WO2010057962A2 - Conjugués de crf ayant des demi-vies prolongées - Google Patents

Conjugués de crf ayant des demi-vies prolongées Download PDF

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WO2010057962A2
WO2010057962A2 PCT/EP2009/065513 EP2009065513W WO2010057962A2 WO 2010057962 A2 WO2010057962 A2 WO 2010057962A2 EP 2009065513 W EP2009065513 W EP 2009065513W WO 2010057962 A2 WO2010057962 A2 WO 2010057962A2
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crf
conjugate
administered
peg
conjugates
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WO2010057962A3 (fr
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William Henry
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Neutron Limited
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    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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 conjugates of corticotropin-releasing factor (CRF) having an increased half-life and stability as compared to unmodified CRF.
  • CRF corticotropin-releasing factor
  • Corticotropin-Releasing Factor is an endogenous 41 amino acid peptide first identified in 1981 as the major hypothalamic hormone responsible for stimulation of the pituitary-adrenal axis (Vale, W., et al, Science 213:1394-1397 (1981)).
  • CRF can be obtained from natural sources, expressed recombinantly, or produced synthetically.
  • CRF has been shown to have a peripheral, non-endocrine function mediated biological activity as a potent inhibitor of edema and inflammation (Wei, E. T. et al., Ciba Foundation Symposium 172:258-276 (1993)).
  • CRF corticotrop(h)in-releasing hormone
  • corticoliberin corticorelin
  • the CRF neuropeptide was first isolated from extracts of ovine hypothalami (OCRF; Vale, W., et al., Science 213:1394-1397 (1981)) and has subsequently been identified and isolated from the hypothalamus of numerous other mammals including rat (rCRF; Rivier, J., et al., Proc. Natl. Acad. Sci. USA 80:4851-4855 (1983)), porcine (PCRF; Schally, A., et al., Proc. Natl Acad. Sci. USA 78:5197-5201 (1981) and human (hCRF; Shibahara, S., et al., EMBO J. 2:775-779 (1983)).
  • OCRF ovine hypothalami
  • CRF has been shown to be a safe and useful pharmaceutical agent for a variety of different applications in humans.
  • CRF in vivo administration of CRF has been extensively employed to help elucidate the cause of hyper- and hypo-cortisolemic conditions in humans and is an extremely useful diagnostic and investigative tool for various other disorders affecting the hypothalamic-pituitary-adrenal axis, including endogenous depression and Cushing's disease (Chrousos, G., et al, JV. Eng. J. Med. 310:622 (1984) and Lytras, N., et al, Clin. Endocrinol. 20:71 (1984)).
  • CRF in vivo administration of CRF is useful to test corticotropic function of the anterior pituitary in all cases in which an impairment of the anterior pituitary function is suspected.
  • CRF hypothalamus-pituitary-adrenal
  • CRF also possesses in vivo antiinflammatory activity.
  • CRF prevents vascular leakage induced by a variety of inflammatory mediators that appear to act selectively on post-capillary venules in skin.
  • CRF also inhibits injury- and inflammatory mediator-induced leakage from capillaries in muscle, cerebral micro-vessels, and lung alveolar capillaries.
  • CRF peptide In light of the novel anti-inflammatory activity of the CRF peptide, numerous clinical indications are evident. For example, clinical indications for which the CRF peptide may find use include rheumatoid arthritis, edema secondary to brain tumors or irradiation for cancer, edema resulting from stroke, head trauma or spinal cord injury, post-surgical edema, asthma and other respiratory diseases and cystoid macular edema of the eye. [0009]
  • One of the challenges of many polypeptides used in disease treatment is that they have a relatively short half-life after administration. Proteins introduced into the blood are rapidly cleared from the mammalian subject by the kidneys.
  • the present invention relates to conjugates of CRF that have been modified to include a moiety that protects CRF from degradation and prolongs the half-life of CRF.
  • the CRF conjugates of the invention have an increased half-life which results in a dose-sparing effect and less frequent administration.
  • An example of a CRF conjugate is CRF that has been modified to include moieties such as polyethylene glycol covalently bound to CRF.
  • the invention provides for CRF conjugates comprising
  • the CRF component of the CRF conjugate has the sequence identified as human CRF identified in Figure 1.
  • the sequence of CRF may be modified or derivatized to include one or more changes in the amino acid sequence, including, but not limited to, insertions, deletions or substitutions.
  • the sequence of CRF has been modified to include one or more cysteine residues.
  • the sequence of CRF may include cysteine as a substitution of one or more of the existing residues of CRF.
  • the cysteine residue may be incorporated as an addition to the existing sequence of CRF.
  • cysteine residues may be inserted within the sequence of CRF, or the cysteine residue may be added to the amino or carboxy terminus of the sequence. In still another embodiment, cysteine residues are added to the amino and carboxy termini of the sequence. When two or more cysteine residues are present, one or more disulfide bonds may form between cysteine residues, thus forming a cyclic peptide.
  • the polyethylene glycol moiety may be covalently bound to CRF through one or more of the cysteine residues.
  • the polyethylene glycol moiety may be covalently bound through one or more of the existing 41 amino acids of CRF, including, but not limited to, lysine, histidine, arginine, aspartic acid, glutamic acid, serine, as well as the N-terminus or C-terminus of the CRF polypeptide.
  • the CRF conjugate may have polyethylene glycol moieties attached via one or more lysine residues.
  • the CRF conjugates of the invention include CRF which has been modified to include one or more polyethylene glycol polymers through a multitude of different sites in the CRF sequence.
  • the CRF conjugate comprises two PEG moieties bound to two cysteine residues.
  • the CRF-PEG conjugate comprises one or more PEG groups simultaneously bound to two cysteine residues in a cysteine added variant of CRF.
  • the CRF-PEG conjugate comprises one or more PEG groups simultaneously bound to two cysteine residues that form a disulfide bond in a cysteine added variant of CRF.
  • conjugates may be produced via reductive cleavage of a disulfide bond, followed by a reaction in which the PEG moiety becomes bound to both thio groups.
  • the resulting CRF conjugate contains a PEG moiety that bridges two sulfurs that had formed a disulfide bond.
  • the CRF conjugate contains a PEG bound to both the C-terminal and N-terminal cysteine residues of a cysteine added variant of CRF.
  • a polyethylene glycol polymer is conjugated to a cysteine added variant of CRF according to general formula I:
  • both -S- are from cysteine residues in a cysteine added variant of CRF, which in certain embodiments form a disulfide bond
  • Q represents a linking group, which is a direct bond, alkylene (preferably C 1-1O alkylene), alkenylene, -O-alkylene-, -O-alkylene-O,
  • -Q NH)-NR 1 -, -C(O)-N(R ] )-O-, -C(O)-N(RVC(O)-, -C(O)-N(R 1 )-, -C(O)-, -N(R 1 )-,
  • aryl group includes phenyl, benzoyl and naphthyl groups
  • heteroaryl group includes pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, primidine and purine
  • each R 1 , R 2 , R 3 , or R 4 is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heterocyclyl
  • the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • Substituents which may be present on an optionally substituted aryl or heteroaryl group include, for example, one or more of the same or different substituents selected from
  • -C N-NR 1 COR, -N + R 3 , -N + H 3 , -N + HR 2 , -N + H 2 R, halogen, for example fluorine or chlorine,
  • each R or R 1 independently represents hydrogen or an alkyl (preferably C 1-6 ) or an aryl (preferably phenyl) group.
  • the presence of electron withdrawing substituents is especially preferred.
  • CRF dimer Two cysteine added variants of CRF may be bound together via a disulfide bond, to form a CRF dimer.
  • the CRF dimer may be conjugated to a polyethylene glycol containing moiety.
  • the CRF dimer conjugate is bound to PEG through the disulfide bond that binds the two CRF polypeptides together.
  • a polyethylene glycol polymer is conjugated to two cysteine added variants of CRF according to general formula III:
  • both -S- are from cysteine residues in a cysteine added variant of CRF, which in certain embodiments form a disulfide bond
  • Q represents a linking group, which is a direct bond, alkylene (preferably C 1-I o alkylene), alkenylene, -O-alkylene-, -O-alkylene-0-, -O-alkylene-NR 1 -, -NR 2 -alkylene-, - NR 2 -alkylene-O-, -NR ⁇ alkylene-NR 1 -, -C(O)O-alkylene-, -C(O)O-alkylene-O-, -C(O)O-alkylene-NR 1 -, -C(O)NR 2 -alkylene-, -C(O)NR 2 -alkylene-O-, -C(O)NR 2 -alkylene-NR'-, -C(NR 3 R 4 )NR 2 -alkylene
  • R 2 , R 3 , or R 4 is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heterocyclyl; and wherein the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • Substituents which may be present on an optionally substituted aryl or heteroaryl group include, for example, one or more of the same or different substituents selected from
  • -C N-NR 1 COR, -N + R 3 , -N + H 3 , -N + HR 2 , -N + H 2 R, halogen, for example fluorine or chlorine,
  • each R or R 1 independently represents hydrogen or an alkyl (preferably C] -6 ) or an aryl (preferably phenyl) group.
  • the presence of electron withdrawing substituents is especially preferred.
  • PEG is conjugated to CRF according to formula IV:
  • a polyethylene glycol polymer is conjugated to a biological molecule to which one or more cysteine residues have been incorporated into the sequence of the biological molecule.
  • the biological molecule is a naturally occurring polypeptide or peptide which does not contain a cysteine residue, but in the context of the invention has been modified to incorporate a cysteine residue. Examples of such biological molecules include, but are not limited to, CRF, insulin, glucagon, and interferon.
  • the biological molecule is a synthetic polypeptide that does not contain a cysteine residue, but in the context of this invention has been modified to incorporate a cysteine residue.
  • the sequence of the biological molecule may be modified or derivatized to include one or more cysteine residues, including, but not limited to, insertions, or substitutions.
  • the sequence of the biological molecule may include cysteine as a substitution of one or more of the existing residues of the biological molecule.
  • the cysteine residues may be inserted within the sequence of the biological molecule, or the cysteine residue may be added to the amino and/or carboxy termini. In another embodiment, cysteine residues are added to both the amino and carboxy termini of the sequence of the biological molecule.
  • both -S- are from cysteine residues in the biological molecule containing two or more cysteines, which in certain embodiments form a disulfide bond
  • Q represents a linking group, which is a direct bond, alkylene (preferably Ci -I0 alkylene), alkenylene,
  • -Q NH)-NR 1 -, -C(O)-N(R')-O-, -C(O)-N(R 1 K(O)-, -C(O)-N(R 1 )-, -C(O)-, -N(R 1 )-,
  • aryl group includes phenyl, benzoyl and naphthyl groups
  • heteroaryl group includes pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, primidine and purine
  • each R , R , R , or R is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heterocyclyl; and wherein the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF.
  • Such biological activities include, for example, the ability to stimulate the release of ACTH, the ability to treat tumor or to inhibit edema in vivo, and the ability to bind to CRF receptors.
  • the biological activity of CRF conjugates may be determined using the assays described herein.
  • the conjugates of the present invention have an increased circulating half-life, plasma residence time and/or decreased clearance.
  • the CRF conjugates have increased clinical activity in vivo as compared to unmodified CRF.
  • the conjugates of the invention have improved potency, stability, area under the curve and circulating half-life.
  • the CRF conjugates of the invention have an improved pharmacokinetic profile as compared to unmodified CRF.
  • the CRF conjugates of the invention may show an improvement in one or more parameters of the pharmacokinetic profile, including AUC, C max , clearance (CL), half-life, and bioavailability as compared to unmodified CRF.
  • the CRF conjugates are useful for the treatment of tumors in a patent.
  • the CRF conjugates are useful for treatment or managing cancer in a patent.
  • cancer refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells.
  • cancer encompasses a disease involving both pre-malignant and malignant cancer cells.
  • the CRF conjugates can be used to present tumor progression in a patient.
  • the term "tumor progression" means that the tumor growth is inhibited, stopped or reversed, that the size or Volume of the tumor remains the same or decreases, and/or no additional metastases of the tumor remains the same or decreases, and/or no additional metastases of the tumor are formed in other parts of the body.
  • the CRF conjugates are useful for treating brain edema in patients in need thereof.
  • such brain edema may be the result of injury or disease to the brain.
  • the present invention relates to methods of treating brain edema resulting from primary or metastasis brain tumors, comprising administering CRF conjugates to patients in need thereof.
  • the CRF conjugates of the invention are useful in treating patients by reducing inflammation and edema in those patients, comprising administering a therapeutically effective amount of the CRF conjugates and formulations of the invention.
  • the CRF conjugates of the invention are useful in providing vasoprotective effects which may be evidenced as a reduction in edema when administered to patients in need thereof.
  • the methods of administering the CRF conjugates of the invention may be useful in reducing peritumoral brain edema.
  • the administration of CRF conjugates to a patient for the treatment of brain edema may be combined with other therapeutics for the treatment of edema.
  • the CRF conjugates of the invention may be used in combination with steroidal therapeutics for the treatment of brain edema, including, but not limited to glucocorticoids.
  • Glucocorticoid steroids include hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, alderstone and deoxycorticosterone acetate.
  • the other therapeutic may be administered concurrently, prior to or subsequently to the administration of the CRP conjugate.
  • the CRF conjugates may be administered to patients for the treatment of brain edema, wherein the conjugate is administered in a treatment regimen as a steroid sparing agent facilitating steroid taper.
  • the invention also encompasses a method for managing brain edema in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a CRF conjugate and a steroid, wherein the method provides a steroid sparing effect.
  • the present invention further provides a method for providing replacement therapy for steroid therapy in a subject receiving such therapy, which comprises administration of a steroid-sparing amount of a CRF conjugate.
  • the invention also provides a method for treating brain edema, comprising a treatment regimen steroid in combination with a CRF conjugate, whereby total exposure to the steroid is reduced by the administration of the CRF conjugate.
  • the present invention relates to pharmaceutical compositions containing a
  • the CRF conjugate may be formulated with a pharmaceutically acceptable carrier. Due to the increased half-life of the CRF conjugate, the pharmaceutical compositions may contain a lower dose of CRF than typically administered to effectively treat edema.
  • the pharmaceutical formulations of the invention may be formulated for parenteral administration, including, but not limited to, intradermal, subcutaneous, and intramuscular injections, and intravenous or intraosseous infusions.
  • the pharmaceutical formulations of the present invention can take the form of solutions, suspensions, or emulsions that include a CRF conjugate, such as CRF chemically modified with polyethylene glycol, and a pharmaceutically acceptable diluent, adjuvant or carrier, depending on the route of administration.
  • the pharmaceutical compositions of the invention are formulated to deliver a therapeutic dose of the CRF conjugate of the invention.
  • the dose of the CRF conjugates contained in pharmaceutical formulation can range from about 1 ⁇ g to about 10 mg. In certain embodiments the dose of the CRF conjugate can range from about 0.1 mg to about 5 mg, or from about 0.3 mg to about 2 mg. In certain embodiments, the dose of the CRF conjugate can be about 0.3 mg, about 0.5 mg, about 1 mg, about 2 mg, about 4 mg or about 5 mg.
  • the conjugates of the invention can be used in the same manner as unmodified CRF. However because of the improved properties of the CRF conjugates, the pharmaceutical formulations of the invention can be administered less frequently than the unmodified CRF. For example, the CRF conjugates may be administered once weekly instead of the once daily for unmodified CRF.
  • the present invention also encompasses dosing regimens, wherein the CRF derivatives may be administered once a day, once every two, three or four days, or once a week to effectively treat edema. Decreased frequency of administration is expected to result in improved patient compliance leading to improved treatment outcomes, as well as improved patient quality of life.
  • FIG. 1 shows the amino acid sequences of the human and rat CRF peptides as compared to that of the ovine CRF peptide. Amino acids are presented as their standard one- letter designations. Amino acids in the ovine sequence which are presented in bold font and are underlined are those that differ from the human/rat CRF sequence. 5- DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is based on conjugates of CRF that have been modified to include a moiety that results in a form of CRF that has an increased circulating half-life or plasma residence time as compared to unmodified CRF.
  • the present invention is also related to methods of preparing such conjugates.
  • the present invention further relates to methods of using such conjugates for treating tumor or cancer in patients.
  • the present invention also relates to methods of using such conjugates for reducing inflammation and edema in patients.
  • the CRF conjugates of the present invention have an improved pharmacokinetic profile as compared to unmodified CRF.
  • the CRF conjugates of the invention may show an improvement in one or more parameters of the pharmacokinetic profile, including AUC, C max , clearance (CL), half-life, and bioavailability as compared to unmodified CRF.
  • the CRF conjugates of the present invention include CRF with an unmodified amino acid sequence as is shown in Figure 1, wherein one or more residues are covalently bound to polyethylene glycol.
  • CRF conjugates of the present invention also include cysteine added variants of CRF, where one or more cysteine residues have been inserted into one of the CRF amino acid sequences shown in Figure 1 , or substituted for one or more residues of one of the CRF sequences shown in Figure 1.
  • the conjugated cysteine added variants of CRF include CRF sequences with cysteine residues added at the N-terminus, the C-terminus, or both the N-terminus and C-terminus of one of the amino acid sequences shown in Figure 1.
  • two cysteine residues may together form a disulfide bond, thus forming a cyclic peptide.
  • a cysteine residue at the C-terminus of the CRF sequence forms a disulfide bond with a cysteine residue at the N-terminus.
  • the CRF conjugates of the present invention can be used to treat cancer by administering to a patient in need thereof a therapeutically acceptable amount of a CFR conjugate.
  • the CRF conjugates of the present invention can be used to prevent tumor progression of administering to a patient in need thereof a therapeutically acceptable amount of a CFR conjugate.
  • the CRF conjugates of the present invention can be used to treat edema by administering to a patient in need thereof a therapeutically acceptable amount of a CRF conjugate.
  • Another aspect of the invention is a method of treating edema comprising administering to a patient in need thereof a pharmaceutical composition comprising CRF chemically modified with polyethylene glycol and a pharmaceutically acceptable diluent, adjuvant or carrier.
  • Another aspect of the invention is a method for treating brain edema comprising administering CRF conjugate, wherein the conjugate is administered in a treatment regimen as a steroid sparing agent facilitating steroid taper.
  • Another aspect of the invention is a method for managing brain edema in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CRF conjugate and a steroid, wherein the method provides a steroid sparing effect.
  • Another aspect of the invention is a method for providing replacement therapy for steroid therapy in a subject receiving such therapy, the method comprising administration of a steroid-sparing amount of CRF conjugate.
  • Another aspect of the invention is a method for treating brain edema, comprising a treatment regimen steroid in combination with a CRF conjugate, whereby total exposure to the steroid is reduced by the administration of the CRF conjugate.
  • area under the curve or "AUC,” as used herein in the context of administering a peptide drug to a patient, is defined as total area under the curve that describes the concentration of drug in systemic circulation in the patient as a function of time from zero to infinity.
  • the term "clearance” or "renal clearance” is defined as the volume of plasma that contains the amount of drug excreted per minute.
  • corticotropin releasing factor As used herein, the terms "corticotropin releasing factor,” "CRF,”
  • corticotrop(h)in-releasing hormone have a functional definition and refer to peptides which share one or more of the biological activities of the native, intact CRF peptide. Such biological activities include, for example, the ability to stimulate the release of ACTH, the ability to inhibit edema in vivo and the ability to bind to CRF receptors.
  • Each of the above terms is intended to denote the 41 amino acid human, rat, ovine, sheep, goat, porcine and fish corticotropin releasing factor peptides and CRF peptides from other mammals, whether isolated from natural source extraction and purification, from recombinant cell culture systems or synthesized using peptide synthesis technology. These terms are also intended to denote other CRF-related peptides which share one or more of the biological activities of the native CRF peptides, such as urocortin (Vaughan, J., et al., Nature 378:287-292 (1995); Donaldson, C. J., et al., Endocrinology 137(5):2167-2170 (1996); and Tumbull, A.
  • the CRF peptides employed in the formulations of the present invention are preferably synthesized using solid- or solution-phase peptide synthesis techniques, however, other sources of the CRF peptide are readily available to the ordinarily skilled artisan.
  • the amino acid sequences of the human, rat and ovine CRF peptides are presented in Figure 1.
  • corticotropin releasing factor and "CRF” likewise cover biologically active CRF equivalents; e.g., peptides differing in one or more amino acids in the overall amino acid sequence as well as substitutional, deletional, insertional and modified amino acid variants of CRF which substantially retain the biological activity normally associated with the intact CRF peptide.
  • CRF conjugate refers to a CRF polypeptide that has been modified to include a moiety that results in an improved pharmacokinetic profile as compared to unmodified CRF.
  • the improvement in the pharmacokinetic profile may be observed as an improvement in one or more of the following parameters: potency, stability, area under the curve and circulating half-life.
  • cyste added variant of CRF refers to CRF that has been modified by the insertion of one or more cysteine residues into the unmodified CRF sequence shown in Figure 1, or the substitution of one or more of the amino acid residues in the CRF polypeptide sequence shown in Figure 1, for cysteine residues.
  • half-life or "Xm,” in the context of administering a peptide drug to a patient, is defined as the time required for plasma concentration of a drug in a patient to be reduced by one half. There may be more than one half-life associated with the peptide drug depending on multiple clearance mechanisms, redistribution, and other mechanisms well known in the art.
  • alpha and beta half-lives are defined such that the alpha phase is associated with redistribution, and the beta phase is associated with clearance.
  • protein drugs that are, for the most part, confined to the bloodstream, there can be at least two clearance half-lives.
  • the precise impact of PEGylation on alpha phase and beta phase half-lives will vary depending upon the size and other parameters, as is well known in the art. Further explanation of "half-life” is found in Pharmaceutical Biotechnology (1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp 101 120).
  • a "patient in need thereof” refers to a patient who has been diagnosed with a condition that may be treated by CRF, e.g. , tumor, cancer, or brain edema.
  • the patient is a human patient.
  • the term "pharmaceutically acceptable" when used in reference to the formulations of the present invention denotes that a formulation does not result in an unacceptable level of irritation in the subject to whom the formulation is administered by any known administration regimen. What constitutes an unacceptable level of irritation will be readily determinable by those of ordinary skill in the art and will take into account erythema and eschar formation as well as the degree of edema associated with administration of the formulation.
  • the term “residence time,” in the context of administering a peptide drug to a patient, is defined as the average time that drug stays in the body of the patient after dosing.
  • the terms “treat,” “treating” or “treatment of mean that the severity of a subject's condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is an inhibition or delay in the progression of the condition and/or delay in the progression of the onset of disease or illness.
  • the terms “treat,” “treating” or “treatment of also means managing the disease state, e.g., tumor, cancer, or brain edema.
  • a "sufficient amount” or an “amount sufficient to” achieve a particular result refers to an amount of CRF conjugate that is effective to produce a desired effect, which is optionally a therapeutic effect (i. e., by administration of a therapeutically effective amount).
  • a "sufficient amount” or “an amount sufficient to” can be an amount that is effective to reduce the amount of steroid required to manage the tumor, cancer, or brain edema.
  • a “therapeutically effective” amount is an amount that provides some improvement or benefit to the subject.
  • a “therapeutically effective” amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom.
  • Clinical symptoms associated with the disorder that can be treated by the methods of the invention are well-known to those skilled in the art. Further, those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF. Such biological activities include, for example, the ability to stimulate the release of ACTH, the ability to treat tumor or cancer, the ability to inhibit edema in vivo and the ability to bind to CRF receptors. The biological activity of CRF conjugates may be determined using the assays described herein. [0056] Compared to unmodified CRF (i.e., CRF without a PEG attached), the conjugates of the present invention have an increased circulating half-life, plasma residence time and/or decreased clearance. In an embodiment of the invention, the CRF conjugates have increased clinical activity in vivo as compared to unmodified CRF.
  • the conjugates of the invention have improved potency, stability, area under the curve and circulating half-life.
  • the CRF conjugates of the invention have an improved pharmacokinetic profile as compared to unmodified CRF.
  • the CRF conjugates of the invention may show an improvement in one or more parameters of the pharmacokinetic profile, including AUC, C max , clearance (CL), half-life, and bioavailability as compared to unmodified CRF.
  • CRF to be modified in accordance with the invention may be obtained and isolated from natural sources.
  • CRF to be modified in accordance with the invention may be expressed recombinantly.
  • CRF to be modified in accordance with the invention may be synthetically produced.
  • the CRF component of the CRF conjugate has the sequence identified as human CRF identified in Figure 1.
  • the CRF component of the CRF conjugate has the sequence identified as rat or ovine CRF identified in Figure 1.
  • the sequence of CRF may be modified or derivatized to include one or more changes in the amino acid sequence, including, but not limited to, insertions, deletions or substitutions.
  • the sequence of CRJF has been modified to include one or more cysteine residues.
  • the sequence of CRF may include cysteine as a substitution of one or more of the existing residues of CRF.
  • the cysteine residue may be incorporated as an addition to the existing sequence of CRF.
  • the cysteine residues may be inserted within the sequence of CRF, the cysteine residue may be added to the amino or carboxy terminus of the sequence, or a cysteine residue may be added at both the amino and carboxy termini.
  • a CRF-PEG conjugate containing a PEG bound to one or more functional groups of the naturally occurring CRF polypeptide leads to increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo.
  • CRF may be modified by covalently binding a polyethylene glycol polymer through one or more of its 41 -amino acids including, but not limited to, lysine, histidine, arginine, aspartic acid, glutamic acid, serine, as well as the N-terminal ⁇ -amino and C-terminal carboxylate groups of the protein.
  • Polyethylene glycol polymer units can be linear or branched.
  • the CRF-PEG conjugate may be delivered intravenously or subcutaneously via injection.
  • Another aspect of the invention is a CRF-PEG conjugate where a polyethylene glycol polymer is bound to the lysine residue.
  • the ⁇ -amino group of lysine in CRF can be readily PEGylated by a variety of techniques, including, but not limited to, alkylation and acylation.
  • Another aspect of the invention is a CRF conjugate, where a polyethylene glycol polymer is bound to the N-terminal ⁇ -amino group.
  • the N-terminal ⁇ -amino residue of CRF can form a PEG conjugate by a variety of techniques including, but not limited to, alkylation or acylation of the N-terminal ⁇ -amino group.
  • cysteine added variants of CRF that contain one or more PEG conjugated cysteine residues that have been substituted for naturally occurring residues in the CRF polypeptide sequence.
  • Cysteine substituted CRF can be produced recombinantly by expressing DNA with point mutations that result in the substitution of a cysteine for a residue in naturally occurring CRF.
  • the codon TCT which codes for serine
  • TGC which codes for cysteine
  • CRF is produced via synthetic means, in the course of the synthesis it is possible to substitute a cysteine residue in place of one or more residues that naturally occur in CRF.
  • the cysteine can then be selectively conjugated to a polyethylene glycol polymer.
  • cysteine added variants of CRF that contain one or more PEG conjugated cysteine residues that have been inserted into the naturally occurring CRF sequence shown in Figure 1. If CRF is produced recombinant! y, this can be done by inserting one or more cysteine codon(s) into the DNA sequence that codes for CRF. In solid phase protein synthesis, cysteines are added at any point of the protein synthesis by introducing an additional cysteine residue where desired. The cysteine can then be selectively bound to a polyethylene glycol polymer.
  • Another aspect of the invention is a cysteine added variant of CRF that contains a PEG conjugated cysteine residue inserted at the N-terminus.
  • Another aspect of the invention is a CRF conjugate that contains a PEG bound to a cysteine residue inserted at the C-terminus.
  • Another aspect of the invention is a CRF conjugate that contains PEG bound to cysteine residues inserted at both the N-terminus and the C-terminus.
  • a cysteine residue at the C-terminus of the CRF sequence forms a disulfide bond with a cysteine residue at the N-terminus.
  • the CRF-PEG conjugate comprises one or more PEG groups simultaneously bound to two cysteine residues in a cysteine added variant of CRF. In another embodiment, the CRF-PEG conjugate comprises one or more PEG groups simultaneously bound to two cysteine residues that form a disulfide bond in a cysteine added variant of CRF. These conjugates may be produced via reductive cleavage of a disulfide bond, followed by a reaction in which the PEG moiety becomes bound to both thio groups. The resulting CRF conjugate contains a PEG moiety that bridges two sulfurs that had formed a disulfide bond. In a specific embodiment, the CRF conjugate contains a PEG bound to both the C-terminal and N-terminal cysteine residue of a cysteine added variant of CRF.
  • a polyethylene glycol polymer is conjugated to a cysteine added variant of CRF according to general formula I:
  • both -S- are from cysteine residues in a cysteine added variant of CRF, which in certain embodiments form a disulfide bond
  • Q represents a linking group, which is a direct bond, alkylene (preferably C 1-1O alkylene), alkenylene, -O-alkylene-, -O-alkylene-O-,
  • -Q NH)-NR 1 -, -C(O)-N(RVo-, -C(O)-N(RVC(O)-, -C(O)-N(R 1 )-, -C(O)-, -N(R 1 )-,
  • aryl group includes phenyl, benzoyl and naphthyl groups
  • heteroaryl group includes pyridine, pyrrole, furan, pyran, imidazole, pyrazole, oxazole, pyridazine, primidine and purine
  • each R 1 , R 2 , R 3 , or R 4 is independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heterocyclyl
  • the linkage to the polymer may be by way of a hydrolytically labile bond, or by a non-labile bond.
  • Substituents which may be present on an optionally substituted aryl or heteroaryl group include for example one or more of the same or different substituents selected from -
  • -C N-NR 1 COR, -N + R 3 , -N + H 3 , -N + HR 2 , -N + H 2 R, halogen, for example fluorine or chlorine,
  • each R or R' independently represents hydrogen or an alkyl (preferably Ci -6 ) or an aryl (preferably phenyl) group.
  • the presence of electron withdrawing substituents is especially preferred.
  • CRF dimer Two cysteine added variants of CRF may be bound together via a disulfide bond,o form a CRF dimer.
  • the CRF dimer may be conjugated to a polyethylene glycol containing moiety.
  • the CRF dimer conjugate is bound to PEG through the disulfide bond that binds the two CRF polypeptides together.
  • a polyethylene glycol polymer is conjugated to two cysteine added variants of CRF according to general formula III:
  • both -S- are from cysteine residues in a cysteine added variant of CRF, which in certain embodiments form a disulfide bond
  • Q represents a linking group, which is a direct bond, alkylene (preferably C 1- Io alkylene), alkenylene, -O-alkylene-, -O-alkylene-0-, -O-alkylene-NR 1 -, -NR 2 -alkylene-, - NR 2 -alkylene-O-, -NR 2 -alkylene-NR ] -, -C(O)O-alkylene-, -C(O)O-alkylene-O-, -C(O)O-alkylene-NR'-, -C(O)NR 2 -alkylene-, -C(O)NR 2 -alkylene-O-, -C(O)NR 2 -alkylene-NR 1 -, -C(NR 3 R 4 )NR 2
  • R is hydrogen or alkyl. In certain embodiments, R is hydrogen. In certain embodiments, R 1 is alkyl.
  • R is hydrogen or alkyl. In certain embodiments, R is hydrogen. In certain embodiments, R 2 is alkyl.
  • R 3 is hydrogen or alkyl.
  • R J is hydrogen. In certain embodiments, R is alkyl.
  • R 4 is hydrogen or alkyl. In certain embodiments, R is hydrogen. In certain embodiments, R 4 is alkyl.
  • Q is alkenylene, -O-alkylene-, -O-alkylene-0-,
  • Q is -O-(CH 2 ) n -, -O-(CH 2 ) n -O-, -0-(CH 2 ) H -NR 1 -, -NR 2 -
  • n is an integer of 1 , 2, 3, 4, 5, or 6.
  • n is 1.
  • n is 2.
  • n is 3.
  • Pn certain embodiments, n is 4.
  • n is 5.
  • n is 6.
  • Q is an alpha amino acid derivate.
  • alpha amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • Q is a beta amino acid derivative.
  • suitable beta amino acids is beta alanine.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF. Such biological activities include, for example, the ability to stimulate the release of ACTH, the ability to treat tumor or cancer, the ability to inhibit edema in vivo and the ability to bind to CRF receptors.
  • the biological activity of CRF conjugates may be determined using the assays described herein.
  • the CRF conjugates provided herein have a molecule weight sufficient to avoid renal filtration.
  • the CRF conjugates provided herein have a molecule weight of no less than about 60 kDa.
  • the CRF conjugates provided herein have a molecule weight from about 60 to 62 kDa.
  • polyethylene glycol polymers that will form conjugates with CRF polypeptides.
  • linear PEG polymers that contain a single polyethylene glycol chain, and there are branched or multi-arm PEG polymers.
  • Branched polyethylene glycol contains 2 or more separate linear PEG chains bound together through a unifying group.
  • two PEG polymers may be bound together by a lysine residue.
  • One linear PEG chain is bound to the ⁇ -amino group, while the other PEG chain is bound to the ⁇ -amino group.
  • the remaining carboxyl group of the lysine core is left available for covalent attachment to a protein.
  • Both linear and branched polyethylene glycol polymers are commercially available in a range of molecular weights.
  • a CRF-PEG conjugate contains one or more linear polyethylene glycol polymers bound to CRF, wherein each PEG having a molecular weight between about 2 kDa to about 100 kDa.
  • a CRF- PEG conjugate contains one or more linear polyethylene glycol polymers bound to CRF, wherein each branched PEG has a molecular weight between about 5 kDa to about 40 kDa.
  • a CRF-PEG conjugate contains one or more linear polyethylene glycol polymers bound to CRF, wherein each linear PEG has a molecular weight between about 10 kDa.
  • the CRF-PEG conjugate has a molecule weight sufficient to avoid renal filtration. In certain embodiments, the CRF-PEG conjugate has a molecule weight of no less than about 60 kDa. In certain embodiments, he CRF-PEG conjugate has a molecule weight from about 60 to 62 kDa. [0085]
  • a CRF-PEG conjugate of this invention may contain one or more branched polyethylene glycol polymers bound to CRF, wherein each branched PEG has a molecular weight between about 2 kDa to about 100 kDa.
  • a CRF- PEG conjugate contains one or more branched polyethylene glycol polymers bound to CRF, wherein each branched PEG has a molecular weight between about 5 kDa to about 40 kDa.
  • a CRF-PEG conjugate contains one or more branched polyethylene glycol polymers bound to CRF, wherein each branched PEG has a molecular weight between about 10 kDa.
  • the CRF-PEG conjugate has a molecule weight sufficient to avoid renal filtration.
  • the CRF-PEG conjugate has a molecule weight of no less than about 60 kDa.
  • he CRF-PEG conjugate has a molecule weight from about 60 to 62 kDa.
  • CRF can be conjugated with polyethylene glycol, without the modification of the original 41 residue polypeptide chains. Both the lysine ⁇ -amino group and the N-terminal ⁇ - amino group can be PEGylated by alkylation and acylation as demonstrated below. [0087] The ⁇ -amino group of lysine is a commonly used group for PEG conjugation of proteins, and CRF contains a single lysine residue. The PEG conjugation of lysine via its ⁇ - amino group may be accomplished by methods including, but not limited to, acylation and alkylation. When a PEG- Aide Hyde reacts with an amino group, a Schiff base is formed. Harris and Herat (U.S. Pat. No.
  • PEG conjugation of the ⁇ -amino group of lysine via acylation is a technique known in the art for conjugating PEG polymers to the ⁇ -amino group of lysine residues, such as the lysine residue in CRF.
  • PEG reagents are N- hydroxysuccinimidyl (NHS) esters of PEG as shown by Veronese, F. M. Biomaterials. 22(2001): 405-417.
  • Other PEG acylation reagents are PEG-p-nitophenylcarbonate and PEG- trichlorophenylcarbonate in Veronese F.M. et. al. Appl. Biochem.
  • CRF-PEG conjugates synthesized by acylation can be purified and isolated by methods known in the art, including gel filtration or size exclusion chromatography.
  • N-terminal ⁇ -amino group can be selectively bound to polyethylene glycol polymers, as taught in Kinstler (U.S. Pat. Nos. 6,586,398) incorporated herein by reference in its entirety.
  • N-terminal PEGylation is reductive alkylation with a PEG aldehyde, in a procedure similar to that described earlier.
  • a large excess methoxy PEG aldehyde can be mixed with the CRF protein in a buffered solution of pH 4-6.
  • Sodium cyanoborohydride is added to the mixture, and the desired CRF-PEG conjugates can be isolated and purified by methods known in the art.
  • the N-terminal amino group can also be modified by acylation with an activated NHS ester of PEG.
  • To a slightly basic buffered solution of CRF can be added a large excess of the PEG ester of NHS. After the reaction is complete, the CRF-PEG conjugate can be isolated and purified by methods known in the art. 5.2.2. Insertion and Substitution of Cysteine Residues
  • CRF derivatives where cysteines have been inserted or substituted can be produced by recombinant means using techniques known in the art. Expression of the desired cysteine substituted or inserted derivative may be done in either eukaryotic or bacterial cells by methods used by Shaw (U.S. Pat. No. 5,166,322) incorporated herein by reference in its entirety, for IL-3 cysteine added variants. Modifications to the naturally occurring CRF protein can be accomplished site directed PCR-based mutagenesis.
  • Cox III (U.S. Pat. No. 7,214,779) incorporated herein by reference in its entirety, discloses cysteine added variants of granulocyte-macrophage colony stimulating factor (GCSF) that are produced by recombinant means.
  • GCSF granulocyte-macrophage colony stimulating factor
  • Cysteine added variants of CRF can also be made by synthetic methods. Cysteine residues can be substituted for another amino acid residue during the course of the synthesis. By adding an additional step to the solid phase synthesis of CRF, a cysteine residue can also be inserted where desired in the polypeptide sequence. In solid phase synthesis, the cysteine may be added to the C-terminus of the CRF sequence at the first step of the synthesis. Alternatively, the cysteine may be added to the N-terminus of the CRF sequence, at the last step of solid phase synthesis. By adding a cysteine residue at the first and last steps of the solid phase synthesis, cysteine residues would be present at the C-terminus and N-terminus of the resulting cysteine added variant of CRF. A disulfide bond between the two cysteines may further result.
  • a conjugate attack by the thiol on the activated terminal vinyl group of PEG-vinylsulphone yields the PEGylated cysteine residue.
  • the cysteine thiol displaces a leaving group, such as chloro, bromo, iodo, or tosyl, via a nucleophilic attack to yield the PEG conjugated cysteine residue.
  • a cysteine residue reacts with PEG- ⁇ ; ⁇ -unsaturated carbonylPEG, via a thiol addition to the activated double bond in a Michael addition reaction.
  • Two cysteine groups that together form a disulfide bond may also be PEGylated selectively by using the technique shown in scheme 2.
  • the native disulfide bond is first reduced.
  • One of the resulting thiols from this bond can nucleophilically attack an electrophilic group, such as a 1,4-addition to an enone. This is followed by the departure of a leaving group, such as, e.g. a sulfone.
  • a leaving group such as, e.g. a sulfone.
  • the subsequent elimination to a second enone, followed by 1 ,4-addition by the remaining thiol leads to the bridged disulfide with a PEG group attached.
  • the chemistry as shown in scheme 2 can also be used to couple with a CRF having a single reactive cysteine.
  • the second reactive site on the bifunctional PEG can be masked by the addition of a second thiol compound, such as methyl mercaptan or beta mercaptoethanol.
  • X Cl, Br, 1, or -OSO 2 R'; R 1 is alkyl or aryl.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF. Such biological activities include, for example, the ability to stimulate the release of ACTH, the ability to inhibit edema in vivo and the ability to bind to CRF receptors.
  • the biological activity of CRF conjugates may be determined using biological assays known in the art, or the assay described in section 6.3.
  • the present invention is directed to methods for treating or managing cancer by administering a CRF conjugate to a human subject.
  • the methods of the disclosure can be used to prevent tumor progression.
  • a method for preventing tumor progression in a human by administering a composition comprising a CRF conjugate to a human diagnosed with or potentially having the tumor.
  • a method for preventing tumor progression in a human by administering a composition comprising a CRF conjugate, wherein the CRF conjugate is administered at a dose effective to inhibit tumor progression, to a human diagnosed with or potentially having the tumor.
  • a human diagnosed with a tumor refers to a human in which a neoplastic growth of a tissue which may be either benign or malignant exists and/or has been detected. In some embodiments, the term refers to a cancer patient.
  • a human that potentially has a tumor refers to a human showing symptoms or abnormal tissue growth that are associated with a tumor, in such a human the tumor may have been detected or a physician has reasonable belief that the tumor exists based on clinical presentation.
  • a treatment regimen for prevention of tumor progression in a human by administering a composition comprising a CRF conjugate to a human potentially having the tumor; and monitoring tumor progression in the human.
  • monitoring refers to methods that can be used to determine tumor growth, an increase in tumor size or volume, and/or formation of metastases. These methods comprise imaging technologies including X-ray radiography, computer tomography, and magnetic resonance imaging (MRI); the detection of biomarkers; biopsy procedures; and any other method known to a person of skill in the art, which may be used to determine tumor growth, an increase in tumor size or volume, and/or formation of metastases.
  • the CRF conjugate can be administered over a period of time that exceeds three days, such as for five days or more, for seven to fourteen days or more, for two or three weeks or more, or for a year or more.
  • the CRF conjugate can be administered continuously over that time or may be administered intermittently over that time.
  • the administered dose of a CRF conjugate can be delivered as a single dose (e.g., a single bolus injection) or intermittently by multiple injections or infusions.
  • the administered dose of a CRF conjugate is delivered over a period of time (e.g., continuous infusion).
  • Administration of the CRF conjugate may be continued or repeated until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.
  • the CRF conjugate can be administered either subcutaneously or intravenously. In one embodiment, the CRF conjugate is administered intravenously. In some embodiments, the CRF conjugate is administered by intravenous infusion at a rate of 4 ⁇ g/kg/hr to 100 ⁇ g/kg/hr. In other embodiments, the CRF conjugate is administered by subcutaneous injection. The amount of the CRF conjugate injected may vary. In certain embodiments, the amount of the CRF conjugate administered subcutaneously or intravenously may be in the range of 2 ⁇ g/kg to 1,000 ⁇ g/kg.
  • the total daily dose of the CRF conjugate administered to a patient diagnosed with a tumor may exceed 1 mg.
  • the total daily dose of the CRF conjugate is in the range of 0.5 mg to 20 mg.
  • the total daily dose of the CRF conjugate is in the range of 1 mg to 20 mg.
  • the total daily dose of the CRF conjugate is in the range of 2.5 mg to 20 mg.
  • the total daily dose of the CRF conjugate is in the range of 4 mg to 20 mg.
  • the tumor is a brain tumor.
  • the brain tumor may be a glioblastoma, glioma, ependymoma, astrocytoma, medulloblastoma, neuroglioma, oligodendroglioma or meningioma.
  • the brain tumor may be a secondary brain tumor or a brain metastasis.
  • cancers and tumors that can be treated in accordance with the methods provided herein include, but are not limited to bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, head and neck cancer, liver cancer, lung cancer (both small cell and non-small cell), melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, and uterine cancer.
  • bladder cancer breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, head and neck cancer, liver cancer, lung cancer (both small cell and non-small cell), melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma (including osteosarcoma), skin cancer (including squamous
  • the methods encompass treating or managing colon, pancreas, breast, mesothelioma, cholangiocarcinoma, leiomyosarcoma, liposarcoma, melanoma, nasopharyngeal, neuroendocrine, ovarian, renal, salivary gland, small cell lung cancer, or spindle cell carcinoma.
  • the tumor or cancer to be treated has relapsed or recurred.
  • the term "relapsed” or “recurred” refers to a situation where patients who have had a remission of cancer after therapy have a return of cancer cells.
  • the tumor or cancer to be treated has become refractory or resistant.
  • the term "refractory” or “resistant” refers to a circumstance where patients, even after intensive treatment, have residual cancer cells in their body.
  • the CRF conjugate can be used with or combined with other pharmacologically active compounds ("second active agents"). It is believed that certain combinations work synergistically in the treatment of particular types of cancers.
  • the CRF conjugate can also work to alleviate adverse effects associated with certain second active agents, and some second active agents can be used to alleviate adverse effects associated with CRF.
  • the methods provided herein comprise administering CRF conjugates in combination with one or more second active agents, and optionally in combination with radiation therapy or surgery.
  • the administration of CRF conjugates and the second active agents to a patient can occur simultaneously or sequentially by the same or different routes of administration.
  • the suitability of a particular route of administration employed for a particular active agent will depend on the active agent itself (e.g., whether it can be administered orally without decomposing prior to entering the blood stream) and the disease being treated.
  • Recommended routes of administration for the second active agents are known to those of ordinary skill in the art. See, e.g., Physicians ' Desk Reference, 1755- 1760 (56 th ed., 2002).
  • the second active agent is administered intravenously or subcutaneously and once or twice daily in an amount of from about 1 to about 1 ,000 mg, from about 5 to about 500 mg, from about 10 to about 375 mg, or from about 50 to about 200 mg.
  • the second active agent is rituximab, oblimersen (GENASENSE ® ), GM-CSF, G-CSF, EPO, taxotere, irinotecan, dacarbazine, transretinoic acid, topotecan, pentoxifylline, ciprofloxacin, dexamethasone, vincristine, doxorubicin, COX-2 inhibitor, IL2, IL 8, ILl 8, IFN, Ara-C, vinorelbine, or a combination thereof.
  • the second active agent is etoposide, daunomycin, actinomycin D, mitomycin C, cisplatin, carboplatin, premetrexed, methotrexate, Ara-C, 5-Fu, wortmannin, geldanamycin, gemcitabin, or a combination thereof.
  • kits for treating or managing hematologic malignancies which comprise administering the CRF conjugate in conjunction with (e.g., before, during or after) conventional therapy including, but not limited to, surgery, immunotherapy, biological therapy, radiation therapy, or other non-drug based therapy presently used to treat or manage cancer.
  • conventional therapy including, but not limited to, surgery, immunotherapy, biological therapy, radiation therapy, or other non-drug based therapy presently used to treat or manage cancer.
  • CRP may provide additive or synergistic effects when given concurrently with conventional therapy.
  • the second active agent is co-administered with the CRF conjugate or administered with 1-50 hours delay. In certain embodiments, the CRF conjugate is administered first followed by administration with the second active agent with 1-50 hours delay. In certain embodiments, the second active agent is administered first followed by administration of the CRF conjugate with 1-50 hours delay.
  • the CRF conjugate is administered in an amount from 1 ⁇ g/kg to 1,000 ⁇ g/kg, from 1 ⁇ g/kg to 100 ⁇ g/kg, from 2 ⁇ g/kg to 80 ⁇ g/kg, from 2 ⁇ g/kg to 50 ⁇ g/kg, from 4 ⁇ g/kg to 40 ⁇ g/kg, or from 5 ⁇ g/kg to 20 ⁇ g/kg alone or in combination with a second active agent disclosed herein, prior to, during, or after the use of conventional therapy.
  • the second active agent is temozolomide.
  • the daily dose of temozolomide is from about 1 to about 5,000 mg, from about 1 to about 1,000 mg, or from about 10 to 500 mg per day. In certain embodiments, the daily dose of temozolomide is about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 83 mg, about 90 mg, about 98 mg, about 105 mg, about 112 mg, about 120 mg, about 128 mg, about 135 mg, about 143 mg, about 150 mg, about 158 mg, about 165 mg, about 173 mg, about 180 mg, about 188 mg, about 195 mg, about 200 mg, about 210 mg, about 220 mg, about 225 mg, about 240 mg, about 255 mg, about 260 mg, about 270 mg, about 280 mg, about 285 mg, about 300 mg, about 315 mg, about 320 mg, about 330 mg, about 340 mg, about 345 mg, about 360 mg, about 375 mg, about 380 mg, about 400 mg, about 420 mg, about
  • temozolomide is administered in an amount ranging from about 10 to about 500 mg/m 2 /day, from about 50 to about 250 mg/m /day, or about 75 to about 200 mg/m 2 /day. In certain embodiments, temozolomide is administered in an amount of about 10 mg/m 2 /day, about 20 mg/m 2 /day, about 30 mg/m 2 /day, about 40 mg/m 2 /day,
  • the administered dose can also be expressed in units other than as mg/m 2 /day.
  • doses for parenteral administration can be expressed as mg/kg/day.
  • One of ordinary skill in the art would readily know how to convert doses from mg/m 2 /day to mg/kg/day given either the height or weight of a subject or both (see, www. fda. gov/cder/cancer/animalframe .htm) .
  • temozolomide is cyclically administered. In certain embodiments, temozolomide is administered daily in a single or divided doses for five days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, eight weeks, ten weeks, fifteen weeks, or twenty weeks, followed by a rest period of about 1 day to about ten weeks. In certain embodiments, temozolomide is administered daily in a single or divided doses for five days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, or eight weeks with a rest period of 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 23, 24, 25, 26, 28, 29 or 30 days. In certain embodiments, the rest period is 7 days. In certain embodiments, the rest period is 14 days.
  • the rest period is 23 days. In certain embodiments, the rest period is a period that is sufficient for bone marrow recovery. In certain embodiments, the rest period is a period that is sufficient for neutrophil recovery. In certain embodiments, the rest period is a period that is sufficient for platelet recovery.
  • the frequency, number, and length of dosing cycles can be increased or decreased.
  • temozolomide is administered daily for four weeks, followed by six cycles of maintenance treatment. In certain embodiments, temozolomide in cycle 1 is administered once daily for five days, followed by a rest period of twenty- three (23) days.
  • temozolomide in each of cycles 2 to 6 is administered once daily for five days, followed by a rest period that is sufficient for neutrophil and platelet recovery.
  • each of cycles 2 to 6 starts when absolute neutrophil count (ANC) exceeds 1.5 x 10 9 /L and the platelet count exceeds 100 x 10 /L.
  • the administration of temozolomide during cycles 1 to 6 may be discontinued if ANC is below 1 x 10 9 /L or platelet count is below 50 x 10 9 /L.
  • the dosage in each cycle can be increased or decreased.
  • temozolomide is administered orally at 75 mg/m 2 daily for 42 days concomitant with 400 focal radiotherapy (60 Gy administered in 30 fractions) followed by maintenance treatment.
  • temozolomide is administered for an additional 6 cycles of maintenance treatment.
  • cycle 1 temozolomide is administered at 150 mg/m 2 once daily for 5 days followed by 23 days without treatment.
  • the dose is escalated to 200 mg/m 2 , if the common toxicity criteria (CTC) non-hematologic toxicity for cycle 1 is no greater than Grade 2 (except for alopecia, nausea, and vomiting), absolute neutrophil count (ANC) is no less than 1.5 x 10 9 /L, and the platelet count is no less than 100 x 10 9 /L.
  • CTC common toxicity criteria
  • ANC absolute neutrophil count
  • the dose remains at 200 mg/m 2 per day for the first 5 days of each subsequent cycle except if toxicity occurs. If the dose was not escalated at cycle 2, escalation should not be done in subsequent cycles.
  • the daily dose of temozolomide is adjusted according to neutrophil and platelet counts.
  • suitable second active agents include large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organometallic or organic molecules).
  • large molecule active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies, particularly therapeutic antibodies to cancer antigens.
  • Typical large molecule active agents are biological molecules, such as naturally occurring or artificially made proteins. Proteins that are particularly useful in the methods and compositions provided herein include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunologically active poietic cells in vitro or in vivo.
  • interleukins such as IL-2 (including recombinant IL- II ("rIL2") and canarypox IL-2), IL-IO, IL- 12, and IL-18
  • interferons such as interferon alfa- 2a, interferon alfa-2b, interferon alfa-nl, interferon alfa-n3, interferon beta-I a, and interferon gamma-I b
  • GM-CF and GM-CSF GM-CF and GM-CSF
  • EPO EPO
  • Particular proteins that can be used in the methods and compositions include, but are not limited to: filgrastim, which is sold in the United States under the trade name NEUPOGEN ® (Amgen, Thousand Oaks, CA); sargramostim, which is sold in the United States under the trade name LEUKINE ® (Immunex, Seattle, WA); and recombinant EPO, which is sold in the United States under the trade name EPOGEN (Amgen, Thousand Oaks, CA).
  • Recombinant and mutated forms of GM-CSF can be prepared as described in U.S. patent nos. 5,391,485; 5,393,870; and 5,229,496; all of which are incorporated herein by reference.
  • Recombinant and mutated forms of G-CSF can be prepared as described in U.S. patent nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; all of which are incorporated herein by reference.
  • mutants and derivatives e.g., modified forms
  • mutants include, but are not limited to, proteins that have one or more amino acid residues that differ from the corresponding residues in the naturally occurring forms of the proteins.
  • mutants include proteins that lack carbohydrate moieties normally present in their naturally occurring forms (e.g., nonglycosylated forms).
  • derivatives include, but are not limited to, pegylated derivatives and fusion proteins, such as proteins formed by fusing IgGl or IgG3 to the protein or active portion of the protein of interest. See, e.g., Penichet, M. L. and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).
  • Antibodies that can be used in combination with the CRF conjugate include monoclonal and polyclonal antibodies.
  • Examples of antibodies include, but are not limited to, trastuzumab (HERCEPTIN ® ), rituximab (RITUXAN ® ), bevacizumab (AVAST ⁇ NTM), pertuzumab (OMNITARGTM), tositumomab (BEXXAR ® ), edrecolomab (PANOREX ® ), and G250.
  • CRF can also be combined with or used in combination with, anti-TNF- ⁇ antibodies.
  • Large molecule active agents may be administered in the form of anti-cancer vaccines.
  • cytokines such as IL- 2, G-CSF, and GM-CSF
  • Second active agents that are small molecules can also be used to alleviate adverse effects associated with the administration of the CRF conjugate. However, like some large molecules, many are believed to be capable of providing a synergistic effect when administered with (e.g., before, after or simultaneously) the CRF conjugate. Examples of small molecule second active agents include, but are not limited to, anti-cancer agents, antibiotics, immunosuppressive agents, and steroids.
  • anti-cancer agents include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor
  • anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP
  • Specific second active agents include, but are not limited to, rituximab, oblimersen (GENASENSE ), remicade, docetaxel, celecoxib, melphalan, dexamethasone (DECADRON ® ), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temozolomide (TEMODAR "), carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, ARISA ® , taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEG INTRON- A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, lip
  • the second active agent is etoposide, daunomycin, actinomycin D, mitomycin C, cisplatin, carboplatin, premetrexed, methotrexate, Ara-C, 5-Fu, wortmannin, gemcitabin, geldanamycin or a combination thereof.
  • the methods provided herein comprise: a) administering to a patient in need thereof, a dose of about 1 mg to 20 mg of a CRF conjugate; and b) administering a therapeutically effective amount of a supportive care agent.
  • the supportive care agent refers to any substance that treats, prevents or manages an adverse effect from CRF treatment.
  • the supportive care agent is any substance that treats, prevents or manages an adverse effect from CRF treatment and is administered according to the appropriate dosing regimen for that substance.
  • different supportive care agents for treating nausea have different dosing regimen. While some are administered prophylactically, others are coadministered with a CRF conjugate while still others are administered after the administration of a CRF conjugate.
  • Illustrative examples of supportive care agents their doses and dosing regimens are found in The Physician's Desk Reference. 5.5 Methods of Treating Edema
  • the present invention is also directed to methods of treating edema.
  • the methods described herein include methods of treating edema comprising administering to a patient in need thereof a pharmaceutical composition comprising a CRF conjugate.
  • the CRF conjugate is CRF chemically modified with polyethylene glycol.
  • the present invention is also directed to methods of treating brain edema comprising administering CRF conjugate, wherein the conjugate is administered in a treatment regimen as a steroid sparing agent facilitating steroid taper.
  • the methods described herein include methods for managing brain edema in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CRF conjugate and a steroid, wherein said method provides a steroid sparing effect.
  • the CRF conjugates described here can be co-administered with any steroid including glucocorticoids, which are a class of steroid hormones characterized by an ability to bind with the Cortisol receptor.
  • Glucocorticoids steroids include hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, aldosterone and deoxycorticosterone acetate.
  • the methods described herein include methods for treating brain edema comprising a treatment regimen comprising administering to in a patient in need thereof a steroid in combination with a CRF conjugate, whereby total exposure to the steroid is reduced by the administration of the CRF conjugate.
  • the present invention also includes methods for providing replacement therapy for steroid therapy in a subject receiving such therapy, said method comprising administration of a steroid-sparing amount of CRF conjugate.
  • the total daily dose of the CRF conjugates described herein, such as CRF chemically modified with polyethylene glycol can range from 1 ⁇ g to 10 mg. In certain embodiments the total daily dose of CRF conjugate can be 0.1 mg to 5 mg, or 0.3 mg to 2 mg. For example, the total daily dose of CRF chemically modified with polyethylene glycol can be about 0.3 mg, about 0.5 mg, about 1 mg, about 2 mg, about 4 mg or about 5 mg.
  • the CRF conjugate can be administered once a day or multiple times a day until the desired daily dose of the CRF conjugate is reached.
  • 0.5 mg or 1.0 mg of a CRF conjugate can be administered 4 times a day to achieve a total daily dose of 2 mg or 4 mg of the CRF conjugate.
  • routes of administration of the CRF conjugate include parenteral routes such as, but not limited to, intradermal, subcutaneous, and intramuscular injections, and intravenous or intraosseous infusions.
  • the compositions of the present invention can take the form of solutions, suspensions, emulsions that include a CRF conjugate, such as CRF chemically modified with polyethylene glycol, and a pharmaceutically acceptable diluent, adjuvant or carrier, depending on the route of administration.
  • the CRF conjugates described herein can be administered by subcutaneous injection in an amount of 0.1 ⁇ g/kg to 1000 ⁇ g/kg.
  • CRF conjugates can be administered subcutaneously in an amount of 1 ⁇ g/kg to 500 ⁇ g/kg, or 2 ⁇ g/kg to 100 ⁇ g/kg, or 2 ⁇ g/kg to 80 ⁇ g/kg, or 4 ⁇ g/kg to 40 ⁇ g/kg, or 5 ⁇ g/kg to 20 ⁇ g/kg.
  • CRF conjugates can be administered in 10 ⁇ g/kg, 30 ⁇ g/kg, 60 ⁇ g/kg, 100 ⁇ g/kg and 300 ⁇ g/kg doses.
  • the CRF conjugates described herein can be administered by subcutaneous injection in an amount of 1 ⁇ g to 100 mg.
  • CRF conjugates can be administered subcutaneously in an amount of 1 ⁇ g to 80 mg, 10 ⁇ g to 50 mg, 100 ⁇ g to 40 mg, 300 ⁇ g to 10 mg, 600 ⁇ g to 1 mg, and 800 ⁇ g to 1 mg.
  • CRF conjugates can be administered subcutaneously in 100 ⁇ g, 300 ⁇ g, 600 ⁇ g, 1 mg, 2 mg, 4 mg and 5 mg doses.
  • the CRF conjugates administered subcutaneously can be administered once a day or multiple times a day.
  • the dosages of CRF conjugates administered subcutaneously can be administered every hour, every two hours, every three hours, every four hours, every six hours, every eight hours or every 12 hours.
  • the CRF conjugates can be administered once every two, three, four, five or six days.
  • the CRF conjugates can be administered once a week, once every two, three or four weeks or once a month. Dosages of CRF conjugates that are administered once a week or longer can be administered in the form of a depot.
  • the CRF conjugates can be administered by intravenous infusion in an amount of 0.1 ⁇ g/kg/h to 100 ⁇ g/kg/h.
  • CRF conjugates can be administered intravenously in an amount of 1 ⁇ g/kg/h to 100 ⁇ g/kg/h, or 2 ⁇ g/kg/h to 80 ⁇ g/kg/h, or 2 ⁇ g/kg/h to 50 ⁇ g/kg/h, or 4 ⁇ g/kg/h to 40 ⁇ g/kg/h, or 5 ⁇ g/kg/h to 20 ⁇ g/kg/h.
  • the CRF conjugates can be administered intravenously in an amount of 1 ⁇ g/kg to 1000 ⁇ g/kg.
  • CRF conjugates can be administered intravenously in an amount of 1 ⁇ g/kg to 100 ⁇ g/kg, or 2 ⁇ g/kg to 80 ⁇ g/kg, or 2 ⁇ g/kg to 50 ⁇ g/kg, or 4 ⁇ g/kg to 40 ⁇ g/kg, or 5 ⁇ g/kg to 20 ⁇ g/kg.
  • CRF conjugates can be administered in 0.5 ⁇ g/kg to 1 ⁇ g/kg, or 2 ⁇ g/kg to 8 ⁇ g/kg, or 4 ⁇ g/kg to 8 ⁇ g/kg, or 5 ⁇ g/kg doses.
  • the CRF conjugates described herein can be administered intravenously over a period of an hour or less than an hour. In certain embodiments the CRF conjugates can be administered intravenously over a period of one hour or more.
  • the dosages of CRF chemically modified with polyethylene glycol administered intravenously, discussed above can be administered over a period of 10 min., 30 min., 45 min., one hour, two hours, four hours, eight hours, 12 hours, 24 hours, 48 hours or 72 hours.
  • Dosing regimens include administration of the CRF conjugates of the invention every other day or once weekly to a patent suffering from edema resulting from disease or injury to the brain or nervous system.
  • the present invention relates to pharmaceutical compositions containing a
  • the CRF conjugate may be formulated with a pharmaceutically acceptable carrier. Due to the increased half-life of the CRF conjugate, the pharmaceutical compositions may contain a lower dose of CRF than typically administered to effectively treat edema.
  • the pharmaceutical formulations of the invention may be formulated for parenteral administration, including, but not limited to, intradermal, subcutaneous, and intramuscular injections, and intravenous or intraosseous infusions.
  • the pharmaceutical formulations of the present invention can take the form of solutions, suspensions, emulsions that include a CRF conjugate, such as CRF chemically modified with polyethylene glycol, and a pharmaceutically acceptable diluent, adjuvant or carrier, depending on the route of administration.
  • the pharmaceutical compositions of the invention are formulated to deliver a therapeutic dose of the CRF conjugate of the invention.
  • the dose of the CRF conjugates contained in pharmaceutical formulation can range from 1 ⁇ g to 10 mg. In certain embodiments the dose of the CRF conjugate can range from 0.1 mg to 5 mg, or 0.3 mg to 2 mg. In certain embodiments, the dose of the CRF conjugate can be about 0.3 mg, about 0.5 mg, about 1 mg, about 2 mg, about 4 mg or about 5 mg.
  • the present invention is also directed to methods of treating edema by administering to a patient in need thereof a CRF conjugate and an additional therapeutic agent.
  • the additional therapeutic agent can be any agent that can alleviate edema or when in combination with the CRF conjugate, improve the conjugate's effect on edema or wherein the CRF conjugate can improve the effect of the additional therapeutic agent on the edema.
  • Suitable additional therapeutic agents include anti-inflammatory agents such as, but not limited to, corticosteroids.
  • Corticosteroids include glucocorticoids and mineralocorticoids such as alclometasone, aldosterone, amcinonide, beclometasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone, desoxycortone, dexamethasone, difiorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fiuocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocor
  • Suitable additional agents also include diuretics such as loop diuretics, osmotic diuretics proximal diuretics, distal convoluted tubule diuretics and cortical collecting tubule diuretics.
  • suitable diuretics include, but are not limited to, glucose, mannitol, bumetanide, ethacrynic acid, furosemide, torsemide, amiloride, spironolactone, triamterene, bendroflumethiazide, hydrochlorothiazide, acetazolamide, dorzolamide, Phosphodiesterase, chlorthalidone, caffeine, metolazone or a combination thereof.
  • Additional agents that can be co-administered with the CRF conjugate include anti-neoplastic, antiproliferative, anti-miotic agents such as paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones, methotrexate, azathioprine, adriamycin and mutamycin; endostatin, angiostatin and thymidine kinase inhibitors, cladribine, taxol and its analogs or derivatives, paclitaxel as well as its derivatives, analogs or paclitaxel bound to proteins.
  • anti-neoplastic, antiproliferative, anti-miotic agents such as paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones, methotrexate, azathioprine, adriamycin and mutamycin
  • CRF conjugates described herein can be co-administered with other anti-cancer treatments such as, radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy.
  • the CRF conjugate and the additional therapeutic agent can be administered sequentially or simultaneously. If administered sequentially, the order of administration is flexible. For instance, the CRF conjugate can be administered prior to administration of the additional therapeutic agent. Alternatively, administration of the additional therapeutic agent can precede administration of the CRP conjugate.
  • each composition is preferably pharmaceutically suitable for administration.
  • the CRF conjugate and the therapeutic agent if administered separately, can be administered by the same or different modes of administration.
  • the CRF conjugates of the invention can be readily synthesized using synthetic methods known in the art.
  • the following synthetic examples demonstrate the syntheses of CRF-PEG conjugates, including CRF-PEG conjugates of cysteine added variants of CRF.
  • the alkylation of the ⁇ -amino group of the lysine residue in hCRF can be accomplished via reductive alkylation using PEG-propionaldehyde as the PEGylation agent.
  • Human-CRF (1 mg) is stirred with an excess of PEG-propionaldehyde (3 mg) and a slight molar excess of sodium cyanoborohydride at room temperature in pH 9 borate buffer. High pH is used to avoid reduction of the aldehyde before Schiff base formation.
  • the mixture undergoes dialysis against phosphate buffered saline.
  • the CRF-PEG conjugate migrates to the top phase, while the unmodified CRF migrates to the bottom phase.
  • the desired CRF-PEG conjugate may be further isolated by gel filtration chromatography.
  • reagents that can be employed to covalently bind a cysteine residue to polyethylene glycol.
  • This example employs PEG-maleimide or maleimido-PEG as the PEGylation reagent.
  • a cysteine added variant of CRF is diluted to 200 ⁇ g/mL in 20 niM Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES) pH 6.75 buffer, 0.6 M NaCl, and 1% glycerol.
  • PEG-maleimide or maleimido-PEG is diluted to 200 ⁇ g/mL in 20 niM Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES) pH 6.75 buffer, 0.6 M NaCl, and 1% glycerol.
  • PPES Piperazine-l,4-bis(2-ethanesulfonic acid)
  • Maleimido-PEG (l ⁇ l) is dissolved in a
  • the maleimdo-PEG may be diluted until the desired concentration is reached for reaction, and then it is added to the solution of CRP. Up to a 20-fold excess of maleimido-PEG may be used. The reaction is allowed to occur at room temperature for one hour, but the reaction may also occur at 4°C with longer reaction times. Upon completion the resulting cysteine added variant CRF-PEG conjugate may be purified by gel filtration chromatography.
  • the disulfide bond can be reduced using phosphine-containing reducing reagent, including tributylphosphine.
  • phosphine-containing reducing reagent including tributylphosphine.
  • the conjugation of the reduced cys-hCRF-cys and ⁇ -methoxy- ⁇ -4-[2,2-bis[(p-tolylsulfonyl)- methyl] acetyl] benzamide derived from poly(ethylene)glycol can be carried out in the presence of the phosphine-containing reducing reagent.
  • the partially reduced cys-hCRF-cys is dissolved in argon purged pH 8 ammonia solution.
  • the polymer conjugating reagent, ⁇ - methoxy- ⁇ -4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide derived from poly(ethylene)glycol is also dissolved in ammonia solution and the resulting solution is added to the Factor IX solution.
  • the PEG eppendorf is washed with fresh ammonia solution and this is also added to the main reaction eppendorf.
  • reaction eppendorf is then closed under argon and heated at 37°C for approximately 24 h and then allowed to cool to room temperature.
  • the cooled reaction solution is then analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE).
  • the N-terminus of unmodified hCRF is a serine residue, so it is to the ⁇ -amino group of serine that a cysteine residue is bound.
  • a cysteine residue protected by S-2,4,6- trimethoxybenzyl (Tmob) is added to a solution of N-terminal deprotected CRF in a solution of dichloromethane/DMF in a ratio of 3 : 1.
  • the coupling reaction can be monitored by the ninhydrin test for completion. Once complete, the solid phase is washed with dichloromethane and methanol, and an additional wash with DMF can be performed after this coupling step, to yield the solid phase coupled intermediate above.
  • cysteine is the last amino acid added. Once coupled, removal from the solid support is accomplished with anhydrous trifluroacetic acid, followed by universal deprotection of all of the protecting groups on side chains, yielding an N-terminal cysteine added variant of CRF.
  • the final polypeptide can be isolated by gel filtration chromatography. Insertions and substitutions of additional cysteine residues may be accomplished by similar preparations in the desired locations of the CRF polypeptide.
  • hCRF-cys or cys-hCRF-cys The C-terminus of unmodified hCRF is an isoleucine residue, so it is to the ⁇ - amino group of the C-terminal cysteine that an isoleucine residue is bound, A cysteine residue protected by S-2,4,6-trimethoxybenzyl (Tmob) is attached to the resin polymer.
  • Isoleucine is added to the solution with DCC and lH-benzo[d][l,2,3]triazol-l-ol in dichloromethane/DMF in a ratio of 3 : 1.
  • the coupling reaction can be monitored by the ninhydrin test for completion. Once complete, the solid phase is washed with dichloromethane and methanol, and an additional wash with DMF can be performed after this coupling step, to yield the solid phase coupled intermediate above.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF.
  • the biological activity of the CRF conjugates may be determined using the bioassays described herein.
  • the CRF conjugates may have the same level of biologic activity as compared to unmodified CRF. Alternatively, the CRF conjugates may have lower levels of biologic activities when compared to unmodified CRF.
  • the following is a bioassay for CRF.
  • the bioassay is to be based upon the binding of radio-labeled human CRF to its receptor on the cellular membrane of AtT-20 cells, a mouse pituitary cell line, or cells derived from the AtT-20 parental cell line.
  • the assay is a competitive binding radio-receptor assay (RRA) that can discriminate between human CRF and closely related molecules.
  • RRA radio-receptor assay
  • Whole cells or homogenized cell membrane preparations may be used in the assay.
  • a competitive binding RRA was developed using 100 ⁇ l of membrane preparation, 100 ⁇ l of radio-labeled human CRF as tracer, and 100 ⁇ l of either buffer or competitor.
  • the data obtained is expressed as Percent B/Bo, where B is the corrected CPM for the sample and Bo is the corrected CPM for the total binding tubes (i.e. no competitor).
  • B is the corrected CPM for the sample
  • Bo is the corrected CPM for the total binding tubes (i.e. no competitor).
  • This bioassay for CRF is based upon the ability of known membrane receptors for CRF to bind I-Tyr -hCRF and to be displaced by unlabeled competitors.
  • This type of assay is typically called a competitive binding radio-receptor assay (RRA).
  • RRA radio-receptor assay
  • the unlabeled competitors that are of interest are different batches of hCRF (active drug substance), different lots of formulated drug product that contain hCRF, and CRF-related molecules, such as potential impurities in the active drug substance and known degradation products.
  • hCRF active drug substance
  • CRF-related molecules such as potential impurities in the active drug substance and known degradation products.
  • various cell lines have been found to express one or more of the CRF receptor subtypes and have been used to measure the effects of CRF, CRF-related peptides, and various agonists and antagonists.
  • AtT-20 cells a mouse anterior pituitary cell line, has been reported to express only CRF Rl, and when CRF binds, an accumulation in intracellular cAMP and an increase in ACTH secretion are observed.
  • CRF Rl CRF Receptor Type 1
  • CRF R2 CRF Receptor Type 2
  • the two types of receptors share -70% sequence homology and both are coupled to adenylate cyclase. However, the two types of receptors have different tissue distributions and bind ligands with different affinities.
  • urocortin Ucn
  • Ucn II Ucn II
  • Ucn III which is also known as stresscopin.
  • CRF plays a central role in the control of the hypothalamic-pituitary-adrenal axis under stress.
  • Ucn is a 40-amino acid long peptide with 45% sequence homology to CRF that has been cloned from the Edinger- Westphal nucleus
  • Ucn II (with 26% sequence homology to CRF) and III have been identified in human and mouse genomic data banks, and all have potent effects on appetite and on the cardiovascular system.
  • the CRF R2 has at least two and possibly three different splice variants - CRF R2 ⁇ and CRF R2 ⁇ and maybe CRF R2 ⁇ - which are expressed in different tissues and organs.
  • CRF R2 ⁇ is predominately found in the brain including the hypothalamus, lateral septum, raphe nuclei of the mid-brain, olfactory bulb, and pituitary.
  • CRF R2 ⁇ is predominately found in the heart, blood vessels, GI tract, and cardiac and skeletal muscle.
  • CRF binding protein In addition to the receptors, a CRF binding protein has been described that binds native CRF with a higher affinity than do any of the cellular receptors.
  • the CRF binding protein is expressed in the brain and it might function as a regulator of CRF-mediated neurotransmission.
  • CRF and CRF-related peptides exert their effect through a cAMP-dependent protein kinase (PKA) pathway in the anterior pituitary and in AtT-20 cells.
  • PKA protein kinase
  • the connection between the changes in the intracellular cAMP concentration and the stimulation of ACTH secretion results from the interaction between cAMP and the concentration of free calcium ion in the cytosol.
  • cAMP plays two major roles (1) to increase the influx of calcium ion into the cell which stimulates secretion and (2) to potentiate the effects of the increased intracellular calcium level on the secretory apparatus.
  • CRF is reported to be specific for activation through its interaction with CRF Rl type receptors: as reported in the literature, it does not activate cells through either the CRF R2 ⁇ or CRF R2 ⁇ receptor subtypes.
  • the 125 I-Tyr°-CRF is eluted from the column with 50% AcOH and 0.5 mL fractions are collected.
  • the radio-labeled peptide elutes immediately after the void volume of the column in fractions #4 and 5.
  • the two fractions are pooled and the radio-labeled peptide is used without further purification or is purified by reverse phase HPLC if monoiodo-peptide is desired. [0182] 4.
  • the cell pellet is resuspended in a small volume (1.5 - 2.0 mL) of ice-clod PBS with 1%BSA and 20 ⁇ g/mL aprotinin, transferred to a 15 mL glass Dounce homogenizer fitted with a tight pestle on ice, and homogenized by 15 strokes with grinding.
  • the lysed cells are transferred to microfuge tubes and centrifuged at 16,000 x g for 15 min at 4 0 C to collect the particulate membrane fraction. The supernatant is discarded, the particulate fraction is washed by resuspending it in the same ice-cold buffer, and collecting the washed particulate membrane fraction by centrifugation.
  • the membrane fraction is resuspended to a volume equal to 5 x 10 cells per mL based upon the number of cells originally isolated and homogenized.
  • the assay reaction is set up as described above (see 5.b) except 100 ⁇ l of suspended particulate fraction is used instead of whole cells and the buffer is PBS with 1% BSA and 20 ⁇ g/mL aprotinin.
  • the protease inhibitor is included to protect the labeled tracer and competitors from degradation during the overnight incubation.
  • the use of the particulate fraction has improved the reproducibility of the assay in which the displacement of trace by unlabeled CRF is measured in the particulate fraction obtained from the 4 different clones we isolated.
  • the CRF conjugates of the invention have one or more of the biological activities of unmodified CRF, e.g. the ability to competitively bind to the CRF receptor.
  • the CRF conjugates may demonstrate differing levels of activity to unmodified CRF.
  • the CRF conjugates of the invention have an improved pharmacokinetic profile as compared to unmodified CRF.
  • the CRF conjugates of the invention may show an improvement in one or more parameters of the pharmacokinetic profile, including AUC, C max j clearance (CL), half-life, and bioavailability as compared to unmodified CRF.
  • AUC AUC
  • half-life a parameter of the pharmacokinetic profile
  • bioavailability as compared to unmodified CRF.
  • the following is an example of the determination of the pharmacokinetic profile of unmodified CRF when administered subcutaneously and intravenously.
  • the objective of this study was to determine the plasma concentration time profile of hCRF following a single intravenous and a single subcutaneous injection in three groups of Sprague-Dawley Crl:CD ® BR rats.
  • Concentrations of hCRF in the vehicle were 10,100, and 1,000 ⁇ g/mL.
  • a dosage volume of 1 mL/kg for all groups resulted in administered doses of 10,100, and 1,000 ⁇ g/kg of hCRF for all three dose groups.
  • each of the three dose groups consisted of 72 males. Each of these groups was divided into three sets of replicates.
  • Plasma samples were prepared and hCRF concentrations in the plasma samples were determine by an ELISA method.
  • the clearance of intravenously administered hCRF in the rat followed a single exponential pattern and the half-lives were determined to be 9.2, 20.7 and 26.7 minutes for doses of 10,100, and 1,000 ⁇ g/kg, respectively.
  • the pharmacokinetics of hCRF administered either intravenously or subcutaneously is dose proportional between 100 and 1,000 ⁇ g/kg. At the 10 ⁇ g/kg intravenous dose level, the measured hCRF in plasma concentrations approached the detection limits of the ELISA assay. Pharmacokinetic analyses were conducted for this dose group using the limited data obtained.
  • the pharmacokinetic values for the 10 ⁇ g/kg intravenous dose group differ from those for the 100 and 1,000 ⁇ g/kg groups. This may be a function of the limitations of the ELISA assay at these low levels, and/or may be due to the saturation of potential binding sites for hCRF at the higher doses.

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Abstract

La présente invention concerne des conjugués de CRF qui ont été modifiés de manière à comprendre un fragment qui protège CRF contre la dégradation et prolonge la demi-vie de CRF. Les conjugués de CRF de l’invention ont une demi-vie augmentée qui conduit à un effet d’épargne de dose et une administration moins fréquente.
PCT/EP2009/065513 2008-11-19 2009-11-19 Conjugués de crf ayant des demi-vies prolongées WO2010057962A2 (fr)

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US9650331B2 (en) 2012-06-18 2017-05-16 Polytherics Limited Conjugation reagents
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