WO2010048365A2 - Therapeutic peptide bioconjugates - Google Patents

Abstract

The present invention provides for therapeutic bioconjugates comprising a bioselective peptide conjugated to a low-adhesion moiety, that is capable of selectively targeting a ligand of inflammatory tissues. A particular embodiment provides for pharmaceutical composition including an ICAM-I -binding, anti-inflammatory peptide/dextran bioconjugate useful, for example, for limiting inflammatory cell adhesion in the eye. The pharmaceutical composition may be formulated in a topical or injectable medicament for the treatment of inflammation, particularly inflammation of the eye.

Description

THERAPEUTIC PEPTIDE BIOCONJUGATES

RELATED APPLICATIONS

[001 ] This application claims the benefit of U.S. Patent Application Ser. No. 61/107,754, filed October 23, 2009, incorporated herein by reference.

FIELD OF THE INVENTION

[002] The present invention relates to pharmaceutical compositions including small bioselective peptides conjugated to low-adhesion moieties, that are capable of selectively targeting ligands of inflammatory tissues. A particular embodiment provides for an ICAM-I- binding, anti -inflammatory peptide/dextran bioconjugate useful for limiting inflammatory cell adhesion in the eye.

BACKGROUND

[003] 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, whether appropriate (as in the case of immune response to infective threats) or inappropriate (as in the case of autoimmune or allergic responses), 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.

[004] Inflammation in any portion of the body is invariably characterized by a leukocytic infiltrate and an increase in vascular permeability. Leukocytes (neutrophils, eosinophils, monocytes, or lymphocytes) are ordinarily free-flowing within the blood stream, but migrate to a site of inflammation in a series of physiologically orchestrated events. Adhesion molecules, immunoregulatory molecules for the interaction between lymphocytes and vascular endothelium, play a key role in the selective recruitment of different leukocyte populations to inflammatory sites. In uveitis, a common form of visually significant ocular inflammation generally thought to be an autoimmune disease, 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.

[005] As another example, 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. Hence, anti-inflammatory therapy is often employed for these and other ocular inflammatory diseases including iritis, scleritis, and chorioretinitis.

[006] 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.

[007] Current modalities of therapy for ocular inflammatory disease are directed at controlling the hallmarks of inflammation, including the role of adhesion molecules in the selective recruitment of leukocytes to the inflammatory site(s). Therapies may include nonsteroidal anti- inflammatory drugs or corticosteroids delivered topically, periocularly, or systemically. Occasionally, in cases of vision-threatening disease which is unresponsive to such treatment, it may be necessary to employ systemic immunosuppression, in the form of a cytotoxic agent and/or an immunomodulating agent. Although each of these approaches has rationale and success, all have some limitations in efficacy as well as toxicity. Such toxicity is often primarily related to the lack of specificity of the medication.

[008] A novel approach to therapy attempts to inhibit the leukocyte-endothelial interaction. Although this has been demonstrated successfully in some animal models of inflammation, there remains a need for a selective yet robust technology that offers therapeutic alternatives for treating inflammatory conditions of the eye.

SUMMARY OF THE INVENTION

[009] 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.

[010] In one embodiment of the present invention, the bioconjugate is a bioselective peptide/low-adhesive bioconjugate that binds Intercellular adhesion molecule 1 (ICAM-I) and inhibits intercellular adhesion., in which the peptide has the amino acid sequence XiX₂X₃X₄X₅GX₆X₇X₈, (SEQ ID NO:9), wherein X, isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X₅ is Asn or GIn; Xe is Thr or Ser; X₇ is Phe, Tyr or Trp; and X_s is COOH or Cys. In a particular embodiment, the peptide has the amino acid sequence X₁YX₂DNGTFX₃ (SEQ ID NO:8), in which Xi is NH₂ or cys; X₂ is ser (S) or thr (T); X₃ is COOH or cys. [Oi l] In a particular embodiment, 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. In particular embodiments, 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.

[012] 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. For example, the pharmaceutically acceptable formulation comprises a low-adhesive molecule conjugated to a peptide selected from X_IX₂X₃X₄X_SGX₆X₇X_S, (SEQ ID NO:9), wherein X₁ isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X₅ is Asn or GIn; X₆ is Thr or Ser; X₇ is Phe, Tyr or Trp; and Xg is COOH or Cys; X₁YX₂DNGTFX₃ (SEQ ID NO: 8), in which Xi is NH₂ or cys; X₂ is ser or thr; X₃ 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 Thr. In a particular embodiment, the low-adhesive molecule is dextran. In another specific embodiment, the bioconjugate is CYTDNGTF/dextran.

[013] 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). In a particular embodiment, the low-adhesive is dextran. The bioselective peptide portion of the bioconjugate can be XiX₂X₃X₄X₅GX₆X₇X₈, (SEQ ID NO:9), wherein X₁ isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X₅ is Asn or GIn; X₆ is Thr or Ser; X₇ is Phe, Tyr or Trp; and Xg is COOH or Cys; X_JYX₂DNGTFX₃ (SEQ ID NO:8), in which Xi is NH₂ or Cys; X₂ is Ser or Thr; X₃ 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.

BRIEF DESCRIPTION OF THE DRAWINGS [014] Figure 1 presents a scheme for conjugating dextran to a cysteine residue of a peptide.

[015] Figure 2 is a bar graph showing the in vitro inhibition of intercellular adhesion by particular bio conjugates. [016] 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.

DETAILED DESCRIPTION

[017] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[018] Unless otherwise defined, scientific and technical terms used in connection with the antibodies described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[019] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[020] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[021] The present invention provides a different approach and offers a viable and superior treatment solution for inflammation of the tissues of the eye.

[022] Cell adhesion molecules (CAM) have a key role in the inflammatory response. 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. During rolling, 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. In addition, new antiadhesion therapeutic approaches (blocking monoclonal antibodies, soluble receptors, synthetic peptides, peptidomimetics, etc.) are being developed.

[023] 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. 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.

[024] 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. Corneal epithelial and stromal wound healing after injuries or intentional trauma, such as refractive surgery, is a complex process wherein the severity of apoptosis and reactivation of keratocytes is closely correlated with haze formation, corneal edema, neovascularization, and opacity. Penetrating injuries typically heal by deposition of fibrotic "repair tissue" that fills and seals wounds but does not restore normal function. Collagen degradation by corneal fibroblasts is an underlying cause of corneal ulceration and excessive deposition of fibrotic repair tissue can lead to excessive scarring and corneal contracture. In the cornea, fibrotic repair presents special challenges affecting both clarity and shape of the cornea, which is an essential component of the ability of the eye to focus.

[025] Despite these mechanisms to prevent potentially harmful ocular inflammation, this protective system can break down, resulting in sight-threatening inflammatory eye diseases such as uveitis and keratitis. Both infectious and immune mechanisms are important in triggering the breakdown in ocular immune privilege and in the development of various forms of inflammatory eye diseases. Microbial infections of the eye are unfortunately common, such as herpes simplex virus (HSV) keratitis and keratouveitis, Pseudomonas keratitis, ocular onchocerciasis, bacterial endophthalmitis, toxoplasmic retinochoroiditis, and cytomegalovirus (CMV) retinitis. Furthermore, it is becoming increasingly apparent that microbial agents may be important in the pathogenesis of "non-infectious" immune mediated inflammatory diseases, such as HLA-B27 associated acute anterior uveitis inflammation.

[026] The central role of ICAM-I function in corneal immune and inflammatory disease is now well established. For example, 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).

[027] 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.

[028] 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. These studies suggest that this protein may contribute to immunopathological processes within the eye. Nagineni et al., 8(8) Cytokine 622-30 (2006). 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). [029] Additionally, an increased expression of adhesion molecules was found in the iris of patients with uveitis, indicating an immunoregulatory function for adhesion molecules in the pathogenesis of uveitis. Heij et al., 82(4) Br. J. Ophthalmol. 432-37 (1998).

[030] Further, the number of cells expressing adhesion molecules is markedly increased in all vernal keratoconjunctivitis (VKC) specimens, concurrent with a heavy inflammatory infiltrate. Strong ICAM-I expression was induced on the basal epithelial cells and vascular endothelial cells, and was also expressed on stromal mononuclear cells. Abu el-Asrar et al., 81(12) Br. J. Ophthalmol. 1099-06 (1999).

[031] Similarly, 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).

[032] 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. For example, antibodies to selectins, integrins, to ICAM-I, a member of the ICAM family, can reduce inflammation in animal models of uveitis.

[033] 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. Although 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.

[034] 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. In some instances, 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.

[035] The term "bioconjugate" as used herein 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. Methods of conjugation of the bioselective peptide and the cell-adhesion barrier molecules are generally known in the art. 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. In preferred embodiments of the invention, 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.

[036] 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. Examples of polysaccharides include, but are not limited to, agarose, dextran, heparin, chondroitin sulfate, and hyaluronic acid.

[037] Alternatively, 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. In addition, 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. These blocks can be made of, for example, lactic acid, glycolic acid, ε-caprolactone, lactic-co-glycolic acid oligomers, trimethylene carbonate, anhydrides, and amino acids. These examples are not exhaustive; other oligomers may also be used for block copolymers. Additionally, the non- adhesive component may be a solid, such as a colloidal particle. Serum proteins such as albumin may also serve as a low cell-adhesive colloidal component. [038] In a particular embodiment of the present invention, the bioconjugate includes a protein zero peptide, YTDNGTF (SEQ ID NO:3), which has targeted liposomes to ICAM- i present on melanoma cells. Jaafari & Foldvari, 91(2) J. Pharm. Sci. 396-404 (2002). 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. For conjugation to dextran, a cysteine residue was added to the NH₂ end of the peptide, yielding the 8-mer having an amino acid sequence CYTDNGTF (SEQ ID NO:4).

[039] In vitro adhesion assays, described in the Examples below, showed that treatments with a low and high dose CYTDNGTF (SEQ ID NO:4) peptide/dextran bioconjugate resulted in a significant reduction of neutrophil adhesion to inflamed vascular endothelial cells when compared to untreated control samples (Figure 2). Moreover, in a mouse model of inflammatory disease, after topical application as described in the Examples below, this bioconjugate was selectively retained on diseased eyes, but not on healthy eyes. This selective retention was mediated by ICAM-I binding.

[040] 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. For example, 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). Thus, 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).

[041] Moreover, the term "peptide" is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. Hence, the 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.

[042] Included also within the scope of the invention is any 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. The probability that a particular oligonucleotide will, in fact, constitute the actual XXX-encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic or prokaryotic cells expressing an anti-CPAA antibody or portion. Such "codon usage rules" are disclosed by Lathe et al., 183 J. Molec. Biol. 1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding YTDNGTF (SEQ ID NO:3) sequences is identified. [043] Although occasionally an amino acid sequence can be encoded by only a single oligonucleotide, frequently the amino acid sequence can be encoded by any of a set of similar oligonucleotides. Whereas all of the members of this set contain oligonucleotides capable of encoding the peptide and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide, typically only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the protein. [044] The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the YTDNGTF (SEQ ID NO:3) peptide is used to identify the sequence of a complementary oligonucleotide or set of oligo-nucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the YTDNGTF (SEQ ID NO:3)-encoding gene (Sambrook et al., 1989).

[045] A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a YTDNGTF (SEQ ID NO:3) peptide (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the above-described procedure), synthesized, and hybridized by means well known in the art, against a DNA or a cDNA preparation derived from cells which are capable of expressing YTDNGTF (SEQ ID NO:3) peptides or portions of equivalents thereof. 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).

[046] Additionally, DNA synthesis can be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al., 1989, and by Hayrnes et al., in NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (IRL Press, DC, 1985). Hybridization wash conditions can include wash solution of 0.2 x SSC/0.1% SDS and incubation with rotation for 10 min at room temperature, (low stringency wash), wash solution of prewarrned (42⁰C) 0.2 x SSC/0.1% SDS and incubation with rotation for fifteen minutes at 42⁰C (medium stringency wash) and wash solution of prewarmed (68°C) 0.1 x SSC/0.1% SDS and incubation with rotation for fifteen minutes at 68°C (high stringency wash). See Ausubel et al., ANTIBODIES: LAB. MANUAL (Harlow & Lane eds., Cold Spring Harbor Lab., 1988). Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu et al., 82 P.N.A.S. USA 3771-75 (1985)), fibronectin (Suzuki et al., 4 Bur. MoI. Biol. Organ. J. 2519-24 (1985)), the human estrogen receptor gene (Walter et al., 82 P.N.A.S. USA 7889-93 (1985)), tissue-type plasminogen activator (Pennica et al., 301 Nature 214 21 (1983)) and human term placental alkaline phosphatase complementary DNA (Keun et al., 82 P.N.A.S. USA 8715-19 (1985)).

[047] It is also intended that the 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. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the peptides.

[048] For example, one class of substitutions is conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and GIu; substitution between the amide residues Asn and GIn; exchange of the basic residues Ly s and Arg; replacements among the aromatic residues Phe, Tyr, and the like. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., 247 Science 1306-10 (1990).

[049] Variant or agonist peptides may be fully functional or may lack some function in one or more activities. Fully functional variants typically contain only conservative variations or variations in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region. [050] Amino acids that are essential for function can be identified by methods known in the art. such as site-directed mutagenesis or alanine-scanning mutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as epitope binding or in vitro ADCC activity. Sites that are critical for ligand- receptor binding can also be determined by structural analysis such as crystallography, nuclear magnetic resonance, or photoaffinity labeling. Smith et al., 224 J. MoI. Biol. 899-904 (1992); de Vos et al., 255 Science 306-12 (1992).

[051 ] Moreover, polypeptides often contain amino acids other than the twenty "naturally occurring" amino acids. Further, many 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.

[052] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as PROTEINS-STRUCTURE & MOLECULAR PROPERTIES (2nd ed., T. E. Creighton, W. H. Freeman & Co., NY, 1993). Many detailed reviews are available on this subject, such as by WOLD, POSTTRANSLAΉONAL COVALENT MODIFICATION OF PROTEINS, 1-12 (Johnson, ed., Acad. Press, NY, 1983); Seifter et al., 182 Meth. Enzymol. 626-46 (1990); and Rattan et al., 663 Ann. N. Y. Acad. Sci. 48-62 (1992). Accordingly, 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.

[053] As noted previously, this invention also includes the peptides for preparation of bioconjugate that have been modified with an additional N-terminal or C-terminal cysteine residue. In another embodiment, the nucleic acid sequences encoding the peptides are modified to express the additional cysteine residue(s). Once the peptide is modified by addition of an N-terminal or C -terminal cysteine residue, it 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. In that regard, the peptides of the present invention may be described as the peptide having the amino acid sequence X_IYX₂DNGTFX₃ (SEQ ID NO: 8), wherein X₁ is NH₂ or cys; X₂ is ser (S) or thr (T); X₃ is COOH or cys. With conservative amino acid sequences, the peptides of the present invention encompass those with the formula XiX₂X₃X₄XsGX₆X₇Xg, (SEQ ID N0:9), wherein X₁ isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X5 is Asn or GIn; Xg is Thr or Ser; X₇ is Phe, Tyr or Trp; and X₈ is COOH or Cys. One or more termini of the peptide may include one or more additional amino acid residues.

[054] As noted previously, there is evidence that ICAM-I is expressed at higher amounts in the vitreous than in the peripheral blood of uveitis patients. Martin et al., 216 Ophthalmologica 203-08 (2002). Hence, delivery of an anti-ICAM-1 composition directly to the eye may have little systemic effect.

[055] 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.

[056] Several embodiments of the present invention provide for pharmaceutical compositions formulated for treating eye inflammation. The eye inflammation may be associated with any number of conditions, including but not limited to iritis, 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.

[057] In one aspect, 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.

[058] In use, at least one drop of 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. [059] The term "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.

[060] The topical eye formulation may further include one or more gelling or thickening agents, such as polyvinyl alcohol, methyl cellulose. CARBAPOL® resin, or hyaluronic acid, or another biocompatible, pharmaceutically acceptable excipient which are well-known in the art,

[061] 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.

[062] In another embodiment of the present invention, 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. In yet another embodiment of the present invention, the additional active agent may also be at least one of nanobodies; antibodies, portions or fragments of antibodies; signaling pathway inhibitors; transcription factor inhibitors; receptor antagonists or agonists; small molecules; oligonucleotides or nucleic acids; fusion proteins, peptides or protein fragments; antibiotics; antivirals; transcription factor inhibitors or activators; receptor antagonists or agonists; small molecules; and allosteric regulators such as allosteric modulators of ceil surface receptors including G-protein coupled receptors (GPCR), cell surface receptor internalization inducers, and GPCR inverse agonists.

[063] In that regard, the term "active agent" is defined broadly as having an impact on a living system such as a cell, nerve, or tissue. For example, 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.

EXAMPLES Example 1. Peptide dextran conjugate

[064] 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. The pH was adjusted to pH 8.0-pH 8.5 with triethanolamine (TEA). 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.

Example 2. In vitro ICAM-I -mediated leukocyte cell adhesion assay

[065] Human aortic endothelial cell (HAEC) 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 α (TNF-α, 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. Shear stress for these experiments was fixed at 1 dyne/cm². MPRO neutrophil cultures (ATCC# CRL-1 1422) were resuspended in prewarmed HBSS to a concentration of 0.5x10⁶ 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.

[066] An observation area was chosen, the recording equipment was started, and flow was initiated. A few minutes of data was recorded at the fixed shear rate (1 dyne/cm ), Treatments with both a low (b l-l-.l) and a high dose (b 1-1-1) CYTDNGTF peptide/dextran bioconjugate resulted in a significant reduction of neutrophil adhesion to inflamed vascular endothelial cells when compared to untreated control samples (pc 1) (Figure 2).

Example 3. Retention of ICAM-I -targeting bioconjugates on the corneal surface

[067] The evaluation of the retention of ICAM- 1 targeting bioconjugates on the corneal surface utilized 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 μm fluorescent latex beads. These beads were then suspended in pH 7.4 buffered physiologic saline solution. One drop of suspension was placed on each cornea and imaged using a stereomicroscope equipped with epifiuorescence illumination. After five minutes, all tissues were washed with plain saline solution. Figure 3 shows representative photomicrographs from the studies. There was no retention of bioconjugate on healthy cornea after only one wash with saline solution (Figure 3B). On diseased corneas, however, bioconjugate remained on the surface after multiple washes (Figures 3D, 3E), These findings indicate that bioconjugate was selectively retained on diseased eyes after topical application, and this selective retention was mediated by ICAM-I binding.

Claims

CLAIMS We claim:

1. A pharmaceutical composition formulated for placement in the eye comprising a peptide/low-adhesive bioconjugate that binds ICAM-I and inhibits intercellular binding, wherein said peptide has the amino acid sequence X1X2X3X4X5GX6X7X8, (SEQ ID NO:9); wherein Xi isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X₅ is Asn or GIn; X₆ is Thr or Ser; X₇ is Phe, Tyr or Trp; and X₈ is COOH or Cys.

2. The pharmaceutical composition of claim 1. wherein the peptide has the amino acid sequence X_IYX₂DNGTFX₃ (SEQ ID NO: 8); wherein Xj is NH₂ or cys; X₂ is ser or thr; X₃ is COOH or cys.

3. The pharmaceutical composition of claim 2, wherein said peptide has the amino acid sequence YXDNGTF (SEQ ID NO:1), wherein X is Ser or Thr.

4. The pharmaceutical composition of claim 2, wherein the peptide has the amino acid sequence CYXDNGTF (SEQ ID N0:2), wherein X is Ser or Thr.

5. The pharmaceutical composition of any of claims 1-4, wherein the low-adhesive of the bioconjugate is dextran.

6. The pharmaceutical composition of any of claims 1-5, further at least one additional active agent.

7. The pharmaceutical composition of claim 6, wherein said active agent is selected from the group consisting of such as glucocorticosteoids; cyclosporines; rapamycins; anti-cytokines; nanobodies; antibodies, portions or antibodies or antibody fragments; signaling pathway inhibitors; transcription factor inhibitors; receptor antagonists or agonists; small molecules; oligonucleotides or nucleic acids; fusion proteins, peptides or protein fragments; antibiotics; antivirals; transcription factor inhibitors or activators; receptor antagonists or agonists; small molecule inhibitors; and allosteric regulators.

8. The pharmaceutical composition of any of claims 1 to 7, wherein said composition is formulated for topical application to the eye.

9. The pharmaceutical composition of any of claims 1 to 7, wherein said composition is formulated for injection into the eye.

10. The use of the pharmaceutical composition of any of claims 1 to 9 in a medicament formulated for treating eye inflammation.

11. The use of the pharmaceutical of claim 10, wherein said medicament is useful for treating eye inflammation is associated with iritis, scleritis, chorioretinitis, uveitis, proliferative vitreoretinopathy, traction retinal detachment, vitreous hemorrhage, retinal inflammation, or Sjogren's or non-Sjogren keratoconjunctivitis sicca.

12. A method for treating inflammation of the eye comprising administering to an eye of mammal in need thereof a pharmaceutical formulation comprising a peptide/low-adhesive bioconjugate that binds ICAM-I and inhibits intercellular binding; wherein said peptide has the amino acid sequence X]X₂X₃X₄XsGX₆X₇X₈, (SEQ ID NO:9); wherein Xi isNH2 or Cys; X₂ is Tyr, Phe or Trp; X₃ is Ser or Thr; X₄ is Asp or GIu; X₅ is Asn or GIn; X₆ is Thr or Ser; X₇ is Phe, Tyr or Trp; and X₈ is COOH or Cys.

13. The method of claim 12 wherein the peptide is selected from the group consisting of X_IYX₂DNGTFX₃ (SEQ ID NO:8), wherein Xi is NH₂ or cys, X₂ is ser or thr, X₃ is COOH or cys; YXDNGTF (SEQ ID NO: 1), where and X is Ser or Thr; and CYXDNGTF (SEQ ID NO:2), wherein X is Ser or Thr.

14. The method of claim 13 wherein the peptide is CYTDNGTF (SEQ ID NO:4).

15. The method of any of claims 12 to 14, wherein said low-adhesive is dextran.