Methods and compositions for targeting agents into and across the blood-barrier and other endothelial cell microvascular barriers

• US 8,026,209 B2
• Filed: 02/10/2004
• Issued: 09/27/2011
• Est. Priority Date: 02/10/2003
• Status: Expired due to Fees

• Current Assignee: 2-BBB Medicines B.V., BBB Holding B.V.
• Original Assignee: BBB Holding B.V.
• Inventors: De Boer, Albertus Gerrit, Brink, Arjen, Gaillard, Pieter Jaap
• Primary Examiner(s): Kolker; Daniel E
• Assistant Examiner(s): BALLARD, KIMBERLY
• Application Number:
  US10/544,991
• Publication Number:
  US 20080213179A1
• Time in Patent Office: 2,786 Days
• Field of Search: None
• US Class Current: 514/1.2
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/6415 Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• CPC Class Codes: A61K 47/6911 the form being a liposome
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/06 Antimigraine agents
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/18 Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/22 Anxiolytics
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 25/28 : for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/06 : Antihyperlipidemics
• CPC Class Codes: A61P 3/08 : for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/08 : Vasodilators for multiple indications
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C07K 14/34 : from Corynebacterium (G)
• CPC Class Codes: C07K 14/47 : from mammals
• CPC Class Codes: C07K 14/52 : Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/035 : containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: Y10S 424/832 : involving bacterial toxin that has modified amino acid sequence
• CPC Class Codes: Y10S 977/714 : Containing protein
• CPC Class Codes: Y10S 977/773 : Nanoparticle, i.e. structure having three dimensions of 100 nm or less
• CPC Class Codes: Y10S 977/801 : Drug
• CPC Class Codes: Y10S 977/808 : Exterior attachment for targeting, e.g. drug targeting

Abstract

The present invention relates to nucleic acids and polypeptides encoded thereby, whose expression is modulated in brain microvascular endothelial cells undergoing early dynamic inflammation-induced changes in blood-brain barrier functionality. Such polypeptides are referred to as lipopolysaccharide-sensitive (LPSS) polypeptides herein. These nucleic acids and polypeptides may be useful in methods for controlling blood-brain barrier properties in mammals in need of such biological effects. This includes the diagnosis and treatment of disturbances in the blood-brain/retina barrier, brain (including the eye) disorders, as well as peripheral vascular disorders. Additionally, the invention relates to the use of anti-LPSS polypeptide antibodies or ligands as diagnostic probes, as blood-brain barrier targeting agents or as therapeutic agents as well as the use of ligands or modulators of expression, activation or bioactivity of LPSS polypeptides as diagnostic probes, therapeutic agents or drug delivery enhancers.

Claims

1. A method of delivering a therapeutic agent into and across an endothelial cell (EC) of a microvascular blood-central nervous system (CNS) barrier in a Diphtheria toxin (DT) sensitive subject in need thereof, comprising:

(a) administering to the subject a moiety that increases binding of DT to DT receptors (DTR) selected from the group consisting of heparin, heparan sulfate, a heparan sulfate proteoglycan, a matrix metalloproteinase inhibitor, BB-94 (batimastat), an inhibitor of ADAM12 or ADAM10, or a MAP/ERK kinase inhibitor;

 

followed by

(b) administering to said subject a therapeutic agent with a pharmaceutically acceptable carrier, which therapeutic agent or carrier is conjugated to a DTR-binding targeting molecule which is selected from the group consisting of

(i) a non-toxic portion of DT polypeptide chain;

(ii) all or a portion of DT B chain; and

(iii) all or a portion of a non-toxic DT mutant CRM197,

 

to constitute a conjugate that specifically binds to the DTR on target tissue cells, such that an effective barrier-entering, barrier-crossing amount of said therapeutic agent enters and crosses the EC microvascular barrier.

2. The method according to claim 1 wherein the DT-sensitive subject is a human.

3. The method of claim 1 wherein the barrier being entered and crossed is the blood-brain barrier (BBB), blood-retina barrier, blood-nerve barrier or blood-spinal cord barrier in a subject with (i) a CNS disease or disorder or (ii) a peripheral disease or disorder with CNS involvement.

4. The method of claim 3 wherein the disease or disorder is:

(a) a neurodegenerative disorder;

(b) a neuropsychiatric disorder;

(c) a CNS disorder selected from the group consisting of a brain tumor, epilepsy, migraine, narcolepsy, insomnia, chronic fatigue syndrome, mountain sickness, encephalitis, meningitis, and AIDS-related dementia;

(d) an angiogenesis-related disorder;

(e) an inflammatory or autoimmune disorder;

(f) age-related macular degeneration;

or

(g) a lysosomal storage disease.

5. The method of claim 1 wherein the therapeutic agent or carrier is directly conjugated to the targeting molecule by:

(a) non-specific or specific protein-protein interaction;

(b) covalent bonding;

(c) non-covalent bonding;

or

(d) coordinating chemical bonding;

which conjugation is optionally effected via a spacer or linker that bridges between the therapeutic agent or carrier and the targeting molecule.

6. The method of claim 5, wherein the therapeutic agent-targeting molecule conjugate or carrier-targeting molecule conjugate is a recombinant fusion or hybrid polypeptide.

7. The method of claim 1 wherein the targeting molecule is an agonist at said DTR.

8. The method of claim 1 wherein the amino acid sequence of the DTR is at least 90% identical with SEQ ID NO:

22.

9. The method of claim 1 wherein said administering is

(a) by continuous intravenous or intraarterial infusion;

or

(b) by bolus injection by an intravenous, intramuscular, intraarterial, or intralesional route.

10. The method according to claim 1, with the proviso that the targeting molecule is not diphtheria toxoid.

11. The method of claim 10 wherein the blood-CNS barrier being entered and crossed is the BBB, blood-retina barrier, blood-nerve barrier or blood-spinal cord barrier.

12. The method of claim 11 wherein the subject has (i) a CNS disease or disorder or (ii) a peripheral disease or disorder with CNS involvement, which disease or disorder is:

(a) a neurodegenerative disorder;

(b) a neuropsychiatric disorder;

(c) a CNS disorder selected from the group consisting of a brain tumor, epilepsy, migraine, narcolepsy, insomnia, chronic fatigue syndrome, mountain sickness, encephalitis, meningitis, and AIDS-related dementia;

(d) an angiogenesis-related disorder;

(e) an inflammatory or autoimmune disorder;

(f) age-related macular degeneration;

or

(g) a lysosomal storage disease.

13. The method of claim 10 wherein the therapeutic agent or carrier is directly conjugated to the targeting molecule by:

(a) non-specific or specific protein-protein interaction;

(b) covalent bonding;

(c) non-covalent bonding;

or

(d) coordinating chemical bonding;

which conjugation optionally includes a spacer or linker that is bound to the therapeutic agent or carrier and the targeting molecule.

14. The method of claim 10, wherein the therapeutic agent-targeting molecule conjugate or carrier-targeting agent conjugate is a recombinant fusion or hybrid polypeptide.

15. The method of claim 10 wherein the targeting molecule is an agonist at said DTR.

16. The method of claim 10 wherein the amino acid sequence of the DTR is at least 90% identical with SEQ ID NO:

22.

17. The method of claim 10 wherein said administering is:

(a) by continuous intravenous or intraarterial infusion, or

(b) by bolus injection by an intravenous, intramuscular, intraarterial, or intralesional route.

18. A method of delivering a therapeutic agent into and across an EC of a microvascular blood-CNS barrier in a subject, comprising administering to a DT-sensitive subject with (i) a CNS disease or disorder or (ii) a peripheral disease or disorder with CNS involvement, a composition comprising:

(a) a therapeutic agent; and

(b) a pharmaceutically acceptable carrier,

wherein the therapeutic agent is encapsulated in a nanocontainer to which is linked a DTR-binding targeting molecule selected from the group consisting of;

(i) a non-toxic portion of DT polypeptide chain;

(ii) all or a portion of DT B chain; and

(iii) all or a portion of a non-toxic DT mutant CRM197,

wherein an effective barrier-entering and barrier-crossing amount of said therapeutic agent enters and crosses the EC microvascular blood CNS barrier.

19. The method of claim 18 wherein the nanocontainer is a nanoparticle, a liposome or a nanogel.

20. The method according to claim 18, wherein the targeting molecule is not diphtheria toxoid.

21. The method of claim 20 wherein the nanocontainer is a nanoparticle, a liposome or a nanogel.

22. A method of delivering a therapeutic agent into and across an EC of a microvascular blood-CNS barrier in a subject with a microvascular disorder, which comprises:

(A) inducing immunological tolerance or unresponsiveness to a DTR-binding targeting molecule by administering to a DT-sensitive subject in need thereof an effective amount of;

(i) free CRM197 or a fragment thereof;

or

(ii) DT B chain or a fragment thereof, and

(B) administering to the subject a therapeutic agent with a pharmaceutically acceptable carrier, which therapeutic agent or carrier is conjugated to a targeting molecule as a conjugate that specifically binds to DTR, wherein the targeting molecule is selected from the group consisting of (i) a non-toxic portion of DT polypeptide chain;

(ii) all or a portion of DT B chain;

and (iii) all or a portion of a non-toxic DT mutant CRM197,

wherein an effective barrier-entering and barrier-crossing amount of said conjugate enters and crosses the EC microvascular barrier.

23. The method according to claim 22 with the proviso that the targeting molecule is not diphtheria toxoid.