WO2020193802A1 - Conjugués polymères et leurs utilisations - Google Patents

Conjugués polymères et leurs utilisations Download PDF

Info

Publication number
WO2020193802A1
WO2020193802A1 PCT/EP2020/058940 EP2020058940W WO2020193802A1 WO 2020193802 A1 WO2020193802 A1 WO 2020193802A1 EP 2020058940 W EP2020058940 W EP 2020058940W WO 2020193802 A1 WO2020193802 A1 WO 2020193802A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
pga
conjugate
fasudil
cancer
Prior art date
Application number
PCT/EP2020/058940
Other languages
English (en)
Inventor
Victoria MORENO MANZANO
Esther GIRALDO REBOLOSO
Raquel REQUEJO AGUILAR
Ana ALASTRUE AGUDO
María Jesús VICENT DOCÓN
Ana ARMIÑAN DE BENITO
Juan José ARROYO-CRESPO
Vicent Josep NEBOT CARDÁ
Oleksandr ZAGORODKO
Snezana Dordevic
Original Assignee
Fundación De La Comunidad Valenciana Centro De Investigación Príncipe Felipe
Fundación Step By Step
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fundación De La Comunidad Valenciana Centro De Investigación Príncipe Felipe, Fundación Step By Step filed Critical Fundación De La Comunidad Valenciana Centro De Investigación Príncipe Felipe
Publication of WO2020193802A1 publication Critical patent/WO2020193802A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to polymer-drug conjugates with ROCK inhibitors, chemotherapeutic, anti-inflammatory and/or neuroprotective agents as well as their pharmaceutical compositions containing the said polymer-drug conjugates and their uses thereof for the treatment of neurodegenerative diseases, spinal cord injury and cancer.
  • Neurological disorders and cancer represent serious health problems, being two of the main leading cause of death and disability in the world today. As a consequence, many efforts and resources are being invested to alleviate and cure these conditions.
  • Neurological disorders are diseases of the central and peripheral nervous system, therefore they affect the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. Many neurological affections are caused by diseases such as Amyotrophic lateral sclerosis, Memory disorders, Multiple Sclerosis, Parkinson ' s disease, etc. On the other side, injuries or trauma can also be the source of other neurological conditions such as Traumatic Brain (TBI) or Spinal Cord Injury (SCI) which leads to the devastating loss of neurological function in the affected area.
  • TBI Traumatic Brain
  • SCI Spinal Cord Injury
  • Cancer is a group of diseases characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighboring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis).
  • Many drugs are now available to be used in the treatment of cancer. However, in many cases the cancer fails to respond to the anti- cancer therapy or its growth and/or metastasis is only slowed. Even when a tumor initially responds to an anti-cancer therapy by decreasing in size or going into remission, the tumor often develops resistance to the drug. For these reasons, there is a need for new anti-cancer agents or improved bioavailable drugs for the treatment of cancers for which there is still no treatment available and for multi-drug resistance cancers.
  • PT Polymer Therapeutics
  • PT-based strategies fail at the preclinical or early-clinical trial stage. Indeed, recent figures have shown that around 90% of all compounds entering Phase I clinical trials do not meet with approval and do not enter the market. Therefore, it seems that the full potential for drug delivery systems has not yet been developed and there remains a need in the state of the art to provide new and improved PTs for the treatment of many diseases of difficult and sometimes ineffective treatment, in particular cancer and neurological diseases.
  • the present invention relates to novel chemical entities comprising a polymeric carrier incorporating covalently linked drug(s) as well as their pharmaceutical compositions containing the said polymer-drug conjugates as pharmaceutical agents and their uses thereof in therapy and, more particularly, but not exclusively, to novel conjugates having attached thereto a targeting moiety, an imaging agent and one or more therapeutic agents to promote axonal growth and for the treatment of neurodegenerative diseases, central nervous system traumatic injuries, inflammatory diseases and cancer.
  • the particular structure of the conjugate allows for the sustained release of the drug after its internalization via an endocytic mechanism or in the inflammation area. This is mediated by the presence of the bio-responsive disulfide containing linker, which is degraded when exposed to the reducing environment found inside the cells or in a damaged tissue. This mechanism of drug delivery exhibits lower toxicity profile compared to other biodegradable linker based conjugates.
  • the invention relates to a polymeric chain conjugate having the formula:
  • x is an integer having a value such that 0.001 £ [x/(x+y)j £ 1 ;
  • y is an integer having a value such that 0 £ [y/(x+y)j £ 0.999;
  • R 1 is selected from the group consisting of H; optionally substituted C1-C500 alkyl; optionally substituted polyethylene glycol; pyroglutamate; polysarcosine; polyoxazolin; and a C-terminal linking point selected from the group consisting of alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals and activated carboxylic groups;
  • R 2 is selected from the group consisting of H; OH; optionally substituted C1- C500 alkyl; optionally substituted polyethylene glycol, polysarcosine, polyoxazolin and a C-terminal linking point selected from the group consisting of alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals and activated carboxylic groups;
  • R 3 is selected from the group consisting of H and CH 3 ;
  • R 4 is selected from the group consisting of -0-, -S-, -COO-, -(CH 2 ) 2 NH-, - (CH 2 ) 3 NH-, -PhO-, -(CH 2 ) 2 NNHNH-, -CONH- , -CH 2 CONH-, -CH 2 CO-, and -CH 2 COO-; wherein at least one occurrence of A is a ROCK inhibitor or a chemotherapeutic agent linked through B, wherein B represents a linker molecule comprising a disulfide group; and wherein the remainder of occurrences of A are linked to the polymeric chain directly or through B and wherein each A is independently selected from an active compound, an imaging agent, a targeting motif, or a combination thereof; wherein the active compound is selected from the group consisting of a ROCK inhibitor, an axonal growth promoter, an anti-inflammatory agent, a glial scar disruptor, a neuroprotective drug, antioxidative agent,
  • At least one occurrence of B represents a linker molecule comprising a disulfide group and the remainder occurrences of B are each independently selected from being absent such that A is linked to the polymeric chain directly, or are each independently selected from a linker molecule comprising one or more functional groups selected from alkynes, azides, disulfides, maleimides, hydrazide, hydrazones, Schiff bases, acetal, aldehydes, carbamates, amides and esters;
  • L is H or CHR 5 R 6 ;
  • R 5 is selected from the group consisting of H and CH3;
  • R 6 is selected from the group consisting of H, linear or branched alkyl C1-C3, OH, SH, COOH, -CH2COOH, -(CH 2 )2NH 2 , -(CH 2 ) 3 NH 2 , -CH 2 SCH 3 , -(CH 2 ) 2 SCH 3 , phenyl, hydroxyphenyl, indolyl, imidazolyl, -(CH 2 ) 2 NNHNH 2 , -CONH 2 , -CH 2 CONH 2 , and SeH and wherein Z is H or CH3.
  • the invention in a second aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one conjugate as defined in the first aspect of the invention and at least one pharmaceutically acceptable excipient.
  • the invention relates to the pharmaceutical composition of the invention for use as a medicament.
  • the invention relates to the pharmaceutical composition of the invention for use in the prevention and/or treatment of a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyloydotic disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyloydotic disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • the invention relates to the pharmaceutical composition of the invention for use in the prevention and/or treatment of cancer.
  • FIGURES Figure 1 General synthetic route for the preparation of Poly-L-glutamate Fasudil conjugates through different linkers amide (PGA-FAS), carbamate (PGA-O-FAS), self- immolative disulphide (PGA-SS-FAS).
  • PGA-FAS linkers amide
  • PGA-O-FAS carbamate
  • PGA-SS-FAS self- immolative disulphide
  • Reaction conditions were: i) H2O, DMTMMCI, Fasudil hydrochloride; ii) DMF, DMTMMBF4, self-immolative disulphide Fasudil derivative (see synthetic methods for details); iii) H2O, DMTMMCI, NH2-(CH2)2-(PEG)2- N3 iv) DMF/H2O, CUSO4, Sodium Ascorbate, carbamate Fasudil derivative (see synthetic methods for details).
  • FIG. 1 Characterization of Poly-L-glutamates Fasudil conjugates.
  • PGA-FAS and PGA-O-FAS a TDA 302 integrated instrument has been used with two columns in succession TSKgel G2500 and G3000PWXL, for PGA-SS-FAS POSTNOVA AF2000 was employed with TSKgel G3000PWXL column (see methods for details).
  • FIG. 3 Characterization of Poly-L-glutamates Fasudil conjugates by UV-Vis and Total Drug Loading (TDL) quantification.
  • UV-Vis was employed for the quantification of Fasudil content within the polypeptide-drug conjugate (figure 3D shows the calibration curve for Fasudil at 323 nm).
  • the PGA-drug conjugates preserved the maximum absorbance characteristics of the parent drug.
  • Figure 4 In vitro effects of Fasudil conjugates on rat neural precursor cells (NPCs) survival, differentiation and neurite outgrowth.
  • B. Quantification of neurite length in Matrigel-adhered NPCs, wherein data is expressed as mean ⁇ SEM (n 3 per group) +++p ⁇ 0.001 vs PGA 12h; ###p ⁇ 0.001 vs F 72h.
  • C Quantification of beta-lll-tubulin, Olig-2 and GFAP positive cells following spontaneous differentiation in rat NPCs, wherein data is expressed as mean ⁇ S.E.M. **p ⁇ 0.01 determined by ANOVA multiple comparison test. Scale bars, 10 mM.
  • FIG. 1 Cell toxicity study of polymer drug conjugates.
  • A MDA-MB-231 cells
  • B MDA- MB-453
  • C IC50 values for free Fasudil and the polymer drug conjugates in both cell lines.
  • FIG. 7 Chromatograms obtained from fasudil (A), ranitidine (B), and blank plasma (C) detected in positive electrospray ionization mode with multiple reaction monitoring scans and by following two mass transitions 292.4 mz 1 99.2 mz 1 and 292.4 129.0 mz 1 for fasudil, and 315.2 mz 1 101.9 mz -1 and 315.2 mz -1 130.0 mz -1 for ranitidine.
  • Figure 8 Percentage of free fasudil in plasma, during stability studies of the PGA-SS- FAS conjugate.
  • Figure 9 A) chromatogram of cell medium after 24h incubation with PGA-SS-FAS. B) control sample.
  • FIG. 10 NSC amplification efficiency from human fetal spinal cord tissue at different gestational stages.
  • VHIR samples collected from Vail d ' Hebron Hospital, from 19 to 21 gestational week;
  • MED samples collected from Clinica Mediterraneo, from 14 to 17 gestational week.
  • FIG. 11 PDL curve from representative human samples collected from VHIR and MED in comparison with the PDL curve of rat embryonic spinal cord NSC (E14,5 RAT).
  • Figure 13 Intracellular Neuronal cell markers in proliferating human NSC.
  • Figure 15 Expression analysis of the neuronal markers PSA-NCAM and CD184 after conditioning the human NSC population by pre-treatment with the nanoconjugate Fasudil-SS-PGA at 50 mM final concentration during 24 hours incubation in growing conditions.
  • FIG. 16 PGA-SS-Fasudil (F) Conditioning treatment of human NPC in the presence of LPA (lysophosphatidic acid).
  • LPA lysophosphatidic acid
  • the LPA (+) induces retraction of the neurites in the control conditions, but this effect is prevented by the treatment with the nanoconjugate (50 pM for 24 hours).
  • Histograms on the right side show the quantification of the length of the neurites in the nestin positive cells induced by the treatment of PGA-SS-F in the presence (+) or not (-) or LPA. Data is represented as mean ⁇ SE; * p ⁇ 0.05 vs control.
  • FIG. 17 In vivo preliminary cell transplantation of human NPCs in Spinal Cord Injury rat model. Left panel represent a summary of the employed experimental design. Right panels: representative images after immunostaining using anti-human mitochondria antibody and NeuN for neuron detection; GFAP for astrocyte staining. And DAPI was used to counterstaining all cell nuclei. The white arrow indicates the positive signal for anti-human mitochondria, co-labeling with NeuN (neuronal marker), indicating the survival and Integration of the human transplanted cells, keeping a neuronal cell fate in vivo.
  • NeuN neuronal marker
  • FIG. 18 Cell viability studies at 72h by MTT assay following treatment with PGA-Fas and PGA-O-Fas in 4T1 (A) and MDA-MB-231 (B), and PGA-SS-Fas in MDA-MB-453 (C), MDA-MB-231 , (D) and 4T1 (E) cell lines.
  • A) Migration of cells at t 0h.
  • E Migration of cells at 24h under PGA-SS-Fas treatment.
  • F Migration of cells at 24 h under Fas treatment.
  • FIG. 20 Intrathecal in vivo administration of PGA-SS-F inhibits Rho/Rock activity, triggers neuroprotective and neuroregenerative signalling pathways in acute phase after SCI in a rat model.
  • D Quantification of pAkt and rQ8K3b (involved in cell survival).
  • FIG. 21 PGA-SS-F promote axonal growth in rat dorsal root ganglia (DRG) explants.
  • FIG. 23 PGA-SS-F overcomes neurite retraction following ROCK activation and promotes neuronal fate, axonal growth and neuroplasticity markers.
  • A) Quantification of the longest neurite was measured using NeuronJ. Data is expressed as mean ⁇ SEM (n 6 per group) ***p ⁇ 0.001 vs control group; +++p ⁇ 0.001 vs all groups; ###p ⁇ 0.001 vs with LPA+F50; &&&p ⁇ 0.001 vs LPA+F100 determined by ANOVA multiple comparison test.
  • G Quantification of cyst area in hematoxilin-eosin staining of transversal sections of SCs showed reduced cysts formation following 8 weeks post-treatment.
  • Figure 25 Structure of PGA-Din conjugates employing various linking strategies.
  • Figure 26 Synthetic scheme of PGA-PP-Din.
  • FIG. 27 31 P NMR spectra of Din-phosphate (A), Din-PP-(Fmoc-ethanolamine) (B), Din- PP-(ethanolamine) (C), and PGA-PP-Din (D).
  • Figure 28 DOSY NMR spectra of PGA-PP-Din.
  • Figure 29 1 H NMR spectra of dinaciclib and PGA-SS-Din.
  • FIG. 30 Toxicity curves for dinaciclib and corresponding conjugates in MDA-MB-231 (up) and ZR-75-1 (down).
  • the present invention relates to novel polymeric conjugates linked at least to an active compound, an imaging agent, a targeting motif, or a combination thereof as well as their pharmaceutical compositions containing the said polymer-drug conjugates as pharmaceutical agents and their uses to promote axonal growth, for the treatment of neurodegenerative diseases, central nervous system traumatic injuries, inflammatory diseases and cancer.
  • the invention relates to a polymeric chain conjugate having the formula:
  • x is an integer having a value such that 0.001 £ [x/(x+y)j £ 1 ;
  • y is an integer having a value such that 0 £ [y/(x+y)j £ 0.999;
  • R 1 is selected from the group consisting of H; optionally substituted C1-C500 alkyl; optionally substituted polyethylene glycol; pyroglutamate; polysarcosine; polyoxazolin; and a C-terminal linking point selected from the group consisting of alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals and activated carboxylic groups;
  • R 2 is selected from the group consisting of H; OH; optionally substituted C1- C500 alkyl; optionally substituted polyethylene glycol, polysarcosine, polyoxazolin and a C-terminal linking point selected from the group consisting of alkyne, azide, thiol, activated thiols, halides, alkenes, activated esters, activated alcohols, protected amines, maleimide group, acetals and activated carboxylic groups;
  • R 3 is selected from the group consisting of H and CH3;
  • R 4 is selected from the group consisting of -0-, -S-, -COO-, -(CH2)2NH-, - (CH 2 )3NH-, -PhO-, -(CH 2 ) 2 NNHNH-, -CONH- , -CH 2 CONH, -CH 2 CO-, and -CH 2 COO-; wherein at least one occurrence of A is a ROCK inhibitor or a chemotherapeutic agent linked through B, wherein B represents a linker molecule comprising a disulfide group; and wherein the remainder of occurrences of A are linked to the polymeric chain directly or through B and wherein each A is independently selected from an active compound, an imaging agent, a targeting motif, or a combination thereof; wherein the active compound is selected from the group consisting of a ROCK inhibitor, an axonal growth promoter, an anti-inflammatory agent, a glial scar disruptor, a neuroprotective drug, antioxidative agent, chemotherapeutic
  • At least one occurrence of B represents a linker molecule comprising a disulfide group and the remainder occurrences of B are each independently selected from being absent such that A is linked to the polymeric chain directly, or are each independently selected from a linker molecule comprising one or more functional groups selected from alkynes, azides, disulfides, maleimides, hydrazide, hydrazones, Schiff bases, acetal, aldehydes, carbamates, amides and esters;
  • L is H or CHR 5 R 6 ;
  • R 5 is selected from the group consisting of H and CH 3 ; wherein R 6 is selected from the group consisting of H, linear or branched alkyl C1-C3, OH, SH, COOH, -CH2COOH, -(CH 2 )2NH 2 , -(CH 2 ) 3 NH 2 , -CH 2 SCH 3 , -(CH 2 ) 2 SCH 3 , phenyl, hydroxyphenyl, indolyl, imidazolyl, -(CH 2 ) 2 NNHNH 2 , -CONH 2 , -CH 2 CONH 2 , and SeH; and wherein Z is H or CH 3 .
  • R 1 is selected from the group consisting of H, optionally substituted C1-C500 alkyl, optionally substituted polyethylene glycol, and pyroglutamate; preferably selected from the group consisting of H, unsubstituted C1-C500 alkyl, unsubstituted polyethylene glycol, and pyroglutamate; more preferably H.
  • R 2 is selected from the group consisting of H, OH, optionally substituted C1-C500 alkyl and optionally substituted polyethylene glycol; preferably from the group consisting of H, OH, unsubstituted C1-C500 alkyl, and unsubstituted polyethylene glycol; more preferably OH.
  • R 3 is H.
  • R 4 is selected from the group consisting of -CONH- , - CH 2 CONH-, -CH 2 CO-, and -CH 2 COO-; preferably selected from the group consisting of -CH 2 CONH- and -CH 2 CO-; more preferably -CH 2 CONH-.
  • At least one occurrence of A is fasudil linked through B, wherein B represents a linker molecule comprising a disulfide group; preferably each A is fasudil linked through B, wherein B represents a linker molecule comprising a disulfide group
  • L is CHR 5 R 6 .
  • R 5 is H.
  • R 6 is selected from the group consisting of COOH and -CH 2 COOH; preferably -CH 2 COOH.
  • Z is H.
  • R 1 , R 3 , R 5 and Z are H;
  • R 2 is OH;
  • R 4 is -CH2CONH-;
  • at least one occurrence of A is fasudil linked through B, wherein B represents a linker molecule comprising a disulfide group;
  • L is CHR 5 R 6 ; and
  • R 6 is -CH2COOH.
  • R 1 , R 3 , R 5 and Z are H; R 2 is OH; R 4 is -CH2CONH-; each occurrence of A is fasudil linked through B, wherein B represents a linker molecule comprising a disulfide group; L is CHR 5 R 6 ; and R 6 is -CH2COOH.
  • R ' is to be understood as a group that is, or is not, substituted in one or more available positions with one or more suitable groups such as OR', SR', SOR', SO2R', OSO2R', SOsR', NO2, N(R ') 2 , N(R')COR', N(R')S0 2 R ', CN, Cl, Br, COR ', CO2R', C0 2 -M + , OCOR ', OCO2R', OCONHR ', OCON (R') 2 , CONHR', CON(R')2, straight or branched C1-C6 alkyl, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ob-Op aryl and 5- to 12-membered heterocyclyl, wherein each of the R 'groups is independently selected from the group consisting of hydrogen, linear or branched Ci- O
  • Activated esters, alcohols, thiols or carboxylic groups are known to the person skilled in the art. They are defined as an ester, alcohol, thiol or carboxylic group comprising a good leaving group, wherein the ester, alcohol, thiol or carboxylic group is bound to said good leaving group. Good leaving groups are known to the person skilled in the art, especially in the field of the polypeptide synthesis.
  • linker refers to a group or groups that covalently links 2 molecules.
  • the linker may itself be biologically active.
  • the linker may be a bioresponsive disulfide group or contain at least one bioresponsive disulfide group.
  • the term“disulfide” refers to a functional group with the structure R-S-S-R', preferably -(CH 2 ) 2 -S-S-(CH 2 ) 2 -0-C0-.
  • the linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups.
  • bioresponsive refers to a chemical link cleavable under specific physiological or external triggers (for example, and without limitation, pH, reactive oxygen species, reductive environment, specific enzymes, glucose, light, temperature, etc).
  • physiological or external triggers for example, and without limitation, pH, reactive oxygen species, reductive environment, specific enzymes, glucose, light, temperature, etc.
  • the disulfide linkages are unstable in a reductive environment as the disulfide bond is readily cleaved in favor of corresponding thiol groups.
  • Polymers with disulfide cross-links degrade when exposed to cysteine or glutathione, reductive amino-acid based molecules present at intracellular concentrations 50-1000 fold greater than those of the extracellular medium
  • the expressions“active compound”,“active agent”, “therapeutic agent” or“drug” are used herein indistinctly and refer to molecules or atoms bound to the conjugate of the invention to produce a conjugate which is useful for therapy. They are physiologically or pharmacologically active substances that act locally or systemically in the body.
  • An active agent is a substance used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • the active agent can be selected from small agents (i.e.
  • biomolecules such as peptides, (apolipo)proteins, antigens, antibodies, Fab or fragment antigen-binding, nucleic acids, oligosaccharide, aptamer, polysaccharide, oligonucleotides (e.g., antisense oligonucleotides or interference RNAs, i.e., small interfering RNA (siRNA)) double stranded oligonucleotide (DNA).
  • oligonucleotides e.g., antisense oligonucleotides or interference RNAs, i.e., small interfering RNA (siRNA)
  • DNA double stranded oligonucleotide
  • Additional therapeutic agents may include drugs, prodrugs, toxins, enzymes, enzymes that activate prodrugs to drugs, enzyme-inhibitors, nucleases, hormones, hormone antagonists, immunomodulators, e.g., cytokines, i.e, interleukins, such as interleukin-2, lymphokines, interferons and tumor necrosis factor, chelators, boron compounds, photoactive agents or dyes, radioisotopes or radionuclides.
  • cytokines i.e, interleukins, such as interleukin-2, lymphokines, interferons and tumor necrosis factor
  • chelators boron compounds
  • photoactive agents or dyes e.g., photoactive agents or dyes, radioisotopes or radionuclides.
  • Suitable active ingredients include without limitation amiloride, aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1 , busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dendrotoxin, dexamethasone, d iethy I sti I bestrol , dinaciclib, palbociclib, doxorubicin and analogs thereof, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol,
  • Suitable radionuclides may include 18F, 32P, 33P, 45Fi, 47Sc, 52Fe, 59Fe, 62Cu, 64Cu, 61 Cu, 67Ga, 68Ga, 75Se, 77 As, 86Y, 89Sr, 89Zr, 90Y, 94Tc, 94mTc, 99Mo, 99mTc, 105Pd, 105Rh, 111Ag, 111 ln, 1231, 1241, 1251, 131 l,142Pr, 143Pr, 149Pm, 153Sm, 154- 158Gd, 161Th, 166Dy, 166Ho, 169Er, 175Lu, 177Lu, 186Re, 188Re, 189Re, 194lr, 198Au, 199Au, 211At, 211 Pb 212B ⁇ , 212Pb, 213B ⁇ , 223Ra, 225Ac, or mixtures thereof
  • Suitable enzymes may include carboxyl esterases, glucuronidases, carboxypeptidases, beta-lactamases, phosphatases, nucleases, proteases, lipases, and mixtures thereof.
  • Suitable photoactive agents and dyes include agents for photodynamic therapy, such as a photosensitizer, such as benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AISPc) and lutetium texaphyrin (Lutex).
  • a photosensitizer such as benzoporphyrin monoacid ring A (BPD-MA), tin etiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AISPc) and lutetium texaphyrin (Lutex).
  • the active agent is selected from the group consisting of a ROCK inhibitor, an axonal growth promoter, an anti inflammatory agent, a glial scar disruptor, a neuroprotective drug, antioxidative agent, chemotherapeutic agent, tyrosine kinase inhibitor, cell-cycle inhibitor, exosome-release modulator and combinations thereof.
  • ROCK refers to a serine/threonine kinase that acts downstream of Rho
  • ROCK-I also referred to as RO ⁇ or p160ROCK
  • ROCK-II also referred to as ROKa or Rho kinase
  • ROCK inhibitor is a compound that inhibits both or either one of the ROCKs, it is not particularly limited, and includes classic ROCK inhibitors based on isoquinoline and pyridine/pyrrolopyridine derivatives, indazole based ROCK inhibitors, pyridine, pyrrolopyridine and pyrimidine based ROCK inhibitors, pyrazole based ROCK inhibitors, methylenephenyl substituted pyrazole based ROCK inhibitors, benzimidazole, benzothiazole, indole and indazole substituted pyrazole derivatives as ROCK inhibitors, quinazolinone substituted pyrazole based ROCK inhibitors, thiophene substituted pyrimidine based and aminofurazane based ROCK inhibitors, ROCK inhibitors based on 6- and 7- substituted isoquinolines and isoquinolinones, boron derivatives as ROCK inhibitors, amakem ' s soft ROCK
  • ROCK inhibitors A complete list of the above mentioned types of ROCK inhibitors can be found in Feng Y et al, J Med Chem. 2016 Mar 24;59(6):2269-300. Additional suitable inhibitors include those described in, for example, International Publication Nos. W01998006433, W02000078351 , International Publication No. W02001017562, W02002076977, W02003082808, W02005035506, W02005074643 and others.
  • Additional suitable inhibitors include, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines, Indazoles, pyridinealkene derivatives, ROKa inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4- pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA- 1037, Ki-23095, quinazoline, Ripasudil, RKI-1447, GSK429286A, Thiazovivin,
  • siRNA RNA interference inducing nucleic acid
  • competitive peptides for example, antagonist peptides, inhibitory antibodies, antibody-ScFV fragments, dominant negative variants and expression vectors thereof.
  • ROCK inhibitors since other low molecular compounds are known as ROCK inhibitors, such compounds or derivatives thereof can be also used in the present invention (for example United State Patent Application Nos. 20050209261 , 20050192304, 20040014755, 20040002508, 20040002507,
  • the term“axonal growth” refers to the ability of a modulating agent to enhance the extension (e.g., regeneration) of axons and/or the reestablishment of nerve cell connectivity.
  • Axonal growth as used herein include all neurite sprouting as part of axonal growth promotion and neurite sprouting which occurs after an axon is damaged and neurite sprouting which occurs in conjunction with the extension of the axon.
  • Axonal growth as used herein includes axonal regeneration in severed neurons which occurs at, or near, the site at which the axon was severed.
  • the term“neural cell” as used herein is meant to include cells from both the central nervous system (CNS) and the peripheral nervous system (PNS). Exemplary neural cells of the CNS are found in the gray matter of the spinal cord or the brain and exemplary neural cells of the PNS are found in the dorsal root ganglia.
  • Agents that can be used to promote axonal growth include bcl modulating agents such as lithium, lithium chloride, lithium acetate, lithium carbonate, lithium citrate and lithium sulfate.
  • lithium chloride LiCI
  • Numerous salts of lithium are commercially available for treating certain manic-depressive illnesses. Compounds having structural similarities to lithium or a salt thereof can also be used.
  • Another agent that upregulates bcl-2 and can be used to promote axonal growth is valproate (valproic acid), which is commercially available (Manjii et al. (2001) Br J Psychiatry Suppl 2001 41 :s107).
  • agents include carbamazepine, lamotrigine, topiramate, gabapentin, primidone, benzodiazepine, clozapine, risperidone, calcium channel blockers, (such as verapamil, diltiazem, nifedipine, and nimodipine), bupropion, serotonin reuptake inhibitor, monoamine oxidase inhibitor, venlalfaxine, nefazodone, tricyclic antidepressants.
  • anti-inflammatory agent refers to substances that reduce inflammation.
  • anti-inflammatory agents include but are not limited to nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, beta- agonists, anticholingeric agents, and methyl xanthines.
  • NSAIDs include, but are not limited to, Ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, Indomethacin, ketoralac, oxaprozin, nabumentone, suhndac, tolmetin, rofecoxib, naproxen, ketoprofen, nabumetone, etc.
  • NSAIDs function by inhibiting a cyclooxgenase enzyme (e.g., COX-I and/or COX-2).
  • a cyclooxgenase enzyme e.g., COX-I and/or COX-2
  • steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone, cortisone, hydrocortisone, prednisone, prednisolone, triamcinolone, azulfdine, and eicosanoids such as thromboxanes, and leukotrienes.
  • glial scar refers to a reactive cellular process involving astrogliosis that occurs after injury to the central nervous system.
  • the glial scar has both beneficial and detrimental effect.
  • the scar prevents axonal extensions via physical and chemical means.
  • Astrocytes form a dense network of gap junctions that generates a physical barrier to axonal regrowth.
  • the astrocytes secrete several growth- inhibitory molecules that chemically prevent axonal extensions.
  • the basal membrane component is expected to generate an additional physical and chemical barrier to axonal extensions.
  • glial scar disruptor as used herein, makes reference to compounds or drugs that suppresses, inhibits or disrupts the glial scar.
  • glial scar disruptors examples include chondroitin-sulfate-ABC endolyase, olomoucine, phosphodiesterase 4 inhibitors such as rolipram, ribavirin and inhibitors of growth factor- b2 and interleukin-6 receptor such as monoclonal antibodies.
  • Neuroprotective is defined herein as an effect which reduces, arrests, or ameliorates nervous insult & is protective, by preventing neuronal cell death when the nervous tissue has suffered an insult.
  • Neuroprotective agent or drug is herein defined as treatment medication, or formulary drug containing a nervous insult treatment dose of treatment medication effective in reducing, arresting, or ameliorating nervous insult & provides protection to nervous tissue that has suffered nervous insult.
  • Neuroprotective agents that may be linked to the polymer to form the conjugate of the invention include, e.g., anticonvulsive agents, antiparkinsonian drugs, caspase inhibitors, superoxide dismutase mimetics, calcium chelators, calcium channel blockers, gamma-aminobutyric acid (GABA) receptor agonists, antagonists, and uptake inhibitors and enhancers, p38 mitogen-activated protein kinase (MAPK) antagonists, ERK-MAPK antagonists, cytochrome C chelating antibodies; APAF-1 (apoptotic protease activating factor-1) inhibitors, AIFs (apoptosis inhibiting factors), PARP (poly (ADP-ribose) polymerase) inhibitors, anti-epileptic agents, immunosuppressants, anti-inflammatory agents, non-steroidal anti-inflammatory drugs (NSAIDs), anti-cancer agents, statins, cannabinoids, anti-virals, noo
  • Suitable anti-epileptic agents that can be used in the conjugates include, e.g, sodium channel inhibitors, for example, phenytoin, carbamazepine, oxcarbazepine; GABA receptor modulators, for example, phenobarbital, tiagabine, vigabatrin (g-vinyl GABA, a GABA transaminase inhibitor), gabapentin; agents that reduce calcium currents, T currents, for example, ethosuccimide and zonisamide; and those that exert unknown or multiple effects, for example, piracetam, levetiracetam, aniracetam, nefiracetam or topiramate.
  • sodium channel inhibitors for example, phenytoin, carbamazepine, oxcarbazepine
  • GABA receptor modulators for example, phenobarbital, tiagabine, vigabatrin (g-vinyl GABA, a GABA transaminase inhibitor), gab
  • Suitable nootropic agents include sedrafinil, centrophenoxine, deprenyl, dehydroepiandrosterone (DHEA), dimethylaminoethanol (DMAE), Gingko Biloba, piracetam, pyroglutamate, Vinpocetine, and xanthinol nicotinate.
  • the neuroprotective agent in the conjugates of the invention can additionally include an agonist of a muscarinic receptor.
  • agonists include Alvameline, Arecoline, Cevimeline, PD-151832, Milameline, CI-979A, RU35926, Sabcomeline, SB 202026, SCH-217443, SDZ-210-086, SR 46559A, Talsaclidine, WAL 2014-FU, Tazomeline, Xanomeline, and YM 796.
  • neurotrophic factors are neuroprotective in brain, but do not cross the blood-brain barrier. These factors are suitable for use in the conjugate of the invention and include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-a, TGF-b, vascular endothelial growth factor (VEGF), interleukin-l receptor antagonist (IL-1 ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin, persephin, interleukins, granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF, netr
  • neuroprotective agents include serofendic acid, PBAS ((5-pentafluorobenzyl) aminosalicylic acid), cannibinoids, such as CB-1 antagonists (Sanofi), and CB-2 agonists (Mak Scientific), anadamide and aminoadamantane derivatives.
  • Antioxidant or“antioxidative agent” as used herein, is referred to substances that reduce the levels of reactive oxygen species, for instance preventing the formation of such reactive oxygen species or removing them before they produce any damage.
  • Suitable antioxidants include but are not limited to free radical eliminators, iron chelating agents, small molecule antioxidants and antioxidant enzymes and the like.
  • Suitable iron chelating agents include but are not limited to deferoxamine, deferiprone, dithiocarbamate, ethylenediaminetetraacetic acid and the like.
  • Suitable small molecule antioxidants include but are not limited to compounds of hydralazine, glutathione, ascorbic acid (vitamin C), vitamin E, cysteine, N-acetyl-cysteine, b-carotene, ubiquinone, ubiquinol-10, tocopherols, coenzyme Q, superoxide dismutase mimetics such as, for example, 2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPO), DOXYL, PROXYL nitroxide compounds; 4-hydroxy-2,2,6,6-tetramethyl-l-piperidinyloxy (TEMPOL), M-40401 , M- 40403, M-40407, M-40419, M-40484, M-40587, M-40588 and the like.
  • vitamin C vitamin E
  • cysteine N-acetyl-cysteine
  • b-carotene ubiquinone
  • ubiquinol-10 ubiquinol-10
  • Suitable antioxidant enzymes include but are not limited to superoxide dismutase, catalase, glutathione peroxidase, NADPH oxidase inhibitors such as, for example, apocynin, aminoguanidine, ONO 1714, SI7834 (a benzo(b)pyran-4-one derivative) and the like; xanthine oxidase inhibitors such as, for example, allopurinol, oxypurinol, amflutizole, diethyldithiocarbamate, 2-styrylchromones, crisine, luteolin, kaempferol, quercetin, myricetin, isorhamnetin, benzophenones such as 2,2',4,4'-tetrahydroxybenzophenone, 3,4,5,2',3',4'-hexahydroxybenzophenone and 4,4'-dihydroxybenzophenone; benzothiazinone analogs such as 2-amino-4H-
  • antioxidants are described in more detail in the literature, such as in Goodman and Gilman, The Pharmacological Basis of Therapeutics (9th Edition), McGraw-Hill, 1995; and The Merck Index on CD-ROM, Thirteenth Edition; and in STN Express, file phar and file registry.
  • the antioxidants are ascorbic acid, vitamin E, apocynin, hydralazine compounds or superoxide dismutase mimetics.
  • chemotherapeutic agent is used herein to make reference to antiproliferative agents, antimetastatic agents of antiangiogenic agents.
  • Antiproliferative agent is understood herein as an agent that is capable of promoting cell death and which has the capacity for reducing growth, stopping growth or destroying cells and, in particular, cells that proliferate quickly and, even more in particular, tumour cells.
  • Cell death may be caused by any mechanism such as, for example, apoptosis, although it is not limited to this cause, by inhibition of metabolism, interference with the cytoskeleton organization or the chemical modification of DNA. It comprises any agent including small sized organic molecules, peptides or oligonucleotides.
  • Antiproliferative agents include but are not limited to (i) antimetabolites such as folic acid antimetabolites (aminopterin, denopterin, methotrexate, edatrexate, trimetrexate, nolatrexed, lometrexol, pemetrexed, raltitrexed, piritrexim, pteropterin, leucovorin, 10-propargyl-5,8- dideazafolate (PDDF, CB3717)), purine analogs (cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine) and pyrimidine analogs (capecitabine, cytarabine or ara-C, decitabine, fluorouracil, 5-fluorouracil, doxifluridine, floxuridine and gemcitabine) (ii) natural products, such as antitumor antibiotics and mitotic inhibitors such vinca alkaloids such as vindes
  • NSC 33410 and allocolchicine (NSC 406042); halichondrin B (NSC 609395); dolastatin 10 (NSC 376128); maytansine (NSC 153858); rhizoxin (NSC 332598); epothilone A, epothilone B; discodermolide; estramustine; nocodazole; (iii) hormones and antagonist thereof, such tamoxifen, toremifene, anastrozole, arzoxifene, lasofoxifene, raloxifene, nafoxidine, fulvestrant, aminoglutethimide, testolactone, atamestane, exemestane, fadrozole, formestane, letrozole, goserelin, leuprorelin or leuprolide, buserelin, histrelin, megestrol and fluoxymesterone; (iv) biological agents, such as viral
  • antiangiogenic agent is understood as a chemical or biological substance which inhibits or reduces the formation of new blood vessels, i.e., angiogenesis.
  • Antiangiogenic agents that can be conjugated with the polymer of the invention include, without limitation, an antiangiogenic agent selected from the group of paclitaxel, 2- methoxyestradiol, prinomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC- 1088, dextromethorphan acetic acid, dimethylxanthenone acetic acid, endostatin, IM- 862, marimastat, penicillamine.
  • PTK787/ZK 222584 R Pi.4610, squalamine lactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291 , SU6668, anti-VEGF antibodies, Medi-522 (Vitaxin I I ), CAI, interleukin 12, IM862, amiloride, angiostatin, KI-3 angiostatin, KI-5 angiostatin, Captopril, DL-alpha- difluoromethylornithine, DL-alpha-difluoromethylornithine HCI, endostatin, fumagillin, herbimycin A, 4-Hydroxyphenylretinamide, juglone, laminin, lam in in hexapeptide, laminin pentapeptide, lavendustin A, medroxyprogesterone, minocycline, placenta rib
  • Antimetastatic agent is understood as a chemical or biological substance which inhibits or reduces metastasis, i.e., the distance propagation, fundamentally by the lymphatic or blood stream, of the cancer causing cells, and the growth of new tumors in the destination sites of said metastasis.
  • Antimetastatic agents that can be conjugated with the polymer of the invention include, without limitation, any cytotoxic agent capable of acting as an antimetastatic agent , such as alkylating agents, antimetabolites such as 5-fluorouracil, permetrexed (MTA) , raltitrexed (TDX), platinum cytotoxic agents such as cisplatin or oxaliplatin, topoisomerase inhibitors, antimicrotubule agents, anthracyclines, plant alkaloids, GTPase inhibitors, angiogenesis inhibitors, matrix metalloproteinase inhibitors, inhibitors of the cell cycle regulating kinases, such as the eye1 in-dependent kinases and cyclin (CDK) inhibitors, such as palbociclib or dinaciclib, Wnt signaling inhibitors, inhibitors of the E2F transcription factor, histone deacetylase inhibitors, AKT kinase or ATPase inhibitors.
  • tyrosine kinase inhibitor is used herein as substances that block the action of enzymes tyrosine kinases.
  • Tyrosine kinases are a part of many cell functions, including cell signaling, growth, and division. These enzymes may be too active or found at high levels in some types of cancer cells, and blocking them may help keep cancer cells from growing.
  • Tyrosin kinase inhibitors can block one type of tyrosine kinase or more than one type.
  • tyrosin kinase inhibitors include without limitation axitinib, bosutinib, dasatinib, erlotinib, imatinib, nilotinib, pazotanib, imatinib, gefitinib, lapatinib, sunitinib, sorafenib, neratinib, vatalanib, genistein and herbimycin.
  • mitotic inhibitors make reference to drugs that inhibit cell division or mitosis.
  • Mitotic inhibitors bind to tubulin and inhibit its polymerization into microtubules.
  • mitotic inhibitors include without limitation vincristine, eribulin, paclitaxel, paclitaxel protein-bound, docetaxel, estramustine, etoposide, ixabepilone, cabazitaxel, vincristine liposome, vinorelbine, vinblastine, teniposide.
  • exosome-release modulator makes reference to drugs able to inhibit or attenuate exosome production by cancer cells. This exosomes may be involved in transporting oncoproteins and other immune suppressive molecules in order to promote tumorigenesis and metastasis. Exosome-release modulators can exercise their action by targeting microtubules assembly and stability and endosomal-sorting pathway or by using inhibitors of the proton pump. Examples of exosome-release modulators include without limitation ketotifen, Cl-amidine, bisindolylmaleimide-l, imipramine, Y27632, calpeptin, Mbq ⁇ , cytochalasin D and chlorpromazine.
  • imaging agent and “contrast agent”, are used herein interchangeably and refer to a biocompatible compound, the use of which facilitates the differentiation of different parts of the image, by increasing the contrast between those different regions of the image.
  • contrast agents thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging).
  • Suitable contrast agent include, without limitation, contrast agents for Radionuclide imaging, for computerized tomography, for Raman spectroscopy, for Magnetic resonance imaging (MRI) and for optical imaging.
  • Contrast agents for radionuclide imaging include iodine 123, technicium 99, indium 11 1 , rhenium 188, rhenium 186, copper 67, iodine 131 , yttrium 90, iodine 125, astatine 21 1 , gallium 67, iridium 192, cobalt 60, radium 226, gold 198, cesium 137 and phosphorus 32 ions.
  • fluorogenic agents include gadolinium and renographin.
  • paramagnetic ions include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (H)3 copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holraium (III) and erbium (III) ions.
  • Contrast agents for optical imaging include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye and the various other fluorescent compounds disclosed herein.
  • Contrast agent for magnetic resonance imaging apparatus gadolinium chelates, manganese chelates, chromium chelates, 19F and iron particles.
  • MRI contrast agents include complexes of metals selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).
  • targeting motif refers to a molecule which has affinity, or binds to a molecule on the surface of a target cell, for example a targeting moiety functions as an agent that homes in on or preferentially associates or binds to a particular tissue, cell type, receptor, infecting agent or other area of interest.
  • Examples of a targeting moiety include, but are not limited to, an antibody, an antigen binding fragment of an antibody, an aptamer, an antigen, a ligand, a receptor, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative, a hormone, e.g., estradiol or histamine, a hormone-mimic, e.g., morphine, or other compound having binding specificity for a cellular target.
  • a targeting moiety include, but are not limited to, an antibody, an antigen binding fragment of an antibody, an aptamer, an antigen, a ligand, a receptor, one member of a specific binding pair, a polyamide including a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a steroid or steroid derivative
  • At least one occurrence of A in the conjugate of the invention is a curcuminoid, N-acetylgalactosamine-4-sulfatase, N-acetylgalactosamine-6- sulfatase or Chondroitin ABC lyase and the ROCK inhibitor is selected from the group consisting of Fasudil, Thiazovivin, Ripasudil and Netersudil.
  • curcumin means one or more curcuma derived components comprising natural curcuminoids, such as curcumin, bisdemethoxycurcumin, demethoxycurcumin and/or synthetically derived bis-o-demethyl curcumin and/or other demethylated curcuminoids or derivatives.
  • curcuminoid encompasses natural curcumin (diferuloylmethane, feruloyl (1 E,6E)-1 ,7-bis (4-hydroxy-3-methoxyphenyl)-1 ,6-heptadiene-3,5-dione) and structurally-derived compounds.
  • Curcuminoids contains three major derivative coumpounds: curcumin (diferuloylmethane, curcumin I, CAS number: 458-37-7), demethoxycurcumin (4-hydroxycinnamoyl(feroyl)methane, curcumin II, CAS number: 24939-17-1), and bisdemethoxycurcumin (Bis(4-hydroxycinnamoyl)methane, curcumin III, CAS number: 24939-16-0).
  • curcumin diiferuloylmethane, curcumin I, CAS number: 458-37-7
  • demethoxycurcumin (4-hydroxycinnamoyl(feroyl)methane, curcumin II, CAS number: 24939-17-1
  • bisdemethoxycurcumin Bis(4-hydroxycinnamoyl)methane, curcumin III, CAS number: 24939-16-0.
  • Those compounds have the formula as follows: I (R
  • Curcuminoids can exist in at least two tautomeric forms, keto and enol. They may differ from curcumin by the number of ethylene units (one or two), the number of conjugated aromatic rings (one or two), the number of phenol moieties on each ring (one or two), the number and nature of additional substituents (usually alkyloxy derivatives, methoxy in curcumin) or by a combination of the foregoing.
  • N-acetylgalactosamine-4-sulfatase also known as chondroitinase, arylsulfatase B, acetylgalactosamine 4-sulfatase or N-acetylgalactosamine 4-sulfate sulfohydrolase within the context of the present invention refers to an enzyme with systematic name N-acetyl-D-galactosamine-4-sulfate 4-sulfohydrolase. This enzyme catalyses the hydrolysis of the 4-sulfate groups of the N-acetyl-D-galactosamine 4- sulfate units of chondroitin sulfate and dermatan sulfate. It also acts on N- acetylglucosamine 4-sulfate.
  • N-acetylgalactosamine-6-sulfatase also known as chondroitin sulfatase, chondroitinase, galactose-6-sulfate sulfatase, acetylgalactosamine 6-sulfatase, N- acetylgalactosamine-6-sulfate sulfatase, and N-acetylgalactosamine 6-sulfatase, refers to an enzyme that catalyzes the chemical reaction of cleaving off the 6-sulfate groups of the N-acetyl-D-galactosamine 6-sulfate units of the macromolecule chondroitin sulfate and, similarly, of the D-galactose 6-sulfate units of the macromolecule keratan sulfate. This enzyme participates in glycosaminoglycan degradation and degradation of glycan structures.
  • chondroitin ABC lyase also known as chondroitinase, chondroitin ABC eliminase or chondroitinase ABC makes reference to an enzyme which catalyses the the degradation of polysaccharides containing 1 ,4-beta-D-hexosaminyl and 1 ,3-beta-D- glucuronosyl or 1 ,3-alpha-L-iduronosyl linkages to disaccharides containing 4-deoxy- beta-D-gluc-4-enuronosyl groups.
  • This enzyme acts on chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate.
  • Thiazovivin is used to refer to the “crude thiazol surviving” N-benzyl-2- (pyrimidin-4-ylamino) thiazole-4-carboxamide.
  • Thiazovivin is a selective small molecule inhibitor of Rho-associated Kinase (ROCK). Thiazovivin promotes survival and cloning efficiency of human pluripotent stem cells after enzymatic dissociation into single cells. It has been shown to increase reprogramming efficiency when combined with inhibitors of the TGF-b and MEK signaling pathways.
  • ROCK Rho-associated Kinase
  • Ripasudil or “K-115” ((S)-4-Fluoro-5-((2-methyl-1 ,4-diazepan-1- yl)sulfonyl)isoquinoline), as used herein, is a derivative of fasudil. Ripasudil is a rho kinase inhibitor drug.
  • Netarsudil ([4-[(2 ⁇ S ⁇ )-3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2- yl]phenyl]methyl 2,4-dimethylbenzoate) , refers to a Rho kinase inhibitor with norepinephrine transport inhibitory activity.
  • At least one occurrence of A is Fasudil.
  • Fasudil an isoquinoline derivative 1-(5-isoquinolinesulfonyl)homopiperazine and its derivatives, hydroxyl-fasudil and dimethyl-fasudil are known in the art, and described, e.g., in U.S. Pat. No. 4,678,783. Fasudil and the fasudil derivatives may be used in their free forms or as salts thereof.
  • the salt is a non-toxic salt which is pharmacologically accepted and may be formed by addition of an acid.
  • the acid there may be mentioned such inorganic acids as hydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid and such organic acids as acetic acid, citric acid, tartaric acid, lactic acid, succinic acid, fumaric acid, maleic acid and methanesulfonic acid.
  • the salt is a hydrochloride, hydrochloride hemihydrate, hydrochloride hydrate, hydrochloride trihydrate, phosphate monobase, phosphate dibase, phosphate, hydrogen sulfate, sulfate, mesylate, ethylsulfonate, maleate, fumarate, or tartrate.
  • the fasudil or fasudil derivative is in form of a hydrochloride, hydrochloride hemihydrates or hydrochloride hydrate, as, e.g., described in EP 1 110 553 and U.S. Pat. No. 5,942,505.
  • fasudil and its derivatives may be used in form of their active metabolites formed in vivo. Such active metabolites are known in the art.
  • fasudil or its derivative is in the form of a hydrochloride.
  • At least one occurrence of A is chemotherapeutic agent which belongs to the family of cyclin-dependent kinase (CDK) inhibitors.
  • CDK cyclin-dependent kinase
  • a cyclin-dependent kinase (CDK) inhibitor can refer to an agent that downregulates, decreases or suppresses the amount and/or activity of cyclin-dependent kinase.
  • Suitable CDK inhibitors for use in the present invention include, without limitation, dinaciclib, palbociclib, ribociclib, abemaciclib, alvociclib, seliciclib, and the like.
  • the CDK inhibitor is dinaciclib or palbociclib, more preferably dinaciclib.
  • At least one occurrence of A is selected from the group consisting of fasudil and dinaciclib.
  • the polymeric chain of the conjugate of the invention has: a) a molecular weight between 3,000 and 300,000 Da, wherein the molecular weight is preferably measured by size exclusion chromatography coupled with Multi-angle light scattering technique.
  • the polymeric chain of the conjugate of the invention has a degree of polymerization comprised between 20 and 2000, wherein the degree of polymerization is preferably measured by 1 H-NMR spectroscopy.
  • the polymeric chain of the conjugate of the invention has a molecular weight between In 1-300KDa, preferably between 3 and 100 kDa and even more preferably between 5-50 kDa.
  • the molecular weight is measured by size exclusion chromatography coupled with Multi-angle light scattering technique.
  • the degree of polymerization is measured by 1 H-NMR spectroscopy
  • molecular weight relates to the average molar mass of a molecule. Unlike small molecules, the molecular weight of a polymer is not one unique value. Rather, a given polymer will have a distribution of molecular weights depending for example on the way the polymer is produced. Therefore, as it is used herein, the term molecular weight for polymers refers to the distribution of molecular weight, or of the average molecular weight. Methods to determine the molecular weight are known by the skilled person and include, without limitation, 1 H-NMR and size exclusion chromatography coupled with Multi-angle light scattering.
  • the conjugate of the invention or any embodiment thereto has a weight ratio between A and the entire conjugate comprised between 1 to 70% w/w, preferably 1 to 50% w/w, even more preferably 1 to 25% w/w.
  • L is a charged amino acid with a metallic counterion, preferably a sodium cation.
  • the invention in a second aspect relates to a pharmaceutical, hereinafter pharmaceutical composition of the invention, comprising at least one conjugate as defined in the first aspect of the invention and at least one pharmaceutically acceptable excipient.
  • an appropriate amount of the conjugate of the invention can be formulated with pharmaceutically acceptable excipients and/or carriers to obtain a pharmaceutical composition.
  • pharmaceutical composition refers to a formulation which has been adapted to administer a predetermined dose of one or several therapeutically useful agents to a cell, a group of cells, an organ, a tissue or an animal in which a direct or indirect therapeutic effect of the related conjugate is sought.
  • the pharmaceutical composition of the invention contains a pharmaceutical effective amount of a composition, which is understood herein as an amount capable of providing a therapeutic effect, and which can be determined by the person skilled in the art by commonly used means.
  • the amount of the conjugate of the invention will vary depending upon the subject and the particular mode of administration.
  • dosages may also be determined with guidance from Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.
  • the pharmaceutical compositions are prepared by conventional means with one or more pharmaceutically acceptable excipients.
  • “Pharmaceutically acceptable excipient” is understood as a therapeutically inactive substance to be used for incorporating the active ingredient and which is acceptable for the patient from a pharmacological toxicological point of view and for the pharmaceutical chemist who manufactures it from a physical/chemical point of view with respect to the composition, formulation, stability, acceptation of the patient and bioavailability.
  • compositions can be prepared by means of the conventional methods known in the state of the art ("Remington: The Science and Practice of Pharmacy", 20th edition (2003) Genaro A.R., ed., Lippincott Williams & Wilkins, Philadelphia, US).
  • the pharmaceutical composition may need the use of carriers for its delivery.
  • Suitable pharmaceutical carriers are known in the art and are preferably those listed in the US or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
  • the composition if desired, can also contain minor amounts of pH buffering agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E W Martin.
  • Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic conjugate preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
  • the pharmaceutical composition of the invention may be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as lyophilized preparations, liquids solutions or suspensions, injectable and infusible solutions, etc.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • a composition that includes a conjugate according to the invention can be delivered to a subject by a variety of routes.
  • routes include intrastriatal, intracerebroventricular, intrathecal, intraparenchymal (e.g., in the striatum), intranasal, ocular delivery, or topical administration.
  • the composition can also be delivered systemically, e.g., by intravenous, subcutaneous or intramuscular injection, which is particularly useful for delivery of the conjugates to peripheral neurons.
  • intraventricular administration may also be adequate.
  • a preferred route of delivery is directly to the brain, e.g., into the ventricles or the hypothalamus of the brain, or into the lateral or dorsal areas of the brain.
  • the pharmaceutical composition of the invention may comprise a plurality of different conjugates, wherein the different conjugates comprise different drugs, targeting moieties of probes for diagnosis.
  • the pharmaceutical compositions may comprise at least 2, at least 3, at least 4, at least 5, at least 6 and more different conjugates comprising each a different drug, targeting moiety or probe for diagnosis.
  • the conjugates are formulated in accordance with standard procedure as a pharmaceutical composition adapted for delivered administration to human beings and other mammals.
  • compositions for intravenous or intraventricular administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, gel, polymer, or sustained release formulation.
  • the composition can be formulated with traditional binders and carriers, as would be known in the art.
  • Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals.
  • Various delivery systems are known and can be used to administer a therapeutic of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.
  • therapeutics containing the conjugates of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, thriethylamine, 2-ethylamino ethanol, histidine, procaine or similar.
  • the composition includes, for example, a liposome as described, for example, in U.S. Pat. No. 6,372,250 (Pardridge), and a pharmaceutically acceptable carrier.
  • Liposomes as described herein can deliver biologically active agents across the blood-brain barrier, followed by expression in the brain.
  • the pharmaceutical composition of the invention further comprises stem cells, and more particularly ependymal stem/progenitor cell (NPCs) derived from spinal cords, and mature neurons.
  • stem cells and more particularly ependymal stem/progenitor cell (NPCs) derived from spinal cords, and mature neurons.
  • NPCs ependymal stem/progenitor cell
  • stem cells refers to biological cells that can differentiate into other types of cells and can divide to produce more of the same type of stem cells.
  • stem cells In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues.
  • ependymal progenitor/stem cells are referred to multipotent stem cells found in the surrounding the ependymal canal of the spinal cord.
  • the pharmaceutical composition of the invention further comprises an additional drug.
  • Suitable drugs to combine with the conjugate of the invention in the same pharmaceutical composition are those already defined in the first aspect of the invention when describing the possible active compounds linked to the polymeric chain of the conjugate of the invention. Therefore, the descriptions and examples of the active compounds already mentioned when defining the first aspect of the invention are equally applicable to the additional drug that is combined with the conjugate of the invention in the same pharmaceutical composition as described in the present embodiment.
  • suitable active agents or drugs to combine with the conjugate of the invention in the same pharmaceutical composition are ROCK inhibitors, axonal growth promoters, anti inflammatory agents, glial scar disruptors, neuroprotective drugs, antioxidative agents, chemotherapeutic agents, tyrosine kinase inhibitors, cell-cycle inhibitors, exosome- release modulators and combinations thereof.
  • the additional drug present in the pharmaceutical composition of the invention together with the conjugate of the invention is a chemotherapeutic agent.
  • chemotherapeutic agent is used herein to refer to antiproliferative agents, antimetastatic agents or antiangiogenic agents. Suitable examples of chemotherapeutic agents have been provided above in the present application when defining the first aspect of the invention and are equally applicable to the present embodiment.
  • antiproliferative agents include but are not limited to (i) antimetabolites such as folic acid antimetabolites (aminopterin, denopterin, methotrexate, edatrexate, trimetrexate, nolatrexed, lometrexol, pemetrexed, raltitrexed, piritrexim, pteropterin, leucovorin, 10-propargyl-5,8-dideazafolate (PDDF, CB3717)), purine analogs (cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine) and pyrimidine analogs (capecitabine, cytarabine or ara-C, decitabine, fluorouracil, 5-fluorouracil, doxifluridine, floxuridine and gemcitabine) (ii) natural products, such as antitumor antibiotics and mitotic inhibitors such vinca alkaloids
  • NSC 33410 and allocolchicine (NSC 406042); halichondrin B (NSC 609395); dolastatin 10 (NSC 376128); maytansine (NSC 153858); rhizoxin (NSC 332598); epothilone A, epothilone B; discodermolide; estramustine; nocodazole; (iii) hormones and antagonist thereof, such tamoxifen, toremifene, anastrozole, arzoxifene, lasofoxifene, raloxifene, nafoxidine, fulvestrant, aminoglutethimide, testolactone, atamestane, exemestane, fadrozole, formestane, letrozole, goserelin, leuprorelin or leuprolide, buserelin, histrelin, megestrol and fluoxymesterone; (iv) biological agents, such as viral
  • Antiangiogenic agents that can be incorporated to the pharmaceutical composition of the invention include, without limitation, an antiangiogenic agent selected from the group of paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic acid, dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, penicillamine.
  • an antiangiogenic agent selected from the group of paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY 12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic acid, dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, penicillamine.
  • PTK787/ZK 222584 R Pi.4610, squalamine lactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3, neovastat, BMS-275291 , SU6668, anti-VEGF antibodies, Medi-522 (Vitaxin I I ), CAI, interleukin 12, IM862, amiloride, angiostatin, KI-3 angiostatin, KI-5 angiostatin, Captopril, DL-alpha-difluoromethylornithine, DL-alpha-difluoromethylornithine HCI, endostatin, fumagillin, herbimycin A, 4-Hydroxyphenylretinamide, juglone, laminin, lam in in hexapeptide, laminin pentapeptide, lavendustin A, medroxyprogesterone, minocycline, placenta
  • Antimetastatic agents that can be added to the pharmaceutical composition of the invention together with the conjugate of the invention include, without limitation, any cytotoxic agent capable of acting as an antimetastatic agent , such as alkylating agents, antimetabolites such as 5-fluorouracil, permetrexed (MTA) , raltitrexed (TDX), platinum cytotoxic agents such as cisplatin or oxaliplatin, topoisomerase inhibitors, antimicrotubule agents, anthracyclines, plant alkaloids, GTPase inhibitors, angiogenesis inhibitors, matrix metalloproteinase inhibitors, inhibitors of the cell cycle regulating kinases, such as the eye1 in-dependent kinases and cyclin (CDK) inhibitors inhibitors such as dinaciclib or palbociclib, Wnt signaling inhibitors, inhibitors of the E2F transcription factor, histone deacetylase inhibitors, AKT kinase or ATPase inhibitors.
  • the invention relates to the conjugate or the pharmaceutical composition of the invention for use as a medicament.
  • “Medicament” is understood as a pharmaceutical composition comprising the conjugate of the invention alone or in combination with a compound useful in the treatment of neurodegenerative diseases, cancer and related conditions such as angiogenesis or metastasis, or with a compound useful in the treatment of a disease associated with inflammation.
  • the conjugate and the pharmaceutical composition of the invention are used in the prevention and/or treatment of a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • Another aspect relates to the use of the conjugate or the pharmaceutical composition of the invention in the manufacture of a medicament for the prevention and/or treatment of a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing.
  • Another aspect relates to a method for the prevention and/or treatment of a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing, comprising administering to a subject in need thereof the conjugate or the pharmaceutical composition of the invention.
  • a condition selected from a neurodegenerative disorder, central nervous system traumatic injuries, neurological disease, disorder related to aging, neuro-inflammation, demyelinating disorder, multiple sclerosis, amyotrophic lateral sclerosis disease, spinal cord injury, immune disorder, inflammatory disorders, rare diseases and wound healing
  • prevention relates to the capacity to prevent, minimize or hinder the onset or development of a disease or condition, in particular cancer, before its onset. Prevention may be complete (e.g. the total absence of pathological condition in a subject). The prevention may also be partial, such that for example the occurrence of pathological condition in a subject is less than that which would have occurred without the present invention. Prevention also refers to reduced susceptibility to a clinical condition.
  • treatment comprises any type of therapy, which aims at terminating, preventing, ameliorating and/or reducing the susceptibility to a clinical condition as described herein.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal, including a human.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • It covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) increasing survival time; (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (d) inhibiting the disease, i.e., arresting its development (e.g., reducing the rate of disease progression); and (e) relieving the disease, i.e., causing regression of the disease.
  • the neurodegenerative diseases may be selected from the list that comprises, without being limited thereto, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia, post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodic limb movement pathologies, restless legs syndrome, attention deficit hyperactivity disorders, Huntington's disease, progressive supranuclear palsy, Pick's disease, fronto-temporal dementia and neuromuscular diseases.
  • central nervous system traumatic injuries are spinal cord injury, and traumatic brain injury,
  • the invention relates to the conjugate or pharmaceutical composition according to any of the precedent aspects for use in the treatment of spinal cord injury.
  • the invention relates to the use of the conjugate or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of spinal cord injury.
  • the invention in another particular embodiment, relates to a method for the treatment of spinal cord injury comprising administering to a subject in need thereof the conjugate or pharmaceutical composition of the invention.
  • spinal cord injury refers to a clinical state that presents peripheral motor, sensory and autonomous nervous system paralysis below the site of injury to the spinal cord parenchyma from trauma such as dislocation-fracture of the spine as a result of, for example, a traffic accident or a fall from a high place.
  • the invention relates to a method for the treatment of traumatic brain injury comprising administering to a subject in need thereof the conjugate or pharmaceutical composition of the invention.
  • TBI traumatic brain injury
  • causes include falls, vehicle collisions and violence.
  • conjugate and the pharmaceutical composition of the invention are used in the treatment and/or prevention of cancer.
  • the invention relates to the use of the conjugate or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment and/or prevention of cancer.
  • the invention in another aspect, relates to a method for the treatment and/or prevention of cancer comprising administering to a subject in need thereof the conjugate or pharmaceutical composition of the invention.
  • cancer refers to a broad group of diseases involving unregulated cell growth and which are also referred to as malignant neoplasms.
  • the term is usually applied to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighboring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body.
  • tumours are classified as being either benign or malignant: benign tumours are tumours that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumours are tumours that are capable of spreading by invasion and metastasis.
  • Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. Cancers usually share some of the following characteristics: sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and eventually metastasis. Cancers invade nearby parts of the body and may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers are classified by the type of cell that the tumour cells resemble, which is therefore presumed to be the origin of the tumour.
  • cancer or tumor examples include without limitation, breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, hepatobiliary and liver tumors.
  • tumors whose clinical outcome may be predicted with the methods of the invention include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangio sarcoma, hematoma, hepatoblastoma, leukaemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma.
  • the tumor/cancer is selected from the group of acrallentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytictumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, germ cell tumors, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma,
  • the tumor/cancer include intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, glioblastoma, small cell cancer, and non-small cell cancer, preferably non-small cell lung cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer and breast cancer.
  • the cancer is selected from lung cancer, colon cancer, melanoma, pancreatic cancer, prostate cancer, glioma, bladder cancer, ovarian cancer, hepatobiliary cancer, breast cancer and lymphoma.
  • the cancer is a primary tumor or cancer metastasis.
  • primary tumor refers to a tumor that originated in the location or organ in which it is present and did not metastasize to that location from another location.
  • cancer is understood as the propagation of a cancer from the organ where it started to a different organ. It generally occurs through the blood or lymphatic system.
  • the cancer cells spread and form a new tumor, the latter is called a secondary or metastatic tumor.
  • the cancer cells forming the secondary tumor are like those of the original tumor. If a breast cancer, for example, spreads (metastasizes) to the lung, the secondary tumor is formed of malignant breast cancer cells.
  • the disease in the lung is metastatic breast cancer and not lung cancer.
  • solid tumors and their metastasis in particular breast cancer, prostate cancer, lung cancer, ovarian cancer, glioblastoma, melanoma, hepatocarcinoma, renal carcinomas.
  • the conjugate or pharmaceutical composition of the invention can be used for the treatment and/or prevention of ER positive (ER+) breast cancer, ER negative (ER- ) breast cancer, PR positive (PR+) breast cancer, PR negative (PR-) breast cancer, HER2 positive (HER2+) breast cancer (cancer over-expressing HER2), HER2 negative (HER2-) breast cancer (cancer expressing normal levels of HER2 or under-expressing HER2 or not expressing a detectable level of HER2), hormone receptor negative breast cancer, i.e.
  • breast cancer with neither of estrogen nor progesterone receptors abbreviated by ER-/PR- breast cancer
  • triple negative breast cancer i.e. breast cancer with neither of estrogen nor progesterone receptors and with normal expression/under-expression (or with the absence of detectable level of expression) of HER2 (abbreviated by ER-/PR-/HER2- breast cancer), luminal subtype A breast cancer, luminal subtype B breast cancer or normal-like breast cancer.
  • the pharmaceutical composition for use in the treatment of the conditions defined herein may include a composition comprising the conjugate of the invention or a pharmaceutical composition comprising the conjugate of the invention together with an additional drug as mentioned when describing the pharmaceutical compositions of the invention.
  • the additional drug present in the pharmaceutical composition of the invention exerts a therapeutic effect different from the therapeutic effect exerted by the conjugate of the invention.
  • the additional drug and the conjugate of the invention present in the pharmaceutical composition synergically interact to achieve the desired therapeutic effect.
  • the pharmaceutical composition of the invention further comprising and additional drug is used in the treatment of cancer and the additional drug is a chemotherapeutic agent or an agent directed to alleviate of treat a condition associated with cancer.
  • the chemotherapeutic agent present in the pharmaceutical composition of the invention for use in the treatment of cancer synergically interact with the conjugate of the invention to achieve the desired therapeutic effect.
  • the invention also provides a method for the preparation of the conjugates of the invention, the method comprising the steps of:
  • x, y, R 1 , R 2 , R 3 , R 4 , L and Z are as defined above in respect of the conjugates of the invention and wherein P is a first reactive group and with a compound having the structure A-S-S-Q, wherein A is as defined in claim 1 and wherein Q is a second reactive group which can react with P to form a covalent bond, said contacting being carried out under conditions adequate for the formation of the bond between A and B and
  • the covalent bond which is formed following the reaction of the P and Q groups is an amide group or an ester group. In a more preferred embodiment, the covalent bond which is formed following the reaction of the P and Q groups is an amide group. In another embodiment, the amide group or the ester group is formed by a reaction between an activated carboxyl group and an amine group or a hydroxyl group, respectively. In another embodiment, the amide group is formed by a reaction between an activated carboxyl group and an amine group. In this case, the P and Q groups are, respectively, an activated carboxyl group and an amine group. In another embodiment, the P and Q groups are, respectively, an amine group and an activated carboxyl group.
  • Primary amines are preferred over secondary amines for the formation of amide linkages.
  • Methods for the formation of amide bonds are known in the art.
  • the coupling reaction may take place in the presence of an activating agent and/or a coupling reagent.
  • Suitable activating and coupling agents for amide bond formation are known in the art and include those normally used in peptide synthesis.
  • Such activating agents are normally designed to react with a carboxylic acid to form an activated compound which then reacts with an amine to form the desired amide bond.
  • Such activating agents can also be used in the reaction of carboxylic acids with hydroxyls to form the desired ester bond, preferably with a primary hydroxyl group.
  • Suitable activating agents include 1-hydroxybenzotriazole (HOBt), HOBt-6- sulfonamidomethyl resin HCI, HOOBt (hydroxy-3, 4-dihydro-4-oxo-1 , 2, 3-benzotriazine), HOSu (N-hydrosxysuccinimide), HOAt (1-hydroxy-7-aza-1 H-benzotriazole), ethyl 2- cyano-2- (hydroxyimino)acetate and propylphosphonic anhydride (T3P).
  • HOBt 1-hydroxybenzotriazole
  • HOBt-6- sulfonamidomethyl resin HCI HOOBt (hydroxy-3, 4-dihydro-4-oxo-1 , 2, 3-benzotriazine)
  • HOSu N-hydrosxysuccinimide
  • HOAt 1-hydroxy-7-aza-1 H-benzotriazole
  • ethyl 2- cyano-2- (hydroxyimino)acetate propyl
  • Suitable coupling agents include carbodiimides such as DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide) and EDC HCI (N-(3- dimethylaminopropyl)-N’- ethylcarbodiimideHCI).
  • carbodiimides such as DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide) and EDC HCI (N-(3- dimethylaminopropyl)-N’- ethylcarbodiimideHCI).
  • Suitable coupling reagents include BOP ((benzotriazol-1-yloxy)- tris(dimethylamino)phosphonium hexafluorophosphate), PyPOB (benzotriazol-1-yl- oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (bromo-tripyrrolidino- phosphonium hexafluorophosphate), PyAOP ((7-azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyOxim ((ethyl cyano(hydroxyimino)acetato-02)tri-1- pyrrolidinylphosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy)- l,2,3-benzotriazin-4(3H)-one), TBTU (2-(1 H-benzotriazole-1-yl)-
  • the Q reactive group can be directly connected to the disulfide group or can be connected by a spacer region.
  • Suitable spacer regions that can be used in the present invention include, without limitation, a short spacer group such as (C1- C12)alkyl, (C2-C12)alkenyl, aryl, aralkyl, heteroaryl or a long-chain spacer group may be chosen from polymeric radicals of formula -W-(CH 2 -) k -W'-, wherein k is an integer between 13 and 3000, and W and W are reactive groups able to react with amino, carboxyl, hydroxy or thio groups and wherein one or more of the non-adjacent CH2 groups may independently be replaced by aryl, heteroaryl, -CH-CH-, -C-C-, or a hydrophilic (or polar) group selected from -0-, -CO-, -CO-O-, -O-CO-, -NR'-,
  • Preferred spacer groups include hydrophilic polymeric radicals (with an increased affinity for aqueous solutions), i.e. polymers containing repeating structural units that comprise one or more of the above hydrophilic (or polar) groups in their alkylene backbone.
  • hydrophilic polymeric radicals include polyoxy(C2-C3)alkylenes (e.g. polyethylene glycol (PEG) or polypropylene glycol (PPG)), polysaccharides (e.g. dextran, pullulan, chitosan, hyaluronic acid), polyamides (e.g. polyamino acids, semisynthetic peptides and polynucleotides); polysialic acid, polyesters (e.g. polylactide (PLA), polylactid-co-glycolid (PLGA)), polycarbonates, polyethyleneimines (PEI), polyimides, polyvinyl acetate (PVA).
  • PEG polyethylene glycol
  • PPG polypropy
  • a preferred spacer is "PEG” or "polyethylene glycol”, which encompasses any water- soluble poly(ethylene oxide).
  • PEG means a polymer that contains a majority, e.g. >50 percent, of subunits that are -CH2CH2O-.
  • PEGs for use in the present invention may preferably comprise one of the two following structures: "-0(CH2CH20) m -" or "- CH 2 CH 2 (CH 2 CH 2 0) m -CH 2 CH 2 -,” where m is 3 to 3000, and the terminal groups and architecture of the overall PEG may vary. As indicated above, depending on its use, PEG may be in end-capped form.
  • the end capping group is generally a carbon-containing group typically comprised of 1-20 carbons and is preferably alkyl (e.g., methyl, ethyl or benzyl) although saturated and unsaturated forms thereof, as well as aryl, heteroaryl, cyclyl, heterocyclyl, and substituted forms of any of the foregoing are also envisioned.
  • the end capping group is generally a carbon-containing group typically comprised of 1-20 carbon atoms and an oxygen atom that is covalently bonded to the group and is available for covalently bonding to one terminus of the PEG.
  • the group is typically alkoxy (e.g., methoxy, ethoxy or benzyloxy) and with respect to the carbon-containing group can optionally be saturated and unsaturated, as well as aryl, heteroaryl, cyclyl, heterocyclyl, and substituted forms of any of the foregoing.
  • non-end-capped terminus is typically a hydroxyl, amine or an activated group that can be subjected to further chemical modification when PEG is defined as "- CH2CH2CH2(CH2CH20) m -CH 2 CH2-"
  • end-capping group can also be a silane.
  • R4 contains a terminal -COO-, group, P is DMTMM and Q is an amino group.
  • R4 is -CH2-COO-, in which case P is a DMTMM ((4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl-morpholinium chloride)) group and Q is an amino group.
  • Oregon Green was purchased from invitrogen.
  • Deuterated dimethyl sulfoxide (d6-DMSO) and Deuterium oxide were purchased from Deutero GmbH.
  • Ultrapure water with resistivity of 18 MQ.cm was used in all aqueous preparations (Milli-Q® ultrapure).
  • Preparative SEC was performed using either Sephadex® G-25 or Sephadex® LH-20 and were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden).
  • UV-ViS Ultraviolet-Visible spectroscopy
  • UV-VIS measurements were performed using JASCO V-630 spectrophotometer at 25 °C with 1.0 cm matched quartz cells and with spectral bandwidth of 0.5 nm. Determination of total FASUDIL content by UV Spectroscopy. Fasudil or Fasudil derivatives were first prepared for use as calibration standards. A stock solution of Fasudil HCI in MilliQ water was prepared (1 mg/ml_). To obtain a calibration curve samples were diluted using MilliQ water to give a concentration range of 1 ,5-20 pg/mL. The total drug loading of the conjugates (0.5 mg/ml) was determined by measuring the absorbance at 323 nm in milliQ H2O. PGA in the same concentration range as the conjugates analysed (0-5 mg/ml_) was used as blank.
  • Solid-liquid extraction was used to determine the free Fasudil content in the PGA-drug conjugates. 2mg of the conjugates were suspended in MeOH (HPLC grade) and sonicated for 30 minutes. Then, the suspension was filtered (0.22pm) into HPLC vials for analysis. Two reference standards were employed in every experiment.
  • NMR spectra were recorded at 27 °C (300 K) on an Avance III 500 MHz Bruker spectrometer equipped with a 5 mm TBI broadband probe or a 300 UltrashieldTM from Bruker (Billerica MA, USA). Data were processed with the software Mestrenova (Bruker GmbH, Düsseldorf, Germany). Samples were prepared tipically at 5mg/ml in deuterated solvents.
  • MALDI-TOF 1 mI_ of sample was spotted into the MALDI target plate. After droplets were air-dried at room temperature, 0.5 pL of matrix (5 mg/mL CHCA (Sigma) in 0.1 % TFA-ACN/H2O (1 : 1 , v/v)) was added and allowed to air-dry at room temperature.
  • the samples were analyzed in a 4700 Proteomics Analyzer (ABSciex) in positive reflectron mode (3000 shots every position) in a suitable range of mass.
  • the samples were also analyzed in a 4700 Proteomics Analyzer (ABSciex) in linear mode (3000 shots every position) in a suitable range of mass.
  • a matrix sample was analyzed under the same experimental conditions.
  • CD Spectroscopy was performed with a J-815 CD Spectrometer (JASCO Corporation) using a Peltier thermostated cell holder (PTC-423, JASCO Corporation) with a recirculating cooler (JULABO F250, JASCO Corporation).
  • a nitrogen flow ( ⁇ 2.7 L-min 1 ) was lead through the spectrometer and controlled with a nitrogen flow monitor (Afriso Euro-Index).
  • DLS measurements were performed using a Malvern ZetasizerNanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°. Solutions were sonicated for 10 min and allowed to age for the required time, filtered through a 0.45 pm cellulose membrane filter and measured. Size distribution was measured (radius, nm) for each polymer per triplicate with n> 3 measurements.
  • the objective was the conjugation of 10% Fasudil (molar relative to glutamate residues).
  • PGM OO-OH 51 mg, 0.395 mmol
  • DMTMMCI 21.8 mg, 0.079 mmol, 0.2 eq to Glutamic acid units (GAU)
  • GAU Glutamic acid units
  • PGA-O-FAS was obtained via copper catalyzed click-chemistry involving the alkine- fasudil derivative (step iv figure 1) and the azide functionalized polyglutamate (figure 1).
  • step iv figure 1 the alkine- fasudil derivative
  • step iv figure 1 the azide functionalized polyglutamate
  • the conjugate was dissolved in 0.5 M aqueous sodium hydrocarbonate solution and dialyzed against water (membrane cut off 500-1000 Da) for two days. Solution was freeze-dried. The conjugate was dissolved in water and purified with VivaSpin for 4 times in order to removed residual low molecular weight impurities. (235 mg, 84%). 1 H NMR characterization shown in figure 2A.
  • SD rats (-200 g) from Charles River and SD-Tg(GFP)2BalRrrc from Rrrc (University of Missouri Columbia, Columbia, MO, USA) were bred at the Animal Experimentation Unit of the Research Institute Principe Felipe (Valencia, Spain). The maintenance and use of all animals was in accordance with the National Guide for the Care and Use of Experimental Animals Committee (Animal Care Committee of the Research Institute Principe Felipe) (Real Decreto 1201/2005).
  • NPCs or GFP-NPCs were harvested from neonatal (P5-8) Sprague Dawley rats and SD- Tg(GFP)2BalRrrc, respectively.
  • eGFP+/+ homozygote transgenic rats were used for cell transplantation experiments and eGFP-/- rats for in vitro assays.
  • Dorsal root ganglion (DRG) neurons were prepared from adult SD eGFP-/- rats. DRGs were dissected and digested with collagenase (at 100 pg/ml for 3 hours)/ trypsin (at 0.25% for 15 minutes) treatment and then mechanically dissociated (Owen D E and Egerton J, Methods Mol. Biol. 2012, 846, 179e187).
  • DRG neurons were plated into Poly-L-lysine-laminin- coated coverslips at a density of 12.5 x 104 cell/cm 2 .
  • the cultures were pulsed with 5 mM cytosine arabinoside for 2 days.
  • isolated NPCs were plated on 8 day-old DRG neuron culture at a density of 2.5 x 10 4 cells/cm 2 with DRG medium.
  • Cells (NPCs and DRG) were cultured for 72 hours to allow axonal growth. Treatments were performed on 4 days-old neurons cultures or co-cultures.
  • Cell toxicity and viability analysis was performed by DAPI stained nuclei counting in a minimum of 100 cells per condition and replicate. Axonal length was determined using ImageJ software of neurons immunostained with Tau and/or MAP2 (all measurements were carried out at least in three different culture preparations).
  • MDA-MB-231 and MDA-MB-453 cell lines were grown in DMEM/F-12 medium supplemented with 10% v/v fetal bovine serum (FBS). Cells were maintained at 37°C in an atmosphere of 5% CO2 and 95% air and underwent weekly passage. Cells were seeded in P96 plates at 7500 cell/well and 10000 cell/well in the case of MDA-MB-231 cells and MDA-MB-453 respectively, and allowed to adhere for 24h. Then, the medium was replaced by fresh complete medium containing Fasudil or PGA-SS-Fasudil at different concentrations.
  • FBS v/v fetal bovine serum
  • cell viability was measured using the Cell Titer 96 Aqueous non-radioactive cell proliferation assay (Promega, Corporation, Spain, UE) in accordance with the manufacturer’s instructions. The optical density of each well was measured at 490 nm using a Wallac 1420 Workstation (PerkinElmer, MA, USA). Cell viability was expressed as a percentage of the viability of untreated control cells. Three independent assays were performed.
  • HBSS Hank's Balanced Salt Solution
  • the slices were transferred onto the Millicell-CM (Millipore) membranes placed in 6-well plates pre-equilibrated with 1 ml_ of culture medium (pH 7.2, 50% DMEM, 10mM HEPES, 25% HBSS, 25% horse serum (Gibco), 2mmol/L L- glutamine, 5mg/ml_ glucose, 1 % amphotericine B, and 0.4% penicillin/streptomycin).
  • the cultures started to grow in a regular, 25% horse serum medium gradually replaced (from 4th until 7th day) by serum free (SF), defined-solution-based medium.
  • the SF medium contained DMEM/F12, 10mM HEPES, 25% HBSS, 2mmol/L L-glutamine, 5mg/ml_ glucose, 1 % amphotericin B and 0.4% penicillin-streptomycin, N2A (1 : 10; Gibco), and the B27 (1 : 100; Gibco) supplements.
  • the cultures were grown in saturated humid conditions and 5% CO2, at 37°C for 2 weeks [Reynods BA et al. , Nat.
  • Treatments were administered 6 days before the samples were fixed for immunohistochemical analysis (different slices of at least three animals were used per treatment).
  • Intrathecal drug delivery of PGA-SS-F or Fasudil was performed through a catheter introduced immediately bellow the thoracic injured segment (as we previously described in (Alastrue-Agudo, Erceg et al. 2014)).
  • 10 pi of PGA or PGA-SS-F (6mM) to reach a final concentration of 300 mM of free FAS in the CSF was delivered by the catheter and then removed.
  • 48 h after SCI and drug administration animals were sacrificed, and CSF and spinal cord tissue were collected for further analysis.
  • DRG explants were dissected from 5 days-old Sprague-Dawley GFP-/- rats. DRG were placed on poly-L-Lysine/laminin coated coverslips and cultured for 10 days in Neurobasal medium, supplemented with B27, 1 mM NAC, 2mM Glutamax, 10% FBS and 1 % penicillin/streptomycin. 48 h after plating of PGA-SS-F or Fasudil were added to the culture media and the explants were incubated for 8 additional days. Then, tissues were fixed with 4% PFA for immunohistochemically analysis (three different slices for at least three animals were used per experimental condition).
  • SC slices were dissected from 5 days-old Sprague-Dawley GFP-/- rats. SCs were cleaned up from meningeal layers and cut with Mcllwain Tissue Chopper in 350 pm thick in transverse and parasagittal longitudinal plane. Immediately after, the slices were transferred onto Millicell-CM® (Millipore) inserts and placed into 6-wells plates with 1 ml of culture medium (50% MEM, 25% HBSS, 25% Horse serum, 2 mM Glutamax, 1 mM NAC, 0.5% NaHC03 and 1 % penicillin/streptomycin).
  • culture medium 50% MEM, 25% HBSS, 25% Horse serum, 2 mM Glutamax, 1 mM NAC, 0.5% NaHC03 and 1 % penicillin/streptomycin).
  • Total protein was extracted from SC tissues or cell cultures using a lysis buffer containing 50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.02 % NaN3, 0.1 SDS, 1 % NP40, 1 mM EDTA, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mM PMSF, 1 x Protease Inhibitor Cocktail (Roche Diagnostics, San Diego, CA, USA). Protein concentrations of the supernatant were determined using BCA and stored at -80C. Equal amount proteins (30 pg/well) were separated in 10% SDS-PAGE and transferred to a PVDF membrane.
  • the membrane was blocked with 1 % bovine serum albumin in TBS with 0.1 % Tween-20 for 1 h at room temperature and incubated at 4 °C overnight with the following primary antibody solutions: pAKT (1 :1000 4060, Cell Signalling), pGSK3B (1 :1000 8566S, Cell
  • EPAC-2 (1 :1000 sc-28326, Cell Signalling
  • TrkB (1 : 1000 sc-377218, Santa Cruz)
  • Bag-1 (1 :1000 sc-376848, Santa Cruz)
  • pCREB (1 :1000 sc-81486 Santa Cruz
  • Bcl-2 (1 : 1000 2870S, Cell Signalling
  • BDNF (1 :500 sc-655114, Santa Cruz
  • cAMP (1 :500 ab24851 , Abeam
  • caspase-3 (1 :1000 9662S Cell Signalling
  • pERK (1 :1000 4370, Cell Signalling
  • pMYPT (1 : 1000 ABS45 Millipore
  • pmTOR (1 :1000 2971 Cell Signalling).
  • cysts/cavities evaluation every fifth collected section was stained with HE in an automatized station (Autostainer XL Leica) and scanned in an Aperio Versa scanner (Leyca Biosystems). Images of approximately 12 mm length of the SC (including the epicentre of the lesion) were analysed with the Image Scope software. The total cyst/cavity area was normalized to the total analysed spinal cord tissue. Immunofluorescence double staining’s by following the previous detailed methodology was performed using beta- 111 -tu bu I i n , for neuronal fibers detection, GFAP, for glial scar analysis and Olig2 and NeuN, for oligodendrocyte and neurons quantification. All samples were counterstained by incubation with DAPI.
  • Chromatographic analysis was performed using an Exion LC system comprising of a Pump, AC Autosampler, AC Column oven, and AB Sciex QTRAP 4500 (Sciex, Singapore).
  • the initial mobile phase composition was acetonitrile: 0.1 % formic acid (20:80, v/v). All runs were performed using a Phenomenex Luna®-C18 column (30 mm length, 4.6 mm internal diameter, 5 pm particle size), which was maintained at 40 °C.
  • the injection volume was 10 pi, while the flow rate of the mobile phase was set at 0.4 ml/min.
  • Fasudil and ranitidine were detected in positive electrospray mode, with multiple reactions monitoring scan and by following two mass transitions 292.4 m/z 99.2 m/z and 292.4 129.0 m/z for fasudil, and 315.2 m/z 101.9 m/z and 315.2 m/z 130.0 m/z for ranitidine ( Figure 7)).
  • the LC-MS/MS method was developed using an experimental design approach, where the percentage of acetonitrile, percentage of formic acid, and flow rate from LC conditions and five factors from MS (ion spray voltage (3000-5500 V), the temperature of ion source (300-750 °C), curtain gas pressure (20-40 psi), nebulizer (Gas 1) gas pressure (20-60 psi) and auxiliary (Gas 2) gas pressure (40- 80 psi)) were selected as factors that may have a significant effect on ionization efficiency.
  • MS ion spray voltage (3000-5500 V)
  • the temperature of ion source 300-750 °C
  • curtain gas pressure (20-40 psi
  • auxiliary (Gas 2) gas pressure 40- 80 psi)
  • LC-MS Liquid chromatography-mass spectrometry
  • LC-MS/MS conditions were the same as for the analysis of fasudil in plasma stability study. Nevertheless, the developed LC-MS and extraction method was tested again for linearity, selectivity, limit of quantification (LOQ), limit of detection (LOD), recovery and matrix effects since the matrix was changed.
  • LOQ limit of quantification
  • LOD limit of detection
  • LC-MS Liquid chromatography-mass spectrometry
  • the cells were cultured with PGA-SS-FAS at 2 different concentration (50 and 100mM of fasudil). The first sample was obtained after 24h of incubation, while the 3 additional samples after three washing cycles. The cell medium obtained from cell cultures without the treatment with PGA-SS-FAS was obtained in the same way as the previous samples and was used as control sample. 50 mM DTT was added to 200 pL of cell medium samples to induce the release of total FAS from PGA-SS-FAS conjugate. Then, 500 mI of acetonitrile were added to 200 mI of cell medium for protein precipitation, followed by vortex-mixing for 30 s and centrifugation at 11 ,000 rpm for 5 min.
  • Fasudil was attached to the polypeptidic backbone employing different linkers including an amide (PGA-FAS) a carbamate (PGA-O-FAS) and disulphide self-immolative (PGA-SS-FAS) linkers ( Figure 1). Fasudil was modified chemically to introduce the different linkers (PGA-O-FAS and PGA-SS-FAS, (see synthetic methods). Molecular weight for polypeptide backbone was 15000 Da, and the targeted drug molar content was 10%. Synthetic methodology was optimized and proven to be reproducible and scalable (up to g scale) yielding the desired products with high purity.
  • PGA-SS-FAS _ Self-immolative disulphide _ 79 _ 54 _ 7,7 0,1 _ 5 -23,4 determined by UV-Vis. determined by HPLC (UV-Vis) and calculated as % of the total drug loading. c Hydrodynamic radius and z-potential measured via DLS instrumentin 1 mM KCI. Dinaciclib polymer conjugates were also analyzed by NMR (Figs. 27- 29) proving compound identity and supporting stable conjugation. We used UV-Vis to determine TDL, and HPLC to determine free drug content (see Table below). Overall, we obtained conjugates with 3.4-5.2 % modification (w/w) and extremely low content of the free drug (usually below the LOD of HPLC).
  • Example 6 Anticancer activity of PGA-SS-Fasudil and free Fasudil.
  • Example 7 Plasma stability of the PGA-SS-FAS conjugate
  • Example 8 Total fasudil content in cerebral spinal fluid (CSF)
  • the concentration of total fasudil in CSF samples was determined with the LC-MS method previously described 48 h after the treatment with PGA-SS-FAS, was found to be 68.4 ⁇ 23.4 ng/ml which represent less than 1 % of fasudil left in the cerebrospinal fluid compared to the dose administered.
  • Example 9 Total fasudil content in cell medium
  • the concentration of total fasudil in cell medium samples was determined with the LC- MS method previously described. 26 % and 26.6 % of total fasudil was found in the cell medium after 24 h incubation with 100 mM and 50 pM of fasudil (in form of PGA-SS- FAS), respectively. These results suggest that the fasudil conjugate is internalized relatively fast. After 3 washing cycles, the level of total fasudil in the cell medium was below the limit of quantification of the method. Additionally, the chromatograms of control samples (figure 9B) did not show a peak at retention time of fasudil (figure 9A) which confirms the selectivity of the developed method.
  • PSA-NCAM has been considered as a pure neuronal cell marker for those multipotent and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro (Kim DS et al, PLoS One. 2012;7(7):e39715).
  • NCAM-PSA was expressed in all of the tested samples however, CD 184 was not expressed in most of them (Figure 12).
  • the expression of PSA-NCAM significantly decreased in higher passages as is shown in Figure 12 from sample MED 3 ( Figure 12; light grey).
  • NSC population was almost 100% positive for Sox2, Notchl (intracellular activated peptide), Doublecortin (DCX) and nesting in both set of cultures, showing no significant differences depending on the gestational stage ( Figure 13).
  • Notchl intracellular activated peptide
  • DCX Doublecortin
  • the final therapeutic product will be the NSC population pre-conditioned by the treatment with the nanoconjugate PGA-SS-Fasudil 50 mM 24 hours before cell transplantation, as is represented in Figure 14.
  • Expression of neuronal markers PSA-NCAM and CD184 was carried out after conditioning the NSC population by pre-treatment with the nanoconjugate Fasudil-SS- PGA at 50 mM final concentration during 24 hours incubation in growing conditions.
  • the Fasudil nanonjugate F-SS-PGA pre-treatment in the human NSC induces the neuritogenesis and axonal elongation effect as previously found in the rodent cells ( Figure 16, lower panels).
  • this effect due to the effective Rho-inhibitory activity of the nanoconjugate, is maintained in the presence of LPA (lisophosphatidic acid) which mimics the activation of the Rho/Rock signalling during spinal cord injury (figure 16, upper panels).
  • the spinal cord injury was induced by a moderate contusion at the level of the T8 thoracic segment applying a force of 200 kdyn with the Horizon Impactor automated device.
  • a sub-acute phase when the first phase of massive peripheral infiltration has decreased, we transplanted a million cells intramedullary by intraparenchymal injection, distributed in two points, 2mm rostral and caudal to the injury, in the axis axial, and at two points from ventral to dorsal. After transplantation, the animals were treated daily with the cyclosporine immunosuppressant.
  • Nuclear ROCKII signal correlated negatively with estrogen receptor (ER) and progesterone receptor (PR) expression and positively with HER2 overexpression.
  • HER2 activates multiple signaling pathways, including ERK1/2, MAPK, and PI3K/Akt.
  • ERK1/2 is an upstream effector of GTP-activating proteins and guanine exchange factors, which in turn activate ROCK.
  • Example 12 Intrathecal in vivo administration of PGA-SS-F inhibits Rho/Rock activity, triggers neuroprotective and neuroregenerative signalling pathways in acute phase after SCI
  • Example 13 PGA-SS-F promote axonal growth in DRG explants
  • PGA-SS-F increased maximal axonal length and number of axons arising from the explant. Quantification of the mean fluorescence intensity of GAP43 showed significant difference between PGA-SS-F and control and Fasudil treated DRGs ( Figure 21 C).
  • Example 14 PGA-SS-F promotes neuroprotection and neuroregeneration in spinal cord organotypic ex vivo cultures
  • PGA-SS-F promotes length of phosphor neurofilaments in organotypic cultures of neonatal rat spinal cords ( Figures 22A-22E). Lesioned organotypic cultures of longitudinal slices of SC treated with PGA-SS-F showed bundles of fibers crossing the lesion site reconnecting both stumps ( Figure 22F, white arrow).
  • Example 15 PGA-SS-F overcomes neurite retraction following ROCK activation and promotes neuronal fate, axonal growth and neuroplasticity markers
  • Example 17 In vivo treatment of PGA-SS-F and NPCs preserves neuronal fibers and promote improve gait analysis.
  • Example 18 Cell Viability studies with PGA-Dinaciclib Conjugates.
  • the conjugate bearing an ester linker displayed extremely low toxicity in both cell lines compared to free dinaciclib, with an observed decrease of more than four orders of magnitude (Fig. 30).
  • PGA-O-Din ester linker
  • Lysosomal enzymes can cleave biphosphate linkers, and we observed a decrease in toxicity for PGA-PP-Din by approximately 1-2 orders of magnitude when compared to the free drug in both cell lines, most probably because of different cell uptake.
  • Example 19 Drug Release Kinetics Of Selected Pga-Dinaciclib Conjugates.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Hospice & Palliative Care (AREA)
  • Molecular Biology (AREA)
  • Psychiatry (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne de nouvelles entités chimiques comprenant un support polymère incorporant un ou des médicament(s) lié(s) de manière covalente ainsi que leurs compositions pharmaceutiques contenant lesdits conjugués polymère-médicament en tant qu'agents pharmaceutiques et leurs utilisations en thérapie et, plus particulièrement, mais pas exclusivement, de nouveaux conjugués ayant fixé à celui-ci une fraction de ciblage, un agent d'imagerie et un ou plusieurs agents thérapeutiques pour favoriser la croissance axonale et pour le traitement de maladies neurodégénératives et inflammatoires, de lésions traumatiques du système nerveux central et du cancer.
PCT/EP2020/058940 2019-03-28 2020-03-30 Conjugués polymères et leurs utilisations WO2020193802A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP19382225 2019-03-28
EP19382225.1 2019-03-28

Publications (1)

Publication Number Publication Date
WO2020193802A1 true WO2020193802A1 (fr) 2020-10-01

Family

ID=66175377

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2020/058940 WO2020193802A1 (fr) 2019-03-28 2020-03-30 Conjugués polymères et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2020193802A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114377139A (zh) * 2022-03-11 2022-04-22 四川省医学科学院·四川省人民医院 载体、药物递送系统及其应用
CN114907337A (zh) * 2021-02-08 2022-08-16 四川大学 靶向cdk4或cdk6的共价抑制剂及其应用

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4678783A (en) 1983-11-04 1987-07-07 Asahi Kasei Kogyo Kabushiki Kaisha Substituted isoquinolinesulfonyl compounds
WO1998006433A1 (fr) 1996-08-12 1998-02-19 Yoshitomi Pharmaceutical Industries, Ltd. MEDICAMENTS COMPRENANT UN INHIBITEUR DE LA Rho KINASE
US5942505A (en) 1995-07-03 1999-08-24 Asahi Kasei Kogyo Kabushiki Kaisha 1-(5-isoquinolinesulfonyl)homopiperazine hydrochloride hydrates
WO2000078351A1 (fr) 1999-06-18 2000-12-28 Mitsubishi Pharma Corporation Promoteurs de l'osteogenese
WO2001017562A1 (fr) 1999-09-02 2001-03-15 Yamanouchi Pharmaceutical Co., Ltd. Agents promoteurs de l'osteogenese
EP1110553A1 (fr) 1998-08-10 2001-06-27 Asahi Kasei Kabushiki Kaisha Preparations orales, a liberation prolongee, a base de chlorhydrate de fasudil
US6372250B1 (en) 2000-04-25 2002-04-16 The Regents Of The University Of California Non-invasive gene targeting to the brain
WO2002076976A2 (fr) 2001-03-23 2002-10-03 Bayer Corporation Inhibiteurs de rho-kinase
WO2002076977A2 (fr) 2001-03-23 2002-10-03 Bayer Corporation Inhibiteurs de rho-kinase
WO2003059913A1 (fr) 2002-01-10 2003-07-24 Bayer Healthcare Ag Inhibiteurs de la rho-kinase
WO2003062227A1 (fr) 2002-01-23 2003-07-31 Bayer Pharmaceuticals Corporation Inhibiteurs de kinase rho
WO2003062225A1 (fr) 2002-01-23 2003-07-31 Bayer Pharmaceuticals Corporation Derives pyrimidine en tant qu'inhibiteurs de kinase rho
WO2003082808A1 (fr) 2002-04-03 2003-10-09 Sumitomo Pharmaceuticals Company, Limited. Derives de benzamide
WO2004039796A1 (fr) 2002-10-28 2004-05-13 Bayer Healthcare Ag Phenylaminopyrimidine substituee par heteroaryloxy et utilisee en tant qu'inhibiteur de kinase
WO2005035506A1 (fr) 2003-10-15 2005-04-21 Ube Industries, Ltd. Nouveau derive d'indazole
WO2005074643A2 (fr) 2004-01-30 2005-08-18 Smithkline Beecham Corporation Composes chimiques
JP2009149728A (ja) * 2007-12-19 2009-07-09 Noguchi Inst ポリエーテルの製造方法
WO2014187654A1 (fr) * 2013-05-20 2014-11-27 Fundación Comunidad Valenciada "Centro De Investigación Príncipe Felipe" Conjugues de medicaments polymeres pour le traitement de l'amylose

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4678783A (en) 1983-11-04 1987-07-07 Asahi Kasei Kogyo Kabushiki Kaisha Substituted isoquinolinesulfonyl compounds
US4678783B1 (en) 1983-11-04 1995-04-04 Asahi Chemical Ind Substituted isoquinolinesulfonyl compounds
US5942505A (en) 1995-07-03 1999-08-24 Asahi Kasei Kogyo Kabushiki Kaisha 1-(5-isoquinolinesulfonyl)homopiperazine hydrochloride hydrates
WO1998006433A1 (fr) 1996-08-12 1998-02-19 Yoshitomi Pharmaceutical Industries, Ltd. MEDICAMENTS COMPRENANT UN INHIBITEUR DE LA Rho KINASE
EP1110553A1 (fr) 1998-08-10 2001-06-27 Asahi Kasei Kabushiki Kaisha Preparations orales, a liberation prolongee, a base de chlorhydrate de fasudil
WO2000078351A1 (fr) 1999-06-18 2000-12-28 Mitsubishi Pharma Corporation Promoteurs de l'osteogenese
WO2001017562A1 (fr) 1999-09-02 2001-03-15 Yamanouchi Pharmaceutical Co., Ltd. Agents promoteurs de l'osteogenese
US6372250B1 (en) 2000-04-25 2002-04-16 The Regents Of The University Of California Non-invasive gene targeting to the brain
WO2002076976A2 (fr) 2001-03-23 2002-10-03 Bayer Corporation Inhibiteurs de rho-kinase
WO2002076977A2 (fr) 2001-03-23 2002-10-03 Bayer Corporation Inhibiteurs de rho-kinase
US20030087919A1 (en) 2001-03-23 2003-05-08 Bayer Corporation Rho-kinase inhibitors
US20030125344A1 (en) 2001-03-23 2003-07-03 Bayer Corporation Rho-kinase inhibitors
WO2003059913A1 (fr) 2002-01-10 2003-07-24 Bayer Healthcare Ag Inhibiteurs de la rho-kinase
US20040014755A1 (en) 2002-01-10 2004-01-22 Dhanapalan Nagarathnam Rho-kinase inhibitors
US20050192304A1 (en) 2002-01-23 2005-09-01 Dhanapalan Nagarathnam Rho-kinase inhibitors
US20040002508A1 (en) 2002-01-23 2004-01-01 Bayer Corporation Rho-kinase inhibitors
US20040002507A1 (en) 2002-01-23 2004-01-01 Bayer Corporation Rho-kinase inhibitors
WO2003062225A1 (fr) 2002-01-23 2003-07-31 Bayer Pharmaceuticals Corporation Derives pyrimidine en tant qu'inhibiteurs de kinase rho
WO2003062227A1 (fr) 2002-01-23 2003-07-31 Bayer Pharmaceuticals Corporation Inhibiteurs de kinase rho
US20050209261A1 (en) 2002-01-23 2005-09-22 Dhanapalan Nagarathnam Rho-kinase inhibitors
WO2003082808A1 (fr) 2002-04-03 2003-10-09 Sumitomo Pharmaceuticals Company, Limited. Derives de benzamide
WO2004039796A1 (fr) 2002-10-28 2004-05-13 Bayer Healthcare Ag Phenylaminopyrimidine substituee par heteroaryloxy et utilisee en tant qu'inhibiteur de kinase
WO2005035506A1 (fr) 2003-10-15 2005-04-21 Ube Industries, Ltd. Nouveau derive d'indazole
WO2005074643A2 (fr) 2004-01-30 2005-08-18 Smithkline Beecham Corporation Composes chimiques
JP2009149728A (ja) * 2007-12-19 2009-07-09 Noguchi Inst ポリエーテルの製造方法
WO2014187654A1 (fr) * 2013-05-20 2014-11-27 Fundación Comunidad Valenciada "Centro De Investigación Príncipe Felipe" Conjugues de medicaments polymeres pour le traitement de l'amylose

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Goodman and Gilman, The Pharmacological Basis of Therapeutics", 1995, MCGRAW-HILL
"Remington: The Science and Practice of Pharmacy", 2003, LIPPINCOTT WILLIAMS & WILKINS
ALASTRUE-AGUDO, ERCEG METHODS MOL BIOL., vol. 1210, 2014, pages 23 - 35
BONNICIKAPFHAMMER, EUR J NEUROSCI, vol. 27, no. 10, May 2008 (2008-05-01), pages 2483 - 92
BRUNING, A.FRIESE, K.BURGES, A.MYLONAS, I., BREAST CANCER RES., vol. 12, 2010, pages R45
CHEMICAL ABSTRACTS, Columbus, Ohio, US; abstract no. 24939-16-0
EDUARDO FERNÁNDEZ ORTUÑO: "EFFECT OF A FASUDIL NANOCONJUGATE AND TRANSPLANTATION OF NEURAL PRECURSORS ON NEURAL REGENERATION IN A SPINAL CORD INJURY CHRONIC MODEL. ROLE OF AMPK SIGNALING IN THE EFFECT OF FASUDIL OVER NEURITE GROWTH AND DIFFERENTIATION IN NEURAL PRECURSORS", July 2019 (2019-07-01), XP055704445, Retrieved from the Internet <URL:https://riunet.upv.es/bitstream/handle/10251/124736/Fern%C3%A1ndez%20-%20Efecto%20de%20un%20nanoconjugado%20de%20Fasudil%20y%20del%20trasplante%20de%20precursores%20neurales%20sobre%20....pdf?sequence=1&isAllowed=y> [retrieved on 20200612] *
FAULITRILLO C.: "Tratado de Farmacia Galenica", 1993
FENG Y ET AL., J MED CHEM., vol. 59, no. 6, 24 March 2016 (2016-03-24), pages 2269 - 300
HAMERS, LANKHORST ET AL., J NEUROTRAUMA, vol. 18, 2001, pages 1707 - 1711
JOHN J NEMUNAITIS ET AL: "A first-in-human, phase 1, dose-escalation study of dinaciclib, a novel cyclin-dependent kinase inhibitor, administered weekly in subjects with advanced malignancies", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, vol. 11, no. 1, 16 October 2013 (2013-10-16), pages 259, XP021164418, ISSN: 1479-5876, DOI: 10.1186/1479-5876-11-259 *
KAKINOHANA O ET AL., CELL TRANSPLANT, vol. 21, no. 12, 2012, pages 2603 - 19
KIM DS ET AL., PLOS ONE, vol. 7, no. 7, 2012, pages e39715
KOCH JAN CHRISTOPH ET AL: "ROCK inhibition in models of neurodegeneration and its potential for clinical translation", PHARMACOLOGY AND THERAPEUTICS, ELSEVIER, GB, vol. 189, 3 April 2018 (2018-04-03), pages 1 - 21, XP085442703, ISSN: 0163-7258, DOI: 10.1016/J.PHARMTHERA.2018.03.008 *
LI YIWEN ET AL: "Glutathione-triggered dual release of doxorubicin and camptothecin for highly efficient synergistic anticancer therapy", COLLOIDS AND SURFACES. B, BIOINTERFACES, ELSEVIER, AMSTERDAM, NL, vol. 169, 17 May 2018 (2018-05-17), pages 273 - 279, XP085417242, ISSN: 0927-7765, DOI: 10.1016/J.COLSURFB.2018.05.025 *
MANJII ET AL., BR J PSYCHIATRY SUPPL, vol. 41, 2001, pages s107
MOL. CELL. BIOL., vol. 4, 2003, pages 446 - 456
OWEN D EEGERTON J, METHODS MOL. BIOL., vol. 846, 2012, pages 179e187
REQUEJO-AGUILAR RAQUEL ET AL: "Combined polymer-curcumin conjugate and ependymal progenitor/stem cell treatment enhances spinal cord injury functional recovery", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 113, 21 October 2016 (2016-10-21), pages 18 - 30, XP029812725, ISSN: 0142-9612, DOI: 10.1016/J.BIOMATERIALS.2016.10.032 *
REYNODS BA ET AL., NAT. METHODS, vol. 2, no. 5, 2005, pages 333 - 6
SAURABH WADHWA ET AL: "Polypeptide conjugates of d-penicillamine and idarubicin for anticancer therapy", JOURNAL OF CONTROLLED RELEASE, vol. 158, no. 2, 1 March 2012 (2012-03-01), NL, pages 215 - 223, XP055376005, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2011.10.027 *
TORRIJOS SAIZ, LUCIA INÉS: "Precondicionamiento de progenitores neurales con un conjugado de poliglutamato del Fasudil para el tratamiento de la lesión medular aguda", 19 July 2019, UNIVERSITAT POLITÈCNICA DE VALÈNCIA. DEPARTAMENTO DE BIOTECNOLOGÍA - DEPARTAMENT DE BIOTECNOLOGIA, Spain, XP009520967 *
YANGBO FENG ET AL: "Rho Kinase (ROCK) Inhibitors and Their Therapeutic Potential", JOURNAL OF MEDICINAL CHEMISTRY, vol. 59, no. 6, 30 October 2015 (2015-10-30), US, pages 2269 - 2300, XP055535566, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.5b00683 *
YUE, D. ET AL., J. MATER. CHEM. B MATER. BIOL. MED., vol. 2, 2014, pages 7210 - 7221
ZHANG ET AL., MAR. DRUGS, vol. 11, 2013, pages 81 - 98

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114907337A (zh) * 2021-02-08 2022-08-16 四川大学 靶向cdk4或cdk6的共价抑制剂及其应用
CN114907337B (zh) * 2021-02-08 2023-06-02 四川大学 靶向cdk4或cdk6的共价抑制剂及其应用
CN114377139A (zh) * 2022-03-11 2022-04-22 四川省医学科学院·四川省人民医院 载体、药物递送系统及其应用
CN114377139B (zh) * 2022-03-11 2023-07-04 四川省医学科学院·四川省人民医院 载体、药物递送系统及其应用

Similar Documents

Publication Publication Date Title
US10905762B2 (en) Targeted nanoparticle conjugates
Kambhampati et al. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells
Cao et al. Polymeric prodrugs conjugated with reduction-sensitive dextran–camptothecin and pH-responsive dextran–doxorubicin: an effective combinatorial drug delivery platform for cancer therapy
Wu et al. A Core–Shell Albumin Copolymer Nanotransporter for High Capacity Loading and Two‐Step Release of Doxorubicin with Enhanced Anti‐Leukemia Activity
US9889209B2 (en) Nanoconjugates able to cross the blood-brain barrier
Deng et al. Discovery of novel cell-penetrating and tumor-targeting peptide-drug conjugate (PDC) for programmable delivery of paclitaxel and cancer treatment
EP3380495B1 (fr) Composés peptidiques et conjugués peptidiques destinés au traitement du cancer par chimiothérapie à médiation par un récepteur
US9180203B2 (en) Self-assembling drug amphiphiles and methods for synthesis and use
Kim et al. The safe and effective intraperitoneal chemotherapy with cathepsin B-specific doxorubicin prodrug nanoparticles in ovarian cancer with peritoneal carcinomatosis
WO2020193802A1 (fr) Conjugués polymères et leurs utilisations
Giraldo et al. A rationally designed self-immolative linker enhances the synergism between a polymer-rock inhibitor conjugate and neural progenitor cells in the treatment of spinal cord injury
KR102257647B1 (ko) 암 치료용 세포독성제
Li et al. Targeted methotrexate prodrug conjugated with heptamethine cyanine dye improving chemotherapy and monitoring itself activating by dual-modal imaging
Jiang et al. Theranostic heterodimeric prodrug with dual‐channel fluorescence turn‐on and dual‐prodrug activation for synergistic cancer therapy
Shim et al. Comparative study of cathepsin B-cleavable linkers for the optimal design of cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy
Pisarevsky et al. Rational design of polyglutamic acid delivering an optimized combination of drugs targeting mutated BRAF and MEK in melanoma
Zhang et al. Novel synovial targeting peptide-sinomenine conjugates as a potential strategy for the treatment of rheumatoid arthritis
Chen et al. Redox homeostasis modulation using theranostic AIE nanoparticles results in positive-feedback drug accumulation and enhanced drug penetration to combat drug-resistant cancer
Xiao et al. Tumor-Selective Cascade-Amplified Dual-Prodrugs Activation for Synergistic Oxidation-Chemotherapy
EP2805732B1 (fr) Conjugués médicament-polymère pour le traitement de l&#39;amyloïdose
Radhika et al. Blood-brain barrier–its implication in drug transport: Novel strategies in drug delivery to the brain
Li et al. “On/off”-switchable crosslinked PTX-nanoformulation with improved precise delivery for NSCLC brain metastases and restrained adverse reaction over nab-PTX
Freidel Design, synthesis and application of novel disulfide-intercalation agents for the site-selective modification of peptides and proteins
Dhiman et al. Rational design of linkers in polymer–drug conjugates
Singh et al. Tumor–homing peptide iRGD-conjugate enhances tumor accumulation of camptothecin for colon cancer therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20713670

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20713670

Country of ref document: EP

Kind code of ref document: A1