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WO2010048365A2 - Bioconjugués peptidiques thérapeutiques - Google Patents

Bioconjugués peptidiques thérapeutiques Download PDF

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Publication number
WO2010048365A2
WO2010048365A2 PCT/US2009/061604 US2009061604W WO2010048365A2 WO 2010048365 A2 WO2010048365 A2 WO 2010048365A2 US 2009061604 W US2009061604 W US 2009061604W WO 2010048365 A2 WO2010048365 A2 WO 2010048365A2
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peptide
seq
eye
ser
thr
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PCT/US2009/061604
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WO2010048365A3 (fr
Inventor
Stephen P. Massia
Gholam Reza Ehteshami
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Arizona Technology Enterprises
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • Inflammatory eye diseases can affect any part of the eye; from the ocular surface to the retina, the optic nerve, and other orbital structures.
  • the consequences of inflammation in the eye may be sight-threatening because the high fidelity visual system is extremely sensitive to even the slightest loss of clarity or micro-anatomical distortion along the visual axis.
  • Inflammation in any portion of the body is invariably characterized by a leukocytic infiltrate and an increase in vascular permeability.
  • Leukocytes neurotrophils, eosinophils, monocytes, or lymphocytes
  • lymphocytes respond initially to an antigen but then rapidly transition to recruitment of additional lymphocytes and other inflammatory cells that do not necessarily specifically recognize the inciting antigen - leading to tissue destruction.
  • the host inflammatory response to Pseudomonas aeruginosa corneal infection consists primarily of an influx of leukocytes which migrate from the tear film and from the limbal and iridial vasculature into the avascular cornea, but also destroy corneal tissue, despite their being essential to resolving the infection.
  • the result can be devastating for the cornea.
  • anti-inflammatory therapy is often employed for these and other ocular inflammatory diseases including crizopriopriopril, fibroblasts, and chorioretinitis.
  • Dry eye (keratoconjunctivitis sicca or keratitis sicca) is another class of ocular inflammatory disease that afflicts millions. Dry eye is characterized by an inability of the eye to make proper tears, leading to eye dryness, pain, and high patient morbidity; it is the largest reason for visits to eye care providers. Abnormal ocular surface inflammation and tear gland inflammation are the major causes of dry eye.
  • the present invention provides for a novel approach in treating inflammatory conditions of the eye.
  • the invention is a class of anti-inflammatory bioconjugate therapeutics that selectively target and locally bind to inflamed tissue surfaces, thus forming a protective colloid barrier against excessive leukocyte adhesion/infiltration and subsequent tissue injury.
  • the bioconjugate is a bioselective peptide/low-adhesive bioconjugate that binds Intercellular adhesion molecule 1 (ICAM-I) and inhibits intercellular adhesion.
  • the peptide has the amino acid sequence XiX 2 X 3 X 4 X 5 GX 6 X 7 X 8 , (SEQ ID NO:9), wherein X, isNH2 or Cys; X 2 is Tyr, Phe or Trp; X 3 is Ser or Thr; X 4 is Asp or GIu; X 5 is Asn or GIn; Xe is Thr or Ser; X 7 is Phe, Tyr or Trp; and X s is COOH or Cys.
  • the peptide has the amino acid sequence X 1 YX 2 DNGTFX 3 (SEQ ID NO:8), in which Xi is NH 2 or cys; X 2 is ser (S) or thr (T); X 3 is COOH or cys.
  • the bioconjugate contains a bioselective peptide has the amino acid sequence YXDNGTF (SEQ ID NO:1), where X is Ser or Thr, conjugated to a low-adhesive component, which bioconjugate binds specifically to Intercellular adhesion molecule 1 (ICAM-I) and inhibits intercellular adhesion.
  • the bioconjugate is the peptide having the amino acid sequence CYXDNGTF (SEQ ID NO: 2), where and X is Ser or Thr, or an equivalent thereof, conjugated to dextran.
  • Another embodiment of the present invention provides for a medicament suitable for placement in the eye, allowing delivery of a bioconjugate directly to the cornea and conjunctiva, in the form of an topically applicable eye drop that contains at least one antiinflammatory biocojugate in a solution or gel.
  • the pharmaceutically acceptable formulation comprises a low-adhesive molecule conjugated to a peptide selected from X I X 2 X 3 X 4 X S GX 6 X 7 X S , (SEQ ID NO:9), wherein X 1 isNH2 or Cys; X 2 is Tyr, Phe or Trp; X 3 is Ser or Thr; X 4 is Asp or GIu; X 5 is Asn or GIn; X 6 is Thr or Ser; X 7 is Phe, Tyr or Trp; and Xg is COOH or Cys; X 1 YX 2 DNGTFX 3 (SEQ ID NO: 8), in which Xi is NH 2 or cys; X 2 is ser or thr; X 3 is COOH or cys; YXDNGTF (SEQ ID NO:1), wherein X is Ser or Thr; or CYXDNGTF (SEQ ID N0:2), wherein X is Ser or
  • Yet another embodiment of the present invention provides for a method of treating inflammation in the eye comprising administering to the eye a pharmaceutical formulation comprising bioselective peptide/low-adhesive bioconjugate that binds to ICAM-I and inhibits intercellular binding.
  • the pharmaceutical formulation may be prepared for topical application (e.g., eye drops), or injection (e.g., into the vitreous).
  • the low-adhesive is dextran.
  • the bioselective peptide portion of the bioconjugate can be XiX 2 X 3 X 4 X 5 GX 6 X 7 X 8 , (SEQ ID NO:9), wherein X 1 isNH2 or Cys; X 2 is Tyr, Phe or Trp; X 3 is Ser or Thr; X 4 is Asp or GIu; X 5 is Asn or GIn; X 6 is Thr or Ser; X 7 is Phe, Tyr or Trp; and Xg is COOH or Cys; X J YX 2 DNGTFX 3 (SEQ ID NO:8), in which Xi is NH 2 or Cys; X 2 is Ser or Thr; X 3 is COOH or Cys; YXDNGTF (SEQ ID NO: 1), wherein X is Ser or Thr; or CYXDNGTF (SEQ ID NO:2), wherein X is Ser or Thr.
  • Figure 2 is a bar graph showing the in vitro inhibition of intercellular adhesion by particular bio conjugates.
  • Figure 3 shows representative photomicrographs from in vivo experiments comprising bioconjugate immobilized on fluorescent beads.
  • Figure 3A healthy cornea no wash
  • Figure 3B healthy cornea, one wash
  • Figure 3C diseased cornea, no wash
  • Figure 3D diseased cornea, one wash
  • Figure 3E diseased cornea, multiple washes.
  • the present invention provides a different approach and offers a viable and superior treatment solution for inflammation of the tissues of the eye.
  • CAM Cell adhesion molecules
  • Selectins, integrins and immunoglobulin (Ig) gene superfamily adhesion receptors mediate the different steps of the migration of leucocytes from the bloodstream towards inflammatory foci.
  • the activation of endothelial cells (EC) upregulates the expression of several CAM and triggers the interaction of these cells with leucocytes.
  • Selectins are involved in the initial interactions (tethering/rolling) of leucocytes with activated endothelium, whereas integrins and Ig superfamily CAM mediate the firm adhesion of these cells and their subsequent extravasation.
  • leucocytes are activated through the intracellular signals generated by CAM and chemokine receptors.
  • Blockade of the function or expression of CAM has emerged as a new therapeutic target in inflammatory diseases. Different drugs are able to interfere with cell adhesion phenomena.
  • new antiadhesion therapeutic approaches blocking monoclonal antibodies, soluble receptors, synthetic peptides, peptidomimetics, etc.
  • Ocular immune privilege involves certain anatomical, cellular, and soluble factors, such as the blood-ocular barrier and immunosuppressive factors of the aqueous humour, designed to avoid the potential sight-destroying consequences of ocular inflammation.
  • the conjunctiva Besides functioning as an anatomical barrier between the external environment and internal ocular structures, the conjunctiva has a fundamental protective role: it participates ion the formation of tear film and, by secreting mucins, contributes directly to its quality. Additionally, the conjunctiva is the inflammatory and immunological tissue that protects the ocular surface.
  • the conjunctiva is highly vascularized, connected o the lymphatic network, and rich in immunocompetent cells that interact in a continuous, dynamic manner from the most superficial to the deepest ocular structures.
  • the cornea As with the conjunctiva, the cornea is in contact with the external environment. But although contiguous with the conjunctiva, the cornea is a different structure. Only the conjunctiva possesses all the components required to react to potential insults to the cornea. Indeed, the conjunctiva holds powerful reactivity to protect the cornea. Maintenance of corneal transparency and integrity is a major requirement of the defensive mechanisms protecting the ocular surface. The immunoflammatory response is a formidible weapon that can have detrimental effects, however, by damaging healthy bystander structures including the cornea.
  • HSV herpes simplex virus
  • Pseudomonas keratitis Pseudomonas keratitis
  • ocular onchocerciasis Pseudomonas keratitis
  • bacterial endophthalmitis toxoplasmic retinochoroiditis
  • CMV cytomegalovirus
  • Pseudomonas aeruginosa may cause devastating corneal infections.
  • the host inflammatory response to P. aeruginosa corneal infection consists primarily of an influx of polymorphonuclear leukocytes (PMN), which also destroy corneal tissue despite their being essential to resolving the infection.
  • PMN migrate from the tear film and from the limbal and iridial vasculature into the avascular cornea.
  • ICAM-I is a key molecule for PMN recruitment into infected tissue, lmmunostaining of inflamed human ocular tissue suggests that ICAM-I is a key mediator of acute ocular inflammation. Hobden et al., 67(2) Infect. Immun. 972-75 (1999).
  • ICAM-I In addition to its role in corneal alloimmunity, ICAM-I function has been linked to allergic disease and dry eye syndrome: ICAM-I is expressed at significantly higher levels on the conjunctival epithelium of dry eye patients and correlates strongly with human leukocyte antigen-MHC class II, cell surface receptor (HLA-DR) upregulation.
  • HLA-DR cell surface receptor
  • Retinal inflammatory diseases are associated with an upregulation in the expression of intercellular adhesion molecule- 1 (ICAM-I) in cells within the retina and with an increase in soluble ICAM-I within the vitreous.
  • IAM-I intercellular adhesion molecule- 1
  • Leukocyte accumulation in the retina is thought to play a key role in the early stage of diabetic retinopathy.
  • Leukocyte-endothelial adhesion is important for ieukostasis, and high glucose levels may influence adhesion. Hirata et al., 82 (1) Exp. Eye Res. 179-82 (1998).
  • VKC vernal keratoconjunctivitis
  • vitreous levels of ICAM-I proteins are significantly higher in eyes with proliferative vitreoretinopathy (PDR), traction retinal detachment, and vitreous hemorrhage. Identification of any abnormalities in the production and control of specific adhesion molecules could have important implications in the design of new therapeutic regimens to treat and prevent this sight-threatening complication of diabetes mellitus. Limb et al., Investig. Opthalmol. Visual Sci. 2453-57 (1999).
  • a novel approach to anti-inflammatory therapy inhibits the leukocyte-endothelial interaction. This has been demonstrated successfully in animal models of inflammation in the eye or inflammation in other organ systems.
  • antibodies to selectins, integrins, to ICAM-I can reduce inflammation in animal models of uveitis.
  • the present invention is a class of anti-inflammatory /immunosuppressant bioconjugate therapeutics that target selectively, and bind locally, to inflamed tissue surfaces, thus forming a protective colloid barrier against pathologically driven excessive leukocyte adhesion/infiltration and subsequent tissue injury.
  • leukocyte adhesion to tissue surfaces is essential for normal immune system function
  • leukocyte/tissue adhesion plays a major role in a number of pathological processes including septic shock, post-trauma multiple organ failure, ischemic reperfusion injury, transplant rejection, inflammatory diseases, and autoimmune diseases. Therefore, the current invention could be exploited as a locally and/or systemically deliverable therapy that specifically targets leukocyte-adhesive tissues, thereby suppressing pathologically excessive leukocyte-mediated damage to healthy tissues and limiting deleterious outcomes.
  • This class of bioconjugates consists of a bioactive ICAM-I -binding molecule covalently attached to low cell-adhesive macromolecules in a monovalent or multivalent fashion.
  • These bioactive ICAM-I binding molecules comprise a peptide, i.e., a bioselective peptide.
  • the bioselective peptide can be a naturally occurring, chemically synthesized, or recombinant protein.
  • the bioactive component of these bioconjugates can be an ICAM-I -binding peptide that has been synthesized, or enzymatically digested from larger proteins.
  • ICAM-I -binding molecules other than peptides and proteins may also be utilized as the bioactive component of this class of bioconjugates. See U.S. Patent Application Ser. No. 10/716,293.
  • bioconjugate means a compound in which at least two components, a bioselective peptide and a low-adhesive component, are chemically attached. i.e., conjugated.
  • a low-adhesive component is synonymous with a low cell-adhesive component, a non-adhesive polymer, a cell-adhesion-barrier polymer, or low cell-adhesive macromolecule.
  • the specific conjugation method is determined by the choice of non- adhesive barrier molecule and the accepted linking methods to the selected bioselective molecule, preferably a protein or peptide. Both univalent and multivalent conjugation methods are suitable.
  • the conjugation method is selected to produce a bioconjugate that retains the bioselective and blockade abilities of the bioconjugate.
  • the molecules are attached in vitro prior to application to the living tissue. In certain other embodiments the molecules may be designed with appropriate linking groups that cause them to congregate in vivo.
  • the low-adhesive component of the class of bioconjugates described in the current invention may be an oligosaccharide or polysaccharide and includes derivatives thereof derived from microbial species, prokaryotic or eukaryotic organisms; or chemical synthesis may provide the low cell-adhesive components for this bioconjugate class.
  • polysaccharides include, but are not limited to, agarose, dextran, heparin, chondroitin sulfate, and hyaluronic acid.
  • the low-adhesive component may be a hydrophilic polymer, such as poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(acrylic acid), poly(ethylene-co-vinyl alcohol), poly(vinyl pyrrolidone), poly(ethyloxazoline), or poly(ethylene oxide)-co-poly(propylene oxide) block copolymers.
  • the low cell-adhesive component can consist of copolymers, block copolymers, graft copolymers, alternating copolymers, or random copolymers. To confer degradability by hydrolytic or enzymatic means, hydrophilic polymers can be modified with blocks polymerized on each end.
  • the bioconjugate includes a protein zero peptide, YTDNGTF (SEQ ID NO:3), which has targeted liposomes to ICAM- i present on melanoma cells.
  • This peptide in the context of the present invention, was found to exhibit excellent solubility in aqueous solutions, making it superior to some other ICAM-I -binding peptides as a component for a formulation for placement into the eye. Indeed, some other ICAM-I binding peptides are poorly soluble in isotonic aqueous solutions such as those found in the eye.
  • a cysteine residue was added to the NH 2 end of the peptide, yielding the 8-mer having an amino acid sequence CYTDNGTF (SEQ ID NO:4).
  • the present invention encompasses the YTDNGTF (SEQ ID NO:3) peptide and equivalents thereof.
  • Equivalent peptides are those having a functionally equivalent amino acid sequence having selective ICAM-I binding activity.
  • the peptide YSDNGTF (SEQ ID NO:5) wherein the threonine residue has been replaced with serine, is a naturally occurring human variant of peptide YTDNGTF (SEQ ID NO:3) and has equivalent ICAM- 1 -binding activity.
  • a peptide having the amino acids YSDNGTF (SEQ ID NO:6) is the corresponding rat 'protein zero' peptide.
  • Another equivalent peptide is that of bovine, having the sequence YGDNGTF (SEQ ID NO:7).
  • a bioselective peptide of the present invention may also be expressed by the formula YXDNGTF (SEQ ID NO:1), wherein X is Ser (S) or Thr (T).
  • peptide is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics.
  • amino acids of the peptides YTDNGTF (SEQ ID NO:3) or YSDNGTF (SEQ ID NO:6) may be substituted with amino acid analogs peptidomimetics and screened for ICAM-I -binding activity.
  • oligonucleotide sequence that encodes the amino acid sequence of YTDNGTF (SEQ ID NO:3) or an equivalent peptide thereof. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the amino acid.
  • Single stranded oligonucleotide molecules complementary to the "most probable" anti-CPAA region peptide coding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art. See Belagaje et al., 254 J. Biol. Chem. 5765-80 (1979); Maniatis et al., in MOLECULAR MECHANISMS IN THE CONTROL OF GENE EXPRESSION (Nierlich et al., eds., Acad. Press, NY, 1976); Wu et al., 1978; Khorana, 203 Science 614-25 (1979).
  • peptide coding regions for use in the present invention could also be provided by altering existing protein zero genes using standard molecular biological techniques that result in variants (agonists) of the peptides described herein.
  • variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the peptides.
  • polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids.
  • amino acids including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art.
  • Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • the peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code.
  • this invention also includes the peptides for preparation of bioconjugate that have been modified with an additional N-terminal or C-terminal cysteine residue.
  • the nucleic acid sequences encoding the peptides are modified to express the additional cysteine residue(s).
  • the peptide may be conjugated to dextran by providing an amount of activated dextran, then contacting the activated dextran and the modified peptide under conditions whereby the dextran and the modified peptide react to form the bioconjugate.
  • the peptides of the present invention may be described as the peptide having the amino acid sequence X I YX 2 DNGTFX 3 (SEQ ID NO: 8), wherein X 1 is NH 2 or cys; X 2 is ser (S) or thr (T); X 3 is COOH or cys.
  • the peptides of the present invention encompass those with the formula XiX 2 X 3 X 4 XsGX 6 X 7 Xg, (SEQ ID N0:9), wherein X 1 isNH2 or Cys; X 2 is Tyr, Phe or Trp; X 3 is Ser or Thr; X 4 is Asp or GIu; X5 is Asn or GIn; Xg is Thr or Ser; X 7 is Phe, Tyr or Trp; and X 8 is COOH or Cys.
  • One or more termini of the peptide may include one or more additional amino acid residues.
  • An embodiment of this invention provides for a bioresponsive bioconjugate, delivered in a pharmaceutically acceptable vehicle, that will selectively bind to inflamed tissues forming a protective barrier for trauma- or infection-induced hyperactive inflammatory cells that would otherwise promote excessive tissue damage including tissues not injured by trauma.
  • eye inflammation may be associated with any number of conditions, including but not limited to crizotis, scleritis, chorioretinitis, uveitis, or Sjogren's and non-Sjogren keratoconjunctivitis sicca.
  • a specific embodiment provides for the treatment of corneal inflammation subsequent to, or caused by, surgery, infection, injury, or other disease.
  • the present invention relates to a topical formulation for the eye, such as an eye solution or gel, which may comprise a saline-based fluid, and an effective amount of a YTDNGTF/low-adhesive bioconjugate (such as a CYTDNGTF/dextran bioconjugate), wherein when at least one drop of the eye solution is applied to the surface of an eye, the bioconjugate is released to the cornea and conjunctiva of the eye.
  • an eye solution or gel which may comprise a saline-based fluid
  • a YTDNGTF/low-adhesive bioconjugate such as a CYTDNGTF/dextran bioconjugate
  • the topical eye formulation is applied on the exterior surface of the eye. Additional drops can also be applied to the exterior of the eye.
  • the topical eye formulation can also be a gel so that an effective amount of at least one therapeutic compound or agent is delivered to the surface of the eye, the cornea and the conjunctiva, in the form of a topical drop or gel.
  • Saline is referred to a biocompatible physiological solution of sodium chloride (NaCl) in water at a concentration such that it is equivalent in concentration to human tears. Saline may be buffered with a number of compounds to maintain correct pH, and may include a variety of agent for thickening or improving retention on the surface of the eye, such as poly- vinyl alcohol or methyl cellulose, for example.
  • the topical eye formulation may further include one or more gelling or thickening agents, such as polyvinyl alcohol, methyl cellulose.
  • gelling or thickening agents such as polyvinyl alcohol, methyl cellulose.
  • the therapeutic bioconjugate may also be formulated for injection into the eye, such as injection into the vitreous of the eye, for the treatment of, for example, retinal inflammatory diseases.
  • a formulation comprising the therapeutic bioconjugate further include at least one additional therapeutic compound or active agent, such as glucocorticosteoids, cyclosporines, rapamycins, or anti-cytokines for the treatment of inflammatory diseases.
  • additional therapeutic compound or active agent such as glucocorticosteoids, cyclosporines, rapamycins, or anti-cytokines for the treatment of inflammatory diseases.
  • the term "active agent” is defined broadly as having an impact on a living system such as a cell, nerve, or tissue.
  • the active agent can be a chemical agent.
  • the active agent can also be a biological agent.
  • the active agent may comprise at least one known component.
  • the active agent can also be a physical agent.
  • Other examples of active agents include bacterial agents, viral agents, andother microorganisms.
  • the synthetic peptide CYTDNGTF (SEQ ID NO:4) was added to phosphate buffered saline (PBS) with 1.5 mM EDTA at a final concentration of 20 rnM.
  • PBS phosphate buffered saline
  • EDTA triethanolamine
  • Methacroylated dextran (2mM) was then added to the reaction mix and the pH was adjusted again to pH 8.0-pH 8.5 with TEA.
  • AU solutions were maintained under inert conditions to minimize disulfide bond formation.
  • Crosslinking was allowed to proceed at room temperature for 2 hr.
  • the reaction mixture was then dialyzed against deionized water in 25,000 MWCO membrane to remove any unreacted or disulfide bonded peptide.
  • the purified dextran/peptide conjugate was recovered by lyophilization.
  • HAEC Human aortic endothelial cell monolayers were established in 35 mm diameter tissue culture plastic dishes. At 4 hours prior to the assay, normal culture medium was replaced with medium containing tumor necrosis factor ⁇ (TNF- ⁇ , 50 ng/ml). Following the 4 hr incubation period, each sample well got a medium change. Treated sample groups received medium containing l%-6% dextran conjugate. Untreated control samples received normal medium. All samples were then incubated for 30 min prior to the adhesion assay. Circular parallel plate flow chambers (GlycoTech Corp., Gaithersburg, MD) were employed for these studies. These flow chambers fit directly into 35 mm culture dishes.
  • MPRO neutrophil cultures (ATCC# CRL-1 1422) were resuspended in prewarmed HBSS to a concentration of 0.5x10 6 cells/ml. This neutrophil suspension was then loaded into a 25 -ml syringe and mounted on a programmable syringe pump. Flow chambers attached to the loaded syringe pump were mounted on sample dishes containing HAEC monolayers. The flow chambers were then mounted on an inverted microscope with a prewarmed 37°C stage incubator and digital video recording equipment.
  • ICAM- 1 targeting bioconjugates were evaluated on the corneal surface using the MRL/MpJ-Tnfrsf61pr/J mouse strain (gene knockout), which spontaneously acquires ocular inflammation via an autoimmune response.
  • the MRL/MpJ strain may be utilized as a control group that does not acquire disease.
  • Four corneas each were explanted from 5 week- old diseased mice (ICAM-I -expressing) and control mice (no surface ICAM-I expression).
  • ICAM-I targeting bioconjugate containing the peptide CYTDNGTF (SEQ ID NO:4) was synthesized as described above and immobilized on 1 ⁇ m fluorescent latex beads.

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Abstract

La présente invention porte sur des bioconjugués thérapeutiques renfermant un peptide biosélectif conjugué à une fraction de faible adhérence, qui est capable de cibler de manière sélective un ligand de tissus inflammatoires. Un mode de réalisation particulier porte sur une composition pharmaceutique renfermant un bioconjugué peptide/dextrane anti-inflammatoire, de liaison à ICAM-I, utile, par exemple, pour limiter l'adhésion d'une cellule inflammatoire dans l'œil. La composition pharmaceutique peut être formulée dans un médicament topique ou injectable pour le traitement d'une inflammation, en particulier d'une inflammation oculaire.
PCT/US2009/061604 2008-10-23 2009-10-22 Bioconjugués peptidiques thérapeutiques WO2010048365A2 (fr)

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Cited By (4)

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WO2017049001A1 (fr) * 2015-09-15 2017-03-23 University Of Southern California Protection et étanchéification de la barrière de la surface oculaire par la clustérine
US9988568B2 (en) 2015-01-30 2018-06-05 Ecolab Usa Inc. Use of anti-agglomerants in high gas to oil ratio formations
CN112315863A (zh) * 2020-11-10 2021-02-05 山东百奥生物医药有限公司 一种舒缓光电术中疼痛热感的组合物及制备方法和应用
CN115607686A (zh) * 2021-07-14 2023-01-17 广州中医药大学(广州中医药研究院) 一种包载过氧化氢酶的纳米粒及其应用

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Publication number Priority date Publication date Assignee Title
US6849612B2 (en) * 1993-01-21 2005-02-01 Isis Pharmaceuticals, Inc. Oligonucleotide modulation of cell adhesion
US6670321B1 (en) * 1998-12-30 2003-12-30 The Children's Medical Center Corporation Prevention and treatment for retinal ischemia and edema
AU2003294318A1 (en) * 2002-11-15 2004-06-15 Arizona Board Of Regents Arizona State University Therapeutic bioconjugates
US20060241022A1 (en) * 2004-10-06 2006-10-26 Bowen Benjamin P Selectin targeting bioconjugates

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9988568B2 (en) 2015-01-30 2018-06-05 Ecolab Usa Inc. Use of anti-agglomerants in high gas to oil ratio formations
WO2017049001A1 (fr) * 2015-09-15 2017-03-23 University Of Southern California Protection et étanchéification de la barrière de la surface oculaire par la clustérine
CN112315863A (zh) * 2020-11-10 2021-02-05 山东百奥生物医药有限公司 一种舒缓光电术中疼痛热感的组合物及制备方法和应用
CN115607686A (zh) * 2021-07-14 2023-01-17 广州中医药大学(广州中医药研究院) 一种包载过氧化氢酶的纳米粒及其应用

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