug and the marker may be a detectable marker such as a radioactive marker, a green fluorescent protein,

- 34 His-Tag protein or β-galactosidase. Such a factor can be delivered, for example, to the patient's central nervous system.

[0148] The uses, methods, compounds, agents, drugs or medications mentioned herein do not affect the integrity of the patient's blood-brain barrier.

[0149] According to the invention, the peptide is TFFYGGSRGKRNNFKTEEYC.

[0150] The carrier of the invention may be attached to or labeled with a detectable label, such as a radio-imaging agent, e.g., emitting radiation, to detect a disease or disease state, for example using an agent-antibody-carrier-conjugate radio-imaging, in which the antibody binds to the antigen specific to a given disease or condition. In addition to antibodies, other binding molecules that are known to and used by those skilled in the art. Optionally, the carrier of the invention may be attached to a therapeutic / therapeutic agent for treating a disease or condition, or attached to or provided with mixtures thereof. By administering a carrier-agent conjugate to a patient under conditions that allow the agent to transport across the blood-brain barrier, treatment can be influenced.

[0151] The therapeutic agent used herein may be a drug, medicine, radiation-emitting agent, cellular toxin (e.g., a chemotherapeutic agent) and / or a biologically active fragment thereof and / or mixtures thereof, to allow the destruction of cells, or may be an agent for treatment , curing, weakening or inhibiting the disease or condition of the patient being treated, or for improving the patient's condition. The therapeutic agent may be a synthetic product or a product derived from fungi, bacteria, or other microorganisms such as mycoplasma, virus etc., from an animal such as reptile or it may be of plant origin. The therapeutic agent and / or its biologically active fragment may be an enzymatically active agent and / or a fragment thereof, or may act by inhibiting or blocking an important and / or key cell pathway, or by competing with an important and / or key natural cellular component.

[0152] Ind-111, TechNet-99 or a low dose of iodine-131 are examples of suitable radio-imaging radiation emitting factors (detectable radiotracers).

[0153] The detectable markers or markers may be radiolabels, fluorescent markers, active nuclear magnetic resonance spectroscopy markers, luminescent markers, chromophore markers, positron emitting isotopes, PET chemotuminescent and enzymatic markers used in PET scanning. Fluorescent labels include green fluorescent protein (GFP), fluorescein and rhodamine. Chemiluminescent labels include, but are not limited to, luciferase and βgalactosidase. Enzyme labels include peroxidase and phosphatase. His-Tag can also be a detectable tag.

[0154] It is contemplated that the agent may be released from the carrier after it has been transferred across the blood-brain barrier, for example by the breakdown or disruption of enzymatic binding

- chemical between the carrier and the agent. The agent released in this way can function, performing its intended function without the presence of a carrier.

EXAMPLE

Drug conjugation strategies (paclitaxel) [0155] Paclitaxel (TAXOL ™) has 2 strategic conjugation positions (positions C2 'and C7). Fig 2 shows the method of attaching a vector or carrier to palitaxel. Briefly, paclitaxel is reacted with pyridine succinic anhydride for 3 hours at room temperature to attach the succinyl group at the 2 'position. Such 2's succinyl-paclitaxel has a disruptive 2 'ester linkage which, when broken, can easily release succinyl acid. This detachable ester bond can then be used for various modifications with linkers, if desired. The resulting 2'-O-succinyl-paclitaxel undergoes a 9-hour reaction with EDC / NHS in DMSO at room temperature, then a carrier or vector in Ringer / DMSO is added, and this reaction lasts another 4 hours, also at room temperature. The conjugation reaction shown in Fig. 2 is monitored by high performance liquid chromatography (HPLC). Each intermediate, such as paclitaxel, 2'-Osuccinyl-paclitaxel or 2'-O-NHS-succinyl-paclitaxel, is purified and confirmed using various methods such as HPLC, thin film <a href="http://pl.wikipedia.org/wiki/Chromatografia">chromatography</a><a href="http://pl.wikipedia.org/wiki/Chromatografia_cieczowa"> liquid</a>, NMR (exchange 12C or 1H), melting point, mass spectrometry The final conjugate is analyzed by mass spectrometry and polyacrylamide gel electrophoresis in the presence of SDS. This allows the number of paclitaxel molecules attached to each vector to be determined.

[0156] The transcytosis capacity of the aprotinin-paclitaxel conjugate is determined and described below in Table 5.

Table 5

Determining the ability of the aprotinin-taxol conjugate to transcytose across the BBB barrier

<td></td><td>Trancipitation (Pe-3 cm / min)</td><td>Sucrose integrity (Pe-3 cm / min)</td>

<td>Control</td><td></td><td></td>

<td>aprotinin</td><td> 0,2</td><td> 0,28</td>

<td>Aprotinin - Taxol</td><td> 0,21</td><td> 0,24 0,22</td>

<td colspan="3"> Conjugation does not affect the ability of aprotinin to cross the barrier</td>

<td colspan="2"> The barrier's integrity is maintained</td><td></td>

[0157] As shown in Table 5, paclitaxel and aprotinin conjugate were still able to pass the blood-brain barrier of the model in vitro, without compromising the integrity of sucrose, proving that the molecules (also as vectors or carriers) retain their activity after conjugation with a large chemical unit such as paclitaxel.

[0158] To verify that conjugated paclitaxel is still active in vivo, survival studies were performed in a rat brain tumor model. To prepare a brain tumor model, rats were implanted intracerebrally with 50,000 CNS-1 glioblastoma cells. Three days later, animals received vehicle (aprotinin), paclitaxel (5mg / kg) or paclitaxel-aprotinin conjugate (5mg / kg) by intravenous injection. This therapy was used every week until the animals were killed (see Fig. 3). Rats were monitored daily for clinical signs and weight loss. According to the protocol of good practices for animals, the animals were sacrificed when weight loss was observed for 3 consecutive days or if the weight loss was greater than 20% of the initial weight of the animal.

[0159] Using the same experimental protocol, injection of the maximum tolerated dose (54mg / kg) of paclitaxel alone did not manage to extend the life of the mice (Laccabue et al., 2001 Cancer, 92 (12): 3085-92).

[0160] A survival study was also performed in mice with a human brain tumor xenograft. To obtain a brain tumor model in mice, animals were intracerebrally implanted with 50,000 U87 human glioblastoma cells. Three days later, the animals were injected intravenously with paclitaxel-angiopep1 conjugate (5mg / kg) or vehicle. This therapy was used every week until the animals were killed. Rats were monitored daily for clinical signs and weight loss. According to the protocol of good practices for animals, the animals were sacrificed when weight loss was observed for 3 consecutive days or if the weight loss was greater than 20% of the initial weight of the animal. It was observed that the average survival in the control group was 19 ± 2 days. A 20% increase in survival was considered significant for statistical analysis. As seen in Fig. 4, the paclitaxel-angiopep-1 conjugate retained its activity and produced a statistically significant effect. The survival time of the animals that received the paclitaxel-angiopep-1 conjugate increased significantly compared to the control group (p <0.05, n = 8).

[0161] The results obtained in two survival rate studies showed that the conjugation of paclitaxel with the vector increases the chances of survival in animals.

EXAMPLE II

Antibody conjugation strategies [0162] Because proteins generally have several amino groups available for conjugation, it is possible to use amine coupling using sulfoNHS / EDC activation to cross-link therapeutic antibodies with vectors (carriers). The method

This was chosen because it is a fast, simple and reproducible conjugation technique because the conjugate obtained is stable and retains the biological activity of the antibody and has a high conjugation capacity that can be reliably controlled and low non-specific interaction during conjugation procedures.

[0163] Antibodies or antibody fragments (Fab and Fab'2) were attached to the vector to increase their delivery to the brain. After demonstrating that the invention behaves exactly like aprotinin, various conjugation methods were used to combine IgG immunoglobulin with aprotinin.

[0164] Various cross-linking agents such as BS have been tested3 [Bis (sulfosuccinimidyl) suberate], NHS / EDC (N-hydroxysuccinimide and N-ethyl-N '(dimethylaminopropyl) carbodiimide or Sulfo-EMCS [Ne-maleimidocaproic acid] hydrazide for conjugation with IgG. BS3 is a homobifunctional ester of N which targets primary amines. NHS / EDC conjugates primary amine groups to carboxyl groups. Sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS-ester) reactive towards sulfhydryl and amino groups.

[0165] First, the conjugation of IgG to aprotinin was assessed using a BS crosslinking agent3 (Fig. 5) or sulfo-EMCS (Fig. 6).

[0166] The transport of IgG and IgG conjugates across the BBB barrier was tested. Absorption [125I] IgG into the luminal layer of the brain's capillaries in mice was measured by in situ cerebral perfusion method adapted in the inventor's laboratory to study drug absorption into the mouse brain (Dagenais et al., 2000, J. Cereb. Blood Flow Metab. 20 ( 2): 381-386). BBB transport constants were established as previously described by Smith (1996, Pharm. Biotechnol. 8: 285-307). Absorption of IgG is expressed as the volume of distribution (Vd) using the following formula:

Vd = Q * br / C * pf where Q * br is the calculated amount [125I] -IgG or conjugate [125I] -IgG-aprotinin per gram of right hemisphere, C * pf is the concentration of radioactive label measured in the perfusate.

[0167] The results of the experiment indicate that absorption of the conjugate [125I] -IgG-aprotinin in the brain is greater than the absorption of immunoglobulin alone [125I] -IgG (see Fig. 7).

[0168] The conjugation of IgG immunoglobulins with aprotinin increases their accumulation in brain parenchyma in vivo.

EXAMPLE III

Effect of taxol-angiopep-2 conjugate on mouse survival [0169] This study using taxol-angiopep-2 conjugate (referring to peptide No. 97 (angiopep2 is not amidated) was performed to determine whether taxol conjugation and

- 38 angiopep-2 may increase mouse survival. The structure of angiopep-2 is reflected in SEQ ID NO .: 97. In this experiment, mice were implanted intracerebrally with 50,000 U87 glioblastoma cells. Three days after implantation, the animals received vehicle (DMSO / Ronger-Hepes 80:20 by volume (i.e. control)) or taxol-angiopep-2 conjugate (3: 1, i.e. a ratio of 3 taxol molecules per peptide; TxlAn2 ( 5 mg / kg)) by injection into the veins of the tail (Fig. 8). Mice were monitored daily for clinical signs and weight loss. Treatment was used until the animals were killed. As shown in Table 6, it was observed that the average survival in the control group was 18 days, while the average survival in the taxol-angiopep-2 conjugate group was 21 days (Fig. 8). The survival curve for mice treated with the taxol-angiopep-2 conjugate (red) indicates that the average survival increased by as much as 17% (Fig. 8). Statistical analysis also presented in Table 6 indicates that administration of the taxol-angiopep-2 conjugate significantly increases survival, namely by 17% (p-values = 0.048).

Table 6. Summary of survival results

<td>a. Average survival</td><td>Days</td><td>Growth (%)</td><td>Mice (n)</td>

<td>Control</td><td> 18,0</td><td> -</td><td> 7</td>

<td>TxlAn2 conjugate</td><td> 21,0</td><td> 17%</td><td> 7</td>

<td>b. Statistical analysis</td><td>(p values)</td><td></td><td>Statistical differences</td>

<td>Control vs TxlAn2 conjugate</td><td>p = 0.048</td><td></td><td>Yes</td>

SEQUENCES [0170]

SEQ ID NO .:

Peptide No. 5 contains the amino acid sequence specified in SEQ ID NO .: 5 and is amidated at its N-terminus (see example in Figure 9).

Peptide No. 67 contains the amino acid sequence specified in SEQ ID NO .: 67 and is amidated at the N-terminus (see example in Figure 9)

Peptide No. 76 contains the amino acid sequence specified in SEQ ID NO .: 76 and is amidated at the N-terminus (see example in Figure 9)

T FFYGGKRGKRNNFKRAKY

<td> 87</td><td>Τ</td><td>F</td><td>Q</td><td>Υ</td><td>G</td><td>G</td><td>C</td><td>R</td><td>these G</td><td>Κ</td><td>R</td><td>Ν</td><td>Ν</td><td>F</td><td>Κ</td><td>τ</td><td>Ε</td><td>Ε</td><td>Υ</td>

Peptide No. 91 contains the amino acid sequence set out in SEQ ID NO .: 91 and it is amidated at the N-terminus (see example in Figure 9)

ESA E. D CMRTCGGA

103 YGGCRAKRNNFKSAEDCMRTCG

GA

104 GLC QTFVY GG C RA KRNNFKSAE

105 LCQTFVYGGCEAKRNNFKSA

SEQ ID NO .: 106 atgagaccag atttctgcct cgagccgccg tacactgggc cctgcaaagc tcgtatćatc cgttacttct acaatgcaaa ggcaggcctg tgtcagacct tcgtatacgg cggctgca accgcgc

Prepared and verified

Mirosława Ważyńska

Patent Attorney

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Priority claims
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65392805	United States of America	P	2005-02-18
65392805	United States of America	P	2005-02-18
05770546	European Patent Office (EPO)	A	2005-07-21
05770546	European Patent Office (EPO)	A	2005-07-21
11000946	European Patent Office (EPO)	A	2005-07-21
EP20050770546	–	–	–
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